WO2016022958A2 - Tick-associated virus sequences and uses thereof - Google Patents

Tick-associated virus sequences and uses thereof Download PDF

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Publication number
WO2016022958A2
WO2016022958A2 PCT/US2015/044284 US2015044284W WO2016022958A2 WO 2016022958 A2 WO2016022958 A2 WO 2016022958A2 US 2015044284 W US2015044284 W US 2015044284W WO 2016022958 A2 WO2016022958 A2 WO 2016022958A2
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seq
sequence
nos
nucleic acid
tick
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PCT/US2015/044284
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French (fr)
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WO2016022958A3 (en
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W. Ian Lipkin
Rafal TOKARZ
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2016022958A2 publication Critical patent/WO2016022958A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12211—Phlebovirus, e.g. Rift Valley fever virus
    • C12N2760/12221—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • Ticks (class Arachnida, subclass Acari) have been implicated as vectors in a wide range of human and animal diseases worldwide. Approximately 900 species of ticks have been described and taxonomically classified into three families: Argasidae (argasid or soft ticks) Ixodidae (ixodid, or hard ticks) and Nuttalliellidae. Their propensity for feeding on a wide array of hosts, expansive range and long life cycle underscore the importance of tick surveillance for the presence of potential pathogens. Argasid and ixodid ticks combined transmit a greater diversity of viral, bacterial and protozoan pathogens than any other arthropod vector.
  • tick-borne disease is increasing, partly due to increased frequency of endemic tick-borne diseases, as well as the discoveries of new tick-associated agents.
  • tick-associated viruses There is a need to identify tick-associated viruses.
  • an immunogenic composition and a method of treating humans and animals (e.g. tick hosts) having tick-associated viral infections there is a need for immunogenic compositions that protect against tick-associated viruses. This invention addresses these needs.
  • the invention is related to novel tick-associated viruses and isolated nucleic acids sequences and peptides thereof.
  • the invention is also related to antibodies against antigens derived from the tick-associated viruses.
  • the invention is also related to iRNAs which target nucleic acid sequences of the tick-associated viruses.
  • the invention is related to methods for detecting the presence or absence of a tick-associated virus in a tick, an animal or a human.
  • the invention is also related to immunogenic compositions for inducing an immune response against a tick-associated virus or viruses in an animal or human.
  • the invention relates to an isolated nucleic acid having the sequence of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated nucleic acid complementary to the sequence of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated nucleic acid having at least about 60% sequence identity to SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In certain aspects, the invention relates to an isolated nucleic acid having at least about 85% sequence identity to SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • the isolated nucleic acid has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention relates to an isolated nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated nucleic acid having at least about 85% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the isolated nucleic acid has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%), about 99.5%) or about 99.9%> sequence identity to a nucleic acid complementary to the sequence of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated nucleic acid comprising at least 10 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42. [0012] In certain aspects, the invention relates to an isolated nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated nucleic acid which comprises consecutive nucleotides having a sequence complementary to an isolated nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or an isolated nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 85%> identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the isolated nucleic acid comprises at least 10 consecutive nucleotides of a sequence having at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the nucleic acid is a DNA sequence. In some embodiments, the nucleic acid is an RNA sequence. In some embodiments, the nucleic acid is a cDNA.
  • the invention relates to an isolated polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated polypeptide encoded by a nucleic acid described herein.
  • the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated polypeptide having at least about 85 > sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42.
  • the isolated polypeptide has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%), about 99.5%) or about 99.9%> sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated polypeptide having at least about 85% sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the isolated polypeptide has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9%) sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42.
  • the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 85%> identity to the sequence of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42.
  • the isolated polypeptide comprises at least 8 amino acids having at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%), about 99%>, about 99.5%> or about 99.9%> sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 85%> identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the isolated polypeptide comprises at least 8 amino acids having at least about 90%>, about 95.5%>, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%) or about 99.9% sequence identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention relates to an isolated polypeptide having the sequence of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain aspects, the invention relates to an isolated polypeptide having at least about 85% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the isolated polypeptide has at least about 90%>, about 95.5%, about 96%>, about 96.5%>, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 85% identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the isolated polypeptide comprises at least 8 amino acids having at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention relates to an isolated antibody that specifically binds to a polypeptide of the invention (e,g a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a polypeptide encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or sequences complementary thereto).
  • a polypeptide of the invention e,g a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a polypeptide encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or sequences complementary thereto.
  • the invention relates to an immunogenic composition
  • an immunogenic composition comprising at least about 24 consecutive nucleotides from an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or nonisolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42
  • the invention relates to an immunogenic composition
  • an isolated (or non-isolated) polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 ; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention relates to a method of inducing an immune response in an animal, the method comprising administering the immunogenic composition described herein.
  • the invention relates to an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent.
  • the polypeptide is L segment protein, a N protein, a polymerase protein or a nucleocapsid protein.
  • the composition is for protecting an animal against a tick-associated virus.
  • the animal is a human.
  • the composition delays the onset of symptoms associated with a tick-associated virus, or reduces the severity of symptoms of a tick-associated virus.
  • the polypeptide is derived from a polypeptide sequence having at least 90% identity to the amino acid sequence SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the composition further comprises an adjuvant.
  • the invention relates to a method for determining the presence or absence of a tick-associated virus in a biological sample, the method comprising: (a) contacting nucleic acid from a biological sample with at least one primer which is a synthetic nucleic acid of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid
  • nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or
  • the invention relates to a synthetic nucleic acid comprising at least about 10 nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 19,21,23,35,
  • the invention relates to a cDNA oligonucleotide probe comprising from about 10 nucleotides to about 50 nucleotides, wherein at least about 10 contiguous nucleotides are at least 95 % complementary to a nucleic acid target region within the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention relates to a synthetic nucleic acid comprising the nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,
  • the invention relates to a primer set for determining the presence or absence of a tick-associated virus in a biological sample, wherein the primer set comprises at least one synthetic nucleic acid sequence described herein.
  • the invention relates to a method for determining whether or not a sample contains a tick-associated virus, the method comprising: (a) contacting a sample with an antibody that specifically binds to a polypeptide of any of an isolated (or non-isolated) polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80%
  • the invention relates to a method for determining whether or not a sample contains a tick-associated virus, the method comprising: (a) contacting a sample with an antibody that specifically binds to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; or an isolated (or non-isolated)
  • the determining comprises use of a lateral flow assay or ELISA.
  • the invention relates to a method for determining whether or not a biological sample has been infected by a tick-associated virus, the method comprising: determining whether or not a biological sample contains antibodies that specifically bind to a polypeptide of any one of an isolated (or non-isolated) polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having
  • the invention relates to a method for determining whether or not a biological sample has been infected by a tick-associated virus, the method comprising: determining whether or not a biological sample contains antibodies that specifically bind to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; or an isolated (or non-isolated) polypeptid
  • the invention relates to an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent.
  • the polypeptide is L segment protein.
  • the polypeptide is a N protein.
  • the polypeptide is a polymerase protein.
  • the polypeptide is a nucleocapsid protein.
  • the composition is for protecting an animal against a tick-associated virus.
  • the composition further comprises an adjuvant.
  • the composition protecting an animal against a tick-associated virus delays the onset of symptoms associated with a tick- associated virus, or reduces the severity of symptoms of a tick-associated virus.
  • the animal is a human.
  • the invention relates to a method of immunizing an animal against a tick- associated virus, the method comprising administering to the animal an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent.
  • the animal is a human.
  • the invention relates to an interfering RNA (iRNA) comprising at least 15 contiguous nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9,
  • the invention relates to an interfering RNA (iRNA) comprising a sense strand having at least 15 contiguous nucleotides complementary to the anti-sense strand of a gene from a virus comprising a nucleic acid sequence selected from the group of sequences consisting of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • iRNA interfering RNA
  • the invention relates to a method for reducing the levels of a tick- associated virus protein in an animal, viral mRNA in an animal or viral titer in a cell of an animal, the method comprising administering to the animal an iRNA described herein.
  • the invention relates to an isolated virus comprising at least 24 consecutive nucleotides from an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%) sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31
  • the invention relates to an isolated cell comprising at least 8 consecutive amino acids from a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or a polypeptide having at least about 80%> identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention relates to a method for culturing cells comprising: a) infecting a cell with a tick-associated virus, or an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid having at least about 60%> sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs:
  • the invention relates to a method of testing a tick-associated virus vaccine, comprising: a) contacting cells with a tick-associated virus vaccine; b) contacting cells with a tick-associated virus; and c) measuring the number of cells infected with a tick-associated virus.
  • the invention relates to a method of testing a tick-associated virus drug, comprising: a) contacting cells with a tick-associated virus drug; b) contacting cells with a tick- associated virus; and c) measuring the number of cells infected with a tick-associated virus.
  • the invention relates to a method of testing an a tick-associated virus drug, comprising: a) contacting cells with a tick-associated virus; b) contacting cells with an a tick- associated virus drug; and c) measuring the replication of a tick-associated virus.
  • Figure 1 shows a map of tick collection sites.
  • Figure 2 shows a maximum likelihood phylogenetic tree based on the nucleotide sequence of the complete POWV polyprotein. The accession number, geographical location and year of isolation/detection is indicated for each isolate. SEQ ID NO: 1, described herein, is indicated by *. Tick-borne encephalitis virus is shown as an outgroup.
  • Figure 3 shows a maximum likelihood phylogeny of all available nairovirus sequences based on a conserved 150 amino acid fragment of the L protein. Accession numbers are provided next to the virus name. * indicates the South Bay virus described herein.
  • Figures 4A-B show maximum likelihood phylogeny based on complete L (A) and N (B) protein of all nairoviruses with available genomic sequences.
  • Figure 5 shows alignment of the conserved C terminal domain present in all Nairovirus N proteins. conserveed residues are indicated by *. Residues implicated in N function are indicated in bold. The numerical amino acid positions within the N are displayed for each virus.
  • Figure 6 shows comparison of the S segments of SBV and Dugbe virus. The location of the N ORFs are shown in green. Putative additional SBV ORFs are shown in red. Numbers indicate nt position within the segment.
  • Figure 7 shows a schematic of the S segments of BTPV-1, BTPV-2 and ADTPV.
  • the putative N ORFs are indicated in yellow.
  • the putative ORFs corresponding to the genomic location of the NSs are displayed in red.
  • Figures 8A-B show maximum likelihood phylogeny of BTPV- 1 , BTPV-2 and ADTPV estimated from amino acid sequences for phlebovirus N (A) and L (B). Accession numbers for every virus in the tree are indicated. * designates viruses described herein.
  • Figure 9 shows phylogeny of the novel /. scapularis-associated mononegavirus, indicated by *, to viruses within Mononegaviridae.
  • Each terminal branch represents a single type- species representative of a genus.
  • Figure 10 shows a comparison of the tetravirus-like virus replicase from D. variablis to helicoverpa armigera stunt virus. Numbers indicate amino acid position.
  • ticks A wide range of bacterial pathogens have been described in ticks, yet the diversity of viruses in ticks is largely unexplored.
  • Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. Described herein are the sequences of several tick-associated viruses.
  • SBV refers to isolates of the South Bay Virus described herein.
  • BTPV refers to isolates of the blacklegged tick phlebovirus described herein.
  • ADTPV refers to isolates of the American dog tick phlebovirus described herein.
  • ISAV refers to isolates of the Ixodes scapularis associated virus described herein.
  • animal refers to a vertebrate, including, but not limited to, birds, canines, felines, equine, sheep, cattle, poultry or humans.
  • immunogenic composition refers to a composition capable of inducing an immunogenic response in an animal or a cell.
  • immunogenic composition can include include include a vaccine.
  • the present invention provides tick-associated virus nucleic acid sequences. These nucleic acid sequences may be useful for, inter alia, expression of tick-associated virus-encoded proteins or fragments, variants, or derivatives thereof, generation of antibodies against tick- associated virus proteins, generation of primers and probes for detecting tick-associated virus and/or for diagnosing tick-associated virus infection, generating immunogenic compositions against tick-associated viruses, and screening for drugs effective against tick-associated viruses as described herein.
  • the present invention also provides methods for diagnosing tick-associated viral infection.
  • the present invention also provides methods of detecting antibodies against tick- associated viruses in a sample from an animal.
  • the invention is directed to a Powassan virus isolate LI-1 nucleic acid sequence (including cDNA sequences corresponding to Powassan RNA sequences, including mRNA sequences).
  • the complete nucleotide coding sequence (cDNA) of Powassan virus isolate LI-1 polyprotein is shown in SEQ ID NO: 1. Sequence information related to Powassan virus isolate LI-1 is accessible in public databases by GenBank Accession Number KJ746872.
  • SEQ ID NO: 1 is the nucleic acid sequence of the complete coding sequence (cDNA) of Powassan virus isolate LI-1 polyprotein.
  • SEQ ID NO: 2 is the protein sequence of Powassan virus isolate LI-1 polyprotein.
  • the invention is directed to South Bay Virus isolate SBV-H-1 nucleic acid sequence (including cDNA sequences corresponding to SBV-H-1 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of South Bay Virus isolate SBV-H-1 segment L is shown in SEQ ID NO:3. Sequence information related to South Bay virus isolate SBV-H-1 segment L is accessible in public databases by GenBank Accession Number KJ746877.
  • SEQ ID NO: 3 is the nucleic acid sequence (cDNA) of South Bay Virus isolate SBV-H- 1 segment L.
  • SEQ ID NO: 4 is the protein sequence of SBV-H-1 segment L.
  • the invention is directed to South Bay Virus isolate SBV-H-1 nucleic acid sequence (including cDNA sequences corresponding to SBV-H-1 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of South Bay virus isolate SBV-H-1 segment S is shown in SEQ ID NO:5. Sequence information related to South Bay virus isolate SBV-H-1 segment S is accessible in public databases by GenBank Accession Number KJ746878.
  • SEQ ID NO: 5 is the nucleic acid sequence (cDNA) of South Bay Virus isolate SBV-H- 1 segment S.
  • SEQ ID NO : 6 is the protein sequence of SB V-H- 1 N protein.
  • the invention is directed to South Bay Virus isolate H38 nucleic acid sequence (including cDNA sequences corresponding to SBV-H38 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of South Bay Virus isolate H38 segment L is shown in SEQ ID NO:7. Sequence information related to South Bay virus isolate H38 segment L is accessible in public databases by GenBank Accession Number KM048320.
  • SEQ ID NO: 7 is the nucleic acid sequence (cDNA) of South Bay Virus isolate H38
  • SEQ ID NO: 8 is the protein sequence of South Bay Virus isolate H38 segment L.
  • SEQ ID NO: 9 is the nucleic acid sequence (cDNA) of South Bay Virus isolate H38 segment S.
  • SEQ ID NO: 10 is the protein sequence of South Bay virus isolate H38 N protein.
  • the invention is directed to Blacklegged tick phlebovirus-1 isolate RTSl nucleic acid sequence (including cDNA sequences corresponding to BTPV-1 RTSl RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate RTSl segment L is shown in SEQ ID NO: l 1. Sequence information related to Blacklegged tick phlebovirus-1 isolate RTSl segment L is accessible in public databases by GenBank Accession Number KJ746873.
  • SEQ ID NO: 11 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate RTS 1 segment L .
  • SEQ ID NO: 12 is the protein sequence of Blacklegged tick phlebovirus-1 isolate RTSl segment L protein.
  • the invention is directed to Blacklegged tick phlebovirus-2 isolate RTS2 nucleic acid sequence (including cDNA sequences corresponding to BTPV-2 RTS2 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment L is shown in SEQ ID NO: 13. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 segment L is accessible in public databases by GenBank Accession Number KJ746874.
  • SEQ ID NO: 13 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate RTS2 segment L.
  • SEQ ID NO: 14 is the protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment L protein.
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate RTS1 segment S is shown in SEQ ID NO: 15. Sequence information related to Blacklegged tick phlebovirus-1 isolate RTS1 segment S is accessible in public databases by GenBank Accession Number KJ746875.
  • SEQ ID NO: 15 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate RTS1 segment S.
  • SEQ ID NO: 16 is the protein sequence of Blacklegged tick phlebovirus-1 isolate RTS l N protein.
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment S is shown in SEQ ID NO: 17. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 segment S is accessible in public databases by GenBank Accession Number KJ746876.
  • SEQ ID NO: 17 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate RTS2 segment S.
  • the protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 N protein is shown in SEQ ID NO: 18. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 N protein is accessible in public databases by GenBank Accession Number AII01809.
  • SEQ ID NO: 18 is the protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 N protein.
  • the invention is directed to American dog tick phlebovirus isolate ADTPV-1 nucleic acid sequence (including cDNA sequences corresponding to ADTPV-1 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of American dog tick phlebovirus isolate ADTPV-1 segment L is shown in SEQ ID NO: 19. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 segment L is accessible in public databases by GenBank Accession Number KJ746901.
  • SEQ ID NO: 19 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate ADTPV-1 segment L.
  • the protein sequence of American dog tick phlebovirus isolate ADTPV-1 polymerase protein is shown in SEQ ID NO:20. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 polymerase protein is accessible in public databases by GenBank Accession Number AIE42674.
  • SEQ ID NO: 20 is the protein sequence of American dog tick phlebovirus isolate ADTPV-1 polymerase protein.
  • nucleotide cDNA sequence of American dog tick phlebovirus isolate ADTPV-1 segment S is shown in SEQ ID NO:21. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 segment S is accessible in public databases by GenBank Accession Number KJ746902.
  • SEQ ID NO: 21 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate ADTPV-1 segment S.
  • SEQ ID NO: 22 is the protein sequence of American dog tick phlebovirus isolate ADTPV-1 nucleocapsid protein.
  • the invention is directed to Blacklegged tick phlebovirus-1 isolate H12 nucleic acid sequence (including cDNA sequences corresponding to BTPV-1 H12 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate H12 segment L is shown in SEQ ID NO:23. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 segment L is accessible in public databases by
  • GenBank Accession Number KM048313 GenBank Accession Number KM048313.
  • SEQ ID NO: 23 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate H12 segment L.
  • SEQ ID NO: 24 is the protein sequence of Blacklegged tick phlebovirus-1 isolate H12 segment L protein.
  • nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate H12 segment S is shown in SEQ ID NO:25. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 segment S is accessible in public databases by GenBank Accession Number
  • SEQ ID NO: 25 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate H12 segment S nucleic acid sequence.
  • the protein sequence of Blacklegged tick phlebovirus-1 isolate H12 N protein is shown in SEQ ID NO: 26. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 N protein is accessible in public databases by GenBank Accession Number AII01802.
  • SEQ ID NO: 26 is the protein sequence of Blacklegged tick phlebovirus-1 isolate H12 N protein.
  • the invention is directed to Blacklegged tick phlebovirus-2 isolate H5 nucleic acid sequence (including cDNA sequences corresponding to BTPV-2 H5 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate H5 segment L is shown in SEQ ID NO:27. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 segment L is accessible in public databases by GenBank Accession Number KM048315.
  • SEQ ID NO: 27 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate H5 segment L.
  • SEQ ID NO: 28 is the protein sequence of Blacklegged tick phlebovirus-2 isolate H5 segment L protein.
  • the nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate H5 segment S is shown in SEQ ID NO:29. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 segment S is accessible in public databases by GenBank Accession Number KM048316.
  • SEQ ID NO: 29 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate H5 segment S.
  • the protein sequence of Blacklegged tick phlebovirus-2 isolate H5 N protein is shown in SEQ ID NO: 30. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 N protein is accessible in public databases by GenBank Accession Number AII01804.
  • SEQ ID NO: 30 is the protein sequence of Blacklegged tick phlebovirus-2 isolate H5 N protein.
  • the invention is directed to American dog tick phlebovirus isolate FI6 nucleic acid sequence (including cDNA sequences corresponding to ADTPV FI6 RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of American dog tick phlebovirus isolate FI6 segment L is shown in SEQ ID NO: 31. Sequence information related to American dog tick phlebovirus isolate FI6 segment L is accessible in public databases by GenBank Accession Number KM04831 1.
  • SEQ ID NO: 31 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate FI6 segment L.
  • SEQ ID NO: 32 is the protein sequence of American dog tick phlebovirus isolate FI6 segment L protein.
  • nucleotide cDNA sequence of American dog tick phlebovirus isolate FI6 segment S is shown in SEQ ID NO:33. Sequence information related to American dog tick phlebovirus isolate FI6 segment S is accessible in public databases by GenBank Accession Number KM048312.
  • SEQ ID NO: 33 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate FI6 segment S.
  • the protein sequence of American dog tick phlebovirus isolate FI6 N protein is shown in SEQ ID NO: 34. Sequence information related to American dog tick phlebovirus isolate FI6 N protein is accessible in public databases by GenBank Accession Number AII01800.
  • SEQ ID NO: 34 is the protein sequence of American dog tick phlebovirus isolate FI6 N protein.
  • the invention is directed to Deer tick mononegavirales-like virus isolate DTMl nucleic acid sequence (including cDNA sequences corresponding to DTMl RNA sequences, including mRNA sequences).
  • DTMl nucleic acid sequence including cDNA sequences corresponding to DTMl RNA sequences, including mRNA sequences.
  • SEQ ID NO: 35 is the nucleic acid sequence (cDNA) of Deer tick mononegavirales-like virus isolate DTMl polymerase.
  • Deer tick mononegavirales-like virus isolate DTMl polymerase protein is shown in SEQ ID NO: 36. Sequence information related to Deer tick mononegavirales-like virus isolate DTMl polymerase protein is accessible in public databases by GenBank
  • SEQ ID NO: 36 is the protein sequence of Deer tick mononegavirales-like virus isolate DTMl polymerase protein.
  • the invention is directed to Deer tick mononegavirales-like virus isolate FI3 nucleic acid sequence (including cDNA sequences corresponding to DTM FI3 RNA sequences, including mRNA sequences).
  • FI3 nucleic acid sequence including cDNA sequences corresponding to DTM FI3 RNA sequences, including mRNA sequences.
  • mononegavirales-like virus isolate FI3 polymerase is shown in SEQ ID NO: 37. Sequence information related to Deer tick mononegavirales-like virus isolate FI3 polymerase is accessible in public databases by GenBank Accession Number KM048317.
  • SEQ ID NO: 37 is the nucleic acid sequence (cDNA) of Deer tick mononegavirales-like virus isolate FI3 polymerase nucleic acid sequence.
  • Deer tick mononegavirales-like virus isolate FI3 polymerase protein The protein sequence of Deer tick mononegavirales-like virus isolate FI3 polymerase protein is shown in SEQ ID NO: 38. Sequence information related to Deer tick mononegavirales- like virus isolate FI3 polymerase protein is accessible in public databases by GenBank Accession Number AIIO 1805.
  • SEQ ID NO: 38 is the protein sequence of Deer tick mononegavirales-like virus isolate FI3 polymerase protein.
  • the invention is directed to Tick borne tetravirus-like virus isolate
  • FI10 nucleic acid sequence (including cDNA sequences corresponding to ick borne tetravirus-like virus isolate FIIO RNA sequences, including mRNA sequences).
  • the nucleotide cDNA sequence of Tick borne tetravirus-like virus isolate FIIO is shown in SEQ ID NO: 39. Sequence information related to Tick borne tetravirus-like virus isolate FIIO is accessible in public databases by GenBank Accession Number KM048322.
  • SEQ ID NO: 39 is the nucleic acid sequence (cDNA) of Tick borne tetravirus-like virus isolate FIIO.
  • Tick borne tetravirus-like virus isolate FI10 polyprotein The protein sequence of Tick borne tetravirus-like virus isolate FI10 polyprotein is shown in SEQ ID NO:40. Sequence information related Tick borne tetravirus-like virus isolate
  • FI10 is accessible in public databases by GenBank Accession Number AII01815.
  • SEQ ID NO: 40 is the protein sequence of Tick borne tetravirus-like virus isolate FI10 polyprotein.
  • the invention is directed to Ixodes scapularis associated virus 1 isolate K13 nucleic acid sequence (including cDNA sequences corresponding to Ixodes scapularis associated virus 1 isolate K13 RNA sequences, including mRNA sequences).
  • the nucleotide sequence of Ixodes scapularis associated virus 1 isolate K13 is shown in SEQ ID NO: 41.
  • Sequence information related to Ixodes scapularis associated virus 1 isolate K13 is accessible in public databases by GenBank Accession Number KM048318.
  • SEQ ID NO: 41 is the nucleic acid sequence of Ixodes scapularis associated virus 1 isolate K13 partial genome.
  • the invention is directed to Ixodes scapularis associated virus 2 isolate Al nucleic acid sequence (including cDNA sequences corresponding to Ixodes scapularis associated virus 2 isolate Al RNA sequences, including mRNA sequences).
  • the nucleotide sequence of Ixodes scapularis associated virus 2 isolate Al is shown in SEQ ID NO: 42. Sequence information related to Ixodes scapularis associated virus 2 isolate Al is accessible in public databases by GenBank Accession Number KM048319.
  • SEQ ID NO: 42 is the nucleic acid sequence of Ixodes scapularis associated virus 2 isolate Al partial genome.
  • the invention is directed to a tick-associated virus isolated nucleic acid sequence as provided in SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid of SEQ ID NOs:
  • the invention is directed to an isolated nucleic acid complementary to SEQ ID Nos: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention is directed to variants of tick-associated virus nucleic acid sequences having greater that 60% similarity to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to isolated nucleic acid sequence variants of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 50%> to about 55% identity to that of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 55.1 %> to about 60%> identity to that of any of SEQ ID NOs: 1,3,5, 7, 9,
  • 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 60.1% to about 65% identity to that of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 65.1 % to about 70% identity to that of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25, 27,29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 70.1% to about 75% identity to that of any of SEQ ID NOs: 1, 3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 75.1% to about 80% identity to that of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 80.1% to about 85% identity to that of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 85.1% to about 90% identity to that of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 90.1% to about 95% identity to that of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 95.1% to about 97% identity to that of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 97.1% to about 99% identity to that of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 50 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 100 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 200 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 300 consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 400 consecutive nucleotides from SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 500 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 600 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 700 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 900 or more consecutive nucleotides from SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 2000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 3000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 4000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 5000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 6000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 7000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 8000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 9000 or more consecutive nucleotides from SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 10,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 11,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 12,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 13,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42.
  • the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 13,892 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39,41 or 42.
  • the invention is directed to isolated nucleic acid sequences such as primers and probes, comprising nucleic acid sequences of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • primers and probes may be useful for detecting the presence of tick-associated virus of the invention, for example in samples of ticks or samples of bodily fluids such as blood, saliva, or urine from an animal, and thus may be useful in the diagnosis of tick-associated virus infection.
  • Such probes can detect polynucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 in samples which comprise tick-associated virus represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the isolated nucleic acids which can be used as primer and/probes are of sufficient length to allow hybridization with, i.e.
  • nucleic acid sequences of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or a variant thereof.
  • the isolated nucleic acid of the invention which can be used as primers and/or probes can comprise about 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 consecutive nucleotides from any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or sequences complementary to any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • the isolated nucleic acid of the invention which can be used as primers and/or probes can comprise from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and up to about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 consecutive nucleotides from any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or sequences complementary to any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • the invention is also directed to primer and/or probes which can be labeled by any suitable molecule and/or label known in the art, for example but not limited to fluorescent tags suitable for use in Real Time PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAM or 6-FAM probes; radiolabels; and so forth.
  • fluorescent tags suitable for use in Real Time PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAM or 6-FAM probes; radiolabels; and so forth.
  • the invention is also directed to primer and/or probes which can be labeled by any suitable molecule and/or label known in the art, for example but not limited to fluorescent tags suitable for use in Real Time PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAM or 6-FAM probes; radiolabels; and so forth.
  • the invention is also directed to primer and/or probes which can be labeled by any suitable molecule and/or label known in the art
  • oligonucleotide primers and/or probe further comprises a detectable non-isotopic label selected from the group consisting of: a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.
  • the invention is directed to primer sets comprising isolated nucleic acids as described herein, which primer set are suitable for amplification of nucleic acids from samples which comprises tick-associated virus represented by SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42, or variants thereof.
  • Primer sets can comprise any suitable combination of primers which would allow amplification of a target nucleic acid sequences in a sample which comprises tick-associated virus represented by SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or variants thereof.
  • Hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, and can hybridize, for example but not limited to, variants of the disclosed
  • polynucleotide sequences including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides.
  • the precise conditions for stringent hybridization are typically sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%> of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • concentration of denaturing agents used in a hybridization and washing procedure.
  • concentration of denaturing agents e.g., formamide
  • concentration of denaturing agents used in a hybridization and washing procedure.
  • concentration of denaturing agents used in a hybridization and washing procedure.
  • concentration of denaturing agents used in a hybridization and washing procedure.
  • concentration of denaturing agents used in a hybridization and washing procedure.
  • concentration of denaturing agents e.g., formamide
  • a non- limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X sodium chloride/sodium citrate (SSC), 50 mM Tris-HCl (pH 7.5), 1 nM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • Another non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1%> SDS at 50-65 °C. Examples of moderate to low stringency hybridization conditions are well known in the art.
  • Polynucleotides homologous to the sequences illustrated in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions.
  • Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical- chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like.
  • the stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the nucleic acid sequences disclosed herein, and fragments thereof under various conditions of stringency (See, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel (1987) Methods Enzymol. 152: 507-511).
  • hybridization conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al.
  • Tm melting temperature
  • Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson et al. (1985) supra).
  • one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non- complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution.
  • Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time.
  • conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
  • Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments.
  • the stringency can be adjusted either during the hybridization step or in the post-hybridization washes.
  • Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency. As a general guidelines high stringency is typically performed at Tm-5°C to Tm -20°C, moderate stringency at Tm-20°C to Tm-35°C and low stringency at Tm-35°SC to Tm-50°C for duplex>150 base pairs.
  • Hybridization may be performed at low to moderate stringency (25-50°C below Tm), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at Tm-25°C for DNA-DNA duplex and Tm -15°C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
  • High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences.
  • An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1 % N- laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50°C and about 70°C.
  • high stringency conditions are about 0.02 M sodium chloride, about 0.5%> casein, about 0.02%> SDS, about 0.001 M sodium citrate, at a temperature of about 50°C.
  • Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.
  • Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas in certain embodiments high stringency hybridization may be obtained in the presence of at least about 35% formamide, and in other embodiments in the presence of at least about 50% formamide.
  • stringent temperature conditions will ordinarily include temperatures of at least about 30°C, and in other embodiment at least about 37°C, and in other embodiments at least about 42°C with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a certain embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide. In another embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • washing steps that follow hybridization may also vary in stringency; the post- hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt
  • concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, and in certain embodiments less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • the wash conditions may be under conditions of 0.1XSSC to 2.0XSSC and 0.1% SDS at 50-65°C, with, for example, two steps of 10-30 min.
  • One example of stringent wash conditions includes about 2.0XSSC, 0.1% SDS at 65°C and washing twice, each wash step being about 30 min.
  • the temperature for the wash solutions will ordinarily be at least about 25°C, and for greater stringency at least about 42°C.
  • Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3°C to about 5°C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6°C to about 9°C.
  • wash steps may be performed at a lower temperature, e.g., 50°C.
  • An example of a low stringency wash step employs a solution and conditions of at least 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42° C in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Even higher stringency wash conditions are obtained at 65°C-68°C in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-1 OX higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15X or more, is obtained.
  • an animal nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2X or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide.
  • the particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a calorimetric label, a radioactive label, or the like.
  • Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Protein and/or nucleic acid sequence identities (homologies) can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of nucleic acids and proteins the BLAST and BLAST 2.2.2. or FASTA version 3.0t78 algorithms and the default parameters discussed below can be used.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, less than about 0.01, and less than about 0.001.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12:387-395, 1984).
  • ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05 respectively.
  • the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919, 1992).
  • Percent identity in the context of two or more nucleic acids, refers to the percentage of nucleotides that two or more sequences or subsequences contain which are the same.
  • a specified percentage of nucleotides can be referred to such as: 60% identity, 65%, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • Substantially identical in the context of two nucleic acids, refers to two or more sequences or subsequences that have at least of at least 98%>, at least 99% or higher nucleotide identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the invention is directed to expression constructs, for example but not limited to plasmids and vectors which comprise the nucleic acid sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, complementary sequences thereof, and/or variants thereof.
  • expression constructs can be prepared by any suitable method known in the art.
  • Such expression constructs are suitable for viral nucleic acid and/or protein expression and purification.
  • the invention is directed to iRNA molecules which target nucleic acids from a tick-associated virus, for example but not limited to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and variants thereof, and silence a target gene.
  • RNA agent (abbreviation for "interfering RNA agent”) as used herein, is an RNA agent, which can down-regulate the expression of a target gene, e.g. a tick-associated virus gene.
  • An iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre- translational mechanisms.
  • RNAi post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre- translational mechanisms.
  • An iRNA agent can be a double stranded (ds) iRNA agent.
  • a "ds iRNA agent” (abbreviation for "double stranded iRNA agent”), as used herein, is an iRNA agent which includes more than one, and in certain embodiments two, strands in which interchain hybridization can form a region of duplex structure.
  • a "strand” herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g. by a linker, e.g. a polyethyleneglycol linker, to form but one molecule.
  • At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the "antisense strand”.
  • a second strand comprised in the ds iRNA agent which comprises a region complementary to the antisense strand is termed the "sense strand”.
  • a ds iRNA agent can also be formed from a single RNA molecule which is, at least partly; self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region.
  • the term "strand” refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.
  • iRNA agents as described herein can mediate silencing of a gene, e.g., by RNA degradation.
  • RNA is also referred to herein as the RNA to be silenced.
  • a gene is also referred to as a target gene.
  • the RNA to be silenced is a gene product of a tick-associated virus gene.
  • RNAi refers to the ability of an agent to silence, in a sequence specific manner, a target gene.
  • "Silencing a target gene” means the process whereby a cell containing and/or secreting a certain product of the target gene when not in contact with the agent, will contain and/or secret at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent.
  • product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.
  • silencing of a target gene can result in a reduction in "viral titer" in the cell or in the animal, wherein "reduction in viral titer” refers to a decrease in the number of viable virus produced by a cell or found in an organism undergoing the silencing of a viral target gene. Reduction in the cellular amount of virus produced can lead to a decrease in the amount of measurable virus produced in the tissues of an animal undergoing treatment and a reduction in the severity of the symptoms of the viral infection.
  • iRNA agents of the present invention are also referred to as "antiviral iRNA agents”.
  • tick-associated virus gene refers to any one of the genes identified in a tick-associated virus genome, including but not limited to, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention provides methods for reducing viral titer in an animal, by administering to an animal, at least one iRNA which inhibits the expression of a tick-associated virus gene.
  • the invention provides methods for identifying and/or generating antiviral drugs.
  • the invention provides methods for identifying drugs that bind to and/or inhibit the function of tick-associated virus-encoded proteins of the invention, or that inhibit the replication or pathogenicity of tick-associated virus of the invention.
  • Methods of identifying drugs that affect or inhibit a particular drug target such as high throughput drug screening methods, are well known in the art and can readily be applied to the proteins and viruses of the present invention.
  • the invention is directed to a tick-associated virus isolated amino acid sequence as provided in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a tick-associated virus isolated amino acid sequence encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
  • the invention relates to variants of tick-associated virus amino acid sequences having greater that 60% similarity to the sequence of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is also directed to isolated polypeptides and variants and derivatives thereof. These polypeptides may be useful for multiple applications, including, but not limited to, generation of antibodies and generation of immunogenic compositions.
  • the invention is directed to an isolated polypeptides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
  • the invention is also directed to any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • a peptide of at least 8 amino acid residues in length can be recognized by an antibody (MacKenzie et al., (1984) Biochemistry 23,6544-6549).
  • the invention is directed to fragments of the polypeptides described herein, that can, for example, be used to generate antibodies.
  • the invention is directed to polypeptide variants of an isolated polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of the isolated polypeptides of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 50% to about 55%> identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 55.1 %> to about 60% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 60.1% to about 65%> identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 65.1 % to about 70% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide having at least from about 70.1% to about 75% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 75.1% to about 80%> identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 80.1% to about 85% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 85.1%> to about 90% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 90.1% to about 95% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 95.1% to about 97% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 97.1%> to about 99% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to polypeptide variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • Variants of any one of the isolated polypeptides encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 50% to about 55 identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide sequences having at least from about 55.1 %> to about 60%) identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 60.1% to about 65 %> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 65.1 %> to about 70%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide having at least from about 70.1% to about 75%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 75.1% to about 80% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide sequences having at least from about 80.1% to about 85% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to,
  • polypeptide sequences having at least from about 85.1%> to about 90%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 90.1% to about 95% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide sequences having at least from about 95.1% to about 97%o identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • polypeptide sequences having at least from about 97.1% to about 99%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 200 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 250 consecutive amino acids of an isolated
  • polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 is directed to a polypeptide sequence comprising from about 10 to about 300 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • polypeptide sequence comprising from about 10 to about 350 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids of an isolated
  • polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 is directed to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • polypeptide sequence comprising from about 10 to about 700 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 1000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 1500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 2000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 2500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 3000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 3500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 4000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 4536 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 200 to about 50 consecutive amino acids of an isolated
  • polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids of an isolated
  • polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the invention is directed to isolated and purified peptides.
  • the polypeptides of the present invention can be suitable for use as antigens to detect antibodies against SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
  • the polypeptides of the present invention which comprise antigenic determinants can be used in various immunoassays to identify animals exposed to and/or samples which comprise SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs:
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9,
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 200 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 250 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 300 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs:
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9,
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 1000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 1500 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 2000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 2500 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 3000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 3500 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 4000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is directed to a polypeptide sequence comprising from about 10 to about 4536 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 200 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99%) identity to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42.
  • the invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs:
  • polypeptide sequences having from about 50%> to about 99% identity to a polypeptide sequence comprising from about 10 to about 1000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9,
  • the invention is directed to isolated and purified peptides.
  • polypeptides of the present invention can be suitable for use as antigens to detect antibodies against tick-associated virus represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and variants thereof.
  • polypeptides of the present invention which comprise antigenic
  • determinants can be used in various immunoassays to identify animals exposed to and/or samples which comprise tick-associated virus represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and variants thereof.
  • the invention is directed to an antibody which specifically binds to amino acids from the polypeptide of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the antibody is purified.
  • the antibodies can be polyclonal or monoclonal.
  • the antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human.
  • Species specific antibodies avoid certain problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences from other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.
  • the invention is directed to an antibody which specifically binds to amino acids from the polypeptide of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the antibody is purified.
  • the antibodies can be polyclonal or monoclonal.
  • the antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human.
  • Species specific antibodies avoid certain of the problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences form other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.
  • Antibodies can bind to other molecules (antigens) via heavy and light chain variable domains, V H and V L , respectively.
  • the antibodies described herein include, but are not limited to IgY, IgY(AFc)), IgG, IgD, IgA, IgM, IgE, and IgL.
  • the antibodies may be intact immunoglobulin molecules, two full length heavy chains linked by disulfide bonds to two full length light chains, as well as subsequences (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen, or subsequences thereof (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen.
  • Antibodies may comprise full length heavy and light chain variable domains, V H and V L , individually or in any combination.
  • the basic immunoglobulin (antibody) structural unit can comprise a tetramer.
  • Each tetramer can be composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (Vi) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E.
  • the Fab' regions may be derived from antibodies of animal or human origin or may be chimeric (Morrison et al, Proc Natl. Acad. Sci. USA 81, 6851-7855 (1984) both incorporated by reference herein) or humanized (Jones et al., Nature 321, 522-525 (1986), and published UK patent application No. 8707252, both incorporated by reference herein).
  • An antibody described in this application can include or be derived from any mammal, such as but not limited to, a bird, a dog, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof and includes isolated avian, human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted or CDR-adapted antibodies, immunoglobulins, cleavage products and other portions and variants thereof.
  • Any method for producing antibodies can be used to generate the antibodies described herein.
  • Examplary methods include animal inoculation, phage display, transgenic mouse technology and hybridoma techonology.
  • Methods for generating avain antibodies can also be used to generate the antibodies described herein.
  • the egg yolk can be used an antibody source (Altchul et al, Nature Genetics, 1994, 6: 119-129).
  • Altchul et al Nature Genetics, 1994, 6: 119-129.
  • the bursa of Fabricius is the site where B cells undergo gene conversion and are selected for the ability to produce antibodies to antigens. Unlike mammals, the generation of antibody binding specities occurs before hatching rather than throughout their lives.
  • IgY immunoglobulin
  • IgY(AFc) A small version of IgY lacking a full Fc region (IgY(AFc)) is also known to be produced in avians. (Zimmerman,et al, (1971) Biochemistry 10 : 482-488).
  • Antibodies useful in the embodiments of the invention can be derived in several ways well known in the art.
  • the antibodies can be obtained using any of the techniques well known in the art, see, e.g., Ausubel, et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.
  • the antibodies may also be obtained from selecting from libraries of such domains or components, e.g. a phage library.
  • a phage library can be created by inserting a library of random oligonucleotides or a library of polynucleotides containing sequences of interest, such as from the B-cells of an immunized animal or human (Smith, G. P. 1985. Science 228: 1315-1317).
  • Antibody phage libraries contain heavy (H) and light (L) chain variable region pairs in one phage allowing the expression of single-chain Fv fragments or Fab fragments (Hoogenboom, et al. 2000, Immunol Today 21(8) 371-7).
  • the diversity of a phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, human monoclonal antibodies.
  • the heavy (H) chain and light (L) chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an assembled immunoglobulin molecule.
  • either or both the H and L chain encoding genes can be mutagenized in a complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable affinity and neutralization capabilities.
  • Antibody libraries also can be created synthetically by selecting one or more human framework sequences and introducing collections of CDR cassettes derived from human antibody repertoires or through designed variation
  • the positions of diversity are not limited to CDRs but can also include the framework segments of the variable regions or may include other than antibody variable regions, such as peptides.
  • Ribosome display is a method of translating mRNAs into their cognate proteins while keeping the protein attached to the RNA.
  • the nucleic acid coding sequence is recovered by RT-PCR (Mattheakis, L. C. et al. 1994. Proc Natl Acad Sci USA 91, 9022).
  • Yeast display is based on the construction of fusion proteins of the membrane-associated alpha-agglutinin yeast adhesion receptor, agal and aga2, a part of the mating type system (Broder, et al. 1997. Nature Biotechnology, 15:553-7).
  • Bacterial display is based fusion of the target to exported bacterial proteins that associate with the cell membrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng, 79:496-503).
  • phage and other antibody display methods afford the opportunity to manipulate selection against the antigen target in vitro and without the limitation of the possibility of host effects on the antigen or vice versa.
  • antibody subsequences include, for example, Fab, Fab', (Fab') 2 , Fv, or single chain antibody (SCA) fragment (e.g., scFv).
  • Subsequences include portions which retain at least part of the function or activity of full length sequence. For example, an antibody
  • subsequence will retain the ability to selectively bind to an antigen even though the binding affinity of the subsequence may be greater or less than the binding affinity of the full length antibody.
  • an Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
  • An (Fab') 2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • An Fab' fragment of an antibody molecule can be obtained from (Fab') 2 by reduction with a thiol reducing agent, which yields a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
  • An Fv fragment is a fragment containing the variable region of a light chain VL and the variable region of a heavy chain V H expressed as two chains.
  • the association may be non-covalent or may be covalent, such as a chemical cross-linking agent or an intermolecular disulfide bond (Inbar et al, (1972) Proc. Natl. Acad Sci. USA 69:2659; Sandhu (1992) Crit. Rev. Biotech.
  • a single chain antibody is a genetically engineered or enzymatically digested antibody containing the variable region of a light chain VL and the variable region of a heavy chain, optionally linked by a flexible linker, such as a polypeptide sequence, in either VL-linker-VH orientation or in VH-linker-VL orientation.
  • a single chain Fv fragment can be produced by linking two variable domains via a disulfide linkage between two cysteine residues.
  • antibody subsequences such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, provided that the subsequences bind to the antigen to which the intact antibody binds.
  • Antibodies used in the invention include full length antibodies, subsequences (e.g., single chain forms), dimers, trimers, tetramers, pentamers, hexamers or any other higher order oligomer that retains at least a part of antigen binding activity of monomer.
  • Multimers can comprise heteromeric or homomeric combinations of full length antibody, subsequences, unmodified or modified as set forth herein and known in the art. Antibody multimers are useful for increasing antigen avidity in comparison to monomer due to the multimer having multiple antigen binding sites.
  • Antibody multimers are also useful for producing oligomeric (e.g., dimer, trimer, tertamer, etc.) combinations of different antibodies thereby producing compositions of antibodies that are multifunctional (e.g., bifunctional, trifunctional, tetrafunctional, etc.).
  • Antibodies can be produced through chemical crosslinking of the selected molecules (which have been produced by synthetic means or by expression of nucleic acid that encode the polypeptides) or through recombinant DNA technology combined with in vitro, or cellular expression of the polypeptide, and subsequent oligomerization. Antibodies can be similarly produced through recombinant technology and expression, fusion of hybridomas that produce antibodies with different epitopic specificities, or expression of multiple nucleic acid encoding antibody variable chains with different epitopic specificities in a single cell.
  • Antibodies may be either joined directly or indirectly through covalent or non-covalent binding, e.g. via a multimerization domain, to produce multimers.
  • a "multimerization domain” mediates non-covalent protein-protein interactions. Specific examples include coiled-coil (e.g., leucine zipper structures) and alpha-helical protein sequences. Sequences that mediate protein- protein binding via Van der Waals' forces, hydrogen bonding or charge-charge bonds are also can also be used as multimerization domains. Additional examples include basic-helix-loop-helix domains and other protein sequences that mediate heteromeric or homomeric protein-protein interactions among nucleic acid binding proteins (e.g., DNA binding transcription factors, such as TAFs).
  • nucleic acid binding proteins e.g., DNA binding transcription factors, such as TAFs
  • multimerization domain p53 residues 319 to 360 which mediate tetramer formation.
  • human platelet factor 4 which self-assembles into tetramers.
  • extracellular protein TSP4 a member of the thrombospondin family, which can form pentamers.
  • Additional specific examples are the leucine zippers of jun, fos, and yeast protein GCN4.
  • Antibodies may be directly linked to each other via a chemical cross linking agent or can be connected via a linker sequence (e.g., a peptide sequence) to form multimers.
  • a linker sequence e.g., a peptide sequence
  • the antibodies of the present invention can be used to modulate the activity of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, variants or fragments thereof.
  • the invention is directed to a method for treating an animal, the method comprising administering to the animal an antibody which specifically binds to amino acids from the polypeptide of any
  • polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit virus propagation and spreading.
  • the antibodies of the present invention can be used to modulate the activity of any polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants or fragments thereof.
  • the invention is directed to a method for treating an animal, the method comprising administering to the animal an antibody which specifically binds to amino acids from the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • antibody binding to the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit virus propagation and spreading.
  • the antibodies of the invention can be used to purify a
  • the antibodies of the invention can be used to identify expression and localization of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants, fragments or domains thereof. Analysis of expression and localization of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
  • 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 can be useful in determining potential role of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the antibodies of the invention can be used to purify polypeptides of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
  • the antibodies of the invention can be used to identify expression and localization of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, variants, fragments or domains thereof.
  • the antibodies of the invention can be used to purify polypeptides of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants or fragments thereof.
  • the antibodies of the invention can be used to identify expression and localization of the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants, fragments or domains thereof.
  • the antibodies of the present invention can be used in various immunoassays to identify animals exposed to and/or samples which comprise antigens from tick-associated viruses represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
  • the antibodies of the present invention can be used in various immunoassays to identify animals exposed to and/or samples which comprise antigens from tick-associated viruses represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
  • any suitable immunoassay which can lead to formation of antigen-antibody complex can also be used. Variations and different formats of immunoassays, for example but not limited to ELISA, lateral flow assays for detection of analytes in samples, immunoprecipitation, and
  • Luciferase Immunoprecipitation Systems are known in the art.
  • the antigen and/or the antibody can be labeled by any suitable label or method known in the art.
  • enzymatic immunoassays may use solid supports, or immunoprecipitation.
  • Immnunoassays which amplify the signal from the antigen-antibody immune complex can also be used with the methods described herein.
  • the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, as provided by the invention, and variants thereof.
  • the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
  • methods for assaying a sample include, but are not limited to, methods which can detect the presence of nucleic acids, methods which can detect the presence of antigens, methods which can detect the presence of antibodies against antigens from a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, methods which can detect the presence of a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, as provided by the invention, and variants thereof.
  • the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus comprising SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
  • methods for assaying a sample include, but are not limited to, methods which can detect the presence of nucleic acids, methods which can detect the presence of antigens, methods which can detect the presence of antibodies against antigens from polypeptides encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, as provided by the invention, and variants thereof.
  • the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus by detecting the presence of antibodies against antigens from polypeptides encoded by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
  • the antibody that is detected is against a L-segment gene protein (e.g. SEQ ID NOs: 4, 8, 12, 14, 24, 28, or 32).
  • the antibody that is detected is against a N protein (e.g. SEQ ID NO: 6, 10, 16, 18, 26, 30, or 34).
  • the antibody that is detected is against a nucleocapsid protein (e.g. SEQ ID NO: 22).
  • the antibody that is detected is against a polymerase protein (e.g. SEQ ID NO: 36, or 38).
  • antibodies against antigens are detected by immunoassay.
  • complex formation between an antibody to the antigen and the antigen is detected.
  • the present invention provides immunogenic compositions capable of inducing an immune response against a tick-associated virus including a tick-associated virus of the invention comprising SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or comprising a cDNA sequence complementary to the sense or an anti-sense strand of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or comprising a polypeptide encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or a cDNA sequence complementary to the sense or an anti- sense strand of SEQ ID NOs: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or comprising a polypeptide of any of SEQ ID NOs: 2,
  • the immunogenic compositions are capable of ameliorating the symptoms of a tick-associated virus infection and/or of reducing the duration of a tick-associated virus associated disease. In another embodiment, the immunogenic compositions are capable of inducing protective immunity against a disease associated with a tick-associated virus.
  • the immunogenic compositions of the invention can be effective against a tick-associated virus disclosed herein, and may also be cross-reactive with, and effective against, multiple different clades and strains of tick-associated viruses.
  • the present invention provides immunogenic compositions for inducing an immune response in an animal, wherein the composition includes a recombinant or isolated capsid polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent.
  • this immunogenic composition is a vaccine composition.
  • the polypeptide employed in the immunogenic compositions is SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a fragment thereof.
  • polypeptide sequence corresponds to the immunogenic protein in both the whole virus and subunit compositions provided by the present invention.
  • polypeptide sequence refers to polypeptide sequences and includes derivatives of polypeptide sequences, such as including, but not limited to, polypeptide sequences which include Histidine tags, XpressTM tags, signal sequences or other epitope tags at the N- and/or C-terminal ends.
  • the polypeptide composition can be used to protect animals against a tick-associated virus.
  • the present invention also provides a method of immunizing an animal against a tick- associated virus, wherein the method includes administering to the animal the polypeptide composition.
  • the tick-associated virus from which the polypeptide is derived encodes a polypeptide sequence having at least 90% identity to the amino acid sequence SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or fragment thereof.
  • the capsid polypeptide composition includes an adjuvant.
  • immunogenic compositions encompassed by the invention include, but are not limited to, attenuated live viral immunogenic compositions, inactivated (killed) viral immunogenic compositions, and subunit immunogenic compositions.
  • tick-associated viruses of the invention may be attenuated by removal or disruption of those viral sequences whose products cause or contribute to the disease and symptoms associated with tick-associated infection, and leaving intact those sequences required for viral replication. In this way an attenuated tick-associated virus can be produced that replicates in animals, and induces an immune response in animals, but which does not induce the deleterious disease and symptoms usually associated with tick-associated virus infection.
  • tick-associated virus sequences can or should be removed or disrupted, and which sequences should be left intact, in order to generate an attenuated tick-associated virus suitable for use as an immunogenic composition.
  • novel tick-associated viruses of the invention may be also be inactivated, such as by chemical treatment, to "kill” the viruses such that they are no longer capable of replicating or causing disease in animals, but still induce an immune response in an animal.
  • suitable viral inactivation methods known in the art and one of skill in the art can readily select a suitable method and produce an inactivated "killed" tick-associated viruses suitable for use as an immunogenic composition.
  • the immunogenic compositions of the invention may comprise subunit immunogenic compositions.
  • Subunit immunogenic compositions include nucleic acid immunogenic
  • compositions such as DNA immunogenic compositions, which contain nucleic acids that encode one or more viral proteins or subunits, or portions of those proteins or subunits.
  • the nucleic acid is administered to the animal, and the immunogenic proteins or peptides encoded by the nucleic acid are expressed in the animal, such that an immune response against the proteins or peptides is generated in the animal.
  • Subunit immunogenic compositions may also be proteinaceous immunogenic compositions, which contain the viral proteins or subunits themselves, or portions of those proteins or subunits.
  • tick- associated virus sequences disclosed herein may be incorporated into a plasmid or expression vector containing the nucleic acid that encodes the viral protein or peptide.
  • Any suitable plasmid or expression vector capable of driving expression of the protein or peptide in the animal may be used.
  • Such plasmids and expression vectors should include a suitable promoter for directing transcription of the nucleic acid.
  • the nucleic acid sequence(s) that encodes a tick-associated virus protein or peptide may also be incorporated into a suitable recombinant virus for administration to the animal.
  • Suitable viruses include, but are not limited to, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses.
  • vaccinia viruses retroviruses
  • adenoviruses adeno-associated viruses
  • adeno-associated viruses adeno-associated viruses
  • tick- associated virus nucleic acid sequences of the invention are delivered to cultured cells, for example by transfecting cultured cells with plasmids or expression vectors containing tick-associated virus nucleic acid sequences, or by infecting cultured cells with recombinant viruses containing tick- associated virus nucleic acid sequences. Tick-associated virus proteins or peptides may then be expressed in the cultured cells and purified. The purified proteins can then be incorporated into compositions suitable for administration to animals. Methods and techniques for expression and purification of recombinant proteins are well known in the art, and any such suitable methods may be used.
  • Subunit immunogenic compositions of the present invention may encode or contain any of tick-associated virus proteins or peptides described herein, or any portions, fragments, derivatives or mutants thereof, that are immunogenic in an animal.
  • tick-associated virus proteins or peptides described herein or any portions, fragments, derivatives or mutants thereof, that are immunogenic in an animal.
  • One of skill in the art can readily test the immunogenicity of tick-associated virus proteins and peptides described herein, and can select suitable proteins or peptides to use in subunit immunogenic compositions.
  • the immunogenic compositions of the invention comprise at least one tick-associated virus-derived immunogenic component, such as those described herein.
  • compositions may also comprise one or more additives including, but not limited to, one or more pharmaceutically acceptable carriers, buffers, stabilizers, diluents, preservatives, solubilizers, liposomes or immunomodulatory agents.
  • Suitable immunomodulatory agents include, but are not limited to, adjuvants, cytokines, polynucleotide encoding cytokines, and agents that facilitate cellular uptake of tick-associated virus-derived immunogenic component.
  • Immunogenic compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used to induce an immunogenic response.
  • physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used to induce an immunogenic response.
  • These immunogenic compositions may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
  • protein or other active ingredient of the present invention can be in the form of a tablet, capsule, powder, solution or elixr.
  • the immunogenic composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and from about 25 to 90% protein or other active ingredient of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the immunogenic composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the immunogenic composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and from about 1 to 50% protein or other active ingredient of the present invention.
  • protein or other active ingredient of the present invention When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein or other active ingredient solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • One immunogenic composition for intravenous, cutaneous, or subcutaneous injection can contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the immunogenic composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the agents of the invention may be formulated in aqueous solutions, physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with immunogenicly acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Immunogenic preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
  • polyvinylpyrrolidone PVP
  • disintegrating agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Immunogenic preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • Capsules and cartridges may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Immunogenic formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient maybe in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneous ly or intamuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a carrier for hydrophobic compounds of the invention can be a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the co-solvent system may be the VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution.
  • This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • other delivery systems for hydrophobic immunogenic compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein or other active ingredient stabilization may be employed.
  • the immunogenic compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Many of the active ingredients of the invention may be provided as salts with immunogenicly compatible counter ions.
  • Such immunogenicly acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
  • the immunogenic composition of the invention may be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.
  • the immunogenic composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference. [0272] Other additives that are useful in immunogenic composition formulations are known and will be apparent to those of skill in the art.
  • an "immunologically effective amount" of the compositions of the invention may be administered to an animal (including a human).
  • the term “immunologically effective amount” refers to an amount capable of inducing, or enhancing the induction of, the desired immune response in an animal (including a human).
  • the desired response may include, inter alia, inducing an antibody or cell-mediated immune response, or both.
  • the desired response may also be induction of an immune response sufficient to ameliorate the symptoms of a disease associated with a tick-associated virus and/or provide protective immunity in an animal (including a human) against subsequent challenge with a tick-associated virus.
  • An immunologically effective amount may be an amount that induces actual "protection" against tick-associated virus associated diseases, meaning the prevention of any of the symptoms or conditions resulting from tick- associated virus associated disease in animals (including humans).
  • An immunologically effective amount may also be an amount sufficient to delay the onset of symptoms and conditions associated with infection, reduce the degree or rate of infection, reduce in the severity of any disease or symptom resulting from infection, and reduce the viral load of an infected animal (including a human).
  • an effective amount can be determined by conventional means, starting with a low dose of and then increasing the dosage while monitoring the immunological effects. Numerous factors can be taken into consideration when determining an optimal amount to administer, including the size, age, and general condition of the animal, the presence of other drugs in the animal, the virulence of the particular tick-associated virus against which the animal is being vaccinated, and the like. The actual dosage is can be chosen after consideration of the results from various animal studies.
  • the immunologically effective amount of the immunogenic composition may be administered in a single dose, in divided doses, or using a "prime-boost" regimen.
  • compositions may be administered by any suitable route, including, but not limited to parenteral, intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, oral, or intraocular routes, or by a combination of routes.
  • the compositions may also be administered using a "gun" device which fires particles, such as gold particles, onto which compositions of the present invention have been coated, into the skin of an animal.
  • the skilled artisan will be able to formulate the immunogenic composition according to the route chosen.
  • Methods of purification of inactivated virus are known in the art and may include one or more of, for instance gradient centrifugation, ultracentrifugation, continuous-flow
  • ultracentrifugation and chromatography such as ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography. Additional method of purification include ultrafiltration and dialfiltration. See J P Gregersen “Hergori von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutician Biotecnology (eds. O. Kayser and R H Mueller)ticianliche Verlagsgesellschaft, Stuttgart, 2000. See also, O'Neil et al, "Virus Harvesting and Affinity Based Liquid Chromatography. A Method for Virus Concentration and Purification", Biotechnology (1993) 11 : 173-177; Prior et al., "Process Development for Manufacture of
  • purification methods suitable for use in the invention include polyethylene glycol or ammonium sulfate precipitation (see Trepanier et al., "Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration” Journal of Virological Methods (1981) 3(4):201-711; Hagen et al, "Optimization of Polyethylene glycol) Precipitation of Hepatitis Virus Used to prepare VAQTA, a Highly Purified Inactivated Vaccine” Biotechnology Progress (1996) 12:406-412; and Carlsson et al., "Purification of Infectious
  • Pancreatic Necrosis Virus by Anion Exchange Chromatography Increases the Specific Infectivity" Journal of Virological Methods (1994) 47:27-36) as well as ultrafiltration and microfiltration (see Pay et al., Developments in Biological Standardization (1985) 60: 171-174; Tsurumi et al.,
  • Tick-associated virus can be purified using chromatography, such as ion exchange, chromatography. Chromatic purification allows for the production of large volumes of virus containing suspension.
  • the viral product of interest can interact with the chromatic medium by a simple adsorption/desorption mechanism, and large volumes of sample can be processed in a single load. Contaminants which do not have affinity for the adsorbent pass through the column. The virus material can then be eluted in concentrated form.
  • Anion exchange resins that may be used include DEAE, EMD TMAE.
  • Cation exchange resins may comprise a sulfonic acid-modified surface.
  • Viruses can be purified using ion exchange chromatography comprising a strong anion exchange resin (e.g. EMD TMAE) for the first step and EMD-SO 3 (cation exchange resin) for the second step.
  • a metal-binding affinity chromatography step can optionally be included for further purification. (See, e.g., WO 97/06243).
  • a resin such as Fractogel EMD can also be used.
  • This synthetic methacrylate based resin has long, linear polymer chains covalently attached and allows for a large amount of sterically accessible ligands for the binding of biomolecules without any steric hindrance.
  • MCS Matrex Cellufme Sulfate
  • a resin for use in purification method is Matrex Cellufme Sulfate (MCS).
  • MCS consists of a rigid spherical (approx. 45-105 ⁇ diameter) cellulose matrix of 3,000 Dalton exclusion limit (its pore structure excludes macromolecules), with a low concentration of sulfate ester functionality on the 6-position of cellulose.
  • the functional ligand (sulfate ester) is relatively highly dispersed, it presents insufficient cationic charge density to allow for most soluble proteins to adsorb onto the bead surface. Therefore the bulk of the protein found in typical virus pools (cell culture supernatants, e.g. pyrogens and most contaminating proteins, as well as nucleic acids and endotoxins) are washed from the column and a degree of purification of the bound virus is achieved.
  • the rigid, high-strength beads of MCS tend to resist compression.
  • the pressure/flow characteristics the MCS resin permit high linear flow rates allowing high-speed processing, even in large columns, making it an easily scalable unit operation.
  • a chromatographic purification step with MCS provides increased assurance of safety and product sterility, avoiding excessive product handling and safety concerns. As endotoxins do not bind to it, the MCS purification step allows a rapid and contaminant free depyrogenation. Gentle binding and elution conditions provide high capacity and product yield.
  • the MCS resin therefore represents a simple, rapid, effective, and cost-saving means for concentration, purification and depyrogenation.
  • MCS resins can be reused repeatedly.
  • Inactivated viruses may be further purified by gradient centrifugation, or density gradient centrifugation.
  • gradient centrifugation For commercial scale operation a continuous flow sucrose gradient centrifugation would be an option.
  • This method is widely used to purify antiviral immunogenic compositions and is known to one skilled in the art (See J P Gregersen "Hergori von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutician Biotechnology (eds. O. Kayser and R H Mueller)ticianliche Verlagsgesellschaft, Stuttgart, 2000.)
  • Additional purification methods which may be used to purify viruses of the invention include the use of a nucleic acid degrading agent, a nucleic acid degrading enzyme, such as a nuclease having DNase and RNase activity, or an endonuclease, such as from Serratia marcescens, membrane adsorbers with anionic functional groups or additional chromatographic steps with anionic functional groups (e.g. DEAE or TMAE).
  • An ultrafiltration/dialfiltration and final sterile filtration step could also be added to the purification method.
  • the purified viral preparation of the invention is substantially free of contaminating proteins derived from the cells or cell culture and can comprises less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/ ⁇ g virus antigen, and less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/dose.
  • the purified viral preparation can also comprises less than about 20 pg or less than about 10 pg.
  • Tick-associated virus can be used to infect cells.
  • Cells may be cultured in any useful media and any permissive cell or tissues, which may be, or may be derived from any animal cell.
  • a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, rodent cells, avian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells.
  • Cell lines suitable for propagating, growing, or harboring tick-associated virus nucleic acid sequence or for expressing a polypeptide produced by the tick-associated virus nucleic acid sequence include, animal cells, including but are not limited to dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
  • animal cells including but are not limited to dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells,
  • Cell culture media formulations to suitable for culturing cells infected with tick- associated viruses described herein include, but are not limted to, Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof. These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro.TM. SFM, AIM V.R media, HyQ SFM4 MegaVir, EX-CELL Vera SFM, EPISERF, Pro Vera, any 293 or CHO media and analogues and derivatives thereof.
  • the culture media described herein can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamine and biological buffers.
  • One medium is OptiPRO SFM supplemented with L-glutamine and trypsin.
  • the cell culture media can be supplemented with 0.1 to 10 units of trypsin.
  • plant derived equivalents of trypsin e.g. Accutase
  • Cell culture media can be used in the absence or presence of animal-derived components.
  • An example of supplementation with an animal-derived component is gamma-irradiated serum ranging from 0.5- 10% final concentration.
  • An expression vector can be introduced into cells in order to produce proteins (for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40) encoded by nucleotide sequences of the invention (for example SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42).
  • Cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
  • An expression vector can be introduced into cells in order to produce proteins encoded by nucleotide sequences of the invention (for example SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42).
  • Cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
  • a eukaryotic expression vector can be used to transfect cells in order to produce proteins (for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40) encoded by nucleotide sequences of the vector.
  • proteins for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40
  • An exogenous nucleic acid for example any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, a cDNA of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a cDNA complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, fragments, or variants thereof) can be introduced into a cell via a variety of techniques known in the art.
  • a eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences (for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, a cDNA of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a cDNA complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, fragments, or variants thereof).
  • nucleotide sequences for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, a cDNA of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,
  • Mammalian cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
  • An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation.
  • Other methods used to transfect cells can also include calcium phosphate precipitation, modified calcium phosphate precipitation, polybrene precipitation, microinjection liposome fusion, and receptor-mediated gene delivery.
  • Cells to be infected with a tick-associated virus or nucleic acids thereof for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, a cDNA of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or a cDNA complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, fragments, or variants thereof) can be primary and secondary cells, which can be obtained from various tissues and include cell types which can be maintained and propagated in culture.
  • a tick-associated virus or nucleic acids thereof for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, a cDNA of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
  • Various culturing parameters can be used with respect to the host cell being cultured.
  • Appropriate culture conditions for mammalian cells are well known in the art or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2 nd Ed., Rickwood, D. and Hames, B.D., eds. (Oxford University Press: New York, 1992)), and vary according to the particular cell selected.
  • Commercially available medium can be utilized.
  • Non- limiting examples of medium include, for example, Dulbecco's Modified Eagle Medium (DMEM, Life Technologies), Minimal Essential Medium (MEM, Sigma, St. Louis, MO); HyClone cell culture medium (HyClone, Logan, Utah); and serum-free basal epithelial medium (CellnTech).
  • Cell medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
  • an energy source usually in the form of a carbohydrate such as glucose
  • all essential amino acids and usually the basic set of twenty amino acids plus cysteine
  • vitamins and/or other organic compounds required at low concentrations (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
  • the medium also can be supplemented electively with one or more components from any of the following categories: (1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, epidermal growth factor and fibroblast growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example, pluronic polyol; and (8) galactose.
  • salts for example, magnesium, calcium, and phosphate
  • hormones and other growth factors such as, serum, insulin, transferrin, epidermal growth factor and fibroblast growth factor
  • protein and tissue hydrolysates for example peptone or peptone mixture
  • induced epithelial cells are stably maintained in cell culture for at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 20 passages, at least 25 passages, or at least 30 passages.
  • the cells suitable for culturing according to the methods of the present invention can harbor introduced expression vectors (constructs), such as plasmids and the like.
  • the expression vector constructs can be introduced via transformation, microinjection, trans fection, lipofection, electroporation, or infection.
  • the expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate
  • transcriptional and translational control elements can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in J.
  • cells that have been infected with a tick-associated virus or contain nucleic acids thereof can express a variety of markers that distinguish them from uninfected cells. Expression of markers can be evaluated by a variety of methods known in the art. The presence of markers can be determined at the DNA, RNA or polypeptide level.
  • the method can comprise detecting the presence of a marker gene polypeptide expression.
  • Polypeptide expression includes the presence of a marker gene
  • polypeptide sequence or the presence of an elevated quantity of marker gene polypeptide as compared to non-epithelial cells.
  • polypeptide expression maybe evaluated by methods including, but not limited to, immunostaining, FACS analysis, or Western blot. These methods are well known in the art (for example, US Patent 8,004,661, US Patent 5,367,474, US Patent 4,347,935) and are described in T.S. Hawley & R.G. Hawley, 2005, Methods in Molecular Biology Volume 263: Flow Cytometry Protocols, Humana Press Inc; LB. Buchwalow & W.BoEcker, 2010, Immunohistochemistry: Basics & Methods, Springer, Medford, MA; O.J. Bjerrum & N.H.H. Heegaard, 2009, Western Blotting:
  • the method can comprise detecting the presence of nucleic acids (for example any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, a cDNA of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a cDNA complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, fragments, or variants thereof).
  • RNA expression includes the presence of an RNA sequence, the presence of an RNA splicing or processing, or the presence of a quantity of RNA.
  • in situ hybridization can be used to detect tick-associated virus nucleic acids.
  • tick-associated viruses and immunogenic compositions described herein can be be produced in cells. Production of the tick-associated viruses and immunogenic compositions described herein may also be accomplished on any useful media and permissive cell or tissues, which may be derived from animal cell lines.
  • a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, fish cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells, such as recombinant cells expressing a virus.
  • production of the tick-associated viruses and immunogenic compositions can be in any cell type, including but not limited to animal cells.
  • Cell lines suitable for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limited to animal cells.
  • cell lines suitable for producing the tick-associated viruses and immunogenic compositions described herein also include dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
  • the cell culture system for producing the tick-associated viruses and immunogenic compositions described herein can be a traditional adherent monolayer culture. Alternatively, suspension and microcarrier cell culture systems can also be utilized. [0298] Vessels for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limted to, roller bottles. For example, alternatively, other useful cell culture formats include flasks, stacked modules and stir tanks.
  • Production of the tick-associated viruses and immunogenic compositions can also be performed using a recombinant expression system that expresses tick-associated virus, a tick- associated virus protein, a fragment of a tick-associated virus protein or a variant of a tick- associated virus protein.
  • the expression system can comprise any suitable plasmid or a linear expression construct known in the art.
  • the immunogenic compositions described herein can comprise an inactivated or killed tick-associated virus vaccine.
  • Inactivated immunogenic composition can made by methods well known in the art. For example, once tick-associated virus is propagated to high titers, tick- associated virus antigenic mass could be obtained by methods well known in the art. For example, the tick-associated viral antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate tick-associated virus antigenic mass to produce immunogenic compositions.
  • Tick-associated virus may be inactivated by treatment with formalin (e.g. 0.1-10%), betapropriolactone (BPL) (e.g. 0.01-10%), or with binary ethyleneimine (BEI) (e.g. 1-10 mM), or using other methods known to those skilled in the art.
  • formalin e.g. 0.1-10%
  • BPL betapropriolactone
  • BEI binary ethyleneimine
  • Attenuation leading to modified live immunogenic compositions can also be used in conjunction with the compositions and methods described herein.
  • Methods of attenuation suitable for use with the viruses described herein include continuous passaging in cell culture, continuous passaging in animals, various methods for generating genetic modifications and ultraviolet or chemical mutagenesis.
  • Attenuation of tick-associated virus may be achieved through cold-adaptation of an tick- associated virus strain.
  • Cold-adapted tick-associated virus strains may be produced by methods which includes passaging a wild-type tick-associated virus, followed by selection for tick- associated virus that grows at a reduced temperature.
  • Cold-adapted tick-associated virus can be produced, for example, by sequentially passaging a wild-type tick-associated virus in embryonated cells or chicken eggs at progressively lower temperatures, thereby selecting for certain members of tick-associated virus mixture which stably replicate at the reduced temperature.
  • a cold-adapted tick-associated virus strain may exhibit a temperature sensitive phenotype.
  • a temperature sensitive cold-adapted tick-associated virus replicates at reduced temperatures, but no longer replicates at certain higher growth temperatures at which the wild-type tick-associated virus will replicate.
  • a temperature at which a temperature sensitive tick-associated virus will grow is referred to herein as a "permissive" temperature for that temperature sensitive tick-associated virus, and a higher temperature at which the temperature sensitive tick-associated virus will not grow, but at which a corresponding wild-type tick-associated virus will grow, is referred to herein as a "non-permissive" temperature for that temperature sensitive tick-associated virus.
  • a cold-adapted tick-associated virus may also be produced through recombinant means.
  • one or more specific mutations associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, can be identified and are introduced back into a wild-type tick- associated virus strain using a reverse genetics approach.
  • Reverse genetics entails can be performed using RNA polymerase complexes isolated from tick-associated virus -infected cells to transcribe artificial tick-associated virus genome segments containing the mutation(s),
  • RNA segment(s) incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes.
  • Attenuation of an tick-associated virus may be achieved by serial passaging of a wild- type tick-associated virus strain in cell culture.
  • a tick-associated virus strain can be passaged in a variety of cell systems until its ability to produce disease is lost whilst its immunogenic character is fully retained. Once inoculated into the host, tick-associated virus may be capable of multiplication to some extent.
  • attenuated tick-associated virus compositions can be prepared from cell line that has been attenuated by serial passage including serial passage at sub-optimal temperatures to a state where it is no longer capable of causing disease, but still capable of eliciting a protective immune response.
  • Suitable attenuated tick-associated virus strains may also be obtained by serial passaging to obtain an over-attenuated strain.
  • the "over-attenuation" means that the number of passages for attenuation has been substantially greater than what is normally necessary for the removal of pathogenicity.
  • the attenuated tick-associated virus retains its antigenicity after these numerous passages so that its immunogenic ability is not impaired.
  • Such strains produce practically no symptoms or side effects when administered, and thus are safe and efficacious vaccines.
  • Dose sizes of the immunogenic compositions described herein can be in the range of about 2.0 to 0.1 ml depending on the route of administration, but dose sizes are not limited to this range.
  • for inactivated tick-associated virus compositions can contain suitable TCID50 levels of virus prior to inactivation.
  • the antigen content in tick-associated virus preparation can have, but is not limited to, a titer of between 10 to 10,000 units/ml as the amount administered per dose.
  • immunogenic compositions described herein are described herein.
  • a therapeutically effective dose can be determined by one of skill in the art.
  • a therapeutically effective dose can be determined by one of skill in the art. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan.
  • An animal or a human can be inoculated with the immunogenic compositions or formulations described herein to generate an immune response.
  • inoculation can be perfomed on animals that are at least 1, 2, 3, 4, 5, 6, or more weeks or older.
  • the animals can receive one or more dosages.
  • two or more dosages can be administered to the animal 3-4 weeks apart.
  • the administration can be by subcutaneous injection. Intramuscular, intradermal, or oral routes of administration can also be used to administer the immunogenic compositions or formulations described herein.
  • Example 1 Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses
  • ticks are reservoirs to a wide range of viruses and suggests that discovery and characterization of tick-borne viruses will have implications on viral taxonomy and may provide insight into ticktransmitted diseases.
  • Ticks are implicated as vectors of a wide array of human and animal pathogens. To better understand the extent of tick-borne diseases, it is crucial to uncover the full range of microbial agents associated with ticks. The current knowledge of the diversity of tick-associated viruses is limited, in part due to the lack of investigation of tick viromes. In this study the virome of three tick species from the United States were examined. It was found that ticks are hosts to highly divergent viruses across several taxa, including ones previously associated with human disease. The data described herein underscore the diversity of tick-associated viruses and provides the foundation for further studies into viral etiology of tick-borne diseases.
  • Ticks (class Arachnida, subclass Acari) have been implicated as vectors in a wide range of human and animal diseases worldwide (1-10). Approximately 900 species of ticks have been described and taxonomically classified into three families: Argasidae (argasid or soft ticks) Ixodidae (ixodid, or hard ticks) and Nuttalliellidae (11). Their propensity for feeding on on a wide array of hosts, expansive range and long life cycle underscore the importance of tick surveillance for the presence of potential pathogens. Argasid and ixodid ticks combined transmit a greater diversity of viral, bacterial and protozoan pathogens than any other arthropod vector (12). The worldwide incidence of tick-borne disease is increasing, partly due to increased frequency of endemic tick-borne diseases, as well as the discoveries of new tick-associated agents (13).
  • tick-borne disorders In the United States (US), bacterial agents are implicated in the majority of tick-borne disorders. Lyme disease, caused by Borrelia burgdorferi represents the most frequently reported tick-borne illness (14). Other bacterial agents, such as Anaplasma, Ehrlichia, Rickettsia, other Borrelia species, as well as the protozoan Babesia contribute to the overall spectrum of tick-borne disease (1, 6, 7, 14-16). Conversely, viral causes are diagnosed in only a fraction of tick-borne disease cases (14). Despite considerable insights into the diversity of tick bacteriomes, our understanding of tick-associated viruses is still limited. Traditional viral isolation and identification methods using tissue culture have isolated several tick-associated viruses but few have been characterized thus far.
  • tick-borne viruses In comparison to bacterial and protozoan agents, the literature associated with tick-borne viruses in the Americas is also limited. Most of the literature to date on suspected viral tick-borne pathogens focuses on those found in Europe, Asia and Africa (3, 17). Powassan and Colorado tick-fever viruses have been historically recognized as the only human tick-borne viral pathogens in the US (3, 18).
  • Heartland virus a new pathogenic tick-borne virus was isolated from patients in Missouri and characterized by high-throughput sequencing (HTS) (19). Heartland virus was shown to be phylogenetically similar to severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne virus isolated from ticks and humans in China in 2009 (10).
  • SFTSV thrombocytopenia syndrome virus
  • tick-borne viruses as well as the dearth of data on tick viromes suggests a need for viral surveillance and discovery in ticks. Although it seems plausible that there are tick-borne viruses that have not been amenable to isolation via tissue culture, as far as is known, no extensive culture-independent studies have been attempted to examine tick viromes.
  • Each tick pool was homogenized in 500 ⁇ of PBS, followed by purification through 0.45 ⁇ filter.
  • the filtrate (237 ⁇ ) was treated with 1.5 ⁇ RNase A for 15 minutes, followed by Turbo DNase (7.5 ⁇ ), Benzonase (1.8 ⁇ ) and 2.7 ⁇ of 1M MgC12 for 45 minutes. All nuclease treatment was performed at room temperature. 250 ⁇ of the nuclease treated filtrate was added to 750 ⁇ of Nuclisens buffer and total nucleic acid (TNA) was extracted using the EasyMag extraction platform (Biomerieux). TNA from each pool was eluted in a 35 ⁇ volume.
  • TNA TNA (11 ⁇ ) from each tick pool was subjected to first and second strand cDNA synthesis with Super Script III reverse transcriptase (Invitrogen) and Klenow Fragment (New England Biolabs), respectively. Random primers (303 ⁇ 4/ ⁇ 1) (Invitrogen) were used in both assays. Ion ShearTM Plus Reagents Kit (Life Technologies) was used for double stranded cDNA fragmentation at 37°C for 25 min. Agencourt® AMPure® XP (Beckman Coulter) reagent (1.8 x sample volume) was used for DNA purification. Ion XpressTM Adapters and unique Ion
  • XpressTM Barcodes (Life Technologies) were ligated to fragmented material by using the Ion Plus Fragment Library kit (Life Technologies). Ligation was performed at 25°C for 15 min and 72°C for 5 min. Ligated and nick repaired products were purified with Agencourt® AMPure® XP (0.85 x sample volume) and amplified according to manufacturers' instructions with Platinum®PCR Super Mix High Fidelity from the Ion Plus Fragment Library kit. Amplified products were purified as previously described for the ligation reaction. Agilent® High Sensitivity DNA Kit was used for library quantitation on the BioanalyzerTM 2100 instrument. The concentration of each barcoded library was approximately 50nM.
  • Ion OneTouchTM 200 Template Kit v2 (Life Technologies) was used to bind barcoded libraries to Ion SphereTM particles (ISPS).
  • Emulsion PCR of DNA linked ISPS was performed on the Ion OneTouchTM 2 instrument (Life Technologies).
  • Ion OneTouchTM ES instrument was used to isolate template -positive ISPS.
  • Ion PGMTM Sequencing 200 Kit v2 (Life Technologies) was used for sequencing of templated ISPS which were loaded on the Ion 316TM Chip for further processing on the Ion Personal Genome Machine® (PGMTM) System (Life Technologies). Approximately 600,000 reads were obtained for each library.
  • the de -multiplexed reads were preprocessed by trimming primers and adaptors, length filtering, and masking of low complexity regions (WU-BLAST 2.0).
  • the remaining reads were subjected to homology search using BLASTn against a database consisting of ribosomal and genomic metazoan sequences. Following the processing, the remaining reads amounted to 116,946 (pool 1) and 131,676 reads (pool 2) for /, scapularis, 30,353 (pool 1) and 29,334 (pool 2) for D. variabilis and 297,175 for the A. amblyomma pool.
  • the host-subtracted reads were assembled using the Newbler assembler (454, v2.6). Contigs and singletons were then subjected to a homology search against the entire GenBank database using BLASTn and the viral GenBank database using BLASTx. Contigs and singletons with similarity to viral sequences from the BLASTx analysis were again subjected to a homology search against entire GenBank database to correct for biased e-values. For potential viral candidates, close relatives were used to identify low homology regions in the genome from
  • cDNA generated from adult /. scapularis for a previous study was used as template for PCR (23).
  • the ticks were collected in 2008 from four separate locations in New York State: two from Suffolk county (Heckscher State Park, Fire Island) and two from Westchester County (Kitchawan Nature Preserve, Blue Mountain Reservation) ( Figure 1).
  • cDNA from individual ticks was screened by PCR for each novel virus identified by HTS.
  • cDNA from individual virus-positive ticks were used to generate viral genomic sequence equivalent to the sequences obtained by HTS.
  • the sequences were obtained by generating overlapping PCR products using HTS sequences as a reference for primer design and sequence assembly. All PCR products were verified by dideoxy sequencing.
  • Arthropod genomes frequently contain integrated fragments of archaic RNA viral genomes (27-29).
  • the genome of /. scapularis contains numerous sequences of viral origin, designated endogenous viral elements, or EVEs (27).
  • EVEs endogenous viral elements
  • results of RT-PCR vs DNA PCR were compared as an additional indication of the template source i.e., authentic viral nucleic acid vs. tick genomic DNA. Consistent with a model where these novel virus sequences represent authentic RNA viruses and not EVEs, products were obtained with RT-PCR but not with PCR.
  • POWV lineage II has been detected in /. scapularis throughout the Northeast (23, 32, 33). In this HTS study, approximately 0.8% of all filtered reads from /. scapularis pool 1 were from POWV lineage II. Assembly of all reads and contigs from this pool resulted in assembly of 98% of the 10.8 Kb POWV genome.
  • Nucleotide (nt) comparison of the complete polyprotein coding sequence indicated that the nt sequence of this virus (designated strain LI-1) is 99.4% identical to isolate NSF001 (GenBank accession number HM44059) obtained from Nantucket, MA in 1996 and clustered with viruses in a subclade of POWV lineage II isolated from the Northeast ( Figure 2) (33).
  • the nt identity to the subclade of lineage II made up of isolates from the Midwestern United States was 93.4%.
  • the genus Nairovirus (family Bunyaviridae) is comprised of 37 tick-borne viruses. Its genome consists of three segments of negative sense single-stranded RNA, designated small (S), medium (M), and large (L), that encode the nucleocapsid protein (N), the envelope glycoproteins (Gn and Gc) and an RNA-dependent RNA polymerase (L), respectively (34). Both /. scapularis pools contained multiple contigs with sequence similar to viruses in the Nairovirus genus by BLASTx. Assembly of these Nairovirus-like contigs, using CCHFV as a reference genome, provided >90%> coverage of the S and L segments.
  • SBV South Bay virus
  • SBV H38 The complete S and L sequences from an individual SBV-positive tick (designated SBV H38) were also obtained. Comparison of the genomic nt sequences obtained from HTS and SBV H38 indicated they were 97%> and 99%> identical in the S and L segments, respectively. SBV reads were the predominant viral reads obtained from both /. scapularis pools and accounted for 25 %> of filtered reads from pool 1 and 41% of reads from pool 2.
  • /. scapularis DNA and corresponding cDNA was screened for the presence of SBV. Partial S and L sequences were amplified in SBV-positive tick cDNA, but were absent in the corresponding genomic DNA. Additionally, it was found that /. scapularis genome contains an integrated Nairovirus N gene open reading frame (ORF) (accession number XM002414099), although it was not transcribed in any tick sample. This sequence was more similar to SBV than other nairovirus sequences, likely representing an ancestral integration of an SBV-like nairovirus into the /. scapularis genome.
  • ORF Nairovirus N gene open reading frame
  • SBV contained a seven aa insertion in this region and had ⁇ 45% identity to the next closest virus, distinguishing SBV from other nairoviruses (Table 2). SBV did not cluster with any of the described nairovirus serogroups, but fell outside of all described viruses in this genus ( Figure 3).
  • the L segment of SBV is 13892 nt long and contains a 13611 nt ORF that encodes a 4536 aa protein. This represents the longest known nairovirus open reading frame, over 600 amino acids longer than the L ORF of Dugbe virus.
  • Comparison of SBV to L proteins from other nairoviruses revealed that SBV does not cluster with any of the previously sequenced viruses, and contains only 28% aa identity to the next closest virus (Erve virus) ( Figure 4A, Table 3).
  • the catalytic polymerase domain is located within the region between amino acid (aa) residues 2600 and 3200 and contains all known viral RNA polymerase motifs (pre-motif A, motifs A-E) (36).
  • the nairovirus L proteins may contain several other protein motifs, such as ovarian tumor domain (OTU), topoisomerase domain, zinc finger motif and a leucine zipper motif (36, 37).
  • OTU ovarian tumor domain
  • the OTU domain represents a family of cysteine proteases and it is present in the N terminus of all seven sequenced nairovirus L proteins. In CCHFV this domain may function in modulation of interferon response in host cells (38).
  • SBV has an unusually long S segment comprising 5536 nucleotides (nt) and containing a 1664 nt ORF encoding a putative N protein.
  • the N protein has a number of important functions in negative-sense single-stranded RNA viruses. It binds to genomic RNA forming ribonucleoprotein complexes that associate with the polymerase for viral RNA synthesis. In bunyaviruses, N proteins are also associated with immune response modulation.
  • the N ORF begins at position 865 from the 3' end of the segment and is predicted to encode a 547 aa N protein, representing the second largest Nairovirus N.
  • the S segment of SBV contains additional 3028 nt of sequence following the termination of the N ORF ( Figure 6). This portion of the segment does not display homology to any known sequence and contains several short potential ORFs, the longest consisting of 441 nt, though none contain any identifiable domains.
  • the terminal 1400 nt of the S segment does not contain any potential ORF longer than 170 nt.
  • the genus Phlebovirus is comprised of over 70 viruses isolated primarily from ticks, mosquitoes and sandflies (42). Multiple contigs were identified with homology to phleboviruses by BLASTx in each pool of /. scapularis and D. variabilis. The contigs originating from each tick species were assembled separately to a reference phlebovirus genome (Precarious Point virus). Initial sequence comparison indicated that both viruses likely represented new phlebovirus species, as they were distinct from each other and other phlebovirus sequences in GenBank. The I.
  • BTPV blacklegged tick phlebovirus
  • ADTPV American dog tick phlebovirus
  • PCR analysis of individual ticks indicated that BTPV and ADTPV were highly prevalent in their tick hosts (Table 4). Out of 24 adult /. scapularis from Heckscher state park, 12 (50%o) were positive by PCR for BTPV-1 or BTPV-2 (Table 4). Twelve D. variabilis collected on Fire Island, NY, approximately 16 miles Southeast of Heckscher state park, were screened 9 positive ticks for ADTPV (75%>) were identified. Complete S and L sequences from two individual /. scapularis were iobtained (designated BTPV-1 HI 2, and BTPV-2 H5, accession numbers KM48313-KM48316 (SEQ ID NOs:23, 25, 27, 29)), and ADTPV sequences from one individual D.
  • a typical Phlebovirus S segment is approximately 1700 nts in length and codes for a nucleocapsid (N) and a non-structural protein ⁇ NSs ⁇ in an ambisense orientation (43).
  • the lengths of the putative S segments of BTPV-1 and BTPV-2 were >2400 nt and contained unusually long ORFs of 1581 and 1578 nt in length, respectively ( Figure 7). These would encode putative 526 and 525 aa proteins.
  • the N terminal portions of these putative proteins displayed no homology to any known protein, whereas the C-terminal 250 aa portion exhibited similarity to other Phlebovirus N proteins.
  • the S segment of ADTPV also contained an atypically long ORF of 1209 nts encoding a putative 402 aa protein. Similar to BTPV, the characteristic Phlebovirus N was identified only within the C-terminal 250 aa sequence of this protein. A putative NSs ambisense ORF was identified, although it was shorter than the ORFs identified in BTPV (151 nt). The aa identity of the putative N of ADTPV to BTPV-1 and BTPV-2 was 24%, and ⁇ 34% to other Phlebovirus N proteins (Table 5).
  • the overall length of the complete L segments of both BTPV-1 and BTPV-2 were 6733 nt and contained 6624 nt ORFs that would encode a 2207 aa L protein.
  • the nt sequence identity of BTPV-1 and BTPV-2 L ORFs was 84% (95.6% aa identity).
  • the length of the ADTV L segment was 6600 nt, with a 6537 nt ORF encoding a 2178 aa protein.
  • the ADTV L protein was 29.6% and 29.2% identical to the L protein of BTPV-1 and BTPV-2, respectively (Table 5).
  • Phleboviruses have traditionally been classified into two groups consisting of sandfly/mosquito-borne and tick-borne viruses (42). At least three phylogenetic clusters of tick- borne phleboviruses have been identified, each comprised of several potential species: the
  • Mononegavirales-like virus [0354] In sequences originating from pool 2 of /. scapularis contigs with weak homology by BLASTx to the L protein RNA-dependent RNA polymerase domain (RdRp) of viruses within the order Mononegavirales were identified. This order represents five families (Bornaviridae,
  • ISAV-1 Ixodes scapularis associated virus-1
  • ISAV-2 Ixodes scapularis associated virus-2
  • variabilis contained sequences with low similarity ( ⁇ 20%) to a wide range of invertebrate single-stranded positive-sense viruses. A 5.3 kb segment of this virus was recovered, encoding a putative >1700 aa replicase. One out of nine tested D. variabilis ticks was positive for this virus, with 96.8% nt and 99.3% aa identity to the HTS sequence (accession number KM048322). The N terminal 1300 aa of this protein contained the methyltransferase, helicase, and RdRp domains ( Figure 10). No putative domain could be identified within the >400 aa C-terminal portion.
  • RNA1 segment (approximately 5.3 kb in length) encodes a replicase containing all aforementioned domains and RNA2 segment (approximately 2.5 kb in length) encodes the capsid (46).
  • RNA2 segment that encodes a protein with low (11% aa) identity to Omegatetravirus capsids were identified. Further classification of this new virus was challenging due to high sequence divergence in combination with a limited number of available Alphatetraviridae sequences. Although this virus was tentatively included within the Alphatetraviridae, due to dissimilarity in sequence and host association, it likely represents a new family of arthropod- associated viruses.
  • Bunyavirus-like sequences predominated in the HTS data described herein.
  • the known Bunyaviridae comprise more than 350 viruses with many of those only recently discovered and characterized (10, 19, 50-52). Relatively few nairoviruses have been discovered; Kupe and Finch Creek viruses were the only new characterized members of this genus reported within the last decade (53, 54). In this study, molecular evidence of a new nairovirus present in ixodid ticks in North America was demonstrated.
  • SBV is the first New World nairovirus with available coding region sequence of the S and L segments.
  • nairoviruses have been shown to have broad geographical distributions and have been isolated throughout the range of their hosts.
  • tick-borne phlebo viruses representing novel clades of tick-borne phlebo viruses.
  • phleboviruses were classified into tick-borne or sandfly/mosquito-borne groups based on vector, genomic, and serological relationships (42). The discovery of these highly divergent viruses along with the recent
  • scapularis with SBV may be significantly higher than with Powassan virus, at least within the geographical range analyzed.
  • Possible explanations for the high prevalence of these viruses may be the result of a high assortment of susceptible viremic hosts resulting in proficient horizontal transmission of the virus to uninfected ticks.
  • the viruses may also be efficiently transmitted by non-viremic hosts, or by "co-feeding" of infected and uninfected ticks.
  • these viruses may be transtadially (maintained throughout life stages) and transovarially transmitted to progeny, resulting in the tick vector also playing a role of a long-term reservoir in tick populations (3).
  • tick-borne viruses can be isolated from more than one tick species.
  • CCHFV for example, has been isolated from over 30 tick species and multiple genera throughout Europe, Africa and Asia, though not all likely represent true vectors (9).
  • HTS analysis suggest tick host specificity, it cannot be ruled out that other tick species may serve as hosts of these viruses.
  • the L segment organization of SBV, ADTPV and BTPV is comparable to the L segments of other nairo- and phleboviruses. Conversely, the organization of the presumed S segments of these viruses is at least partially inconsistent with their putative genome affiliation. In these viruses, the S segments are considerably longer, and in the case of SBV, contain a large portion of sequence lacking identifiable ORFs.
  • the N ORFs in ADTPV and BTPV are longer than typical Phlebovirus N, while the putative NSs ORFs are considerably shorter relative to typical Phlebovirus NSs ORFs and may not encode functional proteins.
  • Another notable characteristic of these viruses is the lack of recognizable glycoprotein- coding segment. Although > 90% of the L and S segments were recovered for SBV and both phleboviruses by HTS and dispelled the possibility of viral integration, it was not possible to identify any sequences with similarity to Bunyaviridae M segments. In addition, large contigs (>300 nt) containing an uninterrupted ORF that was confirmed by PCR to be present in individual S and L segment-positive ticks but absent in virus-negative ticks were not identified. Consensus PCR was also performed with numerous primers targeting Phlebovirus and Nairovirus M segments, without success. To investigate if the depth of sequencing was adequate to detect bunyavirus M segments, a new pool of four adult /.
  • the viruses uncovered here may represent viral lineages that do not have a typical bunyaviral M segment and employ other means for cellular entry.
  • the S and L segments of these viruses may exist in an episome-like form in tick cells. As such, they may not form an infectious virion instead using transovarial transmission or some other means as a vehicle for their dissemination to new hosts.
  • Table 1 Viral sequences identified in I. scapularis and D. variabilis by highthroughput sequencing.
  • tick-associated viruses and immunogenic compositions described herein can be be produced in cells. Production of the tick-associated viruses and immunogenic compositions described herein may also be accomplished on any useful media and permissive cell or tissues, which may be derived from avian or mammalian cell lines derived from human, canine, feline, equine, bovine or porcine cell lines.
  • a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, avian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells, such as recombinant cells expressing a virus.
  • tick-associated viruses and immunogenic compositions can be in any cell type, including but not limited to mammalian cells.
  • Cell lines suitable for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limited to dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NSO, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
  • the cell culture system for producing the tick-associated viruses and immunogenic compositions described herein can be a traditional adherent monolayer culture. Alternatively, suspension and microcarrier cell culture systems can also be utilized.
  • Vessels for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limted to, roller bottles.
  • other useful cell culture formats include flasks, stacked modules and stir tanks.
  • multiplicity of infection (MOI) can be 0.001-0.1 but can range from 0.0001-2.0.
  • the harvest virus from cell culture can be, but is not limited to, any time between day 2 to 5 post-infection, but can range from day 1 to day 7 post-infection.
  • Cell culture media formulations suitable for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limted to, Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof. These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro.TM. SFM, AIM V.RTM. media, HyQ SFM4 MegaVir.TM., EX-CELL. TM. Vera SFM, EPISERF, ProVero, any 293 or CHO media and analogues and derivatives thereof.
  • the culture media described herein can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamine and biological buffers.
  • Preferable medium is OptiPRO.TM. SFM supplemented with L-glutamine and trypsin.
  • the cell culture media can be supplemented with 0.1 to 10 units of trypsin.
  • plant derived equivalents of trypsin e.g. Accutase
  • Cell culture media can be used in the absence or presence of animal-derived components.
  • An example of supplementation with an animal-derived component is gamma- irradiated serum ranging from 0.5-10% final concentration.
  • tick-associated viruses and immunogenic compositions in can also be performed in eggs.
  • tick-associated viruses propagation can be accomplished by inoculating embryonated eggs.
  • 0-12 day old embryonated eggs can be used for tick-assocaited virus propagation.
  • 7-8 day old embryonated eggs can be used for virus growth.
  • the tick-assocaited virus can be inoculated into the amniotic cavity of the egg.
  • the tick-associated viruses will replicate in the cells of the amniotic membrane and large quantities are released back into the amniotic fluid.
  • tick-associated viruses in the amniotic fluid can be harvested after 23-24 days post inoculation.
  • Production of the tick-associated viruses and immunogenic compositions in can also be performed using a recombinant expression system that expresses the tick-associated virus, a tick- associated virus protein, a fragment of a tick-associated virus protein or a variant of a tick- associated virus protein.
  • the expression system can comprise any suitable plasmid or a linear expression construct known in the art.
  • Example 3 Virus Preparation, Attenutation and Inactivation
  • the immunogenic compositions described herein can comprise an inactivated or killed tick- associated virus vaccine.
  • Inactivated immunogenic composition can made by methods well known in the art. For example, once the tick-associated virus is propagated to high titers, the tick-associated virus antigenic mass could be obtained by methods well known in the art. For example, the tick-associated virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate tick-associated virus antigenic mass to produce immunogenic compositions.
  • the tick-associated virus may be inactivated by treatment with formalin (e.g. 0.1-10%), betapropriolactone (BPL) (e.g. 0.01-10%), or with binary
  • ethyleneimine (BEI) (e.g. 1-10 mM), or using other methods known to those skilled in the art.
  • Attenuation leading to modified live immunogenic compositions can also be used in conjunction with the compositions and methods described herein.
  • Methods of attenuation suitable for use with the viruses described herein include continuous passaging in cell culture, continuous passaging in animals, various methods for generating genetic modifications and ultraviolet or chemical mutagenesis.
  • Attenuation of tick-associated virus may be achieved through cold-adaptation of a tick- associated virus strain.
  • Cold-adapted tick-associated virus strains may be produced by methods which includes passaging a wild-type tick-associated virus, followed by selection for tick- associated virus that grows at a reduced temperature.
  • Cold-adapted tick-associated virus can be produced, for example, by sequentially passaging a wild-type tick-associated virus in embryonated cells or chicken eggs at progressively lower temperatures, thereby selecting for certain members of the tick-associated virus mixture which stably replicate at the reduced temperature.
  • a cold-adapted tick-associated virus strain may exhibit a temperature sensitive phenotype.
  • a temperature sensitive cold-adapted tick-associated virus replicates at reduced temperatures, but no longer replicates at certain higher growth temperatures at which the wild-type tick-associated virus will replicate.
  • a temperature at which a temperature sensitive tick-associated virus will grow is referred to herein as a "permissive" temperature for that temperature sensitive tick-associated virus, and a higher temperature at which the temperature sensitive tick-associated virus will not grow, but at which a corresponding wild-type tick-associated virus will grow, is referred to herein as a "non-permissive" temperature for that temperature sensitive tick-associated virus.
  • a cold-adapted tick-associated virus may also be produced through recombinant means.
  • one or more specific mutations associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, can be identified and are introduced back into a wild-type tick- associated virus strain using a reverse genetics approach.
  • Reverse genetics entails can be performed using RNA polymerase complexes isolated from tick-associated virus-infected cells to transcribe artificial tick-associated virus genome segments containing the mutation(s),
  • RNA segment(s) incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes.
  • Attenuation of a tick-associated virus may be achieved by serial passaging of a wild-type tick-associated virus strain in cell culture.
  • the tick-associated virus strain can be passaged in a variety of cell systems until its ability to produce disease is lost whilst its immunogenic character is fully retained.
  • the tick-associated virus Once inoculated into the host, the tick-associated virus may be capable of multiplication to some extent.
  • attenuated tick-associated virus compositions can be prepared from cell line that has been attenuated by serial passage including serial passage at sub- optimal temperatures to a state where it is no longer capable of causing disease, but still capable of eliciting a protective immune response.
  • Suitable attenuated tick-associated virus strains may also be obtained by serial passaging to obtain an over-attenuated strain.
  • the "over-attenuation" means that the number of passages for attenuation has been substantially greater than what is normally necessary for the removal of pathogenicity.
  • the attenuated tick-associated virus retains its antigenicity after these numerous passages so that its immunogenic ability is not impaired.
  • Such strains produce practically no symptoms or side effects when administered, and thus are safe and efficacious vaccines.
  • Dose sizes of the immunogenic compositions described herein can be in the range of about 2.0 to 0.1 ml depending on the route of administration, but dose sizes are not limited to this range.
  • for inactivated tick-assocaited virus compositions can contain suitable TCID50 levels of virus prior to inactivation.
  • suitable TCID50 levels for the immunogenic compositions described herein.
  • the antigen content in the tick- assocaited virus preparation can have, but is not limited to, a titer of between 10 to 10,000 units/ml as the amount administered per dose.
  • a suitable antigen content for the immunogenic compositions described herein can be in the range of about 2.0 to 0.1 ml depending on the route of administration, but dose sizes are not limited to this range.
  • a therapeutically effective dose can be determined by one of skill in the art.
  • a therapeutically effective dose can be determined by one of skill in the art. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan.
  • An animal for example a dog, can be inoculated with the immunogenic compositions or formulations described herein to generate an immune response.
  • inoculation can be perfomed on an animal (e.g. a dog) that is at least 6 weeks or older.
  • the animal e.g. dog
  • the animal can receive one or more dosages.
  • two or more dosages can be administered to the animal (e.g. dog) 3-4 weeks apart.
  • the administration can be by subcutaneous injection.
  • Intramuscular, intradermal, oral, oronasal or nasal routes of administration can also be used to administer the immunogenic compositions or formulations described herein.

Abstract

The invention is directed to isolated tick-associated viruses and isolated nucleic acids sequences and polypeptides thereof. The invention also relates to antibodies against antigens from tick- associated viruses. The invention also relates to iRNAs which target nucleic acid sequences of the tick-associated virus. The invention is related to methods for detecting the presence or absence of tick-associated viruses in an animal. The invention is also related to immunogenic compositions for inducing an immune response against tick-associated viruses in an animal.

Description

TICK-ASSOCIATED VIRUS SEQUENCES AND USES THEREOF
[0001] This application claims the benefit of and priority to U.S. Serial No.: 62/034,722 filed August 7, 2014, the contents of which is hereby incorporated by reference in its entirety.
[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
[0003] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. The issued patents, applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.
Government Support
[0004] The work described herein was supported in whole, or in part, by National Institute of Health Grant No. 1U19AI109761. Thus, the United States Government has certain rights to the invention.
BACKGROUND
[0005] Ticks (class Arachnida, subclass Acari) have been implicated as vectors in a wide range of human and animal diseases worldwide. Approximately 900 species of ticks have been described and taxonomically classified into three families: Argasidae (argasid or soft ticks) Ixodidae (ixodid, or hard ticks) and Nuttalliellidae. Their propensity for feeding on a wide array of hosts, expansive range and long life cycle underscore the importance of tick surveillance for the presence of potential pathogens. Argasid and ixodid ticks combined transmit a greater diversity of viral, bacterial and protozoan pathogens than any other arthropod vector. The worldwide incidence of tick-borne disease is increasing, partly due to increased frequency of endemic tick-borne diseases, as well as the discoveries of new tick-associated agents. There is a need to identify tick-associated viruses. There is also a need for tools and methods for detecting the presence of the tick-associated viruses, as well as protecting tick hosts from tisk-associated viruses. There is also a need for a diagnostic test, an immunogenic composition and a method of treating humans and animals (e.g. tick hosts) having tick-associated viral infections. Additionally, there is a need for immunogenic compositions that protect against tick-associated viruses. This invention addresses these needs. SUMMARY OF THE INVENTION
[0006] The invention is related to novel tick-associated viruses and isolated nucleic acids sequences and peptides thereof. The invention is also related to antibodies against antigens derived from the tick-associated viruses. The invention is also related to iRNAs which target nucleic acid sequences of the tick-associated viruses. The invention is related to methods for detecting the presence or absence of a tick-associated virus in a tick, an animal or a human. The invention is also related to immunogenic compositions for inducing an immune response against a tick-associated virus or viruses in an animal or human.
[0007] In certain aspects, the invention relates to an isolated nucleic acid having the sequence of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0008] In certain aspects, the invention relates to an isolated nucleic acid complementary to the sequence of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0009] In certain aspects, the invention relates to an isolated nucleic acid having at least about 60% sequence identity to SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In certain aspects, the invention relates to an isolated nucleic acid having at least about 85% sequence identity to SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. In some embodiments, the isolated nucleic acid has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0010] In certain aspects, the invention relates to an isolated nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated nucleic acid having at least about 85% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In some embodiments, the isolated nucleic acid has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%), about 99.5%) or about 99.9%> sequence identity to a nucleic acid complementary to the sequence of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0011] In certain aspects, the invention relates to an isolated nucleic acid comprising at least 10 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42. [0012] In certain aspects, the invention relates to an isolated nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0013] In certain aspects, the invention relates to an isolated nucleic acid which comprises consecutive nucleotides having a sequence complementary to an isolated nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or an isolated nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 85%> identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In some embodiments, the isolated nucleic acid comprises at least 10 consecutive nucleotides of a sequence having at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0014] In some embodiments, the nucleic acid is a DNA sequence. In some embodiments, the nucleic acid is an RNA sequence. In some embodiments, the nucleic acid is a cDNA.
[0015] In certain aspects, the invention relates to an isolated polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0016] In certain aspects, the invention relates to an isolated polypeptide encoded by a nucleic acid described herein.
[0017] In certain aspects, the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated polypeptide having at least about 85 > sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42. In some embodiments, the isolated polypeptide has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%), about 99.5%) or about 99.9%> sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0018] In certain aspects, the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. In certain aspects, the invention relates to an isolated polypeptide having at least about 85% sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. In some embodiments, the isolated polypeptide has at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9%) sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0019] In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0020] In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0021] In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 85%> identity to the sequence of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42. In some embodiments, the isolated polypeptide comprises at least 8 amino acids having at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%), about 99%>, about 99.5%> or about 99.9%> sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0022] In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 85%> identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. In some embodiments, the isolated polypeptide comprises at least 8 amino acids having at least about 90%>, about 95.5%>, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%) or about 99.9% sequence identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0023] In certain aspects, the invention relates to an isolated polypeptide having the sequence of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0024] In certain aspects, the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain aspects, the invention relates to an isolated polypeptide having at least about 85% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In some embodiments, the isolated polypeptide has at least about 90%>, about 95.5%, about 96%>, about 96.5%>, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0025] In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0026] In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 85% identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In some embodiments, the isolated polypeptide comprises at least 8 amino acids having at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0027] In certain aspects, the invention relates to an isolated antibody that specifically binds to a polypeptide of the invention (e,g a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a polypeptide encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or sequences complementary thereto).
[0028] In certain aspects, the invention relates to an immunogenic composition comprising at least about 24 consecutive nucleotides from an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or nonisolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%) identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or a polypeptide encoded from any such nucleic acid.
[0029] In certain aspects, the invention relates to an immunogenic composition comprising an isolated (or non-isolated) polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain aspects, the invention relates to an immunogenic composition comprising at least 8 consecutive amino acids of a polypeptide described herein.
[0030] In certain aspects, the invention relates to a method of inducing an immune response in an animal, the method comprising administering the immunogenic composition described herein.
[0031] In certain aspects, the invention relates to an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent. [0032] In some embodiments, the polypeptide is L segment protein, a N protein, a polymerase protein or a nucleocapsid protein. In some embodiments, the composition is for protecting an animal against a tick-associated virus. In some embodiments, the animal is a human. In some embodiments, the composition delays the onset of symptoms associated with a tick-associated virus, or reduces the severity of symptoms of a tick-associated virus. In some embodiments, the polypeptide is derived from a polypeptide sequence having at least 90% identity to the amino acid sequence SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In some embodiments, the composition further comprises an adjuvant.
[0033] In certain aspects, the invention relates to a method for determining the presence or absence of a tick-associated virus in a biological sample, the method comprising: (a) contacting nucleic acid from a biological sample with at least one primer which is a synthetic nucleic acid of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid
complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to any of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; (b) subjecting the nucleic acid and the primer to amplification conditions, and (c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with a tick-associated virus in the sample.
[0034] In certain aspects, the invention relates to a synthetic nucleic acid comprising at least about 10 nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0035] In certain aspects, the invention relates to a cDNA oligonucleotide probe comprising from about 10 nucleotides to about 50 nucleotides, wherein at least about 10 contiguous nucleotides are at least 95 % complementary to a nucleic acid target region within the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0036] In certain aspects, the invention relates to a synthetic nucleic acid comprising the nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%> sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0037] In certain aspects, the invention relates to a primer set for determining the presence or absence of a tick-associated virus in a biological sample, wherein the primer set comprises at least one synthetic nucleic acid sequence described herein.
[0038] In certain aspects, the invention relates to a method for determining whether or not a sample contains a tick-associated virus, the method comprising: (a) contacting a sample with an antibody that specifically binds to a polypeptide of any of an isolated (or non-isolated) polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; and (b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates that the biological sample contains a tick-associated virus.
[0039] In certain aspects, the invention relates to a method for determining whether or not a sample contains a tick-associated virus, the method comprising: (a) contacting a sample with an antibody that specifically binds to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; and (b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates that the biological sample contains a tick-associated virus.
[0040] In some embodiments, the determining comprises use of a lateral flow assay or ELISA.
[0041] In certain aspects, the invention relates to a method for determining whether or not a biological sample has been infected by a tick-associated virus, the method comprising: determining whether or not a biological sample contains antibodies that specifically bind to a polypeptide of any one of an isolated (or non-isolated) polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0042] In certain aspects, the invention relates to a method for determining whether or not a biological sample has been infected by a tick-associated virus, the method comprising: determining whether or not a biological sample contains antibodies that specifically bind to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide having at least about 80%> sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80%> identity to the sequence of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0043] In certain aspects, the invention relates to an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent. In certain embodiments, the polypeptide is L segment protein. In certain embodiments, the polypeptide is a N protein. In certain embodiments, the polypeptide is a polymerase protein. In certain embodiments, the polypeptide is a nucleocapsid protein. In certain embodiments, the composition is for protecting an animal against a tick-associated virus. In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the composition protecting an animal against a tick-associated virus delays the onset of symptoms associated with a tick- associated virus, or reduces the severity of symptoms of a tick-associated virus. In certain embodiments the animal is a human.
[0044] In certain aspects, the invention relates to a method of immunizing an animal against a tick- associated virus, the method comprising administering to the animal an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent. In certain embodiments, the animal is a human.
[0045] In certain aspects, the invention relates to an interfering RNA (iRNA) comprising at least 15 contiguous nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0046] In certain aspects, the invention relates to an interfering RNA (iRNA) comprising a sense strand having at least 15 contiguous nucleotides complementary to the anti-sense strand of a gene from a virus comprising a nucleic acid sequence selected from the group of sequences consisting of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0047] In certain aspects, the invention relates to a method for reducing the levels of a tick- associated virus protein in an animal, viral mRNA in an animal or viral titer in a cell of an animal, the method comprising administering to the animal an iRNA described herein. [0048] In certain aspects, the invention relates to an isolated virus comprising at least 24 consecutive nucleotides from an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60%) sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%> identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0049] In certain aspects, the invention relates to an isolated cell comprising at least 8 consecutive amino acids from a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; or a polypeptide having at least about 80%> identity to the sequence of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0050] In certain aspects, the invention relates to a method for culturing cells comprising: a) infecting a cell with a tick-associated virus, or an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or nonisolated) nucleic acid having at least about 60%> sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60%) identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and b) culturing the cells.
[0051] In certain aspects, the invention relates to a method of testing a tick-associated virus vaccine, comprising: a) contacting cells with a tick-associated virus vaccine; b) contacting cells with a tick-associated virus; and c) measuring the number of cells infected with a tick-associated virus.
[0052] In certain aspects, the invention relates to a method of testing a tick-associated virus drug, comprising: a) contacting cells with a tick-associated virus drug; b) contacting cells with a tick- associated virus; and c) measuring the number of cells infected with a tick-associated virus.
[0053] In certain aspects, the invention relates to a method of testing an a tick-associated virus drug, comprising: a) contacting cells with a tick-associated virus; b) contacting cells with an a tick- associated virus drug; and c) measuring the replication of a tick-associated virus.
BRIEF DESCRIPTION OF THE FIGURES
[0054] Figure 1 shows a map of tick collection sites.
[0055] Figure 2 shows a maximum likelihood phylogenetic tree based on the nucleotide sequence of the complete POWV polyprotein. The accession number, geographical location and year of isolation/detection is indicated for each isolate. SEQ ID NO: 1, described herein, is indicated by *. Tick-borne encephalitis virus is shown as an outgroup.
[0056] Figure 3 shows a maximum likelihood phylogeny of all available nairovirus sequences based on a conserved 150 amino acid fragment of the L protein. Accession numbers are provided next to the virus name. * indicates the South Bay virus described herein.
[0057] Figures 4A-B show maximum likelihood phylogeny based on complete L (A) and N (B) protein of all nairoviruses with available genomic sequences.
[0058] Figure 5 shows alignment of the conserved C terminal domain present in all Nairovirus N proteins. Conserved residues are indicated by *. Residues implicated in N function are indicated in bold. The numerical amino acid positions within the N are displayed for each virus. [0059] Figure 6 shows comparison of the S segments of SBV and Dugbe virus. The location of the N ORFs are shown in green. Putative additional SBV ORFs are shown in red. Numbers indicate nt position within the segment.
[0060] Figure 7 shows a schematic of the S segments of BTPV-1, BTPV-2 and ADTPV. The putative N ORFs are indicated in yellow. The putative ORFs corresponding to the genomic location of the NSs are displayed in red.
[0061] Figures 8A-B show maximum likelihood phylogeny of BTPV- 1 , BTPV-2 and ADTPV estimated from amino acid sequences for phlebovirus N (A) and L (B). Accession numbers for every virus in the tree are indicated. * designates viruses described herein.
[0062] Figure 9 shows phylogeny of the novel /. scapularis-associated mononegavirus, indicated by *, to viruses within Mononegaviridae. Each terminal branch represents a single type- species representative of a genus.
[0063] Figure 10 shows a comparison of the tetravirus-like virus replicase from D. variablis to helicoverpa armigera stunt virus. Numbers indicate amino acid position.
DETAILED DESCRIPTION
[0064] A wide range of bacterial pathogens have been described in ticks, yet the diversity of viruses in ticks is largely unexplored. In the United States, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. Described herein are the sequences of several tick-associated viruses.
[0065] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
[0066] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.
[0067] As used herein, "SBV" refers to isolates of the South Bay Virus described herein.
[0068] As used herein, "BTPV" refers to isolates of the blacklegged tick phlebovirus described herein.
[0069] As used herein, "ADTPV" refers to isolates of the American dog tick phlebovirus described herein.
[0070] As used herein, "ISAV" refers to isolates of the Ixodes scapularis associated virus described herein. [0071] As used herein, the term "animal" refers to a vertebrate, including, but not limited to, birds, canines, felines, equine, sheep, cattle, poultry or humans.
[0072] As used herein, the term immunogenic composition refers to a composition capable of inducing an immunogenic response in an animal or a cell. As used herein, refernce to an
immunogenic composition can include include a vaccine.
[0073] The present invention provides tick-associated virus nucleic acid sequences. These nucleic acid sequences may be useful for, inter alia, expression of tick-associated virus-encoded proteins or fragments, variants, or derivatives thereof, generation of antibodies against tick- associated virus proteins, generation of primers and probes for detecting tick-associated virus and/or for diagnosing tick-associated virus infection, generating immunogenic compositions against tick-associated viruses, and screening for drugs effective against tick-associated viruses as described herein. The present invention also provides methods for diagnosing tick-associated viral infection. The present invention also provides methods of detecting antibodies against tick- associated viruses in a sample from an animal.
[0074] In certain aspects, the invention is directed to a Powassan virus isolate LI-1 nucleic acid sequence (including cDNA sequences corresponding to Powassan RNA sequences, including mRNA sequences). The complete nucleotide coding sequence (cDNA) of Powassan virus isolate LI-1 polyprotein is shown in SEQ ID NO: 1. Sequence information related to Powassan virus isolate LI-1 is accessible in public databases by GenBank Accession Number KJ746872.
[0075] SEQ ID NO: 1 is the nucleic acid sequence of the complete coding sequence (cDNA) of Powassan virus isolate LI-1 polyprotein.
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
[0076] The protein sequence of Powassan virus isolate LI-1 polyprotein is shown in SEQ ID NO:2. Sequence information related to Powassan virus isolate LI-1 is accessible in public databases by GenBank Accession Number AIG95651.
[0077] SEQ ID NO: 2 is the protein sequence of Powassan virus isolate LI-1 polyprotein.
Figure imgf000021_0002
Figure imgf000022_0001
[0078] In certain aspects, the invention is directed to South Bay Virus isolate SBV-H-1 nucleic acid sequence (including cDNA sequences corresponding to SBV-H-1 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of South Bay Virus isolate SBV-H-1 segment L is shown in SEQ ID NO:3. Sequence information related to South Bay virus isolate SBV-H-1 segment L is accessible in public databases by GenBank Accession Number KJ746877.
[0079] SEQ ID NO: 3 is the nucleic acid sequence (cDNA) of South Bay Virus isolate SBV-H- 1 segment L.
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
[0080] The protein sequence of South Bay virus isolate SBV-H-1 segment L is shown in SEQ ID NO:4. Sequence information related to South Bay virus isolate SBV-H-1 segment L protein is accessible in public databases by GenBank Accession Number AII01810.
[0081] SEQ ID NO: 4 is the protein sequence of SBV-H-1 segment L.
Figure imgf000028_0002
Figure imgf000029_0001
Figure imgf000030_0001
[0082] In certain aspects, the invention is directed to South Bay Virus isolate SBV-H-1 nucleic acid sequence (including cDNA sequences corresponding to SBV-H-1 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of South Bay virus isolate SBV-H-1 segment S is shown in SEQ ID NO:5. Sequence information related to South Bay virus isolate SBV-H-1 segment S is accessible in public databases by GenBank Accession Number KJ746878.
[0083] SEQ ID NO: 5 is the nucleic acid sequence (cDNA) of South Bay Virus isolate SBV-H- 1 segment S.
Figure imgf000030_0002
Figure imgf000031_0001
Figure imgf000032_0001
[0084] The protein sequence of South Bay Virus isolate SBV-H-1 N protein is shown in SEQ ID NO:6. Sequence information related to South Bay Virus isolate SBV-H-1 N protein protein is accessible in public databases by GenBank Accession Number AII01798.
[0085] SEQ ID NO : 6 is the protein sequence of SB V-H- 1 N protein.
Figure imgf000032_0002
[0086] In certain aspects, the invention is directed to South Bay Virus isolate H38 nucleic acid sequence (including cDNA sequences corresponding to SBV-H38 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of South Bay Virus isolate H38 segment L is shown in SEQ ID NO:7. Sequence information related to South Bay virus isolate H38 segment L is accessible in public databases by GenBank Accession Number KM048320. SEQ ID NO: 7 is the nucleic acid sequence (cDNA) of South Bay Virus isolate H38
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
[0088] The protein sequence of South Bay Virus isolate H38 segment L is shown in SEQ ID
NO:8. Sequence information related to South Bay Virus isolate H38 segment L protein is accessible in public databases by GenBank Accession Number AII01813.
[0089] SEQ ID NO: 8 is the protein sequence of South Bay Virus isolate H38 segment L.
Figure imgf000038_0002
Figure imgf000039_0001
4501 padqapqpyv tgsalrealk aidfvkkfde iheged
[0090] The nucleotide cDNA sequence of South Bay Virus isolate H38 segment S is shown in SEQ ID NO:9. Sequence information related to South Bay Virus isolate H38 segment S is accessible in public databases by GenBank Accession Number KM048321.
[0091] SEQ ID NO: 9 is the nucleic acid sequence (cDNA) of South Bay Virus isolate H38 segment S.
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
[0092] The protein sequence of South Bay virus isolate H38 N protein is shown in SEQ ID NO: 10. Sequence information related to South Bay virus isolate H38 N protein is accessible in public databases by GenBank Accession Number AII01814.
[0093] SEQ ID NO: 10 is the protein sequence of South Bay virus isolate H38 N protein.
Figure imgf000042_0002
[0094] In certain aspects, the invention is directed to Blacklegged tick phlebovirus-1 isolate RTSl nucleic acid sequence (including cDNA sequences corresponding to BTPV-1 RTSl RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate RTSl segment L is shown in SEQ ID NO: l 1. Sequence information related to Blacklegged tick phlebovirus-1 isolate RTSl segment L is accessible in public databases by GenBank Accession Number KJ746873.
[0095] SEQ ID NO: 11 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate RTS 1 segment L .
Figure imgf000042_0003
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0002
[0096] The protein sequence of Blacklegged tick phlebovirus-1 isolate RTSl segment L protein is shown in SEQ ID NO: 12. Sequence information related to Blacklegged tick phlebovirus-1 isolate RTSl segment L protein is accessible in public databases by GenBank Accession Number AII01806.
[0097] SEQ ID NO: 12 is the protein sequence of Blacklegged tick phlebovirus-1 isolate RTSl segment L protein.
Figure imgf000045_0001
Figure imgf000046_0001
[0098] In certain aspects, the invention is directed to Blacklegged tick phlebovirus-2 isolate RTS2 nucleic acid sequence (including cDNA sequences corresponding to BTPV-2 RTS2 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment L is shown in SEQ ID NO: 13. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 segment L is accessible in public databases by GenBank Accession Number KJ746874.
[0099] SEQ ID NO: 13 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate RTS2 segment L.
Figure imgf000046_0002
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
[0100] The protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment L protein is shown in SEQ ID NO: 14. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 segment L protein is accessible in public databases by GenBank Accession Number AII01807.
[0101] SEQ ID NO: 14 is the protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment L protein.
Figure imgf000049_0002
Figure imgf000050_0002
[0102] The nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate RTS1 segment S is shown in SEQ ID NO: 15. Sequence information related to Blacklegged tick phlebovirus-1 isolate RTS1 segment S is accessible in public databases by GenBank Accession Number KJ746875.
[0103] SEQ ID NO: 15 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate RTS1 segment S.
Figure imgf000050_0001
Figure imgf000051_0001
[0104] The protein sequence of Blacklegged tick phlebovirus-1 isolate RTS l N protein is shown in SEQ ID NO: 16. Sequence information related to Blacklegged tick phlebovirus-1 isolate RTS l N protein is accessible in public databases by GenBank Accession Number AII01808.
[0105] SEQ ID NO: 16 is the protein sequence of Blacklegged tick phlebovirus-1 isolate RTS l N protein.
Figure imgf000051_0002
[0106] The nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate RTS2 segment S is shown in SEQ ID NO: 17. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 segment S is accessible in public databases by GenBank Accession Number KJ746876.
[0107] SEQ ID NO: 17 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate RTS2 segment S.
Figure imgf000051_0003
Figure imgf000052_0001
[0108] The protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 N protein is shown in SEQ ID NO: 18. Sequence information related to Blacklegged tick phlebovirus-2 isolate RTS2 N protein is accessible in public databases by GenBank Accession Number AII01809.
[0109] SEQ ID NO: 18 is the protein sequence of Blacklegged tick phlebovirus-2 isolate RTS2 N protein.
Figure imgf000052_0002
301 varqmkeagv salhkaegsv aggfmdrltl vviglmrgan ldkvrkgmke anranfdvlv
361 khygiqskpv dskaitlprv iatfpgmamd vlkvaelgpv rhadmtamvk dfpremmfsa
421 fpsliprdae gvtepllsay llyqhhvsmv inpdyktwdl qkqkqslegf araameseyv
481 tqrqrvlrll eegwvsvded gkkisltpal apainaaavl yqtrk
[0110] In certain aspects, the invention is directed to American dog tick phlebovirus isolate ADTPV-1 nucleic acid sequence (including cDNA sequences corresponding to ADTPV-1 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of American dog tick phlebovirus isolate ADTPV-1 segment L is shown in SEQ ID NO: 19. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 segment L is accessible in public databases by GenBank Accession Number KJ746901.
[0111] SEQ ID NO: 19 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate ADTPV-1 segment L.
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
[0112] The protein sequence of American dog tick phlebovirus isolate ADTPV-1 polymerase protein is shown in SEQ ID NO:20. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 polymerase protein is accessible in public databases by GenBank Accession Number AIE42674.
[0113] SEQ ID NO: 20 is the protein sequence of American dog tick phlebovirus isolate ADTPV-1 polymerase protein.
1 msnpsslavl vgqnpppnde rffcpgpnvy yydtqdqvip hhtsvrrgaf idvtfetsal
Figure imgf000056_0001
[0114] The nucleotide cDNA sequence of American dog tick phlebovirus isolate ADTPV-1 segment S is shown in SEQ ID NO:21. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 segment S is accessible in public databases by GenBank Accession Number KJ746902.
[0115] SEQ ID NO: 21 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate ADTPV-1 segment S.
Figure imgf000057_0001
[0116] The protein sequence of American dog tick phlebovirus isolate ADTPV-1 nucleocapsid protein is shown in SEQ ID NO:22. Sequence information related to American dog tick phlebovirus isolate ADTPV-1 nucleocapsid protein is accessible in public databases by GenBank Accession Number AIE42675.
[0117] SEQ ID NO: 22 is the protein sequence of American dog tick phlebovirus isolate ADTPV-1 nucleocapsid protein.
Figure imgf000057_0002
Figure imgf000058_0002
[0118] In certain aspects, the invention is directed to Blacklegged tick phlebovirus-1 isolate H12 nucleic acid sequence (including cDNA sequences corresponding to BTPV-1 H12 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate H12 segment L is shown in SEQ ID NO:23. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 segment L is accessible in public databases by
GenBank Accession Number KM048313.
[0119] SEQ ID NO: 23 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate H12 segment L.
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
[0120] The protein sequence of Blacklegged tick phlebovirus-1 isolate H12 segment L protein is shown in SEQ ID NO:24. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 segment L protein is accessible in public databases by GenBank Accession Number AII01801.
[0121] SEQ ID NO: 24 is the protein sequence of Blacklegged tick phlebovirus-1 isolate H12 segment L protein.
Figure imgf000061_0001
[0122] The nucleotide cDNA sequence of Blacklegged tick phlebovirus-1 isolate H12 segment S is shown in SEQ ID NO:25. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 segment S is accessible in public databases by GenBank Accession Number
KM048314.
[0123] SEQ ID NO: 25 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-1 isolate H12 segment S nucleic acid sequence.
Figure imgf000062_0001
[0124] The protein sequence of Blacklegged tick phlebovirus-1 isolate H12 N protein is shown in SEQ ID NO: 26. Sequence information related to Blacklegged tick phlebovirus-1 isolate H12 N protein is accessible in public databases by GenBank Accession Number AII01802.
[0125] SEQ ID NO: 26 is the protein sequence of Blacklegged tick phlebovirus-1 isolate H12 N protein.
Figure imgf000063_0001
[0126] In certain aspects, the invention is directed to Blacklegged tick phlebovirus-2 isolate H5 nucleic acid sequence (including cDNA sequences corresponding to BTPV-2 H5 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate H5 segment L is shown in SEQ ID NO:27. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 segment L is accessible in public databases by GenBank Accession Number KM048315.
[0127] SEQ ID NO: 27 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate H5 segment L.
Figure imgf000063_0002
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0002
[0128] The protein sequence of Blacklegged tick phlebovirus-2 isolate H5 segment L protein is shown in SEQ ID NO:28. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 segment L protein is accessible in public databases by GenBank Accession Number AII01803.
[0129] SEQ ID NO: 28 is the protein sequence of Blacklegged tick phlebovirus-2 isolate H5 segment L protein.
Figure imgf000066_0001
Figure imgf000067_0002
[0130] The nucleotide cDNA sequence of Blacklegged tick phlebovirus-2 isolate H5 segment S is shown in SEQ ID NO:29. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 segment S is accessible in public databases by GenBank Accession Number KM048316.
[0131] SEQ ID NO: 29 is the nucleic acid sequence (cDNA) of Blacklegged tick phlebovirus-2 isolate H5 segment S.
Figure imgf000067_0001
Figure imgf000068_0001
[0132] The protein sequence of Blacklegged tick phlebovirus-2 isolate H5 N protein is shown in SEQ ID NO: 30. Sequence information related to Blacklegged tick phlebovirus-2 isolate H5 N protein is accessible in public databases by GenBank Accession Number AII01804.
[0133] SEQ ID NO: 30 is the protein sequence of Blacklegged tick phlebovirus-2 isolate H5 N protein.
Figure imgf000068_0002
[0134] In certain aspects, the invention is directed to American dog tick phlebovirus isolate FI6 nucleic acid sequence (including cDNA sequences corresponding to ADTPV FI6 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of American dog tick phlebovirus isolate FI6 segment L is shown in SEQ ID NO: 31. Sequence information related to American dog tick phlebovirus isolate FI6 segment L is accessible in public databases by GenBank Accession Number KM04831 1.
[0135] SEQ ID NO: 31 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate FI6 segment L.
Figure imgf000068_0003
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0002
[0136] The protein sequence of American dog tick phlebovirus isolate FI6 segment L protein is shown in SEQ ID NO:32. Sequence information related to American dog tick phlebovirus isolate FI6 segment L protein is accessible in public databases by GenBank Accession Number AII01799.
[0137] SEQ ID NO: 32 is the protein sequence of American dog tick phlebovirus isolate FI6 segment L protein.
Figure imgf000071_0001
Figure imgf000072_0001
[0138] The nucleotide cDNA sequence of American dog tick phlebovirus isolate FI6 segment S is shown in SEQ ID NO:33. Sequence information related to American dog tick phlebovirus isolate FI6 segment S is accessible in public databases by GenBank Accession Number KM048312.
[0139] SEQ ID NO: 33 is the nucleic acid sequence (cDNA) of American dog tick phlebovirus isolate FI6 segment S.
Figure imgf000072_0002
Figure imgf000073_0001
[0140] The protein sequence of American dog tick phlebovirus isolate FI6 N protein is shown in SEQ ID NO: 34. Sequence information related to American dog tick phlebovirus isolate FI6 N protein is accessible in public databases by GenBank Accession Number AII01800.
[0141] SEQ ID NO: 34 is the protein sequence of American dog tick phlebovirus isolate FI6 N protein.
Figure imgf000073_0002
[0142] In certain aspects, the invention is directed to Deer tick mononegavirales-like virus isolate DTMl nucleic acid sequence (including cDNA sequences corresponding to DTMl RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Deer tick
mononegavirales-like virus isolate DTMl polymerase is shown in SEQ ID NO: 35. Sequence information related to Deer tick mononegavirales-like virus isolate DTMl polymerase is accessible in public databases by GenBank Accession Number KJ746903.
[0143] SEQ ID NO: 35 is the nucleic acid sequence (cDNA) of Deer tick mononegavirales-like virus isolate DTMl polymerase.
Figure imgf000073_0003
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0002
[0144] The protein sequence of Deer tick mononegavirales-like virus isolate DTMl polymerase protein is shown in SEQ ID NO: 36. Sequence information related to Deer tick mononegavirales- like virus isolate DTMl polymerase protein is accessible in public databases by GenBank
Accession Number AIE42676.
[0145] SEQ ID NO: 36 is the protein sequence of Deer tick mononegavirales-like virus isolate DTMl polymerase protein.
Figure imgf000076_0001
Figure imgf000077_0002
[0146] In certain aspects, the invention is directed to Deer tick mononegavirales-like virus isolate FI3 nucleic acid sequence (including cDNA sequences corresponding to DTM FI3 RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Deer tick
mononegavirales-like virus isolate FI3 polymerase is shown in SEQ ID NO: 37. Sequence information related to Deer tick mononegavirales-like virus isolate FI3 polymerase is accessible in public databases by GenBank Accession Number KM048317.
[0147] SEQ ID NO: 37 is the nucleic acid sequence (cDNA) of Deer tick mononegavirales-like virus isolate FI3 polymerase nucleic acid sequence.
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
[0148] The protein sequence of Deer tick mononegavirales-like virus isolate FI3 polymerase protein is shown in SEQ ID NO: 38. Sequence information related to Deer tick mononegavirales- like virus isolate FI3 polymerase protein is accessible in public databases by GenBank Accession Number AIIO 1805.
[0149] SEQ ID NO: 38 is the protein sequence of Deer tick mononegavirales-like virus isolate FI3 polymerase protein.
Figure imgf000080_0001
[0150] In certain aspects, the invention is directed to Tick borne tetravirus-like virus isolate
FI10 nucleic acid sequence (including cDNA sequences corresponding to ick borne tetravirus-like virus isolate FIIO RNA sequences, including mRNA sequences). The nucleotide cDNA sequence of Tick borne tetravirus-like virus isolate FIIO is shown in SEQ ID NO: 39. Sequence information related to Tick borne tetravirus-like virus isolate FIIO is accessible in public databases by GenBank Accession Number KM048322.
[0151] SEQ ID NO: 39 is the nucleic acid sequence (cDNA) of Tick borne tetravirus-like virus isolate FIIO.
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0002
[0152] The protein sequence of Tick borne tetravirus-like virus isolate FI10 polyprotein is shown in SEQ ID NO:40. Sequence information related Tick borne tetravirus-like virus isolate
FI10 is accessible in public databases by GenBank Accession Number AII01815.
[0153] SEQ ID NO: 40 is the protein sequence of Tick borne tetravirus-like virus isolate FI10 polyprotein.
Figure imgf000083_0001
[0154] In certain aspects, the invention is directed to Ixodes scapularis associated virus 1 isolate K13 nucleic acid sequence (including cDNA sequences corresponding to Ixodes scapularis associated virus 1 isolate K13 RNA sequences, including mRNA sequences). The nucleotide sequence of Ixodes scapularis associated virus 1 isolate K13 is shown in SEQ ID NO: 41.
Sequence information related to Ixodes scapularis associated virus 1 isolate K13 is accessible in public databases by GenBank Accession Number KM048318.
[0155] SEQ ID NO: 41 is the nucleic acid sequence of Ixodes scapularis associated virus 1 isolate K13 partial genome.
Figure imgf000084_0001
Figure imgf000085_0001
[0156] In certain aspects, the invention is directed to Ixodes scapularis associated virus 2 isolate Al nucleic acid sequence (including cDNA sequences corresponding to Ixodes scapularis associated virus 2 isolate Al RNA sequences, including mRNA sequences). The nucleotide sequence of Ixodes scapularis associated virus 2 isolate Al is shown in SEQ ID NO: 42. Sequence information related to Ixodes scapularis associated virus 2 isolate Al is accessible in public databases by GenBank Accession Number KM048319.
[0157] SEQ ID NO: 42 is the nucleic acid sequence of Ixodes scapularis associated virus 2 isolate Al partial genome.
Figure imgf000085_0002
Figure imgf000086_0001
[0158] In certain aspects, the invention is directed to a tick-associated virus isolated nucleic acid sequence as provided in SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33,35,37,39,41 or 42.
[0159] In certain aspects, the invention is directed to an isolated nucleic acid of SEQ ID NOs:
I, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0160] In certain aspects, the invention is directed to an isolated nucleic acid complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
[0161] In certain aspects, the invention is directed to variants of tick-associated virus nucleic acid sequences having greater that 60% similarity to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0162] In certain aspects, the invention is directed to isolated nucleic acid sequence variants of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 50%> to about 55% identity to that of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 55.1 %> to about 60%> identity to that of any of SEQ ID NOs: 1,3,5, 7, 9,
II, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs:
1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 60.1% to about 65% identity to that of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. Variants of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 65.1 % to about 70% identity to that of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25, 27,29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 70.1% to about 75% identity to that of any of SEQ ID NOs: 1, 3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 75.1% to about 80% identity to that of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 80.1% to about 85% identity to that of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 85.1% to about 90% identity to that of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 90.1% to about 95% identity to that of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 95.1% to about 97% identity to that of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, nucleic acid sequences having at least from about 97.1% to about 99% identity to that of any of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
Programs and algorithms for sequence alignment and comparison of % identity and/or homology between nucleic acid sequences, or polypeptides, are well known in the art, and include BLAST, SIM alignment tool, and so forth.
[0163] In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 50 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 100 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 200 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 300 consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 400 consecutive nucleotides from SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence
complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 500 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 600 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 700 consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 900 or more consecutive nucleotides from SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 2000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 3000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 4000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 5000 or more consecutive nucleotides from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 6000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 7000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 8000 or more consecutive nucleotides from SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 9000 or more consecutive nucleotides from SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence
complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 10,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 11,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 12,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 13,000 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39, 41 or 42. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 13,892 or more consecutive nucleotides from SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a sequence complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39,41 or 42.
[0164] In other aspects the invention is directed to isolated nucleic acid sequences such as primers and probes, comprising nucleic acid sequences of any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. Such primers and/or probes may be useful for detecting the presence of tick-associated virus of the invention, for example in samples of ticks or samples of bodily fluids such as blood, saliva, or urine from an animal, and thus may be useful in the diagnosis of tick-associated virus infection. Such probes can detect polynucleotides of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 in samples which comprise tick-associated virus represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. The isolated nucleic acids which can be used as primer and/probes are of sufficient length to allow hybridization with, i.e. formation of duplex with a corresponding target nucleic acid sequence, a nucleic acid sequences of any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or a variant thereof.
[0165] The isolated nucleic acid of the invention which can be used as primers and/or probes can comprise about 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 consecutive nucleotides from any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or sequences complementary to any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. The isolated nucleic acid of the invention which can be used as primers and/or probes can comprise from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and up to about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 consecutive nucleotides from any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or sequences complementary to any of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. The invention is also directed to primer and/or probes which can be labeled by any suitable molecule and/or label known in the art, for example but not limited to fluorescent tags suitable for use in Real Time PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAM or 6-FAM probes; radiolabels; and so forth. In certain embodiments, the
oligonucleotide primers and/or probe further comprises a detectable non-isotopic label selected from the group consisting of: a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.
[0166] In certain aspects, the invention is directed to primer sets comprising isolated nucleic acids as described herein, which primer set are suitable for amplification of nucleic acids from samples which comprises tick-associated virus represented by SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42, or variants thereof. Primer sets can comprise any suitable combination of primers which would allow amplification of a target nucleic acid sequences in a sample which comprises tick-associated virus represented by SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, or variants thereof. Amplification can be performed by any suitable method known in the art, for example but not limited to PCR, RT-PCR, qPCR, and transcription mediated amplification (TMA). [0167] Hybridization conditions: As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, and can hybridize, for example but not limited to, variants of the disclosed
polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. The precise conditions for stringent hybridization are typically sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%> of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
[0168] Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985) Nature 313:402-404, and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y ("Sambrook"); and by Haymes et al, "Nucleic Acid Hybridization: A Practical Approach", IRL Press, Washington, D.C. (1985), which references are incorporated herein by reference.
[0169] In general, stringency is determined by the temperature, ionic strength, and
concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure. The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate nucleic sequences having similarity to the nucleic acid sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed nucleic acid sequences, such as, for example, nucleic acid sequences having 60%> identity, or about 70%> identity, or about 80%> or greater identity with disclosed nucleic acid sequences.
[0170] Stringent conditions are known to those skilled in the art and can be found in Current Protocols In Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-7.3.6. In certain embodiments, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non- limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X sodium chloride/sodium citrate (SSC), 50 mM Tris-HCl (pH 7.5), 1 nM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. Another non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1%> SDS at 50-65 °C. Examples of moderate to low stringency hybridization conditions are well known in the art.
[0171] Polynucleotides homologous to the sequences illustrated in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical- chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations.
[0172] Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the nucleic acid sequences disclosed herein, and fragments thereof under various conditions of stringency (See, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel (1987) Methods Enzymol. 152: 507-511). With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual" (2nd ed., Cold Spring Harbor Laboratory); Berger and Kimmel, eds., (1987) "Guide to Molecular Cloning Techniques", In Methods in Enzymology: 152: 467-469; and Anderson and Young (1985) "Quantitative Filter Hybridisation." In: Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical Approach. Oxford, IRL Press, 73-111.
[0173] Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (Tm) is defined as the temperature when 50%> of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equation: DNA- DNA: Tm(°C)=81.5+16.6(log [Na+])+0.41(% G+C)-0.62(% formamide)-500/L (1) DNA-RNA: Tm(°C)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C).sup.2-0.5(% formamide)-820/L (2) RNA-RNA: Tm(C)=79.8+18.5(log [Na+])+0.58(%G+C)+0.12(%G+C).sup.2-0.35(% formamide)- 820/L (3), where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of
(guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1°C is required to reduce the melting temperature for each 1% mismatch.
[0174] Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson et al. (1985) supra). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non- complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
[0175] Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency. As a general guidelines high stringency is typically performed at Tm-5°C to Tm -20°C, moderate stringency at Tm-20°C to Tm-35°C and low stringency at Tm-35°SC to Tm-50°C for duplex>150 base pairs. Hybridization may be performed at low to moderate stringency (25-50°C below Tm), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at Tm-25°C for DNA-DNA duplex and Tm -15°C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
[0176] High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1 % N- laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50°C and about 70°C. In certain embodiments, high stringency conditions are about 0.02 M sodium chloride, about 0.5%> casein, about 0.02%> SDS, about 0.001 M sodium citrate, at a temperature of about 50°C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.
[0177] Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas in certain embodiments high stringency hybridization may be obtained in the presence of at least about 35% formamide, and in other embodiments in the presence of at least about 50% formamide. In certain embodiments, stringent temperature conditions will ordinarily include temperatures of at least about 30°C, and in other embodiment at least about 37°C, and in other embodiments at least about 42°C with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a certain embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide. In another embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide. Useful variations on these conditions will be readily apparent to those skilled in the art.
[0178] The washing steps that follow hybridization may also vary in stringency; the post- hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt
concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, and in certain embodiments less than about 15 mM NaCl and 1.5 mM trisodium citrate. For example, the wash conditions may be under conditions of 0.1XSSC to 2.0XSSC and 0.1% SDS at 50-65°C, with, for example, two steps of 10-30 min. One example of stringent wash conditions includes about 2.0XSSC, 0.1% SDS at 65°C and washing twice, each wash step being about 30 min. The temperature for the wash solutions will ordinarily be at least about 25°C, and for greater stringency at least about 42°C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3°C to about 5°C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6°C to about 9°C. For identification of less closely related homolog, wash steps may be performed at a lower temperature, e.g., 50°C.
[0179] An example of a low stringency wash step employs a solution and conditions of at least 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42° C in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Even higher stringency wash conditions are obtained at 65°C-68°C in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art.
[0180] Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-1 OX higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15X or more, is obtained. Accordingly, an animal nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2X or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a calorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
[0181] The sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Protein and/or nucleic acid sequence identities (homologies) can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
[0182] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.2.2. or FASTA version 3.0t78 algorithms and the default parameters discussed below can be used.
[0183] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444, 1988, by computerized implementations of these algorithms (FASTDB (Intelligenetics), BLAST (National Center for Biomedical Information), GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al, (1999 SuppL), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., 1987).
[0184] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm, which is described in Pearson, W. R. & Lipman, D. J., Proc. Natl. Acad. Sci. U.S.A. 85: 2444, 1988. See also W. R. Pearson, Methods Enzymol. 266: 227-258, 1996. Exemplary parameters used in a FASTA alignment of DNA sequences to calculate percent identity are optimized, BL50 Matrix 15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty -12, gap length penalty=-2; and width=16.
[0185] Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al, J. Mol. Biol. 215:402-410, 1990, respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for
Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89: 10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0186] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, less than about 0.01, and less than about 0.001.
[0187] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12:387-395, 1984).
[0188] Another example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J. D. et al, Nucl. Acids. Res.
22:4672-4680, 1994). ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919, 1992).
[0189] "Percent identity" in the context of two or more nucleic acids, refers to the percentage of nucleotides that two or more sequences or subsequences contain which are the same. A specified percentage of nucleotides can be referred to such as: 60% identity, 65%, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
[0190] "Substantially identical," in the context of two nucleic acids, refers to two or more sequences or subsequences that have at least of at least 98%>, at least 99% or higher nucleotide identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
[0191] In other aspects, the invention is directed to expression constructs, for example but not limited to plasmids and vectors which comprise the nucleic acid sequence of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, complementary sequences thereof, and/or variants thereof. Such expression constructs can be prepared by any suitable method known in the art. Such expression constructs are suitable for viral nucleic acid and/or protein expression and purification.
[0192] In certain aspects, the invention is directed to iRNA molecules which target nucleic acids from a tick-associated virus, for example but not limited to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and variants thereof, and silence a target gene.
[0193] An "iRNA agent" (abbreviation for "interfering RNA agent") as used herein, is an RNA agent, which can down-regulate the expression of a target gene, e.g. a tick-associated virus gene. An iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre- translational mechanisms. An iRNA agent can be a double stranded (ds) iRNA agent.
[0194] A "ds iRNA agent" (abbreviation for "double stranded iRNA agent"), as used herein, is an iRNA agent which includes more than one, and in certain embodiments two, strands in which interchain hybridization can form a region of duplex structure. A "strand" herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g. by a linker, e.g. a polyethyleneglycol linker, to form but one molecule. At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the "antisense strand". A second strand comprised in the ds iRNA agent which comprises a region complementary to the antisense strand is termed the "sense strand". However, a ds iRNA agent can also be formed from a single RNA molecule which is, at least partly; self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. In such case, the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.
[0195] iRNA agents as described herein, including ds iRNA agents and siRNA agents, can mediate silencing of a gene, e.g., by RNA degradation. For convenience, such RNA is also referred to herein as the RNA to be silenced. Such a gene is also referred to as a target gene. In certain embodiments, the RNA to be silenced is a gene product of a tick-associated virus gene.
[0196] As used herein, the phrase "mediates RNAi" refers to the ability of an agent to silence, in a sequence specific manner, a target gene. "Silencing a target gene" means the process whereby a cell containing and/or secreting a certain product of the target gene when not in contact with the agent, will contain and/or secret at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent. Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.
[0197] In the anti viral uses of the present invention, silencing of a target gene can result in a reduction in "viral titer" in the cell or in the animal, wherein "reduction in viral titer" refers to a decrease in the number of viable virus produced by a cell or found in an organism undergoing the silencing of a viral target gene. Reduction in the cellular amount of virus produced can lead to a decrease in the amount of measurable virus produced in the tissues of an animal undergoing treatment and a reduction in the severity of the symptoms of the viral infection. iRNA agents of the present invention are also referred to as "antiviral iRNA agents".
[0198] As used herein, a "tick-associated virus gene" refers to any one of the genes identified in a tick-associated virus genome, including but not limited to, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0199] In other aspects, the invention provides methods for reducing viral titer in an animal, by administering to an animal, at least one iRNA which inhibits the expression of a tick-associated virus gene. [0200] In other aspects, the invention provides methods for identifying and/or generating antiviral drugs. For example, in one aspect the invention provides methods for identifying drugs that bind to and/or inhibit the function of tick-associated virus-encoded proteins of the invention, or that inhibit the replication or pathogenicity of tick-associated virus of the invention. Methods of identifying drugs that affect or inhibit a particular drug target, such as high throughput drug screening methods, are well known in the art and can readily be applied to the proteins and viruses of the present invention.
[0201] In certain aspects, the invention is directed to a tick-associated virus isolated amino acid sequence as provided in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0202] In certain aspects, the invention is directed to a tick-associated virus isolated amino acid sequence encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0203] In certain aspects, the invention relates to variants of tick-associated virus amino acid sequences having greater that 60% similarity to the sequence of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0204] The invention is also directed to isolated polypeptides and variants and derivatives thereof. These polypeptides may be useful for multiple applications, including, but not limited to, generation of antibodies and generation of immunogenic compositions. For example, the invention is directed to an isolated polypeptides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34, 36, 38, or 40. For example, the invention is also directed to any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. A peptide of at least 8 amino acid residues in length can be recognized by an antibody (MacKenzie et al., (1984) Biochemistry 23,6544-6549). In certain embodiments, the invention is directed to fragments of the polypeptides described herein, that can, for example, be used to generate antibodies.
[0205] In one aspect, the invention is directed to polypeptide variants of an isolated polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of the isolated polypeptides of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 50% to about 55%> identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 55.1 %> to about 60% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 60.1% to about 65%> identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 65.1 % to about 70% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide having at least from about 70.1% to about 75% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 75.1% to about 80%> identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 80.1% to about 85% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 85.1%> to about 90% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 90.1% to about 95% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 95.1% to about 97% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. Variants of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 include, but are not limited to, polypeptide sequences having at least from about 97.1%> to about 99% identity to that of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0206] In one aspect, the invention is directed to polypeptide variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. Variants of any one of the isolated polypeptides encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 50% to about 55 identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide sequences having at least from about 55.1 %> to about 60%) identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 60.1% to about 65 %> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 65.1 %> to about 70%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide having at least from about 70.1% to about 75%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 75.1% to about 80% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31, 33, 35, 37, 39, 41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide sequences having at least from about 80.1% to about 85% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to,
polypeptide sequences having at least from about 85.1%> to about 90%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to, polypeptide sequences having at least from about 90.1% to about 95% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 include, but are not limited to, polypeptide sequences having at least from about 95.1% to about 97%o identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 include, but are not limited to,
polypeptide sequences having at least from about 97.1% to about 99%> identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0207] The invention is directed to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 200 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 250 consecutive amino acids of an isolated
polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 300 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids of an isolated
polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 1000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 1500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 2000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 2500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 3000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 3500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 4000 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is directed to a polypeptide sequence comprising from about 10 to about 4536 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0208] The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 200 to about 50 consecutive amino acids of an isolated
polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50%) to about 99% identity to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids of an isolated
polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain embodiments, the invention is directed to isolated and purified peptides.
[0209] In certain embodiments, the polypeptides of the present invention can be suitable for use as antigens to detect antibodies against SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof. In other embodiments, the polypeptides of the present invention which comprise antigenic determinants can be used in various immunoassays to identify animals exposed to and/or samples which comprise SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
[0210] The invention is directed to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs:
I, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9,
I I, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 200 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 250 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 300 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs:
I, 3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5, 7, 9,
II, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 1000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 1500 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 2000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 2500 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 3000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 3500 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 4000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is directed to a polypeptide sequence comprising from about 10 to about 4536 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0211] The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 200 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50% to about 99%) identity to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50%> to about 99%> identity to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acidofSEQ IDNOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39, 41 or 42. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs:
I, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. he invention is further directed to polypeptide sequences having from about 50%> to about 99% identity to a polypeptide sequence comprising from about 10 to about 1000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9,
I I, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In certain embodiments, the invention is directed to isolated and purified peptides.
[0212] In certain embodiments, the polypeptides of the present invention can be suitable for use as antigens to detect antibodies against tick-associated virus represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and variants thereof. In other embodiments, the polypeptides of the present invention which comprise antigenic
determinants can be used in various immunoassays to identify animals exposed to and/or samples which comprise tick-associated virus represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, and variants thereof.
[0213] In another aspect, the invention is directed to an antibody which specifically binds to amino acids from the polypeptide of an isolated polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In one embodiment the antibody is purified. The antibodies can be polyclonal or monoclonal. The antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human. Species specific antibodies avoid certain problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences from other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.
[0214] In another aspect, the invention is directed to an antibody which specifically binds to amino acids from the polypeptide of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment the antibody is purified. The antibodies can be polyclonal or monoclonal. The antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human. Species specific antibodies avoid certain of the problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences form other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.
[0215] Antibodies can bind to other molecules (antigens) via heavy and light chain variable domains, VH and VL, respectively. The antibodies described herein include, but are not limited to IgY, IgY(AFc)), IgG, IgD, IgA, IgM, IgE, and IgL. The antibodies may be intact immunoglobulin molecules, two full length heavy chains linked by disulfide bonds to two full length light chains, as well as subsequences (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen, or subsequences thereof (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen. Antibodies may comprise full length heavy and light chain variable domains, VH and VL, individually or in any combination.
[0216] The basic immunoglobulin (antibody) structural unit can comprise a tetramer. Each tetramer can be composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (Vi) and variable heavy chain (VH) refer to these light and heavy chains respectively.
[0217] Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. In particular, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993) for more antibody fragment terminology). While the Fab' domain is defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
[0218] The Fab' regions may be derived from antibodies of animal or human origin or may be chimeric (Morrison et al, Proc Natl. Acad. Sci. USA 81, 6851-7855 (1984) both incorporated by reference herein) or humanized (Jones et al., Nature 321, 522-525 (1986), and published UK patent application No. 8707252, both incorporated by reference herein).
[0219] An antibody described in this application can include or be derived from any mammal, such as but not limited to, a bird, a dog, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof and includes isolated avian, human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted or CDR-adapted antibodies, immunoglobulins, cleavage products and other portions and variants thereof.
[0220] Any method for producing antibodies can be used to generate the antibodies described herein. Examplary methods include animal inoculation, phage display, transgenic mouse technology and hybridoma techonology.
[0221] Methods for generating avain antibodies can also be used to generate the antibodies described herein. In avaians, the egg yolk can be used an antibody source (Altchul et al, Nature Genetics, 1994, 6: 119-129). For a review of preimmune diversification and antibody generation in avians, see Reynaud et al, Cell 40, 283-291, 1985 and Thompson et al, Cell 48, 369-378,1987. In birds, the bursa of Fabricius is the site where B cells undergo gene conversion and are selected for the ability to produce antibodies to antigens. Unlike mammals, the generation of antibody binding specities occurs before hatching rather than throughout their lives. Another difference between avians and mammals is that the major immunoglobulin is IgY rather than IgG. A small version of IgY lacking a full Fc region (IgY(AFc)) is also known to be produced in avians. (Zimmerman,et al, (1971) Biochemistry 10 : 482-488).
[0222] Any methods for producing antibodies in animals can be used to produce the antibodies described herein.
[0223] Antibodies useful in the embodiments of the invention can be derived in several ways well known in the art. In one aspect, the antibodies can be obtained using any of the techniques well known in the art, see, e.g., Ausubel, et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al, eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al, Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).
[0224] The antibodies may also be obtained from selecting from libraries of such domains or components, e.g. a phage library. A phage library can be created by inserting a library of random oligonucleotides or a library of polynucleotides containing sequences of interest, such as from the B-cells of an immunized animal or human (Smith, G. P. 1985. Science 228: 1315-1317). Antibody phage libraries contain heavy (H) and light (L) chain variable region pairs in one phage allowing the expression of single-chain Fv fragments or Fab fragments (Hoogenboom, et al. 2000, Immunol Today 21(8) 371-7). The diversity of a phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, human monoclonal antibodies. For example, the heavy (H) chain and light (L) chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an assembled immunoglobulin molecule. Additionally, either or both the H and L chain encoding genes can be mutagenized in a complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable affinity and neutralization capabilities. Antibody libraries also can be created synthetically by selecting one or more human framework sequences and introducing collections of CDR cassettes derived from human antibody repertoires or through designed variation
(Kretzschmar and von Ruden 2000, Current Opinion in Biotechnology, 13:598-602). The positions of diversity are not limited to CDRs but can also include the framework segments of the variable regions or may include other than antibody variable regions, such as peptides.
[0225] Other target binding components which may include other than antibody variable regions are ribosome display, yeast display, and bacterial displays. Ribosome display is a method of translating mRNAs into their cognate proteins while keeping the protein attached to the RNA. The nucleic acid coding sequence is recovered by RT-PCR (Mattheakis, L. C. et al. 1994. Proc Natl Acad Sci USA 91, 9022). Yeast display is based on the construction of fusion proteins of the membrane-associated alpha-agglutinin yeast adhesion receptor, agal and aga2, a part of the mating type system (Broder, et al. 1997. Nature Biotechnology, 15:553-7). Bacterial display is based fusion of the target to exported bacterial proteins that associate with the cell membrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng, 79:496-503).
[0226] In comparison to hybridoma technology, phage and other antibody display methods afford the opportunity to manipulate selection against the antigen target in vitro and without the limitation of the possibility of host effects on the antigen or vice versa.
[0227] Specific examples of antibody subsequences include, for example, Fab, Fab', (Fab')2, Fv, or single chain antibody (SCA) fragment (e.g., scFv). Subsequences include portions which retain at least part of the function or activity of full length sequence. For example, an antibody
subsequence will retain the ability to selectively bind to an antigen even though the binding affinity of the subsequence may be greater or less than the binding affinity of the full length antibody.
[0228] Pepsin or papain digestion of whole antibodies can be used to generate antibody fragments. In particular, an Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain. An (Fab')2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. An Fab' fragment of an antibody molecule can be obtained from (Fab')2 by reduction with a thiol reducing agent, which yields a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
[0229] An Fv fragment is a fragment containing the variable region of a light chain VL and the variable region of a heavy chain VH expressed as two chains. The association may be non-covalent or may be covalent, such as a chemical cross-linking agent or an intermolecular disulfide bond (Inbar et al, (1972) Proc. Natl. Acad Sci. USA 69:2659; Sandhu (1992) Crit. Rev. Biotech.
12:437).
[0230] A single chain antibody ("SCA") is a genetically engineered or enzymatically digested antibody containing the variable region of a light chain VL and the variable region of a heavy chain, optionally linked by a flexible linker, such as a polypeptide sequence, in either VL-linker-VH orientation or in VH-linker-VL orientation. Alternatively, a single chain Fv fragment can be produced by linking two variable domains via a disulfide linkage between two cysteine residues. Methods for producing scFv antibodies are described, for example, by Whitlow et al, (1991) In: Methods: A Companion to Methods in Enzymology 2:97; U.S. Pat. No. 4,946,778; and Pack et al, (1993) Bio/Technology 11 : 1271.
[0231] Other methods of producing antibody subsequences, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, provided that the subsequences bind to the antigen to which the intact antibody binds.
[0232] Antibodies used in the invention, include full length antibodies, subsequences (e.g., single chain forms), dimers, trimers, tetramers, pentamers, hexamers or any other higher order oligomer that retains at least a part of antigen binding activity of monomer. Multimers can comprise heteromeric or homomeric combinations of full length antibody, subsequences, unmodified or modified as set forth herein and known in the art. Antibody multimers are useful for increasing antigen avidity in comparison to monomer due to the multimer having multiple antigen binding sites. Antibody multimers are also useful for producing oligomeric (e.g., dimer, trimer, tertamer, etc.) combinations of different antibodies thereby producing compositions of antibodies that are multifunctional (e.g., bifunctional, trifunctional, tetrafunctional, etc.).
[0233] Antibodies can be produced through chemical crosslinking of the selected molecules (which have been produced by synthetic means or by expression of nucleic acid that encode the polypeptides) or through recombinant DNA technology combined with in vitro, or cellular expression of the polypeptide, and subsequent oligomerization. Antibodies can be similarly produced through recombinant technology and expression, fusion of hybridomas that produce antibodies with different epitopic specificities, or expression of multiple nucleic acid encoding antibody variable chains with different epitopic specificities in a single cell.
[0234] Antibodies may be either joined directly or indirectly through covalent or non-covalent binding, e.g. via a multimerization domain, to produce multimers. A "multimerization domain" mediates non-covalent protein-protein interactions. Specific examples include coiled-coil (e.g., leucine zipper structures) and alpha-helical protein sequences. Sequences that mediate protein- protein binding via Van der Waals' forces, hydrogen bonding or charge-charge bonds are also can also be used as multimerization domains. Additional examples include basic-helix-loop-helix domains and other protein sequences that mediate heteromeric or homomeric protein-protein interactions among nucleic acid binding proteins (e.g., DNA binding transcription factors, such as TAFs). One specific example of a multimerization domain is p53 residues 319 to 360 which mediate tetramer formation. Another example is human platelet factor 4, which self-assembles into tetramers. Yet another example is extracellular protein TSP4, a member of the thrombospondin family, which can form pentamers. Additional specific examples are the leucine zippers of jun, fos, and yeast protein GCN4.
[0235] Antibodies may be directly linked to each other via a chemical cross linking agent or can be connected via a linker sequence (e.g., a peptide sequence) to form multimers.
[0236] The antibodies of the present invention can be used to modulate the activity of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, variants or fragments thereof. In certain aspects, the invention is directed to a method for treating an animal, the method comprising administering to the animal an antibody which specifically binds to amino acids from the polypeptide of any
polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In certain embodiments, antibody binding to the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit virus propagation and spreading.
[0237] The antibodies of the present invention can be used to modulate the activity of any polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants or fragments thereof. In certain aspects, the invention is directed to a method for treating an animal, the method comprising administering to the animal an antibody which specifically binds to amino acids from the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain embodiments, antibody binding to the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit virus propagation and spreading.
[0238] In other embodiments, the antibodies of the invention can be used to purify a
polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants or fragments thereof. In other embodiments, the antibodies of the invention can be used to identify expression and localization of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants, fragments or domains thereof. Analysis of expression and localization of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 can be useful in determining potential role of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0239] In other embodiments, the antibodies of the invention can be used to purify polypeptides of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, variants or fragments thereof. In other embodiments, the antibodies of the invention can be used to identify expression and localization of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, variants, fragments or domains thereof. Analysis of expression and localization of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 can be useful in determining potential role of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
[0240] In other embodiments, the antibodies of the invention can be used to purify polypeptides of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants or fragments thereof. In other embodiments, the antibodies of the invention can be used to identify expression and localization of the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, variants, fragments or domains thereof.
Analysis of expression and localization of the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 can be useful in determining potential role of the polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0241] In other embodiments, the antibodies of the present invention can be used in various immunoassays to identify animals exposed to and/or samples which comprise antigens from tick- associated viruses represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
[0242] In other embodiments, the antibodies of the present invention can be used in various immunoassays to identify animals exposed to and/or samples which comprise antigens from tick- associated viruses represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and variants thereof.
[0243] Any suitable immunoassay which can lead to formation of antigen-antibody complex can also be used. Variations and different formats of immunoassays, for example but not limited to ELISA, lateral flow assays for detection of analytes in samples, immunoprecipitation, and
Luciferase Immunoprecipitation Systems (LIPS) are known in the art. In various embodiments, the antigen and/or the antibody can be labeled by any suitable label or method known in the art. For example enzymatic immunoassays may use solid supports, or immunoprecipitation.
Immnunoassays which amplify the signal from the antigen-antibody immune complex can also be used with the methods described herein.
[0244] In certain aspects, the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, as provided by the invention, and variants thereof. In certain aspects the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, as provided by the invention, and variants thereof. In certain embodiments, methods for assaying a sample, include, but are not limited to, methods which can detect the presence of nucleic acids, methods which can detect the presence of antigens, methods which can detect the presence of antibodies against antigens from a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, methods which can detect the presence of a polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, as provided by the invention, and variants thereof.
[0245] In certain aspects the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus comprising SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, as provided by the invention, and variants thereof. In certain embodiments, methods for assaying a sample, include, but are not limited to, methods which can detect the presence of nucleic acids, methods which can detect the presence of antigens, methods which can detect the presence of antibodies against antigens from polypeptides encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, as provided by the invention, and variants thereof.
[0246] In certain aspects the invention provides methods for assaying a sample to determine the presence or absence of a tick-associated virus by detecting the presence of antibodies against antigens from polypeptides encoded by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42. In one embodiment the antibody that is detected is against a L-segment gene protein (e.g. SEQ ID NOs: 4, 8, 12, 14, 24, 28, or 32). In one embodiment the antibody that is detected is against a N protein (e.g. SEQ ID NO: 6, 10, 16, 18, 26, 30, or 34). In one embodiment the antibody that is detected is against a nucleocapsid protein (e.g. SEQ ID NO: 22). In one embodiment the antibody that is detected is against a polymerase protein (e.g. SEQ ID NO: 36, or 38). In one embodiment, antibodies against antigens are detected by immunoassay. In one embodiment, complex formation between an antibody to the antigen and the antigen is detected.
[0247] Immunogenic compositions
[0248] In certain aspects, the present invention provides immunogenic compositions capable of inducing an immune response against a tick-associated virus including a tick-associated virus of the invention comprising SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or comprising a cDNA sequence complementary to the sense or an anti-sense strand of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or comprising a polypeptide encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or a cDNA sequence complementary to the sense or an anti- sense strand of SEQ ID NOs: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or comprising a polypeptide of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In one embodiment, the immunogenic compositions are capable of ameliorating the symptoms of a tick-associated virus infection and/or of reducing the duration of a tick-associated virus associated disease. In another embodiment, the immunogenic compositions are capable of inducing protective immunity against a disease associated with a tick-associated virus. The immunogenic compositions of the invention can be effective against a tick-associated virus disclosed herein, and may also be cross-reactive with, and effective against, multiple different clades and strains of tick-associated viruses.
[0249] In other embodiments, the present invention provides immunogenic compositions for inducing an immune response in an animal, wherein the composition includes a recombinant or isolated capsid polypeptide derived from a tick-associated virus; and a pharmaceutically acceptable vehicle or diluent. In one embodiment, this immunogenic composition is a vaccine composition. In one embodiment, the polypeptide employed in the immunogenic compositions is SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a fragment thereof.
[0250] In certain embodiments, the polypeptide sequence corresponds to the immunogenic protein in both the whole virus and subunit compositions provided by the present invention.
[0251] As used herein, the term "polypeptide sequence", and the like, refer to polypeptide sequences and includes derivatives of polypeptide sequences, such as including, but not limited to, polypeptide sequences which include Histidine tags, Xpress™ tags, signal sequences or other epitope tags at the N- and/or C-terminal ends.
[0252] The polypeptide composition can be used to protect animals against a tick-associated virus. Thus, the present invention also provides a method of immunizing an animal against a tick- associated virus, wherein the method includes administering to the animal the polypeptide composition. In certain embodiments, the tick-associated virus from which the polypeptide is derived encodes a polypeptide sequence having at least 90% identity to the amino acid sequence SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or fragment thereof. In one embodiment, the capsid polypeptide composition includes an adjuvant.
[0253] The types of immunogenic composition encompassed by the invention include, but are not limited to, attenuated live viral immunogenic compositions, inactivated (killed) viral immunogenic compositions, and subunit immunogenic compositions.
[0254] The tick-associated viruses of the invention may be attenuated by removal or disruption of those viral sequences whose products cause or contribute to the disease and symptoms associated with tick-associated infection, and leaving intact those sequences required for viral replication. In this way an attenuated tick-associated virus can be produced that replicates in animals, and induces an immune response in animals, but which does not induce the deleterious disease and symptoms usually associated with tick-associated virus infection. One of skill in the art can determine which tick-associated virus sequences can or should be removed or disrupted, and which sequences should be left intact, in order to generate an attenuated tick-associated virus suitable for use as an immunogenic composition.
[0255] The novel tick-associated viruses of the invention may be also be inactivated, such as by chemical treatment, to "kill" the viruses such that they are no longer capable of replicating or causing disease in animals, but still induce an immune response in an animal. There are many suitable viral inactivation methods known in the art and one of skill in the art can readily select a suitable method and produce an inactivated "killed" tick-associated viruses suitable for use as an immunogenic composition. [0256] The immunogenic compositions of the invention may comprise subunit immunogenic compositions. Subunit immunogenic compositions include nucleic acid immunogenic
compositions such as DNA immunogenic compositions, which contain nucleic acids that encode one or more viral proteins or subunits, or portions of those proteins or subunits. When using such immunogenic compositions, the nucleic acid is administered to the animal, and the immunogenic proteins or peptides encoded by the nucleic acid are expressed in the animal, such that an immune response against the proteins or peptides is generated in the animal. Subunit immunogenic compositions may also be proteinaceous immunogenic compositions, which contain the viral proteins or subunits themselves, or portions of those proteins or subunits.
[0257] To make the nucleic acid and DNA immunogenic compositions of the invention tick- associated virus sequences disclosed herein may be incorporated into a plasmid or expression vector containing the nucleic acid that encodes the viral protein or peptide. Any suitable plasmid or expression vector capable of driving expression of the protein or peptide in the animal may be used. Such plasmids and expression vectors should include a suitable promoter for directing transcription of the nucleic acid. The nucleic acid sequence(s) that encodes a tick-associated virus protein or peptide may also be incorporated into a suitable recombinant virus for administration to the animal. Examples of suitable viruses include, but are not limited to, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses. [0258] One of skill in the art could readily select a suitable plasmid, expression vector, or recombinant virus for delivery of tick-associated virus nucleic acid sequences of the invention.
[0259] To produce the proteinaceous immunogenic compositions of the invention, tick- associated virus nucleic acid sequences of the invention are delivered to cultured cells, for example by transfecting cultured cells with plasmids or expression vectors containing tick-associated virus nucleic acid sequences, or by infecting cultured cells with recombinant viruses containing tick- associated virus nucleic acid sequences. Tick-associated virus proteins or peptides may then be expressed in the cultured cells and purified. The purified proteins can then be incorporated into compositions suitable for administration to animals. Methods and techniques for expression and purification of recombinant proteins are well known in the art, and any such suitable methods may be used.
[0260] Subunit immunogenic compositions of the present invention may encode or contain any of tick-associated virus proteins or peptides described herein, or any portions, fragments, derivatives or mutants thereof, that are immunogenic in an animal. One of skill in the art can readily test the immunogenicity of tick-associated virus proteins and peptides described herein, and can select suitable proteins or peptides to use in subunit immunogenic compositions. [0261] The immunogenic compositions of the invention comprise at least one tick-associated virus-derived immunogenic component, such as those described herein. The compositions may also comprise one or more additives including, but not limited to, one or more pharmaceutically acceptable carriers, buffers, stabilizers, diluents, preservatives, solubilizers, liposomes or immunomodulatory agents. Suitable immunomodulatory agents include, but are not limited to, adjuvants, cytokines, polynucleotide encoding cytokines, and agents that facilitate cellular uptake of tick-associated virus-derived immunogenic component.
[0262] Immunogenic compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used to induce an immunogenic response. These immunogenic compositions may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention can be in the form of a tablet, capsule, powder, solution or elixr. When administered in tablet form, the immunogenic composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the immunogenic composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the immunogenic composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and from about 1 to 50% protein or other active ingredient of the present invention.
[0263] When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. One immunogenic composition for intravenous, cutaneous, or subcutaneous injection can contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The immunogenic composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[0264] For oral administration, the compounds can be formulated readily by combining the active compounds with immunogenicly acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Immunogenic preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
[0265] Immunogenic preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[0266] Capsules and cartridges may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0267] Immunogenic formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient maybe in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0268] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneous ly or intamuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A carrier for hydrophobic compounds of the invention can be a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic immunogenic compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.
[0269] The immunogenic compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with immunogenicly compatible counter ions. Such immunogenicly acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
[0270] The immunogenic composition of the invention may be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.
[0271] The immunogenic composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble
monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference. [0272] Other additives that are useful in immunogenic composition formulations are known and will be apparent to those of skill in the art.
[0273] An "immunologically effective amount" of the compositions of the invention may be administered to an animal (including a human). As used herein, the term "immunologically effective amount" refers to an amount capable of inducing, or enhancing the induction of, the desired immune response in an animal (including a human). The desired response may include, inter alia, inducing an antibody or cell-mediated immune response, or both. The desired response may also be induction of an immune response sufficient to ameliorate the symptoms of a disease associated with a tick-associated virus and/or provide protective immunity in an animal (including a human) against subsequent challenge with a tick-associated virus. An immunologically effective amount may be an amount that induces actual "protection" against tick-associated virus associated diseases, meaning the prevention of any of the symptoms or conditions resulting from tick- associated virus associated disease in animals (including humans). An immunologically effective amount may also be an amount sufficient to delay the onset of symptoms and conditions associated with infection, reduce the degree or rate of infection, reduce in the severity of any disease or symptom resulting from infection, and reduce the viral load of an infected animal (including a human).
One of skill in the art can readily determine what is an "immunologically effective amount" of the compositions of the invention without performing any undue experimentation. An effective amount can be determined by conventional means, starting with a low dose of and then increasing the dosage while monitoring the immunological effects. Numerous factors can be taken into consideration when determining an optimal amount to administer, including the size, age, and general condition of the animal, the presence of other drugs in the animal, the virulence of the particular tick-associated virus against which the animal is being vaccinated, and the like. The actual dosage is can be chosen after consideration of the results from various animal studies.
[0274] The immunologically effective amount of the immunogenic composition may be administered in a single dose, in divided doses, or using a "prime-boost" regimen. The
compositions may be administered by any suitable route, including, but not limited to parenteral, intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, oral, or intraocular routes, or by a combination of routes. The compositions may also be administered using a "gun" device which fires particles, such as gold particles, onto which compositions of the present invention have been coated, into the skin of an animal. The skilled artisan will be able to formulate the immunogenic composition according to the route chosen. [0275] Methods of purification of inactivated virus are known in the art and may include one or more of, for instance gradient centrifugation, ultracentrifugation, continuous-flow
ultracentrifugation and chromatography, such as ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography. Additional method of purification include ultrafiltration and dialfiltration. See J P Gregersen "Herstellung von Virussimpfstoffen aus Zellkulturen" Chapter 4.2 in Pharmazeutische Biotecnology (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000. See also, O'Neil et al, "Virus Harvesting and Affinity Based Liquid Chromatography. A Method for Virus Concentration and Purification", Biotechnology (1993) 11 : 173-177; Prior et al., "Process Development for Manufacture of
Inactivated HIV-1", Pharmaceutical Technology (1995) 30-52; and Majhdi et al., "Isolation and Characterization of a Coronavirus from Elk Calves with diarrhea" Journal of Clinical Microbiology (1995) 35(11): 2937-2942.
Other examples of purification methods suitable for use in the invention include polyethylene glycol or ammonium sulfate precipitation (see Trepanier et al., "Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration" Journal of Virological Methods (1981) 3(4):201-711; Hagen et al, "Optimization of Polyethylene glycol) Precipitation of Hepatitis Virus Used to prepare VAQTA, a Highly Purified Inactivated Vaccine" Biotechnology Progress (1996) 12:406-412; and Carlsson et al., "Purification of Infectious
Pancreatic Necrosis Virus by Anion Exchange Chromatography Increases the Specific Infectivity" Journal of Virological Methods (1994) 47:27-36) as well as ultrafiltration and microfiltration (see Pay et al., Developments in Biological Standardization (1985) 60: 171-174; Tsurumi et al.,
"Structure and filtration performances of improved cuprammonium regenerated cellulose hollow fibre (improved BMM hollow fibre) for virus removal" Polymer Journal (1990) 22(12): 1085-1100; and Makino et al., "Concentration of live retrovirus with a regenerated cellulose hollow fibre, BMM", Archives of Virology (1994) 139(l-7):87-96.).
[0276] Tick-associated virus can be purified using chromatography, such as ion exchange, chromatography. Chromatic purification allows for the production of large volumes of virus containing suspension. The viral product of interest can interact with the chromatic medium by a simple adsorption/desorption mechanism, and large volumes of sample can be processed in a single load. Contaminants which do not have affinity for the adsorbent pass through the column. The virus material can then be eluted in concentrated form.
[0277] Anion exchange resins that may be used include DEAE, EMD TMAE. Cation exchange resins may comprise a sulfonic acid-modified surface. Viruses can be purified using ion exchange chromatography comprising a strong anion exchange resin (e.g. EMD TMAE) for the first step and EMD-SO3 (cation exchange resin) for the second step. A metal-binding affinity chromatography step can optionally be included for further purification. (See, e.g., WO 97/06243).
[0278] A resin such as Fractogel EMD can also be used. This synthetic methacrylate based resin has long, linear polymer chains covalently attached and allows for a large amount of sterically accessible ligands for the binding of biomolecules without any steric hindrance.
Column-based liquid affinity chromatography is another purification method that can be used invention. One example of a resin for use in purification method is Matrex Cellufme Sulfate (MCS). MCS consists of a rigid spherical (approx. 45-105 μιη diameter) cellulose matrix of 3,000 Dalton exclusion limit (its pore structure excludes macromolecules), with a low concentration of sulfate ester functionality on the 6-position of cellulose. As the functional ligand (sulfate ester) is relatively highly dispersed, it presents insufficient cationic charge density to allow for most soluble proteins to adsorb onto the bead surface. Therefore the bulk of the protein found in typical virus pools (cell culture supernatants, e.g. pyrogens and most contaminating proteins, as well as nucleic acids and endotoxins) are washed from the column and a degree of purification of the bound virus is achieved.
[0279] The rigid, high-strength beads of MCS tend to resist compression. The pressure/flow characteristics the MCS resin permit high linear flow rates allowing high-speed processing, even in large columns, making it an easily scalable unit operation. In addition a chromatographic purification step with MCS provides increased assurance of safety and product sterility, avoiding excessive product handling and safety concerns. As endotoxins do not bind to it, the MCS purification step allows a rapid and contaminant free depyrogenation. Gentle binding and elution conditions provide high capacity and product yield. The MCS resin therefore represents a simple, rapid, effective, and cost-saving means for concentration, purification and depyrogenation. In addition, MCS resins can be reused repeatedly.
Inactivated viruses may be further purified by gradient centrifugation, or density gradient centrifugation. For commercial scale operation a continuous flow sucrose gradient centrifugation would be an option. This method is widely used to purify antiviral immunogenic compositions and is known to one skilled in the art (See J P Gregersen "Herstellung von Virussimpfstoffen aus Zellkulturen" Chapter 4.2 in Pharmazeutische Biotechnology (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.)
[0280] Additional purification methods which may be used to purify viruses of the invention include the use of a nucleic acid degrading agent, a nucleic acid degrading enzyme, such as a nuclease having DNase and RNase activity, or an endonuclease, such as from Serratia marcescens, membrane adsorbers with anionic functional groups or additional chromatographic steps with anionic functional groups (e.g. DEAE or TMAE). An ultrafiltration/dialfiltration and final sterile filtration step could also be added to the purification method.
[0281] The purified viral preparation of the invention is substantially free of contaminating proteins derived from the cells or cell culture and can comprises less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/μg virus antigen, and less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/dose. The purified viral preparation can also comprises less than about 20 pg or less than about 10 pg. Methods of measuring host cell nucleic acid levels in a viral sample are known in the art. Standardized methods approved or recommended by regulatory authorities such as the WHO or the FDA can be used.
[0282] Tick-associated virus can be used to infect cells. Cells may be cultured in any useful media and any permissive cell or tissues, which may be, or may be derived from any animal cell. As used herein, a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, rodent cells, avian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells. Cell lines suitable for propagating, growing, or harboring tick-associated virus nucleic acid sequence or for expressing a polypeptide produced by the tick-associated virus nucleic acid sequence include, animal cells, including but are not limited to dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
[0283] Cell culture media formulations to suitable for culturing cells infected with tick- associated viruses described herein include, but are not limted to, Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof. These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro.TM. SFM, AIM V.R media, HyQ SFM4 MegaVir, EX-CELL Vera SFM, EPISERF, Pro Vera, any 293 or CHO media and analogues and derivatives thereof. The culture media described herein can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamine and biological buffers. One medium is OptiPRO SFM supplemented with L-glutamine and trypsin. In certain embodiments, the cell culture media can be supplemented with 0.1 to 10 units of trypsin. Alternatively, plant derived equivalents of trypsin (e.g. Accutase) ranging from 2-100 units can also be used in cell culture. Cell culture media can be used in the absence or presence of animal-derived components. An example of supplementation with an animal-derived component is gamma-irradiated serum ranging from 0.5- 10% final concentration.
[0284] An expression vector can be introduced into cells in order to produce proteins (for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40) encoded by nucleotide sequences of the invention (for example SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42). Cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
[0285] An expression vector can be introduced into cells in order to produce proteins encoded by nucleotide sequences of the invention (for example SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a sequence complementary to SEQ ID NOs: 1, 3, 5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35,37,39,41 or 42). Cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
[0286] A eukaryotic expression vector can be used to transfect cells in order to produce proteins (for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40) encoded by nucleotide sequences of the vector.
[0287] An exogenous nucleic acid (for example any of SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, a cDNA of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42 or a cDNA complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, fragments, or variants thereof) can be introduced into a cell via a variety of techniques known in the art.
[0288] A eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences (for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, a cDNA of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a cDNA complementary to SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42, fragments, or variants thereof).
[0289] Mammalian cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
[0290] An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation. Other methods used to transfect cells can also include calcium phosphate precipitation, modified calcium phosphate precipitation, polybrene precipitation, microinjection liposome fusion, and receptor-mediated gene delivery.
[0291] Cells to be infected with a tick-associated virus or nucleic acids thereof (for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, a cDNA of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, or a cDNA complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, fragments, or variants thereof) can be primary and secondary cells, which can be obtained from various tissues and include cell types which can be maintained and propagated in culture.
[0292] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B.D., eds. (Oxford University Press: New York, 1992)), and vary according to the particular cell selected. Commercially available medium can be utilized. Non- limiting examples of medium include, for example, Dulbecco's Modified Eagle Medium (DMEM, Life Technologies), Minimal Essential Medium (MEM, Sigma, St. Louis, MO); HyClone cell culture medium (HyClone, Logan, Utah); and serum-free basal epithelial medium (CellnTech).
[0293] The media described above can be supplemented as necessary with supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired. Cell medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
[0294] The medium also can be supplemented electively with one or more components from any of the following categories: (1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, epidermal growth factor and fibroblast growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example, pluronic polyol; and (8) galactose. Cells maintained in culture can be passaged by their transfer from a previous culture to a culture with fresh medium. In one embodiment, induced epithelial cells are stably maintained in cell culture for at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 20 passages, at least 25 passages, or at least 30 passages.
The cells suitable for culturing according to the methods of the present invention can harbor introduced expression vectors (constructs), such as plasmids and the like. The expression vector constructs can be introduced via transformation, microinjection, trans fection, lipofection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate
transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in J.
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY and in F.M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY.
In one embodiment, cells that have been infected with a tick-associated virus or contain nucleic acids thereof (for example SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, a cDNA of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a cDNA complementary to a SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, fragments, or variants thereof) can express a variety of markers that distinguish them from uninfected cells. Expression of markers can be evaluated by a variety of methods known in the art. The presence of markers can be determined at the DNA, RNA or polypeptide level.
[0295] In one embodiment, the method can comprise detecting the presence of a marker gene polypeptide expression. Polypeptide expression includes the presence of a marker gene
polypeptide sequence, or the presence of an elevated quantity of marker gene polypeptide as compared to non-epithelial cells. These can be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies). For example, polypeptide expression maybe evaluated by methods including, but not limited to, immunostaining, FACS analysis, or Western blot. These methods are well known in the art (for example, US Patent 8,004,661, US Patent 5,367,474, US Patent 4,347,935) and are described in T.S. Hawley & R.G. Hawley, 2005, Methods in Molecular Biology Volume 263: Flow Cytometry Protocols, Humana Press Inc; LB. Buchwalow & W.BoEcker, 2010, Immunohistochemistry: Basics & Methods, Springer, Medford, MA; O.J. Bjerrum & N.H.H. Heegaard, 2009, Western Blotting:
Immunoblotting, John Wiley & Sons, Chichester, UK.
In another embodiment, the method can comprise detecting the presence of nucleic acids (for example any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, a cDNA of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42 or a cDNA complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42, fragments, or variants thereof). RNA expression includes the presence of an RNA sequence, the presence of an RNA splicing or processing, or the presence of a quantity of RNA. These can be detected by various techniques known in the art, including by sequencing all or part of the marker gene RNA, or by selective hybridization or selective amplification of all or part of the RNA. In one embodiment, in situ hybridization can be used to detect tick-associated virus nucleic acids.
[0296] The tick-associated viruses and immunogenic compositions described herein can be be produced in cells. Production of the tick-associated viruses and immunogenic compositions described herein may also be accomplished on any useful media and permissive cell or tissues, which may be derived from animal cell lines. As used herein, a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, fish cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells, such as recombinant cells expressing a virus. For example, production of the tick-associated viruses and immunogenic compositions can be in any cell type, including but not limited to animal cells. Cell lines suitable for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limited to animal cells. In certain embodiments, cell lines suitable for producing the tick-associated viruses and immunogenic compositions described herein also include dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
[0297] The cell culture system for producing the tick-associated viruses and immunogenic compositions described herein can be a traditional adherent monolayer culture. Alternatively, suspension and microcarrier cell culture systems can also be utilized. [0298] Vessels for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limted to, roller bottles. For example, alternatively, other useful cell culture formats include flasks, stacked modules and stir tanks.
[0299] Production of the tick-associated viruses and immunogenic compositions can also be performed using a recombinant expression system that expresses tick-associated virus, a tick- associated virus protein, a fragment of a tick-associated virus protein or a variant of a tick- associated virus protein. The expression system can comprise any suitable plasmid or a linear expression construct known in the art.
[0300] The immunogenic compositions described herein can comprise an inactivated or killed tick-associated virus vaccine. Inactivated immunogenic composition can made by methods well known in the art. For example, once tick-associated virus is propagated to high titers, tick- associated virus antigenic mass could be obtained by methods well known in the art. For example, the tick-associated viral antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate tick-associated virus antigenic mass to produce immunogenic compositions. Tick-associated virus may be inactivated by treatment with formalin (e.g. 0.1-10%), betapropriolactone (BPL) (e.g. 0.01-10%), or with binary ethyleneimine (BEI) (e.g. 1-10 mM), or using other methods known to those skilled in the art.
[0301] In addition to killed tick-associated virus production, various means of attenuation are also possible and are well known and described in the art. Attenuation leading to modified live immunogenic compositions can also be used in conjunction with the compositions and methods described herein. Methods of attenuation suitable for use with the viruses described herein include continuous passaging in cell culture, continuous passaging in animals, various methods for generating genetic modifications and ultraviolet or chemical mutagenesis.
[0302] Attenuation of tick-associated virus may be achieved through cold-adaptation of an tick- associated virus strain. Cold-adapted tick-associated virus strains may be produced by methods which includes passaging a wild-type tick-associated virus, followed by selection for tick- associated virus that grows at a reduced temperature. Cold-adapted tick-associated virus can be produced, for example, by sequentially passaging a wild-type tick-associated virus in embryonated cells or chicken eggs at progressively lower temperatures, thereby selecting for certain members of tick-associated virus mixture which stably replicate at the reduced temperature. A cold-adapted tick-associated virus strain may exhibit a temperature sensitive phenotype. A temperature sensitive cold-adapted tick-associated virus replicates at reduced temperatures, but no longer replicates at certain higher growth temperatures at which the wild-type tick-associated virus will replicate. A temperature at which a temperature sensitive tick-associated virus will grow is referred to herein as a "permissive" temperature for that temperature sensitive tick-associated virus, and a higher temperature at which the temperature sensitive tick-associated virus will not grow, but at which a corresponding wild-type tick-associated virus will grow, is referred to herein as a "non-permissive" temperature for that temperature sensitive tick-associated virus. A cold-adapted tick-associated virus may also be produced through recombinant means. In this approach, one or more specific mutations, associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, can be identified and are introduced back into a wild-type tick- associated virus strain using a reverse genetics approach. Reverse genetics entails can be performed using RNA polymerase complexes isolated from tick-associated virus -infected cells to transcribe artificial tick-associated virus genome segments containing the mutation(s),
incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes.
[0303] Attenuation of an tick-associated virus may be achieved by serial passaging of a wild- type tick-associated virus strain in cell culture. A tick-associated virus strain can be passaged in a variety of cell systems until its ability to produce disease is lost whilst its immunogenic character is fully retained. Once inoculated into the host, tick-associated virus may be capable of multiplication to some extent. For example, attenuated tick-associated virus compositions can be prepared from cell line that has been attenuated by serial passage including serial passage at sub-optimal temperatures to a state where it is no longer capable of causing disease, but still capable of eliciting a protective immune response.
[0304] Suitable attenuated tick-associated virus strains may also be obtained by serial passaging to obtain an over-attenuated strain. The "over-attenuation" means that the number of passages for attenuation has been substantially greater than what is normally necessary for the removal of pathogenicity. The attenuated tick-associated virus retains its antigenicity after these numerous passages so that its immunogenic ability is not impaired. Such strains produce practically no symptoms or side effects when administered, and thus are safe and efficacious vaccines.
[0305] Dose sizes of the immunogenic compositions described herein can be in the range of about 2.0 to 0.1 ml depending on the route of administration, but dose sizes are not limited to this range. For inactivated tick-associated virus compositions can contain suitable TCID50 levels of virus prior to inactivation. The antigen content in tick-associated virus preparation can have, but is not limited to, a titer of between 10 to 10,000 units/ml as the amount administered per dose. One of skill in the art will readily be capable of determining a suitable antigen content for the
immunogenic compositions described herein. [0306] For immunogenic compositions containing modified live tick-associated viruses or attenuated tick-associated viruses, a therapeutically effective dose can be determined by one of skill in the art. For immunogenic compositions containing tick-associated virus subunit antigens, a therapeutically effective dose can be determined by one of skill in the art. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan.
[0307] An animal or a human can be inoculated with the immunogenic compositions or formulations described herein to generate an immune response. In certain embodiments, inoculation can be perfomed on animals that are at least 1, 2, 3, 4, 5, 6, or more weeks or older. In certain embodiments, the animals can receive one or more dosages. In certain embodiements, two or more dosages can be administered to the animal 3-4 weeks apart. In certain embodiments, the administration can be by subcutaneous injection. Intramuscular, intradermal, or oral routes of administration can also be used to administer the immunogenic compositions or formulations described herein.
EXAMPLES
[0308] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
[0309] Example 1: Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses
[0310] A wide range of bacterial pathogens have been described in ticks, yet the diversity of viruses in ticks is largely unexplored. In the United States, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. High-throughput sequencing was used to characterize the viromes of these tick species and identified the presence of Powassan virus and eight novel viruses. These included the most divergent nairovirus described to date, two new clades of tick-borne phleboviruses, a mononegavirus, and viruses with similarity to plant and insect viruses. The analysis revealed that ticks are reservoirs to a wide range of viruses and suggests that discovery and characterization of tick-borne viruses will have implications on viral taxonomy and may provide insight into ticktransmitted diseases. [0311] Ticks are implicated as vectors of a wide array of human and animal pathogens. To better understand the extent of tick-borne diseases, it is crucial to uncover the full range of microbial agents associated with ticks. The current knowledge of the diversity of tick-associated viruses is limited, in part due to the lack of investigation of tick viromes. In this study the virome of three tick species from the United States were examined. It was found that ticks are hosts to highly divergent viruses across several taxa, including ones previously associated with human disease. The data described herein underscore the diversity of tick-associated viruses and provides the foundation for further studies into viral etiology of tick-borne diseases.
[0312] Introduction
[0313] Ticks (class Arachnida, subclass Acari) have been implicated as vectors in a wide range of human and animal diseases worldwide (1-10). Approximately 900 species of ticks have been described and taxonomically classified into three families: Argasidae (argasid or soft ticks) Ixodidae (ixodid, or hard ticks) and Nuttalliellidae (11). Their propensity for feeding on on a wide array of hosts, expansive range and long life cycle underscore the importance of tick surveillance for the presence of potential pathogens. Argasid and ixodid ticks combined transmit a greater diversity of viral, bacterial and protozoan pathogens than any other arthropod vector (12). The worldwide incidence of tick-borne disease is increasing, partly due to increased frequency of endemic tick-borne diseases, as well as the discoveries of new tick-associated agents (13).
[0314] In the United States (US), bacterial agents are implicated in the majority of tick-borne disorders. Lyme disease, caused by Borrelia burgdorferi represents the most frequently reported tick-borne illness (14). Other bacterial agents, such as Anaplasma, Ehrlichia, Rickettsia, other Borrelia species, as well as the protozoan Babesia contribute to the overall spectrum of tick-borne disease (1, 6, 7, 14-16). Conversely, viral causes are diagnosed in only a fraction of tick-borne disease cases (14). Despite considerable insights into the diversity of tick bacteriomes, our understanding of tick-associated viruses is still limited. Traditional viral isolation and identification methods using tissue culture have isolated several tick-associated viruses but few have been characterized thus far. In comparison to bacterial and protozoan agents, the literature associated with tick-borne viruses in the Americas is also limited. Most of the literature to date on suspected viral tick-borne pathogens focuses on those found in Europe, Asia and Africa (3, 17). Powassan and Colorado tick-fever viruses have been historically recognized as the only human tick-borne viral pathogens in the US (3, 18).
[0315] Recently, the Heartland virus, a new pathogenic tick-borne virus was isolated from patients in Missouri and characterized by high-throughput sequencing (HTS) (19). Heartland virus was shown to be phylogenetically similar to severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne virus isolated from ticks and humans in China in 2009 (10). The emergence of novel pathogenic tick-borne viruses as well as the dearth of data on tick viromes suggests a need for viral surveillance and discovery in ticks. Although it seems plausible that there are tick-borne viruses that have not been amenable to isolation via tissue culture, as far as is known, no extensive culture-independent studies have been attempted to examine tick viromes. These studies may not only identify viruses associated with acute disease but could also provide insights into the pathogenesis of more controversial chronic illnesses associated with tick bites (20, 21). Thus, to survey viral diversity, viromes of three human-biting ticks in the US (Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis) were examined by HTS. The analysis and characterization described herein identified eight new viruses and indicates that ticks carry a wide array of previously uncharacterized viral agents.
[0316] Materials and Methods
[0317] Adult /. scapularis, D. variabilis, and A. americanum were collected within a 2 square mile area of Heckscher State Park (Suffolk County, NY) in April 2013 (Figure 1). Ticks were pooled prior to nucleic acid extraction; two pools of /. scapularis (N=30/pool), two pools of D. variablis (N=30/pool) and a single pool of americanum (pool N=25) were prepared. A two-step purification and nuclease treatment protocol was followed prior to extraction to enrich for viral sequences and minimize host, bacterial and fungal template that can compete with virus template in HTS (22). Each tick pool was homogenized in 500 μΐ of PBS, followed by purification through 0.45 μΜ filter. The filtrate (237 μΐ) was treated with 1.5 μΐ RNase A for 15 minutes, followed by Turbo DNase (7.5 μΐ), Benzonase (1.8 μΐ) and 2.7 μΐ of 1M MgC12 for 45 minutes. All nuclease treatment was performed at room temperature. 250 μΐ of the nuclease treated filtrate was added to 750 μΐ of Nuclisens buffer and total nucleic acid (TNA) was extracted using the EasyMag extraction platform (Biomerieux). TNA from each pool was eluted in a 35 μΐ volume.
[0318] Unbiased high-throughput sequencing
[0319] TNA (11 μΐ) from each tick pool was subjected to first and second strand cDNA synthesis with Super Script III reverse transcriptase (Invitrogen) and Klenow Fragment (New England Biolabs), respectively. Random primers (30¾/μ1) (Invitrogen) were used in both assays. Ion ShearTM Plus Reagents Kit (Life Technologies) was used for double stranded cDNA fragmentation at 37°C for 25 min. Agencourt® AMPure® XP (Beckman Coulter) reagent (1.8 x sample volume) was used for DNA purification. Ion XpressTM Adapters and unique Ion
XpressTM Barcodes (Life Technologies) were ligated to fragmented material by using the Ion Plus Fragment Library kit (Life Technologies). Ligation was performed at 25°C for 15 min and 72°C for 5 min. Ligated and nick repaired products were purified with Agencourt® AMPure® XP (0.85 x sample volume) and amplified according to manufacturers' instructions with Platinum®PCR Super Mix High Fidelity from the Ion Plus Fragment Library kit. Amplified products were purified as previously described for the ligation reaction. Agilent® High Sensitivity DNA Kit was used for library quantitation on the Bioanalyzer™ 2100 instrument. The concentration of each barcoded library was approximately 50nM. Libraries were diluted to approximately 45nM and a pool of libraries in equimolar concentrations was prepared. Ion OneTouchTM 200 Template Kit v2 (Life Technologies) was used to bind barcoded libraries to Ion SphereTM particles (ISPS). Emulsion PCR of DNA linked ISPS was performed on the Ion OneTouch™ 2 instrument (Life
Technologies). Ion OneTouchTM ES instrument was used to isolate template -positive ISPS. Ion PGM™ Sequencing 200 Kit v2 (Life Technologies) was used for sequencing of templated ISPS which were loaded on the Ion 316™ Chip for further processing on the Ion Personal Genome Machine® (PGM™) System (Life Technologies). Approximately 600,000 reads were obtained for each library.
[0320] The de -multiplexed reads were preprocessed by trimming primers and adaptors, length filtering, and masking of low complexity regions (WU-BLAST 2.0). The remaining reads were subjected to homology search using BLASTn against a database consisting of ribosomal and genomic metazoan sequences. Following the processing, the remaining reads amounted to 116,946 (pool 1) and 131,676 reads (pool 2) for /, scapularis, 30,353 (pool 1) and 29,334 (pool 2) for D. variabilis and 297,175 for the A. amblyomma pool.
[0321] The host-subtracted reads were assembled using the Newbler assembler (454, v2.6). Contigs and singletons were then subjected to a homology search against the entire GenBank database using BLASTn and the viral GenBank database using BLASTx. Contigs and singletons with similarity to viral sequences from the BLASTx analysis were again subjected to a homology search against entire GenBank database to correct for biased e-values. For potential viral candidates, close relatives were used to identify low homology regions in the genome from
BLASTx; gaps were filled in by PCR using primers specific to the assembled sequence. Genome termini were obtained by 5 ' and 3 ' rapid amplification of cDNA ends (RACE) (Clontech
Laboratories). The final genome sequences were verified by classical dideoxy sequencing using primers designed to generate overlapping PCR products.
[0322] Tick screening
[0323] To determine the authenticity and prevalence of viral sequences in ticks, cDNA generated from adult /. scapularis for a previous study was used as template for PCR (23). The ticks were collected in 2008 from four separate locations in New York State: two from Suffolk county (Heckscher State Park, Fire Island) and two from Westchester County (Kitchawan Nature Preserve, Blue Mountain Reservation) (Figure 1). cDNA from individual ticks was screened by PCR for each novel virus identified by HTS. cDNA from individual virus-positive ticks were used to generate viral genomic sequence equivalent to the sequences obtained by HTS. The sequences were obtained by generating overlapping PCR products using HTS sequences as a reference for primer design and sequence assembly. All PCR products were verified by dideoxy sequencing. Sequences from individual ticks were deposited in GenBank under accession numbers KM048311- KM048322. All genome assemblies and alignments were performed with Geneious v 6.1 and Mega 5.2 programs (24, 25). Phylogenetic trees were constructed with Mega 5.2 using the maximum likelihood method with 1000 bootstrap replications. Amino acid trees were generated using the JTT matrixbased model. Nucleotide trees were generated using the Jukes-Cantor model.
[0324] Results
[0325] Analysis of HTS data revealed the presence of eight previously uncharacterized viruses (Table 1). The most diverse virome was observed in /. scapularis where in addition to Powassan virus, sequences representing six novel viruses were identified. Bunyavirus-like sequences represented more than 50% of the total filtered reads in both /. scapularis pools, and were similar to viruses in the Nairovirus and Phlebovirus genera. Two viruses were identified in each pool of D. variablis; one virus was similar to phleboviruses and the other displayed limited homology to insect viruses. Although the pool of A. americanum generated the most reads of the five analyzed tick pools, a rhabdo virus was the lone virus identified in this tick species. The complete genome characterization of this virus, named Long Island tick rhabdo virus (LITRV) has been reported (26).
[0326] Arthropod genomes frequently contain integrated fragments of archaic RNA viral genomes (27-29). The genome of /. scapularis contains numerous sequences of viral origin, designated endogenous viral elements, or EVEs (27). To test for the authenticity of the novel sequences obtained by HTS, results of RT-PCR vs DNA PCR were compared as an additional indication of the template source i.e., authentic viral nucleic acid vs. tick genomic DNA. Consistent with a model where these novel virus sequences represent authentic RNA viruses and not EVEs, products were obtained with RT-PCR but not with PCR.
[0327] Powassan virus
[0328] Two lineages of Powassan virus (POWV, family Flaviviridae, genus Flavivirus) circulate in the US: lineage I, isolated mainly from /. cookei, and lineage II (also known as deer tick virus), detected predominately in /. scapularis (30). Human infection with viruses from either lineage has been linked with Powassan encephalitis, a severe potentially life-threatening
neurological illness (30, 31). POWV lineage II has been detected in /. scapularis throughout the Northeast (23, 32, 33). In this HTS study, approximately 0.8% of all filtered reads from /. scapularis pool 1 were from POWV lineage II. Assembly of all reads and contigs from this pool resulted in assembly of 98% of the 10.8 Kb POWV genome. Nucleotide (nt) comparison of the complete polyprotein coding sequence (GenBank accession number KJ746872 (SEQ ID NO: l)) indicated that the nt sequence of this virus (designated strain LI-1) is 99.4% identical to isolate NSF001 (GenBank accession number HM44059) obtained from Nantucket, MA in 1996 and clustered with viruses in a subclade of POWV lineage II isolated from the Northeast (Figure 2) (33). The nt identity to the subclade of lineage II made up of isolates from the Midwestern United States was 93.4%.
[0329] Nairovirus
[0330] The genus Nairovirus (family Bunyaviridae) is comprised of 37 tick-borne viruses. Its genome consists of three segments of negative sense single-stranded RNA, designated small (S), medium (M), and large (L), that encode the nucleocapsid protein (N), the envelope glycoproteins (Gn and Gc) and an RNA-dependent RNA polymerase (L), respectively (34). Both /. scapularis pools contained multiple contigs with sequence similar to viruses in the Nairovirus genus by BLASTx. Assembly of these Nairovirus-like contigs, using CCHFV as a reference genome, provided >90%> coverage of the S and L segments. The complete segments were obtained by overlapping PCR and 3' and 5' RACE. The assembled sequences showed low similarity to other members of the genus, suggesting that this virus, provisionally named South Bay virus (SBV) after its geographic location, represents a novel nairovirus species. The complete S and L sequences from an individual SBV-positive tick (designated SBV H38) were also obtained. Comparison of the genomic nt sequences obtained from HTS and SBV H38 indicated they were 97%> and 99%> identical in the S and L segments, respectively. SBV reads were the predominant viral reads obtained from both /. scapularis pools and accounted for 25 %> of filtered reads from pool 1 and 41% of reads from pool 2. However, despite an exhaustive bioinformatics analysis, any contigs or reads with any similarity to Nairovirus M segments were not identified. Attempts at virus isolation by inoculation of /. scapularis SBV PCR positive pools in Vera, Cos7, C6/36 and 297 cell lines were unsuccessful.
[0331] To ascertain whether the SBV sequences represented authentic viral reads, /. scapularis DNA and corresponding cDNA was screened for the presence of SBV. Partial S and L sequences were amplified in SBV-positive tick cDNA, but were absent in the corresponding genomic DNA. Additionally, it was found that /. scapularis genome contains an integrated Nairovirus N gene open reading frame (ORF) (accession number XM002414099), although it was not transcribed in any tick sample. This sequence was more similar to SBV than other nairovirus sequences, likely representing an ancestral integration of an SBV-like nairovirus into the /. scapularis genome. [0332] Conserved genus-specific sequences at the termini of each segment are a feature of all bunyaviruses. In nairoviruses the termini sequence typically consists of UCUCAAAAGA at the 5' end. It was found that the sequences of SBV L and S termini were consistent with the Nairovirus sequence with the exception of a single nt change at the fifth position in this sequence
(UCUCUAAAGA). The complete sequences of the L and S segments were deposited in GenBank under accession numbers KJ746877 (SEQ ID NO:3)-KJ746878 (SEQ ID NO:5) (originating from tick pools) and KM048320 (SEQ ID NO:7-KM048321 (SEQ ID NO:9) (tick H38).
[0333] Phylogeny and L segment analysis
[0334] Of the 37 described viruses assigned to the Nairovirus genus, only seven are completely sequenced and there is no published sequence data for 21 putative members of the genus. For the remaining nine viruses, the only available sequence data consists of a short (<450 nt) fragment within the L segment (35). This region is part of the polymerase catalytic domain, and is highly conserved with no nt insertions or deletions in any of the 16 nairoviruses analyzed to date. Thus, the amino acid (aa) sequence from this region was used to determine the phylogenetic relationships of SBV to the rest of the Nairovirus genus. SBV contained a seven aa insertion in this region and had <45% identity to the next closest virus, distinguishing SBV from other nairoviruses (Table 2). SBV did not cluster with any of the described nairovirus serogroups, but fell outside of all described viruses in this genus (Figure 3).
[0335] The L segment of SBV is 13892 nt long and contains a 13611 nt ORF that encodes a 4536 aa protein. This represents the longest known nairovirus open reading frame, over 600 amino acids longer than the L ORF of Dugbe virus. Comparison of SBV to L proteins from other nairoviruses revealed that SBV does not cluster with any of the previously sequenced viruses, and contains only 28% aa identity to the next closest virus (Erve virus) (Figure 4A, Table 3). In SBV, the catalytic polymerase domain is located within the region between amino acid (aa) residues 2600 and 3200 and contains all known viral RNA polymerase motifs (pre-motif A, motifs A-E) (36).
[0336] In addition to the polymerase domain, the nairovirus L proteins may contain several other protein motifs, such as ovarian tumor domain (OTU), topoisomerase domain, zinc finger motif and a leucine zipper motif (36, 37). The OTU domain represents a family of cysteine proteases and it is present in the N terminus of all seven sequenced nairovirus L proteins. In CCHFV this domain may function in modulation of interferon response in host cells (38). No evidence of the presence of a functional OTU domain in SBV was found; the conserved catalytic residues (D37, C40, and H151 in CCHFV) were absent and overall, the SBV N-terminal region of the L showed little similarity to other nairoviruses. The N terminal topoisomerase motif, zinc finger motif and leucine zipper motifs were also absent. [0337] Analysis of the S segment
[0338] SBV has an unusually long S segment comprising 5536 nucleotides (nt) and containing a 1664 nt ORF encoding a putative N protein. The N protein has a number of important functions in negative-sense single-stranded RNA viruses. It binds to genomic RNA forming ribonucleoprotein complexes that associate with the polymerase for viral RNA synthesis. In bunyaviruses, N proteins are also associated with immune response modulation. In SBV, the N ORF begins at position 865 from the 3' end of the segment and is predicted to encode a 547 aa N protein, representing the second largest Nairovirus N. Only Erve virus, encoding a 630 aa protein contains a longer nucleocapsid sequence (39). The aa identity of the SBV N protein to the other seven sequenced Nairovirus N proteins ranged between 18.8% - 21.6% (Table 2, Figure 4B). Despite the overall high degree of divergence from other nairoviruses, a highly conserved domain present at the C terminal part of the protein was also identified. This domain represents the only area of high conservation amongst all eight viruses. Out of 32 aa in this region, 11 were conserved among all nairoviruses (Figure 5). Two recent analyses of the crystal structure of the CCHFV N protein revealed a number of residues essential for N function. Several of the implicated residues are present within this conserved domain. Carter et al analyzed the interaction of CCHFV N with RNA and identified five residues (K90, K132, Q300, K411, H456) essential for RNA binding (40). A high degree of conservation of these residues between SBV and other nairoviruses was found (Figure 5). In an independent study, analysis by Guo et al revealed that the CCHFV N possesses metaldependent DNA endonuclease activity and implicated seven potential active site residues (41). Four of these (R384, E387, H453, and Q457) are conserved in all nairoviruses including SBV, with both H453 and Q457 part of the conserved C terminal domain. Another residue, K411, was one of the residues implicated as also essential for RNA binding by Carter et al. In the remaining two residues, South Bay and Erve viruses represent the only viruses with aa variations; for residue R298, SBV contains a Y and Erve virus a K, and for Y374 both viruses contain an H.
[0339] The S segment of SBV contains additional 3028 nt of sequence following the termination of the N ORF (Figure 6). This portion of the segment does not display homology to any known sequence and contains several short potential ORFs, the longest consisting of 441 nt, though none contain any identifiable domains. The terminal 1400 nt of the S segment does not contain any potential ORF longer than 170 nt.
[0340] Prevalence of SBV in ticks
[0341] To determine the prevalence and distribution of SBV in ticks we used cDNA generated from individual ticks in 2008 from four locations in two New York State counties (Figure 1). These samples were previously screened for the presence of multiple tick-borne pathogens (23). SBV was detected in all four locations, with a total of 20 out of 100 ticks positive for the virus (Table 4). The highest number of SBV -positive ticks originated from Heckscher State Park (12 out of 26). The SBV sequences were genetically homogeneous, even at different collection sites. Partial S and L sequences obtained from all SBV-positive ticks displayed 98- 100% nt identity among all viruses.
[0342] Phlebovirus
[0343] The genus Phlebovirus is comprised of over 70 viruses isolated primarily from ticks, mosquitoes and sandflies (42). Multiple contigs were identified with homology to phleboviruses by BLASTx in each pool of /. scapularis and D. variabilis. The contigs originating from each tick species were assembled separately to a reference phlebovirus genome (Precarious Point virus). Initial sequence comparison indicated that both viruses likely represented new phlebovirus species, as they were distinct from each other and other phlebovirus sequences in GenBank. The I.
scapularis-associated virus was named the blacklegged tick phlebovirus (BTPV) and the D.
variabilis-associated virus the American dog tick phlebovirus (ADTPV) (GenBank accession numbers KJ746873-KJ746876 (SEQ ID NOs: 11, 13, 15, 17) and KJ746901 (SEQ ID NO: 19) - KJ746902 (SEQ ID NO:21)).
[0344] Complete S and L segments were obtained of both viruses; as was the case with the nairovirus, it was not possible to identify any sequences with similarity to phlebovirus M segments. The typical phlebovirus segment termini sequence AC AC AAAG (5 ' end) was identified at the termini of both viruses, with the exception of a single nt change at the 3' end of BTPV L segment (ACACAAUG). During the genome assembly phase of BTPV, two distinct putative BTPV S and L segments were identified, suggesting the presence of multiple genotypes of this virus. This was confirmed by PCR using cDNA from both tick pools and individual scapularis ticks as template and sequencing of PCR products. The two genotypes, designated BTPV-1 and BTPV-2, were 12% and 15%) divergent in the nt sequence of the S and L segments, respectively.
[0345] PCR analysis of individual ticks indicated that BTPV and ADTPV were highly prevalent in their tick hosts (Table 4). Out of 24 adult /. scapularis from Heckscher state park, 12 (50%o) were positive by PCR for BTPV-1 or BTPV-2 (Table 4). Twelve D. variabilis collected on Fire Island, NY, approximately 16 miles Southeast of Heckscher state park, were screened 9 positive ticks for ADTPV (75%>) were identified. Complete S and L sequences from two individual /. scapularis were iobtained (designated BTPV-1 HI 2, and BTPV-2 H5, accession numbers KM48313-KM48316 (SEQ ID NOs:23, 25, 27, 29)), and ADTPV sequences from one individual D. variabilis (ADTPV H6, accession numbers KM048311 (SEQ ID NO:31) -KM048312 (SEQ ID NO:33). The nt sequence of all segments from individual ticks were 97%>-99%> identical to the sequence obtained by HTS. [0346] S segment
[0347] A typical Phlebovirus S segment is approximately 1700 nts in length and codes for a nucleocapsid (N) and a non-structural protein {NSs} in an ambisense orientation (43). The lengths of the putative S segments of BTPV-1 and BTPV-2 were >2400 nt and contained unusually long ORFs of 1581 and 1578 nt in length, respectively (Figure 7). These would encode putative 526 and 525 aa proteins. The N terminal portions of these putative proteins displayed no homology to any known protein, whereas the C-terminal 250 aa portion exhibited similarity to other Phlebovirus N proteins. Comparison of this C-terminal portion of BTPV-1 and BTPV-2 N revealed they were 91% identical to each other and <26% to other Phlebovirus N proteins. ORFs corresponding to the typical genomic orientation of the Phlebovirus NSs ORF were identified; however, these were unusually short and displayed no homology to any reported NSs protein.
[0348] The S segment of ADTPV also contained an atypically long ORF of 1209 nts encoding a putative 402 aa protein. Similar to BTPV, the characteristic Phlebovirus N was identified only within the C-terminal 250 aa sequence of this protein. A putative NSs ambisense ORF was identified, although it was shorter than the ORFs identified in BTPV (151 nt). The aa identity of the putative N of ADTPV to BTPV-1 and BTPV-2 was 24%, and <34% to other Phlebovirus N proteins (Table 5).
[0349] L segment
[0350] The overall length of the complete L segments of both BTPV-1 and BTPV-2 were 6733 nt and contained 6624 nt ORFs that would encode a 2207 aa L protein. The nt sequence identity of BTPV-1 and BTPV-2 L ORFs was 84% (95.6% aa identity). The length of the ADTV L segment was 6600 nt, with a 6537 nt ORF encoding a 2178 aa protein. The ADTV L protein was 29.6% and 29.2% identical to the L protein of BTPV-1 and BTPV-2, respectively (Table 5).
[0351] Phylogenetic analysis
[0352] Phleboviruses have traditionally been classified into two groups consisting of sandfly/mosquito-borne and tick-borne viruses (42). At least three phylogenetic clusters of tick- borne phleboviruses have been identified, each comprised of several potential species: the
Uukuniemi group, the Bhanja group and the STFS group (19, 44, 45). It was found that BTPV does not cluster with any these groups, and forms a separate monophyletic clade outside all tick-borne and sandfly/mosquito-borne phleboviruses, similar to Gouleako and Cumuto viruses. ADTPV is more similar to viruses within the Uukuniemi group but forms a distinct monophyletic clade outside this group (Figure 8).
[0353] Mononegavirales-like virus [0354] In sequences originating from pool 2 of /. scapularis contigs with weak homology by BLASTx to the L protein RNA-dependent RNA polymerase domain (RdRp) of viruses within the order Mononegavirales were identified. This order represents five families (Bornaviridae,
Filoviridae, Nyamiviridae, Paramyxoviridae, Rhabdoviridae) of monopartite negative single-stand viruses with comparable ORF organization (46). The complete 6444 nt L ORF of this virus (accession number KJ746903 (SEQ ID NO: 35)) was obtained, that encodes a putative 2147 aa protein and identified a Mononegavirales RNA-dependent RNA polymerase (RdRp) mRNA- capping and a virus-capping methyltransferase domains, characteristic of a Mononegavirales-like virus L protein. Homology searches revealed this virus to be very distant from other
Mononegavirales (Figure 9). The greatest similarity was observed with the Nyamanini and Midway viruses (17% aa identity). Nyamanini virus (originally isolated in 1957 in South Africa) and Midway viruses (isolated in 1966 on islands in the Pacific) have frequently been isolated from from argasid ticks and form a distinct lineage in the order Mononegavirales (47). Along with soybean cyst nematode virus both viruses are now included in Nyamiviridae, a new Mononegavirales family (48).
[0355] To determine the prevalence of this virus 47 individual tick samples were screened by PCR (Table 4). One positive tick was detected (FI3, accession number KM048317 (SEQ ID NO: 37)). The complete nt sequence of the FI3 L ORF was 99.2% identical to the assembled sequence (99% aa identity). This result, in concert with the FITS detection in only one tick pool with low genome coverage, suggests that this virus has a low prevalence in tick populations, at least within the geographical area surveyed.
[0356] Invertebrate/plant viruses
[0357] In addition to vertebrate viruses, sequences with similarity to viruses associated with plants and arthropods were identified. Two distinct invertebrate-like viruses were identified in /. scapularis pools, designated ISAV-1 (Ixodes scapularis associated virus- 1), present in both pools, and ISAV-2, identified only in one pool. Partial genomes of both viruses were recovered (2.8 kb for ISAV-1 and 2.3 kb for ISAV-2, respectively), encompassing the majority of a putative protease and RdRp. Analysis of a 255 aa conserved portion of the RdRp indicated that these viruses were 50%> identical to each other and limited homology was observed to described viruses with highest similarity to invertebrate viruses in the genus Sobemo virus (<25% aa similarity). This genus represents positive-sense singlestranded RNA viruses that infect plants, with various arthropods implicated as vectors (46). ISAV-1 virus was identified in 5 out of 20 ticks; however, none of these were positive for ISAV-2. An additional 50 samples were screened, but were unable to identify any ISAV-2 positive ticks. [0358] Both pools of D. variabilis contained sequences with low similarity (<20%) to a wide range of invertebrate single-stranded positive-sense viruses. A 5.3 kb segment of this virus was recovered, encoding a putative >1700 aa replicase. One out of nine tested D. variabilis ticks was positive for this virus, with 96.8% nt and 99.3% aa identity to the HTS sequence (accession number KM048322). The N terminal 1300 aa of this protein contained the methyltransferase, helicase, and RdRp domains (Figure 10). No putative domain could be identified within the >400 aa C-terminal portion. This genome organization was similar to viruses within the Omegatetravirus genus (family Alphatetraviridae) (49). Limited aa identity was found (19%) across the N terminal 1300 aa portion of the replicase of this virus with a representative of this genus (helicoverpa armigera stunt virus). Omegatetraviruses, isolated from moths (order Lepidoptera), have a bi-partite genome. RNA1 segment (approximately 5.3 kb in length) encodes a replicase containing all aforementioned domains and RNA2 segment (approximately 2.5 kb in length) encodes the capsid (46). Sequences for a putative RNA2 segment that encodes a protein with low (11% aa) identity to Omegatetravirus capsids were identified. Further classification of this new virus was challenging due to high sequence divergence in combination with a limited number of available Alphatetraviridae sequences. Although this virus was tentatively included within the Alphatetraviridae, due to dissimilarity in sequence and host association, it likely represents a new family of arthropod- associated viruses.
[0359] Discussion
[0360] In this study, the viromes of /. scapularis, D. variablis and A. americanum were analyzed, tick species frequently associated with pathogen transmission in the US (14). In addition to exploring viral diversity, this work was aimed at uncovering viruses with potential relevance for human disease. Several novel viruses were identified with genetic similarities to pathogens of humans and livestock. While these data do not allow conclusions regarding transmissibility, these discoveries can lay the foundation for such future work.
[0361] Bunyavirus-like sequences predominated in the HTS data described herein. The known Bunyaviridae comprise more than 350 viruses with many of those only recently discovered and characterized (10, 19, 50-52). Relatively few nairoviruses have been discovered; Kupe and Finch Creek viruses were the only new characterized members of this genus reported within the last decade (53, 54). In this study, molecular evidence of a new nairovirus present in ixodid ticks in North America was demonstrated. SBV is the first New World nairovirus with available coding region sequence of the S and L segments. With more than 70% aa divergence from other nairoviruses for these segments it is the most divergent of all nairoviruses analyzed to date. Based on the aa analysis of the polymerase region, SBV forms a distinct phylogenetic lineage outside all currently described nairoviruses and does not cluster into any of the nairovirus serogroups.
[0362] The findings described herein indicate that the Nairovirus genus is significantly more diverse than previously appreciated. An earlier phylogenetic analysis of nairoviruses indicated the presence of two main phylogenetic clades with one clade isolated exclusively from ixodid ticks and the other from argasid ticks (35). The phylogeny is more complex, as SBV represents a previously unidentified, genetically distinct nairovirus clade. The current nairovirus classification scheme, based on a very short sequence fragment and serology is tentative and is further confounded by the lack of complete genome sequence data for any argasid tick-isolated nairovirus. Future genome sequencing of genetically unclassified nairoviruses, especially those isolated from argasid ticks, may uncover viruses similar to SBV that will aid in the establishment of a more accurate classification system for this genus. Interestingly, during the preparation of this manuscript, a new divergent nairovirus was reported from bats in France, although the genome sequence of this virus was not available for comparison with SBV (55).
[0363] The detection of SBV in all four surveyed areas suggests that it may have a broad geographical distribution, perhaps throughout the range of /. scapularis, comparable to B.
burgdorferi and other pathogens vectored by this tick. Other nairoviruses have been shown to have broad geographical distributions and have been isolated throughout the range of their hosts.
[0364] In addition to SBV, viruses in /. scapularis and D. variabilis were identified
representing novel clades of tick-borne phlebo viruses. Historically, phleboviruses were classified into tick-borne or sandfly/mosquito-borne groups based on vector, genomic, and serological relationships (42). The discovery of these highly divergent viruses along with the recent
characterization of the atypical Gouleako and Cumuto viruses underscores the potential need to reassess this classification scheme (56, 57). Additionally, in conjunction with the recent
identification of A. americanum as host of Heartland and Lone Star viruses, the data also suggests that considerable diversity of phleboviruses may exist in tick species within the US (58-60).
[0365] It was discovered that all novel bunyaviruses had high infection rates in ticks. Twenty percent of all ticks from four different locations were infected with SBV. The infection rates with BTPV and ADTPV were also high, although the analysis was limited to ticks from a single site. While these rates are higher than is typically reported for arboviruses, nairovirus infection rates of up to 20% have been reported in ticks (61). The highest rate of infection with SBV was observed in ticks from Heckscher State Park, which was also the origin of the ticks used for the high-throughput sequencing study described here. In contrast, Powassan virus was detected in less than 2% of these same ticks (23). This suggests that the infection rate of /. scapularis with SBV may be significantly higher than with Powassan virus, at least within the geographical range analyzed. Possible explanations for the high prevalence of these viruses may be the result of a high assortment of susceptible viremic hosts resulting in proficient horizontal transmission of the virus to uninfected ticks. The viruses may also be efficiently transmitted by non-viremic hosts, or by "co-feeding" of infected and uninfected ticks. In addition, as shown for some arboviruses, these viruses may be transtadially (maintained throughout life stages) and transovarially transmitted to progeny, resulting in the tick vector also playing a role of a long-term reservoir in tick populations (3).
[0366] It is not known whether SBV, ADTPV, and BTPV are specific to their tick hosts. Some tick-borne viruses can be isolated from more than one tick species. CCHFV, for example, has been isolated from over 30 tick species and multiple genera throughout Europe, Africa and Asia, though not all likely represent true vectors (9). Although the HTS analysis suggest tick host specificity, it cannot be ruled out that other tick species may serve as hosts of these viruses.
[0367] The L segment organization of SBV, ADTPV and BTPV is comparable to the L segments of other nairo- and phleboviruses. Conversely, the organization of the presumed S segments of these viruses is at least partially inconsistent with their putative genome affiliation. In these viruses, the S segments are considerably longer, and in the case of SBV, contain a large portion of sequence lacking identifiable ORFs. The N ORFs in ADTPV and BTPV are longer than typical Phlebovirus N, while the putative NSs ORFs are considerably shorter relative to typical Phlebovirus NSs ORFs and may not encode functional proteins. Although this genome arrangement is uncharacteristic, there has been a precedent for unconventional Phlebovirus S segments illustrated by the lack of an NSs ORF in Gouleako virus (56). Presumably, continued molecular surveillance will lead to the discovery of other bunyaviruses with atypical genome organization.
[0368] Another notable characteristic of these viruses is the lack of recognizable glycoprotein- coding segment. Although > 90% of the L and S segments were recovered for SBV and both phleboviruses by HTS and dispelled the possibility of viral integration, it was not possible to identify any sequences with similarity to Bunyaviridae M segments. In addition, large contigs (>300 nt) containing an uninterrupted ORF that was confirmed by PCR to be present in individual S and L segment-positive ticks but absent in virus-negative ticks were not identified. Consensus PCR was also performed with numerous primers targeting Phlebovirus and Nairovirus M segments, without success. To investigate if the depth of sequencing was adequate to detect bunyavirus M segments, a new pool of four adult /. scapularis ticks positive for both SBV and BTPV by PCR, were selected and analyzed them by Illumina HiSeq. Despite achieving a 10-fold increase in reads, and nearly 100% coverage of S and L segments for both viruses, again it was not possible to identify any sequences representative of potential M segments. Various explanations for this confounding result have been considered. A complicated secondary structure of the M segments of these viruses may inhibit efficient cDNA synthesis and interfere with amplification (and detection). While plausible, it is acknowledged that this confound has not been reported in HTS analysis of other bunyaviruses. Alternatively, these viruses may not have a typical bunyavirus-like M segment. While incompatible with current understanding of bunyaviruses, the viruses uncovered here may represent viral lineages that do not have a typical bunyaviral M segment and employ other means for cellular entry. Finally, it also cannot be excluded that the S and L segments of these viruses may exist in an episome-like form in tick cells. As such, they may not form an infectious virion instead using transovarial transmission or some other means as a vehicle for their dissemination to new hosts.
[0369] The analysis described herein also uncovered a novel mononegavirus in /. scapularis, although due to limited sequence obtained by HTS, the analysis was limited to the L ORF. The data indicates that this virus may be a novel member of the family Nyamiviridae. Of the three viruses in this family, Nyamanini and Midway viruses display a high degree of similarity and are part of the genus Nyavirus, while the more divergent soybean cyst nematode virus is a representative to a yet unnamed Nyamiviridae genus (48, 49, 62). The virus uncovered in this study appears to be the most genetically diverse of this group and presumably constitutes a new genus within Nyamiviridae, although a complete genetic characterization will be required to fully determine its taxonomy.
[0370] The results of this study indicate that ticks harbor a wide array of highly diverse viruses. One constraint of this study was the limited geographical distribution of the sampled ticks as the HTS analysis focused on specimens collected from a single location. While it is anticipated that many of the viruses uncovered by the HTS analysis are distributed along the range of their presumed tick hosts, it is speculated that other agents remain to be discovered and analysis of ticks from diverse geographical areas would reveal greater viral diversity of tick-borne viruses. It is expected that when additional HTS data from diverse arthropod vectors becomes available, it will have a profound impact on viral taxonomy and will allow for more precise elucidation of evolutionary relationships.
[0371] Table 1. Viral sequences identified in I. scapularis and D. variabilis by highthroughput sequencing.
Figure imgf000151_0001
Figure imgf000151_0002
Figure imgf000151_0003
[0374] Table 4. Prevalence of novel viral sequences in individual ticks
Figure imgf000152_0002
[0375] Table 5. L and N protein amino acid percent identity comparison of ISPV and ADTV to select phleboviruses.
Figure imgf000152_0001
º for the N, only the C-termltial 250 amino acid region of BTPV and ADTPV was analyzed
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[0439] Example 2: Production of Immunogenic Compositions [0440] The tick-associated viruses and immunogenic compositions described herein can be be produced in cells. Production of the tick-associated viruses and immunogenic compositions described herein may also be accomplished on any useful media and permissive cell or tissues, which may be derived from avian or mammalian cell lines derived from human, canine, feline, equine, bovine or porcine cell lines. As used herein, a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, avian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells, such as recombinant cells expressing a virus. For example, production of the tick-associated viruses and immunogenic compositions can be in any cell type, including but not limited to mammalian cells. Cell lines suitable for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limited to dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NSO, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
[0441] The cell culture system for producing the tick-associated viruses and immunogenic compositions described herein can be a traditional adherent monolayer culture. Alternatively, suspension and microcarrier cell culture systems can also be utilized.
[0442] Vessels for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limted to, roller bottles. For example, alternatively, other useful cell culture formats include flasks, stacked modules and stir tanks. For viral production, multiplicity of infection (MOI) can be 0.001-0.1 but can range from 0.0001-2.0. The harvest virus from cell culture can be, but is not limited to, any time between day 2 to 5 post-infection, but can range from day 1 to day 7 post-infection.
[0443] Cell culture media formulations suitable for producing the tick-associated viruses and immunogenic compositions described herein include, but are not limted to, Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof. These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro.TM. SFM, AIM V.RTM. media, HyQ SFM4 MegaVir.TM., EX-CELL. TM. Vera SFM, EPISERF, ProVero, any 293 or CHO media and analogues and derivatives thereof. The culture media described herein can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamine and biological buffers. Preferable medium is OptiPRO.TM. SFM supplemented with L-glutamine and trypsin. In certain embodiments, the cell culture media can be supplemented with 0.1 to 10 units of trypsin. Alternatively, plant derived equivalents of trypsin (e.g. Accutase) ranging from 2-100 units can also be used in cell culture. Cell culture media can be used in the absence or presence of animal-derived components. An example of supplementation with an animal-derived component is gamma- irradiated serum ranging from 0.5-10% final concentration.
[0444] Growth or production of the tick-associated viruses and immunogenic compositions in can also be performed in eggs. For example, tick-associated viruses propagation can be accomplished by inoculating embryonated eggs. In certain embodiments, 0-12 day old embryonated eggs can be used for tick-assocaited virus propagation. In certain embodiments, 7-8 day old embryonated eggs can be used for virus growth. The tick-assocaited virus can be inoculated into the amniotic cavity of the egg. In certain embodiments, the tick-associated viruses will replicate in the cells of the amniotic membrane and large quantities are released back into the amniotic fluid. In certain embodiments, tick-associated viruses in the amniotic fluid can be harvested after 23-24 days post inoculation.
[0445] Production of the tick-associated viruses and immunogenic compositions in can also be performed using a recombinant expression system that expresses the tick-associated virus, a tick- associated virus protein, a fragment of a tick-associated virus protein or a variant of a tick- associated virus protein. The expression system can comprise any suitable plasmid or a linear expression construct known in the art.
[0446] Example 3: Virus Preparation, Attenutation and Inactivation
[0447] The immunogenic compositions described herein can comprise an inactivated or killed tick- associated virus vaccine. Inactivated immunogenic composition can made by methods well known in the art. For example, once the tick-associated virus is propagated to high titers, the tick- associated virus antigenic mass could be obtained by methods well known in the art. For example, the tick-associated virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate tick-associated virus antigenic mass to produce immunogenic compositions. The tick-associated virus may be inactivated by treatment with formalin (e.g. 0.1-10%), betapropriolactone (BPL) (e.g. 0.01-10%), or with binary
ethyleneimine (BEI) (e.g. 1-10 mM), or using other methods known to those skilled in the art.
[0448] In addition to killed tick-associated virus production, various means of attenuation are also possible and are well known and described in the art. Attenuation leading to modified live immunogenic compositions can also be used in conjunction with the compositions and methods described herein. Methods of attenuation suitable for use with the viruses described herein include continuous passaging in cell culture, continuous passaging in animals, various methods for generating genetic modifications and ultraviolet or chemical mutagenesis.
[0449] Attenuation of tick-associated virus may be achieved through cold-adaptation of a tick- associated virus strain. Cold-adapted tick-associated virus strains may be produced by methods which includes passaging a wild-type tick-associated virus, followed by selection for tick- associated virus that grows at a reduced temperature. Cold-adapted tick-associated virus can be produced, for example, by sequentially passaging a wild-type tick-associated virus in embryonated cells or chicken eggs at progressively lower temperatures, thereby selecting for certain members of the tick-associated virus mixture which stably replicate at the reduced temperature. A cold-adapted tick-associated virus strain may exhibit a temperature sensitive phenotype. A temperature sensitive cold-adapted tick-associated virus replicates at reduced temperatures, but no longer replicates at certain higher growth temperatures at which the wild-type tick-associated virus will replicate. A temperature at which a temperature sensitive tick-associated virus will grow is referred to herein as a "permissive" temperature for that temperature sensitive tick-associated virus, and a higher temperature at which the temperature sensitive tick-associated virus will not grow, but at which a corresponding wild-type tick-associated virus will grow, is referred to herein as a "non-permissive" temperature for that temperature sensitive tick-associated virus. A cold-adapted tick-associated virus may also be produced through recombinant means. In this approach, one or more specific mutations, associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, can be identified and are introduced back into a wild-type tick- associated virus strain using a reverse genetics approach. Reverse genetics entails can be performed using RNA polymerase complexes isolated from tick-associated virus-infected cells to transcribe artificial tick-associated virus genome segments containing the mutation(s),
incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes.
[0450] Attenuation of a tick-associated virus may be achieved by serial passaging of a wild-type tick-associated virus strain in cell culture. The tick-associated virus strain can be passaged in a variety of cell systems until its ability to produce disease is lost whilst its immunogenic character is fully retained. Once inoculated into the host, the tick-associated virus may be capable of multiplication to some extent. For example, attenuated tick-associated virus compositions can be prepared from cell line that has been attenuated by serial passage including serial passage at sub- optimal temperatures to a state where it is no longer capable of causing disease, but still capable of eliciting a protective immune response. [0451] Suitable attenuated tick-associated virus strains may also be obtained by serial passaging to obtain an over-attenuated strain. The "over-attenuation" means that the number of passages for attenuation has been substantially greater than what is normally necessary for the removal of pathogenicity. The attenuated tick-associated virus retains its antigenicity after these numerous passages so that its immunogenic ability is not impaired. Such strains produce practically no symptoms or side effects when administered, and thus are safe and efficacious vaccines.
[0452] Example 4: Immunogenic Composition Dosages
[0453] Dose sizes of the immunogenic compositions described herein can be in the range of about 2.0 to 0.1 ml depending on the route of administration, but dose sizes are not limited to this range. For inactivated tick-assocaited virus compositions can contain suitable TCID50 levels of virus prior to inactivation. One of skill in the art will readily be capable of determining a suitable TCID50 level for the immunogenic compositions described herein. The antigen content in the tick- assocaited virus preparation can have, but is not limited to, a titer of between 10 to 10,000 units/ml as the amount administered per dose. One of skill in the art will readily be capable of determining a suitable antigen content for the immunogenic compositions described herein.
[0454] For immunogenic compositions containing modified live tick-assocaited virus or attenuated tick-associated virus, a therapeutically effective dose can be determined by one of skill in the art. For immunogenic compositions containing tick-associated virus subunit antigens, a therapeutically effective dose can be determined by one of skill in the art. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan.
[0455] Example 5: Administration of Immunogenic Compositions
[0456] An animal, for example a dog, can be inoculated with the immunogenic compositions or formulations described herein to generate an immune response. In certain embodiments, inoculation can be perfomed on an animal (e.g. a dog) that is at least 6 weeks or older. In certain embodiments, the animal (e.g. dog) can receive one or more dosages. In certain embodiements, two or more dosages can be administered to the animal (e.g. dog) 3-4 weeks apart. In certain embodiments, the administration can be by subcutaneous injection. Intramuscular, intradermal, oral, oronasal or nasal routes of administration can also be used to administer the immunogenic compositions or formulations described herein.

Claims

What is claimed is:
1. An isolated nucleic acid sequence having the sequence of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
2. An isolated nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
3. An isolated nucleic acid having at least about 85% sequence identity to SEQ ID NOs: 1,3,5, 7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
4. The isolated nucleic acid of claim 3, having at least about 90%>, about 95.5%>, about 96%>, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42.
5. An isolated nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs:
1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
6. An isolated nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31,33,35,37,39,41 or 42.
7. An isolated nucleic acid having at least about 85%> sequence identity to a nucleic acid
complementary to the sequence SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33,35,37,39,41 or 42.
8. The isolated nucleic acid of claim 7, having at least about 90%>, about 95.5%>, about 96%>, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35,37,39,41 or 42.
9. The isolated nucleic acid of any of claims 1-8, wherein the nucleic acid is a cDNA.
10. An isolated polypeptide encoded by the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
11. An isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide
encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29, 31,33,35,37,39,41 or 42.
12. An isolated polypeptide having at least about 85%> sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33, 35,37,39,41 or 42.
13. The isolated polypeptide of claim 12, having at least about 90%>, about 95.5%>, about 96%>, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
14. An isolated polypeptide having the sequence of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
15. An isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
16. An isolated polypeptide having at least about 85% sequence identity to the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
17. The isolated polypeptide of claim 16, having at least about 90%>, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
18. An isolated antibody that specifically binds to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
19. An immunogenic composition comprising at least about 24 consecutive nucleotides of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
20. An immunogenic composition comprising at least about 8 consecutive amino acids of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
21. A method for determining the presence or absence of a tick-associated virus in a biological sample, the method comprising:
a) contacting nucleic acid from a biological sample with at least one primer which comprises at least about 10 nucleotides of the isolated nucleic acid of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.,
b) subjecting the nucleic acid and the primer to amplification conditions, and c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with a tick- associated virus in the sample.
22. A synthetic nucleic acid comprising at least about 10 nucleotides of the isolated nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37,39,41 or 42.
23. A synthetic nucleic acid comprising at least about 10 nucleotides complementary to the isolated nucleic acid of SEQ ID NOs: 1,3,5,7, 9, 11, 13, 15, 17, 19,21,23,25,27, 29,31,33,35,37, 39,41 or 42.
24. A cDNA oligonucleotide probe comprising from about 10 nucleotides to about 50 nucleotides, wherein at least about 10 contiguous nucleotides are at least 95 % complementary to a nucleic acid target region within the nucleic acid of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
25. A primer set for determining the presence or absence of a tick-associated virus in a biological sample, wherein the primer set comprises at least one synthetic nucleic acid sequence selected from the group consisting of:
a) a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42; and
b) a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides from a nucleic acids sequence which is complementary to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 42.
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EP3530668A1 (en) * 2018-02-22 2019-08-28 Euroimmun Medizinische Labordiagnostika AG A novel assay for the diagnosis of viral infections
WO2019162454A1 (en) * 2018-02-22 2019-08-29 Euroimmun Medizinische Labordiagnostika Ag Assay for the diagnosis of viral infections
WO2020132418A1 (en) * 2018-12-20 2020-06-25 The Wistar Institute Of Anatomy And Biology Vaccines against powassan virus, and methods of using same
CN116716437A (en) * 2023-06-05 2023-09-08 昆明理工大学 Tick-derived vitex virus genome amplification primer group, amplification method and application

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EP3530668A1 (en) * 2018-02-22 2019-08-28 Euroimmun Medizinische Labordiagnostika AG A novel assay for the diagnosis of viral infections
WO2019162454A1 (en) * 2018-02-22 2019-08-29 Euroimmun Medizinische Labordiagnostika Ag Assay for the diagnosis of viral infections
US11292816B2 (en) 2018-02-22 2022-04-05 Euroimmun Medizinische Labordiagnostika Ag Assay for the diagnosis of viral infections
WO2020132418A1 (en) * 2018-12-20 2020-06-25 The Wistar Institute Of Anatomy And Biology Vaccines against powassan virus, and methods of using same
CN116716437A (en) * 2023-06-05 2023-09-08 昆明理工大学 Tick-derived vitex virus genome amplification primer group, amplification method and application

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