WO2011046634A1 - Infectious clones of torque teno virus - Google Patents

Infectious clones of torque teno virus Download PDF

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Publication number
WO2011046634A1
WO2011046634A1 PCT/US2010/031373 US2010031373W WO2011046634A1 WO 2011046634 A1 WO2011046634 A1 WO 2011046634A1 US 2010031373 W US2010031373 W US 2010031373W WO 2011046634 A1 WO2011046634 A1 WO 2011046634A1
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WIPO (PCT)
Prior art keywords
ttv
seq
ttvgtl
polynucleotide
sequence
Prior art date
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PCT/US2010/031373
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French (fr)
Inventor
Gregory Paul Nitzel
Robert Gerard Ankenbauer
Jay Gregory Calvert
Donna Steuerwald Dunyak
Jacqueline Gayle Marx
Nancee Lois Oien
Douglas Steven Pearce
Mira Ivanova Stoeva
James Richard Thompson
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Pfizer, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/US2009/005662 external-priority patent/WO2010044889A2/en
Application filed by Pfizer, Inc. filed Critical Pfizer, Inc.
Priority to JP2012534181A priority Critical patent/JP2013507918A/en
Priority to BR112012008946A priority patent/BR112012008946A2/en
Priority to CA2775277A priority patent/CA2775277A1/en
Priority to AU2010307250A priority patent/AU2010307250B2/en
Priority to US13/501,371 priority patent/US8846388B2/en
Priority to MX2012004448A priority patent/MX2012004448A/en
Priority to CN2010800566762A priority patent/CN102655871A/en
Priority to EP20100823754 priority patent/EP2488185A4/en
Publication of WO2011046634A1 publication Critical patent/WO2011046634A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10021—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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to novel nucleotide and amino acid sequences of Torque teno virus ("TTV”), including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals.
  • TTV Torque teno virus
  • Vaccines provided according to the practice of the invention are effective against multiple swine TTV genotypes and isolates.
  • Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines.
  • vaccines that comprise, as antigen, the expressed protein of single TTV open reading frames, most particularly from ORF1 or ORF2, and also fragments of the full length ORF1 and ORF2-encoded proteins.
  • TTV Torque Teno Virus
  • transfusion-transmitted virus is generally assigned to the Circoviridae family. It is generally recognized that TTV was first isolated from human transfusion patients (see for example, Nishizawa et al., Biochem. Biophys. Res. Comm. vol. 241 , 1997, pp.92-97). Subsequently, TTV or TTV-like viruses have been identified from other mammals, including swine, and numerous strains or isolates have been published (see for example, McKeown et al. Vet. Microbiol, vol. 104, 2004, pp 1 13-1 17).
  • TTV and TTV-like viruses are very common; however the pathogenesis of TTV, and the contributions it may make to other disease states (for example, those caused by other viruses and bacteria) remains unclear.
  • TTV infections appear to be common in humans, including even in healthy individuals, and such infections are often asymptomatic, and may remain for years.
  • the general inability to propagate the virus in cell culture, and a lack of any clear mechanistic disease models, have made any overall characterization of TTV biology difficult.
  • TTV viremia is elevated in human patients afflicted with other viral diseases, (such as hepatitis or HIV/AIDS), there is also considerable medical literature suggesting that TTVs are, in fact, avirulent, and await any clear actual association with known disease states. See, for example, Biagini et al., Vet. Microbiol, vol. 98, 2004, pp. 95-101 .
  • PDNS porcine circovirus disease
  • TTV infection can potentiate numerous disease states. Accordingly, there is a need for various classes of TTV reagents, such as high affinity antibodies, and for example, peptide fragments of TTV or whole virions that are highly
  • TTV is the principle causative factor of diseases in swine
  • numerous swine diseases either require the presence of more than one virus, or that the primary effect of certain "primary" pathogens is potentiated by TTV infection.
  • numerous diseases of swine can be treated or lessened by administering anti-TTV agents to affected or potentially affected animals.
  • TTV is a small, non-enveloped virus comprised of negative polarity, single- strand circularized DNA.
  • the genome includes three major open reading frames, ORF1 , ORF2 and ORF3, which overlap, and ORF1 encodes the capsid protein.
  • ORF1 encodes the capsid protein.
  • the present invention is directed to recombinant constructs whereby TTV can be propagated in vitro, including via infectious clones. More particularly, the invention is directed to the discovery that effective vaccines can in fact be made from TTV, most particularly when the TTV antigen is the expression product of a single ORF, or a fragment thereof. In a preferred embodiment, the invention provides for ORFI protein vaccines.
  • the present invention provides a method of treating or preventing a disease or disorder in an animal caused by infection with torque teno virus (TTV), including disease states that are directly caused by TTV, and disease states contributed to or potentiated by TTV.
  • TTV torque teno virus
  • the animal treated is a swine.
  • Disease states in swine that may be potentiated by TTV, and which may also be treated or prevented according to the practice of the invention, include those caused by or associated with porcine circovirus (PCV), and porcine reproductive and respiratory syndrome virus (PRRS).
  • PCV porcine circovirus
  • PRRS porcine reproductive and respiratory syndrome virus
  • the present invention also includes the option to administer a combination vaccine, that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-TTV pathogen, with appropriate choice of adjuvant.
  • a combination vaccine that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-TTV pathogen, with appropriate choice of adjuvant.
  • the present invention also provides a diagnostic kit for differentiating between porcine animals vaccinated with the above described TTV vaccines and porcine animals infected with field strains of TTV.
  • ttvg1 -7 SEQ ID NO: 4
  • ttvGT1 -17 SEQ ID NO: 5
  • ttvGT1 -21 SEQ ID NO: 6
  • ttvgtl -27 SEQ ID NO: 3
  • ttvgt1 -178 SEQ ID NO: 7 or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
  • the invention further provides RNA polynucleotide molecules that are the complement of any such DNA polynucleotide sequence, and vectors and plasmids for the expression of any such RNA or DNA polynucleotides, and for TTV virus that is expressed from such nucleotide sequences, wherein said virus is live, or fully or partially attenuated.
  • the invention also provides a DNA vaccine that comprises a
  • the invention provides a polypeptide encoded by any of the open reading frames of the genotype 2 TTV 13 (SEQ ID NO: 1 ) or genotype 2 TTV10 (SEQ ID NO: 2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • the invention also provides a polypeptide encoded by any of the open reading frames of the (all sertotype 1 ) ttvg1 -7 (SEQ ID NO:10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), ttvgt1 -27 (SEQ ID NO: 13), and ttvgtl - 178 (SEQ ID NO:9) ORF1 polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • the present invention provides for such effective vaccines, which preferably comprise a polypeptide resultant from expression of a single TTV open reading frame, or a mixture thereof.
  • the polypeptide is expressed from ORF1
  • preferred mixtures include a combination of the polypeptides of ORF1 and ORF2, and ORF1 and ORF3.
  • polypeptide vaccines wherein the antigen is defined by (a) the first 100 N- terminal amino acids of the capsid protein of TTV13 (SEQ NO:1 ) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto; or (c) an arginine rich region thereof.
  • Figure 2 evidences successful expression of codon-optimized TTVgl ORF1 protein in E. coli, with a 6X His tag for affinity purification
  • Figure 3 provides a vector map for the Chromos construct pcTV-TTV1 -7 ORF1 (plus yeast invertase) expression plasmid from which is expressed (following integration into an artificial chromosome in CHO cells) vaccinating ORF1 protein.
  • Figure 4 provides a phylogenetic tree for various TTV strains including a compilation of percent identities.
  • Figure 5 panels A, B and C provides identification of in-common arginine rich regions of ORF1 proteins as expressed from various TTV isolates.
  • Figure 6 provides a vector map for TTVgl -178 as assembled.
  • Figure 7 demonstrates that Chromos-expressed gITTV ORF1 significantly reduced lung lesions compared to the challenge controls and reduces the magitude and duration of gITTV viremia, again compared to the challenge controls.
  • Figure 8 provides a vector map for the pCR2.1 +TTVg1 -178 construct that contains a ttvg1 -178 strain full length infectious clone.
  • SEQ ID NO:1 provides the genotype gt2 TTV 10 DNA sequence.
  • SEQ ID NO:2 provides the genotype 2 gt2 TTV 13 DNA sequence.
  • SEQ ID NO:3 provides the genotype 1 ttvgt1 -27 DNA sequence.
  • SEQ ID NO:4 provides the genotype 1 ttvgtl -7 DNA sequence.
  • SEQ ID NO:5 provides the genrotype 1 ttvgtl -17 DNA sequence.
  • SEQ ID NO:6 provides the genotype 1 ttvgtl -21 DNA sequence.
  • SEQ ID NO:7 provides the genotype 1 ttvg1 -178 DNA sequence
  • SEQ ID NO:8 provides the amino acid sequence of TTV strain AY823991 ORF1 .
  • SEQ ID NO:9 provides the amino acid sequence of TTV strain ttvgtl -178 ORF1 (TTV genotype 1 ).
  • SEQ ID NO: 10 provides the amino acid sequence of TTV strain ttvgtl -7 ORF1 .
  • SEQ ID NO: 1 1 provides the amino acid sequence of TTV strain ttvgtl -17 ORF1 .
  • SEQ ID NO: 12 provides the amino acid sequence of TTV strain ttvgtl -21 ORF1 .
  • SEQ ID NO: 13 provides the amino acid sequence of TTV strain ttvgtl -27 ORF1 .
  • SEQ ID NO: 14 provides the amino acid sequence of TTV strain gt2 TTV10 ORF1 (genotype 2).
  • SEQ ID NO: 15 provides the amino acid sequence of TTV strain gt2 TTV13 ORF1
  • SEQ ID NO: 16 provides the DNA sequence of known strain AY823991
  • SEQ ID NO: 17 provides the DNA sequence of known strain AY823990
  • SEQ ID NO:18 provides the 76057-3 TTV capsid encoding sequence, codon optimized for E. coli. as cloned into the pUC57 GenScript® vector.
  • SEQ ID NO:19 provides the 76057-4 TTV capsid encoding sequence, codon optimized for E. coli. as cloned into the Invitrogen pET101/D-TOPO® expression plasmid.
  • SEQ ID NO:20 provides the 76057-5 TTV capsid encoding sequence, codon optimized for Saccharomyces cerevisiae as cloned into the pUC57 GenScript® vector.
  • SEQ ID NO:21 provides the DNA sequence for a construct that encodes ttvgtl -7 ORF1 with a yeast invertase expression tag (Yl).
  • SEQ ID NO:22 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
  • SEQ ID NO:23 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
  • SEQ ID NO:24 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
  • SEQ ID NO:25 provides the amino acid sequence of TTV strain AY823990 ORF1 .
  • SEQ ID NOS:26-29 define primer sequences.
  • those familiar with the art will recognize that numerous slightly different abbreviations are commonly used interchangeably for specific serotypes, for example, gITTV, TTVgl , genotype 1 TTV, serotype 1 TTV, gtl TTV, and the like. A similar situation exists for genotype 2.
  • porcine and “swine” are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig.
  • “Mammals” include any warm-blooded vertebrates of the Mammalia class, including humans.
  • infectious DNA molecule is a DNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell.
  • isolated polynucleotide molecule refers to a composition of matter comprising a polynucleotide molecule of the present invention purified to any detectable degree from its naturally occurring state, if any.
  • the nucleotide sequence of a second polynucleotide molecule is "homologous" to the nucleotide sequence of a first polynucleotide molecule , or has "identity" to said first polynucleotide molecule, where the nucleotide sequence of the second polynucleotide molecule encodes the same polyaminoacid as the nucleotide sequence of the first polynucleotide molecule as based on the degeneracy of the genetic code, or when it encodes a polyaminoacid that is sufficiently similar to the polyaminoacid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in practicing the present invention.
  • Homologous polynucleotide sequences also refers to sense and anti-sense strands, and in all cases to the complement of any such strands.
  • a polynucleotide molecule is useful in practicing the present invention, and is therefore homologous or has identity, where it can be used as a diagnostic probe to detect the presence of TTV virus or viral polynucleotide in a fluid or tissue sample of an infected pig, e.g. by standard hybridization or amplification techniques.
  • nucleotide sequence of a second polynucleotide molecule is homologous to the nucleotide sequence of a first polynucleotide molecule if it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the
  • BLASTN algorithm National Center for Biotechnology Information, otherwise known as NCBI, (Bethesda, Maryland, USA) of the United States National Institute of Health).
  • NCBI National Center for Biotechnology Information
  • BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences- a new tool for comparing protein and nucleotide sequences.” (1999) FEMS Microbiol Lett. 174:247-250.]. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1 , and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 89:10915-10919. 1992). The percent identity is then calculated as: Total number of identical matches x 100/ divided by the length of the longer sequence+number of gaps introduced into the longer sequence to align the two sequences.
  • a homologous nucleotide sequence has at least about 75% nucleotide sequence identity, even more preferably at least about 80%, 85%, 90% and 95% nucleotide sequence identity. Since the genetic code is
  • a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.
  • a homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
  • non-silent mutations i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
  • certain conservative amino acid substitutions may be made which are generally recognized not to inactivate overall protein function: such as in regard of positively charged amino acids (and vice versa), lysine, arginine and histidine; in regard of negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and in regard of certain groups of neutrally charged amino acids (and in all cases, also vice versa), (1 ) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and
  • homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above.
  • homologous nucleotide sequences can be determined by hybridization under selected conditions.
  • the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO: 1 (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO: 1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2xSSC/0.1 % SDS at 42°C (see Ausubel et al editors, Protocols in Molecular Biology, Wiley and Sons, 1994, pp.
  • hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe.
  • the hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51 .
  • a second nucleotide sequence is homologous to SEQ ID NO: 1 (or any other sequence of the invention) if it hybridizes to the complement of SEQ ID NO: 1 under highly stringent conditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 xSSC/0.1 % SDS at 68°C, as is known in the art.
  • isolated polynucleotide molecules and the isolated RNA molecules of the present invention include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.
  • ttvg1 -7 SEQ ID NO: 4
  • ttvGT1 -17 SEQ ID NO: 5
  • ttvGT1 -21 SEQ ID NO: 6
  • ttvgtl -27 SEQ ID NO: 3
  • ttvgt1 -178 SEQ ID NO: 7 or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
  • the invention also provides a polypeptide encoded by any of the open reading frames of the genotype 2 TTV13 (SEQ ID NO: 1 ) or genotype 2 TTV10 (SEQ ID NO:2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • the invention also provides a polypeptide encoded by any of the open reading frames of the (all sertotype 1 ) ttvg 1 -7 (SEQ ID NO: 10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), ttvgt1 -27 (SEQ ID NO: 13), and ttvgtl - 178 (SEQ ID NO:9) ORF1 polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • polypeptide is expressed from ORF1 , and preferred mixtures include a combination of the polypeptides of ORF1 and ORF2, and ORF1 and ORF3.
  • TTV polypeptide-based vaccines wherein the antigen is defined by: (a) the first 300 N-terminal amino acids of the ORF1 capsid protein of TTV13 (SEQ NO:1) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto;
  • the DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of the viral genes and their encoded gene products.
  • Knowledge of a polynucleotide encoding a viral gene product of the invention also makes available anti-sense polynucleotides which recognize and hybridize to
  • fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to a specific RNA (as determined by sequence comparison of DNA encoding a viral polypeptide of the invention as well as (ii) those which recognize and hybridize to RNA encoding variants of the encoded proteins.
  • Antisense polynucleotides that hybridize to RNA DNA encoding other TTV peptides are also identifiable through sequence comparison to identify characteristic, or signature sequences for the family of molecules. Such techniques (see Example 8) are further of use in the study of antigenic domains in TTV polypeptides, and may also be used to distinguish between infection of a host animal with remotely related non-TTV members of the Circoviridae.
  • Example 4 provides guidance as to effective codon optimization for enhanced expression in yeast and E. coli for the constructs of the invention.
  • Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release.
  • Diluents include water, saline, dextrose, ethanol, glycerol, and the like.
  • Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
  • Stabilizers include albumin, among others.
  • Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
  • Vaccines of the present invention may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others.
  • adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, MT), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta GA), QS-21 (Cambridge Biotech Inc., Cambridge MA), SAF-M (Chiron, Emeryville CA), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant.
  • Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1 % (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 ⁇ g/ml Quil A, 100 ⁇ g/ml cholesterol, and 0.5% (v/v) lecithin.
  • Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1 % (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 ⁇ g/ml Quil A, and 50 ⁇ g/ml cholesterol.
  • Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
  • Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, with may be additionally lipidated, such as those described in
  • ORFI TTV protein is formulated with 5% AMPHIGEN®.
  • Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention.
  • sustained release formulations include virus, infectious DNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like.
  • biocompatible polymers such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like.
  • the virus, plasmid, or viral vector can be microencapsulated to improve
  • Liposomes can also be used to provide for the sustained release of virus, plasmid, viral protein, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Patent
  • An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, viral protein plasmid or viral vector, and then increasing the dosage while monitoring the effects.
  • An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine.
  • Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like.
  • the actual dosage is preferably chosen after consideration of the results from other animal studies (see, for example, Examples 2 and 3 below).
  • One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination.
  • the timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above.
  • the effective dose amount of virus, protein, infectious DNA molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated.
  • the dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 10 1 to about 10 9 pfu (plaque forming units), more preferably from about 10 2 to about 10 8 pfu, and most preferably from about 10 3 to about 10 7 pfu.
  • the dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about O. ⁇ g to about 100mg, more preferably from about ⁇ g to about 10mg, even more preferably from about 1 ( ⁇ g to about 1 mg.
  • the dose amount of an infectious DNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 ⁇ g to about 100mg, more preferably from about ⁇ g to about 10mg, even more preferably from about 10 ⁇ g to about 1 mg.
  • the dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 10 1 pfu to about 10 9 pfu, more preferably from about 10 2 pfu to about 10 8 pfu, and even more preferably from about 10 3 to about 10 7 pfu.
  • a suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
  • Suitable doses for viral protein or peptide vaccines range generally from 1 to 50 micrograms per dose, or higher amounts as may be determined by standard methods, with the amount of adjuvant to be determined by recognized methods in regard of each such substance.
  • an optimum age target for the animals is between about 1 and 21 days, which at pre-weening, may also correspond with other scheduled vaccinations such as against Mycoplasma hyopneumoniae.
  • a preferred schedule of vaccination for breeding sows would include similar doses, with an annual revaccination schedule.
  • anti-TTV antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, porcine, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a TTV polypeptide of the invention.
  • the term "specific for” indicates that the variable regions of the antibodies of the invention recognize and bind a TTV polypeptide exclusively (i.e., are able to distinguish a single TTV polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) to interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA
  • antibody refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen.
  • Immunoglobulins are serum proteins composed of "light” and “heavy” polypeptide chains having "constant” and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
  • Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab')2, as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences. Diagnostic Kits
  • the present invention also provides diagnostic kits.
  • the kit can be valuable for differentiating between porcine animals naturally infected with a field strain of a TTV virus and porcine animals vaccinated with any of the TTV vaccines described herein.
  • the kits can also be of value because animals potentially infected with field strains of TTV virus can be detected prior to the existence of clinical symptoms and removed from the herd, or kept in isolation away from naive or vaccinated animals.
  • the kits include reagents for analyzing a sample from a porcine animal for the presence of antibodies to a particular component of a specified TTV virus.
  • Diagnostic kits of the present invention can include as a component a peptide or peptides from ORF1 , 2, or 3 which is present in a field strain but not in a vaccine of interest, or vice versa, and selection of such suitable peptide domains is made possible by the extensive amino acid sequencing as provided for in Examples 1 and 2 of the Specification.
  • kits of the present invention can alternatively include as a component a peptide which is provided via a fusion protein.
  • the term "fusion peptide" or "fusion protein” for purposes of the present invention means a single polypeptide chain consisting of at least a portion of a TTV virus protein, preferably of ORF1 , and a heterologous peptide or protein.
  • DNA was purified from porcine serum using a DNA blood mini kit (Qiagen) per manufacturer's protocol. DNA was eluted from the columns in 50 ⁇ _ Tris- EDTA buffer. DNA was then amplified via random primed rolling circle
  • Plasmid DNA was isolated from transformed colonies and digested with EcoRI to confirm presence of an approximately 2.7 kB insert. Four clones (4, 7, 10 and 13) were selected and submitted to ACGT, Inc. for sequencing.
  • TTV13 SEQ ID NO:2
  • TTV10 SEQ ID NO: 1
  • TTV genotype 2 AY823991 DNA sequence
  • TTV13 (137) TCTGCAAAAAAGAGGAAATAAATTTCATTGGCTGGTCCATAAGTCCTCAT
  • TTV10 (148) TCTGCAAAAAAGAGGAAGTAAATGCTATTGGCTAAATCTGAAGTCTTCAT AY823991 (187) TAGAATAATAAAAGAACCAATCAGAAGAACTTCCTCTTTTAGAGTATATA
  • TTV10 (348) CGGAGTCAAGGGGCCTATCGGGCGGGCGGTAATCCAGCGGAACCGGGCCC
  • TTV10 (398) CCC-TCCATGGAGGAGAGATGGCTGACGGTAGCGTACGCCGCCCACGGAT
  • TTV10 (447) TATTCTGCGCCTGCAGTAAGCCCAAAGACCACCTTGAAAAATGCCTTTCC
  • TTV13 (537) AGGTGGAGACGCTACTTTCGATATCGGTATCGACGCGCTCCTCGCCGCCG
  • TTV10 (547) AGGTTCCAGCGCTACTTTCGATATCGGTATAGACGCGCTCCTCGCCGCCG
  • TTV13 (587) CCG CACAAAGGTAAGGAGACGGAGG AGGAAAGCTCCGGTCATAC
  • TTV10 (597) CCGACGCTACAAGGTAAGGAGACGGAGGGTTAAAAAGGCTCCGGTCATTC
  • TTV13 (631) AATGGAACCCTCCTAGCCGGAGGACCTGCCTCATAGAGGGGTTCTGGCCG
  • TTV10 (647) AATGGTTCCCCCCAACAGTCAGAAACTGTTTTATCAAGGGAATCTGGCCG
  • TTV13 (881 TAGACATCCTTGGAGAAACTATATAGTGACTTGGGATCAGGACATTCCTT
  • TTV10 (897 TAGACACACAACCAGATCCTACGTAGTAACATGGGACCAAGACATACCAT
  • TTV13 (931 GTAAACCTTTACCATATCAGAACTTACACCCATTATTAATGCTATTAAAA
  • TTV10 (947 GTAAACCTTTACCATACACAAATTTACATCCATTTGTAATGCTTCTAAAA
  • TTV13 (981 AAACAACACAAATTAGTACTCTCACAACAAAACTGTAACCCTAACAGAAA
  • TTV10 (997 AAACATCATAAAGTAGTTCTAAGCAAACAAGACTGTAATCCTAGAAAAAT
  • TTV13 (1031 ACAAAAACCTGTAACTTTAAAATTCAGACCGCCACCAAAACTAACTTCAC
  • TTV10 (1047 GGACAAACCAGTCACCTTAAAAATAAAGCCACCACCAAAACTCACATCAC
  • TTV13 (1081 AATGGAGACTAAGTAGAGAATTAGCAAAAATGCCACTCATTAGACTAGGA
  • TTV10 (1097 AGTGGAGACTAAGCAGAGAATTATCAAAAATACCGCTCTTAAGACTAGGA
  • TTV13 (1131 GTTAGTTTTATAGACTTAACAGAACCGTGGCTAGAAGGTTGGGGAAATGC
  • TTV10 (1147 GTTTCTTTAATAGACTTCAGAGAACCATGGGTTGAAGGTTTTGGAAATGC
  • TTV10 (1197 ATTCTTTAGTACTTTAGGATATGAAGCAGATAAAAGCAATTTAAAAACAA
  • TTV13 (1231 CAAATTGGTCACAAATTAAATATTACTGGATATATGATACAGGAGTAGGA
  • TTV10 (1247 GCGCTTGGTGCCAATGTAAATACTTCTGGATATATGATACCGGAGTAAAT
  • TTV10 1423 TTTGGAAGATCTGAAAGAGACTTAAAAGCACTAGCAACTTCAAACACAAA
  • TTV10 (1523 CTGTAATAGGATGGGCTAGCAGTAACAACACAGCACAAGATAGTACACAA AY823991 (1577) AACAGTGGTGGATCAACATCAGCTATACAAGGTGGATATGTAG CA
  • TTV13 (1623) TGGGC-AGGAGGACAAGGAAAACTAAATCTAGGAGCAGGATCAATAGGAA
  • TTV10 (1811) TTGCCCTCTTCGGACCCTTGGTAGAAAAAGCAAA-CTGGGAAGGCCTAGA
  • TTV10 (2057) TCCTCAATGCGTGGGATTATGACTATGATGGAATTGTTAGAAAAGACACT
  • TTV10 (2154) GTACCCGCTCGCTGGACCCAAAACAGAGAAATTGCCCTCCTCAGACGAAG
  • TTV13 (2221) ACGAAGAGAGCGTTATCTCAAGCACGAGCAGTGGATCCGATCAAGAA
  • TTV10 (2304 CCTCCAGCATGTCCAGCGACTGGTGAAGAGATTCAGGACCCT ATAGA
  • TTV13 (2365 TAAATACAGAAACCTAGCAGACCCCTCACTCAATGTCACAGGACACATGG
  • TTTV10 (2351 CAAATACAGAAACTTAGCAGACCCCTCATTAAATGTCACAGGACATTTTG
  • TTV10 (2401 AACACTTCTGCCGCTTACACTATAAAAACATAGCAGAAATCAGAGCTAGA
  • TTV10 2451 AATGCCAAAAAAAACCTCAATAAACTATACTTTTCAGACTAAAAGAAG--
  • TTV13 2515 TCCTGTGTCCAATCTATTTTTTTAAACACCCTTCAAAATGGCGGGAGGGA
  • TTV13 2565 CACAAAATGGCGGAGGGACTAAGGG TG N
  • TTV10 2533 TAGCGGGGGGGACC CCCCTGCACCCCCCCATGCGG
  • TTV13 (2638 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
  • TTV10 (2570 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
  • TTV10 (2620 CCCGCGGGGGGGCAAGCCCCCCTGCACCCCCCC
  • TTV 13 shows 92% identity when compared with previously published AY823991 sequence. However, TTV10 only show 76% similarity between either AY823991 or TTV13 and may be considered a separate genotype.
  • TTVlOOrfl (1 MPFHRYRRRRRRPTRRWRRRRFQRYFRYRYRRAPRRRRRYKVRRRRVKKA
  • TTV130RF1 (1 MPYRRYRRRRRRPTRRWRHRRWRRYFRYRYRRAPRRRR-TKVRRR-RRKA
  • TTVlOOrfl 151 DIPCKPLPYTNLHPFVMLLKKHHKVVLSKQDCNPRKMDKPVTLKIKPPPK
  • TTV130RF1 149 DIPCKPLPYQNLHPLLMLLKKQHKLVLSQQNCNPNRKQKPVTLKFRPPPK
  • TTV130RF1 (199 LTSQWRLSRELAKMPLIRLGVSFIDLTEPWLEGWGNAFYSVLGYEAIKEQ
  • TTVlOOrfl (251 LKTSAWCQCKYFWIYDTGV NHVYWMLNKDAGDNAGDLITNQNS
  • TTV130RF1 (249 GHWS WSQIKYYWIYDTGVGNAVYWMLKQDVDDNPGKMASTFKTTQGQH
  • TTVlOOrfl (296 IAHIEQIGEGYPYWLYFFGRSERDLKALATSNTNIRNEFNTNPNSKK
  • TTV130RF1 (299 PNAIDHIELINEGWPYWLYFFGKSEQDIKKEAHS-AEIAREYATNPKSKK
  • TTVlOOrfl (343 LKIAVIGWASS NTAQDSTQGANTPIEGTYLISHVLQTSGH TAGAAQ
  • TTV130RF1 (348 LKIGIVGWASSNFTTPGSSQNSGG-NIAAIQGGYVAWAGGQGKLNLGAGS
  • TTV130RF1 (397 IGNLYQQGWPSNQ WPNTNRDETNFDWGLRSLCILRD MQLGNQELDDEC
  • TTVlOOrfl (440 TMFALFGPLVEKAN-WEGLEKIPELKPELKDYNILMRYNFRFQWGGHGTE
  • TTV130RF1 (447 TMLSLFGPFVEKANPIFATTDPKYFKPELKDYNLIMKYAFKFQWGGHGTE
  • TTVlOOrfl (489 TFKTSIGDPSQIPCPYGPGEAPQHLVRNPSKVHEGVLNAWDYDYDGIVRK
  • TTV130RF1 (497 RFKTTIGDPSTIPCPFEPGDRFHSGIQDPSKVQNTVLNPWDYDCDGIVRK
  • TTV130RF1 (547 DTLKRLLELPTETEEEEKAYPLLGQKTEKEPLSDSDEESVISSTSSGSDQ
  • TTV130RF1 (597 EEETQR--RKHHKPSKRRLLKHLQRWKRMKTL-- Amino Acid Alignment of TTV10 TTV13 ORF with Published Sequence
  • TTV10 ORF demonstrates only 65% homology to the published sequence and may represent a unique phenotype of TTV
  • primers were designed to clone the ORF from TTV10 and TTV13 for expression in baculovirus using the
  • TTV13Rev121 1 5' cgt act cga gtc aca gtg ttt tea tec (SEQ ID NO:26); TTV13For121 1 : 5' eta ggt acc atg cct tac aga cgc tat (SEQ ID NO:27)
  • the TTV insert in pGem was isolated by EcoRI digestion, gel- purified and re-circularized using standard ligation conditions. Following an overnight ligation at 4°C, ligase was inactivated at 65°C, and the reaction was purified using QuiQuick purification kit (Qiagen) following the manufacturer's protocol.
  • TTVORF13 was the PCR amplified using re-circularized TTV13 genomic DNA with Expand Hi-Fidelity® enzyme (Roche) using the above described TTV13 forward and reverse primers (0.15 ⁇ each), 0.2 mM dNTP's in 1 X Hi Fidelity enzyme buffer. PCR conditions were: 1 cycle at 4 min, 95°C; 35 cycles with 94°C, 15 sec denaturation, 55°C, 30 sec anneal, and 68°C 1 .5 min extension; and 1 cycle of 72°C, 7 min extension.
  • TTVORF10 was PCR amplified using re-circularized TTV10 genomic DNA with Expand Hi-Fidelity® enzyme (Roche) using the above described TTV10 forward and reverse primers (0.15 ⁇ each) 0.2 mM dNTP's in 1 X Hi Fidelity enzyme buffer. PCR conditions were: 1 cycle at 4 min, 95®°C; 35 cycles with 94°C, 15 sec denaturation, 56°C, 30 sec anneal, and 68°C 1 .5 min extension; and 1 cycle of 72°C, 7 min extension.
  • PCR products were purified using QiaQuick PCR purification kit (Qiagen) following the manufacturer's protocol. Both PCR TTV10Orf1 anfd TTV130rf1 products and the Gateway entry plasmid, pENTR3C, were digested with Kpnl. Digested DNA was purified using QIAquick PCR amplification kit and
  • TTV10 ORF1 or the TTV130RF1 DNAs were ligated into pENTR3C using standard ligation procedures. Following a 2 hour ligation at room temperature, ligated DNA was used to transform chemically competent E. coli DH5a. Transformed colonies were selected using Kanamycin. Plasmid was purified from transformed E.coli and ORF1 DNA insertion was verified by restriction fragment analysis.
  • pENTR3C plasmids containing TTV10 ORF1 or TTV13 ORF1 were then inserted into Invitrogen destination vectors pDESTI O or pDEST 20 encoding a His6X or a GST protein N-terminal to the TTV Orf1 reading frame.
  • Recombinant pDEST vectors containing the open reading frame of TTV Orf1 were used to transform DHI OBac E. coli.
  • Recombinant bacmid DNA was isolated and used for transfection of SF9 cells following standard protocol.
  • Recombinant baculovirus containing the native Orf1 were isolated by plaque purification.
  • Standard PCR was used to incorporate a BamH1 restriction site upstream from the initiation codon in TTV10 Orf1 or an Xbal restriction site upstream from the initiation codon in TTV Orf13. These constructs were cloned into pFastBac transfer vector and used to transform E. coli DHI OBac. The resultant
  • Recombinant baculovirus containing the native Orf1 were isolated by plaque purification. Confirmation of recombinant baculovirus was performed using PCR.
  • TTVOrfl 0 Full-length TTVOrfl 0 was also cloned into a PGex-6p-1 vector for expression of a GST-fusion protein in a bacterial system.
  • the TTV ORFs contain an arginine rich amino terminus.
  • the arginine rich segment was removed from TTVOrfl 3 at a convenient restriction site (EcoR1 ) located at nucleotide 368 of the Orf1 open reading frame and was in frame with the GST coding region of pGex-6p-1 . This clone resulted in the removal of 100 amino- terminal amino acids containing a highly enriched arginine segment.
  • Total cellular DNA from porcine bone marrow was amplified by rolling circle amplification following procedures described above, except that single- stranded binding protein was added to improve the efficiency of the amplification reaction.
  • Amplification products were digested with EcoR1 , purified using a
  • AY823990 (844 GAAGACCAGTACGGATACCTCGTACAATACGGGGGAGGTTGGGGAAGTGG ttvgl-7 ... (850 GAGGACAACTATGGATACTTAGTACAGTATGGAGGTGGTTGGGGTAGCGG ttvGTl-17... (850 GAAGACAACTACGGCTACTTAGTACAGTACGGAGGAGGTTGGGGGAGCGG ttvGTl-21... (850 GAGGACAACTATGGATACTTAGTACAGTATGGAGGTGGTTGGGGTAGCGG ttvgtl-27... (850 GAGGATCAATACGGATACCTGGTGCAATACGGTGGAGGTTGGGGAAGTGG
  • AY823990 (2491 TTACTAATGGGGGGGGGTCCGGGGGGGGCTTGCCCCCCCGCAAGCTGGGT ttvgl-7 ... (2499 TATTATTTTGGGGGG--TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGGGT ttvGTl-17... (2500 TTATTTGTAGGGGGGG-TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGGGT ttvGTl-21... (2499 TATTACTTTGGGGGGG-TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGTGT ttvgtl-27... (2497 TTATTTTTTGGGGGG--TCCGGGGGGGGCTTGCCCCCCCCCGAAAGCTGGGT
  • TTVgt1 -27 demonstrates the greatest homology with published sequence, AY823990, demonstrating 91 % identity.
  • TTVgt1 -7,17, and 21 demonstrate 85- 87% identity.
  • TTVgt1-7 and TTVgt1 -21 share 99% nucleotide identity Orf1 Amino Acid Alignment
  • Ttgl-170rfl MAPARRWRRGFGRRRRRYRKRRWGWRRRYWRYRPRYRRRRWWRRRRRSV
  • Ttgl-270rfl (1) MAPTRRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV ttgl-210rfl (1) MAFARRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV
  • Ttvgl-70rfl (51) YRRGGRRARPYRISAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD
  • Ttgl-170rfl (51) YRRGGRRARPYRISAFNPKIMRRWIRGWWPILQCLRGQESLRYRPLQWD
  • Ttgl-270rfl (51) YRRGGRRARPYRVSAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD ttgl-210rfl (51) YRRGGRRARPYRISAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD
  • Ttgl-170rfl (101) VEKSWRIKTDLEDNYGYLVQYGGGWGSGEVTLEGLYQEHLLWRNSWSKGN
  • Ttgl-170rfl (151) DGMDLVRYFGCIVYLYPLKDQDYWFWWDTDFKELYAESIKEYSQPSVMMM
  • Ttvgl-70rfl (201) AKRTKIVIARSRAPHRRKVRRIFIPPPSRDTTQWQFQTDFCNRPLFTWAA
  • Ttgl-170rfl (201) AKKTKIVIARSRAPHRRKVRKIFIPPPSRDTTQWQFQTEFCNKPLFTWAA
  • Ttgl-270rfl (201) AKRTRIVIARDRAPHRRKVRKIFIPPPSRDTTQWQFQTDFCNRKLFTWAA ttgl-210rfl (201) AKRTKIVIARSRAPHRRKVRRIFIPPPSRDTTQWQFQTDFCNRPLFTWAA
  • Ttgl-170rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPAQ tgl-270rfl (251) GLIDMQKPFDANGAFRNAWWLEQRTEQGEMKYIELWGRVPPQGDSELPKK ttgl-210rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPLQ
  • Ttvgl-70rfl (301) TEFQKPSGYNPKYYVNPGEEKPIYPVIIYVDMKDQKPRKKYCVCYNKTLN
  • Ttgl-170rfl (301) KEFQKPDGYNPKYYVQAGEEKPIYPIIIYVDKKDQKARKKYCVCYNKTLN
  • Ttgl-270rfl (301) SEFTTAT-DNKNYNVNDGEEKPIYPIIIYVDQKDQKPRKKYCVCYNKTLN ttgl-210rfl (301) TEFQKPSGYNPKYYVNPGEEKPIYPVIIYVDMKDQKPRKKYCVCYNKTLN
  • Ttgl-170rfl (351) RWRAAQASTLKIGDLQGLVLRQLMNQEMTYIWKEGEFTNVFLQRWKGFRL
  • Ttgl-170rfl (551) AASSRALSADTPTEAAQSALLRGDSEKKGEETEETTSSSSITSAESSTEG
  • Ttgl-270rfl (550) PASTRALCADTPTEATQSALLRGDSEKKGEETEETTSSSSITSAESSTEG ttgl-210rfl (551) AASSRALSADTPTEATQSALLRGDSEKKGEETEETSSSSSITSAESSTEG
  • Hydrophobicity plots of the proteins demonstrate 5 areas of hydrophilicity, which may indicate surface-exposed regions that are potentially antigenic. Two of these regions are at the amino terminus and at the carboxy terminus, and are both arginine-rich and highly conserved. A highly conserved hydrophilic region between amino acids 190 and 232 was observed and may potentially serve as antigenic site. The remaining hydrophilic regions between amino acids 295 and 316, and between amino acids 415 and 470 are also be antigenic.
  • the putative start codons for ORF1 and coding region are as follows: ttvgtl -27 nt 517-2435; ttvg1 -7 nt 517- 2435; ttvgtl -17 nt 517—2436; ttvgtl -21 nt 517-2439; ttv10 nt 487-2346; and ttv13 nt 477-2363.
  • the putative start codons for ORF 2 and coding region are as follows: ttvgtl -27 nt 428-646; ttvg1 -7 nt 428-643; ttvgtl -17 nt 428-643; ttvgtl - 21 nt 428-646; ttv10 nt 404-610; and ttv13 nt 394-597.
  • the processes were monitored for cell density and viability, and infection was monitored through monitoring of cell size and virus titration. Protein expression was monitored through SDS-PAGE, Coomassie gel analysis and Western blotting. To ensure proper control, negative and positive controls were maintained throughout all experiments. Although all experiments were able to confirm expression of the target protein, optimal conditions were found when utilizing SF9 cells maintained in ExCell 420 media (Sigma, SAFC) with a cell density of 2x10 6 cells/ml and an MOI of 0.1 , with the process terminated following a three day infection. The majority of the recombinant expressed protein can be located within the cell pellet although some resides in the resultant supernatant.
  • Figure 1A sample lanes were as follows (from right to left)
  • Lane 5 (see the arrows in Figure 1 B) demonstrates a unique reaction to a ⁇ 69kD and 49kD protein in the native TTV ORF1 expression utilizing anti-TTV ORF1 rabbit polyclonal antibody.
  • antigen provided only TTV sequence and was not tagged.
  • Figure 6B sample lanes were as follows (from right to left)
  • a liver was collected aseptically from a caesarean-derived, colostrum deprived (CDCD) pig.
  • the liver tissue was tested for g1 and g2 TTV in unique qPCR assays and confirmed to be positive for only gITTV.
  • a 10% (wt/vol) liver homogenate was then prepared in media containing antibiotics and antimycotics. Finally, the homogenate was clarified by centrifugation, designated as gITTVpO and frozen at -70C. The resulting gITTV homogenate was tested to be free of extraneous viruses, bacteria and mycoplasma via routine testing.
  • the present study was conducted to evaluate the efficacy of three TTV vaccine candidates administered at ⁇ 7 days of age, and again at weaning (-21 days of age) followed by a challenge at ⁇ 5 weeks of age.
  • TTV is a small, non-enveloped virus with a single-stranded circular DNA genome of negative polarity.
  • the genome includes an untranslated region and at least three major overlapping open reading frames.
  • Porcine TTV is ubiquitous and PCR-detection of the virus in serum samples collected from various geographical regions shows prevalence in pigs ranging from 33 to 100%.
  • Krakowka et al., AJVR (2008) 69: 1623-1629 reported that g1 -TTV inoculated pigs had no clinical signs but developed interstitial pneumonia, transient thymic atrophy, membranous
  • formulations can be numerically or statistically differentiated when compared to challenge control groups.
  • seronegative females without a history of disease caused by PRRSV or M hyo were sourced from Lincoln Trail / Puregenic Pork, Alton, IL, and transported to the Pfizer Animal Health Research Farm in Richland, Ml at approximately 3 weeks pre-partum. If necessary, sows were induced to farrow within a 2 or 3 day period using injectable prostaglandin (Lutalyse®). Normal piglets from these sows were allotted to study according to the allotment design. Pigs were randomized to treatment by litter and each litter had at least one piglet assigned to each treatment. Housing: During the vaccination phase, pigs were housed with their mother with no cross-fostering, in BL-2 isolation facilities.
  • Pigs remained housed by litter until the time of 2 nd vaccination.
  • Post-second vaccination pigs were moved to a further facility and housed in two rooms (one room contains NTX (non-vaccinated and non-challenge controls) animals, the second room vaccinates), and each room contains 4 or 8 pigs per pen.
  • NTX non-vaccinated and non-challenge controls
  • Vaccine was masked using a numeric code prior to vaccination. The investigator, vaccine administrator and study personnel were masked to treatment and did not have access to the masking code unless treatment information was required for the welfare of an animal. Investigational Veterinary Products
  • gITTV pass 1 was derived from liver homogenate tested positive (7.6x10e8 to 1.6x10e9 DNA copies/2ml_) for gITTV and negative for g2TTV by qPCR. An appropriate number of bottles were removed from the freezer and thawed shortly before challenge. An aliquot was then removed from one of the bottles, and held for retitration at a later time.
  • Challenge stock was transported on ice to the research facility and maintained on ice during the challenge procedure.
  • a challenge dose equals 2.0ml_ of stock solution (2.0 mL intraperitoneal). The dose was delivered to each pig is therefore expected to be 7.6x10e8 to 1 .6x10e9 DNA copies/2mL. Following challenge, an aliquot of challenge stock was kept for titration to confirm challenge dose.
  • Body Weights All pigs were weighed Day 0, the day of challenge (Day 28) and at necropsy. All weights were recorded.
  • Vaccination At approximately 7 days of age (Day 0), -10 randomly allotted pigs per treatment group (Groups T01 thru T04) were vaccinated as described in Table 1 . Pigs were injected in the right neck with a single dose syringe (2.0 mL intramuscular (IM) dose) of IVP, or a 2 mL IM dose of control according to allotment. A second dose of the same IVP or control was administered in the left neck at the time of weaning (-21 days of age).
  • IM intramuscular
  • Blood Sampling Prior to Day 0, Day 14 (prior to vaccination) and Day 28 prior to challenge (as well as Day 31 , 34, 37, and 40), a blood sample was collected, using 5 mL or 9 mL serum separator tubes (dependant on body weight), from all pigs for gITTV status (qPCR-Pfizer-VMRD Laboratory Sciences). Serum samples were aliquoted by site personnel to at least three separate tubes and were stored at -80 C. Table 2: gITTV qPCR analysis to be performed on sera by time point
  • Rectal Temperatures post challenge were recorded once per day on Day 28 prior to challenge as well as Day 31 , 34, 37, and 40.
  • Necropsies On Day 40 all animals were euthanized and necropsied. Upon necropsy, lung lesions were scored using the following methods: 1 ) a numeric score (0, 1 , 2, 3) and 2) the percentage of consolidation for each lobe (left cranial, left middle, left caudal, right cranial, right middle, right caudal, and accessory) was scored and recorded as percent of lobe observed with lesions. Liver, kidney, thymus and lymph nodes were also scored. A blood sample was also taken prior to euthanasia. Tissues were collected as indicated in the following table:
  • the percentage of total lung with lesions was transformed and analyzed with a general linear mixed model with fixed effects, treatment, and random effect litter. Linear combinations of the parameter estimates were used in a priori contrasts after testing for a significant (P ⁇ 0.10) treatment effect. The 10% level of significance (P ⁇ 0.10) was used to assess statistical differences.
  • qPCR data will be transformed prior to analysis with an appropriate log transformation.
  • the transformed titers will be analyzed using a general linear repeated measures mixed model analysis. Pairwise treatment comparisons will be made at each time point if the treatment or treatment by time point interaction effect is significant (P ⁇ 0.10).
  • Treatment least squares means, 90% confidence intervals, the minimum and maximum will be calculated and back-transformed for each time point. Descriptive statistics, means, standard deviations, and ranges, will be calculated for each treatment and day of study, pre-challenge. Study Results and Discussion
  • T01 Chromos expressed gI TTV ORF1
  • T02 Baculovirus expressed g2TTV ORF1
  • T04 Challenge controls
  • the challenge virus was comprised of infectious gITTV, it may not be surprising that the genotype 2 ORF1 from Baculovirus did not provide very substantially lower lung lesions as compared to the challenge controls. It is however interesting to note that while not substantial, it did offer numerically lower lung lesion scores compared to the challenge controls, thereby indicating that some level of cross protection is possible between different TTV genotypes upon optimization of dose and adjuvant selection.
  • Example 4 Codon optimization and recombinant expression glTTV ORF1 as a full length protein with a 6His tag, and detection thereof by an antibody.
  • the TTVgl nucleotide sequence was submitted to GenScript (Piscataway, New Jersey, USA) for codon optimization and gene synthesis for both E. coli and Saccharomyces cerevisiae. In both cases, the codon optimized gene was cloned into the GenScript pUC57 vector, as product.
  • GenScript OptimumGeneTM codon optimization analysis involves analysis of numerous parameters including codon usage bias,GC content, CpG dinucleotide content, mRNA secondary structure, identification of possible cryptic splicing sites, presence of premature polyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motifs (ARE), inhibition sites (INS), repeat sequences of various kinds (including direct, reverse and dyad), and also restriction sites that may interfere with cloning.
  • Translational performance may be additionally improved via translational initiation Kozak sequences, Shine-Dalgarno sequences, and to increase efficiency of translational termination via stop codons.
  • SEQ ID NO: NOS 18-20 provide TTV capsid gene that were codon optimized for both Escherichia coli (NOS: 18-19) and Saccharomyces cerevisiae (NO: 20).
  • the sequences for E. coli are very similar, however, to clone the gene into the commercial pET101/D-TOPO expression vector (Invitrogen) to create 76057-4 (SEQ ID NO:19), additional CA nucleotides had to be added at the N- terminus.
  • the pET101/D-TOPO expression vector also has a C-terminal V5 tag and 6X-His for purification, although the sequences for 76057-3 (SEQ ID NO: 18) and 76057-4 are otherwise identical.
  • the expressed codon-optimized TTVgl protein is approximately 68 kD in size, relative to the 63kD protein, due to the addition of a 10 amino acid protective peptide at the amino terminus, and 32 amino acids corresponding to the V5 epitope and a 6X His tag at the carboxy terminus ( Figure 2).
  • sequence for 76057-5 (SEQ ID NO: 20) has been codon optimized for S. cerevisiae, and it thus differs slightly from the E. coli sequences. In addition, this sequence lacks a 10 amino acid protective peptide at the N-terminus (which was added to the E. coli sequence), and it also has flanking restriction
  • the protective peptide of ten amino acids was added to N-terminus of the TTVgl sequence for expression in E. coli. since this has been shown to increase protein stablility when fused to the amino terminus. Restriction sites have been engineered such that the peptide can be removed for evaluation of the full length protein. Expression of the codon optimized TTVgl was evaluated in the pET101/D-TOPO vector with and without the protective peptide N-terminal fusion. The TTVgl sequence codon optimized for S. cerevisiae was also subcloned into a pESC-Trp vector with the potential for producing suface-expressed protein in yeast that can be used to elicit an antibody response in vivo.
  • Rabbit polyclonal antibodies were raised against Baculovirus expressed g2 TTV GST-ORF1 protein prepared in Example 2. Two rabbits were
  • the rabbit antibody did not, however, respond to the E.coli expressed g2TTV ORF1 that had the 100 A.A. N-terminal arginine-rich region removed from the amino terminus as described in Example 2. This may suggest that a major antigenic epitope may be in the 100 amino acid region that was missing in the truncated g2 TTV ORF1 , and that there is homology between g1 and g2 TTV in this region.
  • Monoclonal antibodies can be generated against full-length g1 TTV ORF1 , or other g1 TTV antigens.
  • Other potential immunizing antigens include g1 TTV whole virus, g2 TTV GST-ORF1 (Baculo), g1 TTV GST-truncated ORF1 (E coli), and g2 TTV GST-truncated ORF1 (E coli).
  • a peptide library can be generated to identify linear epitopes that are antigenic. For example, 18mer peptides, with a 10AA overlap, can be utilized to cover the TTV genome.
  • the peptides can then be utilized in Western blots or ELISA's to determine their overall reactivity to the gI TTV ORF1 or g2TTV ORF1 monoclonal and/or polyclonal antibodies so that immunogenic domains can further be identified.
  • Rabbit polyclonal antibodies may also be raised against three g1 TTV ORF1 peptides cross-linked to KLH, and subsequently screened using peptide- ovalbumin conjugates.
  • the peptide-KLH conjugates can also be used to produce monoclonal antibodies.
  • multiple g1 TTV ORF1 peptides copies may be conjugated together, including from different strains.
  • peptides were generated (CPC Scientific), they were then conjugated to KLH or ovalbumin (by the Proteos Co).
  • the KLH- conjugated peptides were used for immunization of rabbits, while the Ovalbumin conjugated peptides are used for screening the serum (i.e., to detect antibodies to the peptides and not the carrier protein).
  • TTV (459-479): DFGHHSRFGPFCVKNEPLEFQ (21 aa, pi 6.9)
  • CTWKRLRRMVREQLDRRMDHKRQRLH 26 aa, pi 13
  • Each of the three peptides has a single cysteine residue present in the sequence to enable selective peptide coupling to a carrier protein.
  • a further and highly preferred peptide is constructed by using the peptide sequence corresponding to residues 601 -620 of SEQ ID NO:9 (20AA, pi 13) except that a cysteine residue is used at the N-terminus in replacement for Asn 601 .
  • This peptide is also likely surface exposed in the native protein.
  • the Chromos ACE system is a protein expression platform that consists of three main components.
  • the first component is a neutral, functional mammalian artificial chromosome called the Platform ACE, which resides in the genetic material of a modified Chinese Hamster Ovary (CHO) cell line.
  • the second component is the ACE targeting vector, which is a plasmid used for loading target genes onto the Platform ACE.
  • the third element is a site-specific, unidirectional integrase, which catalyzes the direct and specific loading of the target gene onto the Platform ACE. Additional information concerning the ACE System can be found of the website of Chromos Molecular Systems, Inc. of Canada, or by contacting the company directly at 604-415-7100 where the technology is available for license.
  • the Chromos ACE system has a number of significant advantages over traditional protein production platforms. The first of these is speed. The first of these is speed.
  • Chromos ACE system allows for the rapid, efficient and reproducible insertion of selected genes.
  • the second advantage is expression. High level constituitive protein expression is achieved over time.
  • a third advantage is stability.
  • the Chromos ACE system allows selective and controlled protein expression. Briefly, restriction sites were added to both ends of the TTV7 ORF1 gI DNA using PCR. Additionally, the sequence for yeast invertase was added to the 5' end of a separate PCR preparation. The amplified sequences were then treated with restriction enzymes and sub-cloned into the plasmid pCTV927. The DNA sequence was verified by ACGT Inc.
  • CHk2 (Chinese Hamster Ovary) cells were then transfected with the plasmids using Lipofectamine 2000 (Invitrogen), and selective pressure was added using hygromycin B. Ten single-cell clones were analyzed for TTV protein production using SDS PAGE and Western Blotting. More specifically, the ACE Targeting Vector pCTV-TTV7ORF1 +YI was generated as follows (see Figure 3).
  • the gene TTV7ORF1 was obtained as a PCR product.
  • a primer was designed to contain the yeast invertase secretion signal and the restriction site EcoRV at the 5' end of the gene.
  • a second primer was designed to contain the restriction site Kpnl at the 3' end of the gene.
  • the plasmids pCTV-TTV7ORF1 +YI and pSIO343, which coded for TTV7ORF1 /yeast invertase and the unidirectional lambda integrase, respectively, were transfected into the Chk2 cell line, which contained the Platform ACE.
  • the transfected cells were named Chk2-TTV7ORF1 +YI . These cells were seeded in 96-well plates and monitored for the formation of single-cell clones. Media containing Hygromycin was added to each 96-well plate to select for cell clones that contained the ACE targeting vector. Once single-cell clones were identified, twelve of them were expanded into 24-well plates, and then to 6-well plates.
  • Figures 4 and 5 provides a 7-way amino acid alignment of ORF1 (capsid proteins) from 5 TTV gt1 viruses of the present invention and two TTV gt2 (or gt2-like) viruses of the invention.
  • ORF1 capsid proteins
  • TTV10 and TTV13 TTV10 and TTV13
  • TTV fragments (1900bp and 2200bp), which together span the entire TTV circular genome, were separately cloned into separate pCR 2.1 TA (Invitrogen) cloning vectors.
  • PCR primers were designed using the consensus sequence that was generated from strains of the present invention (ttvgt1 -27, -7, -17 and -21 ), and also from published sequences (AY823990(g1 ) and AB076001 -(Sd-TTV31 )).
  • Primer pairs that correspond to the sequence at 680s and 2608a or 1340s and 764a were used to amplify PCR products from DNA that was extracted from liver homogenate samples of pigs infected with TTV challenge strain. These PCR fragments were cloned into Invitrogen's pCR2.1 - TOPO TA vector using directions that were supplied with the kit. Clones were subsequently used to generate DNA sequences across the entire 2880 base genome and the sequence was found to be 86% homologous to published sequences GQ120664.1 and AY823990.1 .
  • gI TTV is a single-stranded DNA (ssDNA) virus. Fragments of gI TTV are converted to double-stranded DNA (dsDNA) using polymerase chain reaction (PCR). The dsDNA fragments of gI TTV are then cloned into pUC-based plasmid cloning vectors and transformed into E. coli. The fragments of gITTV are less than 1 full-length dsDNA equivalent of the gI TTV genome. Amplification of gI TTV dsDNA concatemers.
  • Concatemers of full-length g1 TTV dsDNA genome equivalents are generated using ⁇ 29 polymerase amplification kits (e.g., illustra TempliPhi).
  • Full-length gITTV dsDNA fragments are generated by digestion of the concatemers at appropriate restriction endonucleases (RE) sites.
  • RE restriction endonucleases
  • These full-length gI TTV dsDNA fragments can be cloned into plasmid vectors.
  • the concatemers or the uncloned fragments (resulting from RE digestion) can be used without immediate cloning in subsequent molecular biology constructions (see below).
  • Tandem duplications of the gI TTV genome Plasmid constructs encoding tandem duplications of the gI TTV genome are next generated. The tandem duplications in the constructs are approximately greater than 1 .2 copies of full- length dsDNA equivalents of the the gI TTV genome.
  • the tandem duplications in plasmids are generated using (1 ) subcloning employing appropriate RE sites, (2) PCR assembly of tandem duplications, or (3) other molecular biology methods.
  • the templates for the generation of the tandem duplications are the gI TTV dsDNA fragments and/or the full-length gI TTV dsDNA clones (yielded by ⁇ 29 polymerase amplification).
  • tandem duplication plasmid constructs are not identical to the gITTV virus.
  • the tandem duplication constructs are dsDNA while the virus is ssDNA, the constructs encode >1 .2 full- length dsDNA equivalents of the gI TTV genome while the virus has only one full- length equivalent, the construct contains interrupting plasmid sequences while the virus has only viral sequences.
  • the tandem duplication plasmid constructs are introduced into pigs (by inoculation, injection, electroporation, or other methods of introduction) or introduced into tissue culture cells (by transfection, electroporation, or other methods of introduction) where the plasmid construct recombines at homologous sequences to regenerate a unit-length dsDNA equivalent of the gI TTV genome.
  • the dsDNA equivalent of the gITTV genome is a presumed replicative intermediate of the gI TTV viral life cycle. The presence of this presumed dsDNA replicative intermediate will lead to the production of the bona fide ssDNA gI TTV.
  • Plasmid constructs directing in vivo transcription of gITTV ORFs can be made, such as the fusion of transcriptional promoters (e.g., CMV) to gITTV ORFs.
  • plasmid constructs directing the in vitro generation of gITTV ORF transcripts can be made, such as the fusion of transcriptional promoters (e.g., T7) to gITTV ORFs followed by use of in vitro transcription kits.
  • Either gITTV ORF-expressing plasmids or gITTV ORF- expressing RNA transcripts can be co-injected into pigs or co-transfected into cells along with the tandem duplication plasmid constructs to yield gITTV virus.
  • gITTV virus production Detection of gITTV virus production. To date, whole gITTV virus cannot be propagated in tissue culture cells. The generation of gITTV virus is detected by immune reagents (e.g., a-g1TTV antibody) or by molecular methods (e.g., qPCR).
  • immune reagents e.g., a-g1TTV antibody
  • molecular methods e.g., qPCR
  • Total DNA was isolated (DNEasy Blood and Tissue Kit, Qiagen, Valencia, CA ) from a frozen liver homogenate sample (200 microliters) derived from a prior TTV challenge study.
  • the DNA was then PCR-amplified using forward and reverse primers selected to overlap at the unique EcoRI site of the swine TTV 1 - 178 genome.
  • the forward primer: for TTVg1 -178 was selected as positions 1399 to1428 (ACGG... CCAA) from SEQ ID NO:7
  • the reverse primer: was selected to correspond to base positions 1443 (5')-to 1416 (3') ATAT... TTGT (opposite strand) from SEQ ID NO:7.
  • PCR conditions were as follows: 1 cycle of denaturation at 94°C for 1 minute; 35 cycles of 94°C, 30 seconds; 55°C, 30 sec; 72°C, 3 minutes; followed by a final 10 minute extension at 72°C.
  • the resultant ⁇ 2.8kb fragment was cloned into pCR2.1 vector using a TOPO TA cloning kit. (Invitrogen, Carlsbad, CA). Upon sequence verification, this plasmid (named pCR2.1 +TTV_178) was found to contain the entire TTV 1 -178 genome sequence.
  • pCR2.1 +TTV_178 vector was then linearized with EcoRI in order to release the full length TTV genome, which was then transfected into human embryonic kidney (293), baby hamster kidney (BHK-21 ), swine testicular (ST) and porcine kidney (PK) cells lines using Lipofectin (Invitrogen, Carlsbad, CA). The transfection was allowed to proceed for a total of 5 days at which time the cells were fixed, and then used for IFA staining to determine if the TTV DNA provided expression of ORF 1 protein.
  • IFA staining was accomplished with rabbit polyclonal sera that was raised against a peptide corresponding to a C- terminal region of the capsid protein (residues 601 -620 in SEQ ID NO:9), except that the N terminal residue thereof (Asn 601 ) was replaced with a cysteine residue.
  • the results indicate that the TTV-transfected DNA successfully expressed the ORF-1 protein in at least 293, BHK-21 , ST and PK cells lines.

Abstract

The present invention is directed to novel nucleotide and amino acid sequences of Torque teno virus ("TTV"), including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals. Vaccines provided according to the practice of the invention are effective against multiple swine TTV genotypes and isolates. Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines. Particularly important aspects of the invention include vaccines that provide TTV ORF1 protein, or peptide fragments thereof, as antigen.

Description

Infectious Clones of Torque Teno Virus Field of the Invention
The present invention is directed to novel nucleotide and amino acid sequences of Torque teno virus ("TTV"), including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals. Vaccines provided according to the practice of the invention are effective against multiple swine TTV genotypes and isolates. Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines. Of particular importance, there are disclosed vaccines that comprise, as antigen, the expressed protein of single TTV open reading frames, most particularly from ORF1 or ORF2, and also fragments of the full length ORF1 and ORF2-encoded proteins.
Background of the Invention
Torque Teno Virus ("TTV"), also referred to as transfusion-transmitted virus, is generally assigned to the Circoviridae family. It is generally recognized that TTV was first isolated from human transfusion patients (see for example, Nishizawa et al., Biochem. Biophys. Res. Comm. vol. 241 , 1997, pp.92-97). Subsequently, TTV or TTV-like viruses have been identified from other mammals, including swine, and numerous strains or isolates have been published (see for example, McKeown et al. Vet. Microbiol, vol. 104, 2004, pp 1 13-1 17).
Subsequent work as shown that TTV and TTV-like viruses are very common; however the pathogenesis of TTV, and the contributions it may make to other disease states (for example, those caused by other viruses and bacteria) remains unclear. For example, TTV infections appear to be common in humans, including even in healthy individuals, and such infections are often asymptomatic, and may remain for years. In addition, the general inability to propagate the virus in cell culture, and a lack of any clear mechanistic disease models, have made any overall characterization of TTV biology difficult. Notwithstanding that TTV viremia is elevated in human patients afflicted with other viral diseases, (such as hepatitis or HIV/AIDS), there is also considerable medical literature suggesting that TTVs are, in fact, avirulent, and await any clear actual association with known disease states. See, for example, Biagini et al., Vet. Microbiol, vol. 98, 2004, pp. 95-101 .
In regard of swine, the situation is similar. There is considerable work suggesting that TTV infection is associated with, and contributes to, numerous diseases such as porcine circovirus disease (and its various clinical
manefestations, such as postweaning multisystemic wasting syndrome and respiratory disease complicated by lung lesions), and PRRSV- associated disease (porcine respiratory and reproductive syndrome virus). See for example published international patent applications WO 2008/150275 and WO
2008/127279. Krakowka et al. also report on an often fatal disease in pigs referred to as PDNS (porcine dermatitis and neuropathy syndrome) which is described as a manifestation of disseminated intravascular coagulation, and for which combined infection by serotype 1 TTV and PRRSV virus was possibly implicated (Am. J. Vet Res, vol 69(12), 2008, pp. 1615-1622. PDNS disease was also correlated with porcine circovirus disease (notably PCV-2) and also with bacterial infections. Accordingly, while considerable work has been
accomplished, there remains little work that definitively correlates porcine TTV infection with specific pathologies. Nonetheless, it has become reasonably clear that TTV infection can potentiate numerous disease states. Accordingly, there is a need for various classes of TTV reagents, such as high affinity antibodies, and for example, peptide fragments of TTV or whole virions that are highly
immunizing, both to further our understanding of overall TTV biology and to vaccinate, directly or indirectly, against numerous disease states to which TTV may contribute. Thus, although the possibility exists that TTV is the principle causative factor of diseases in swine, it seems more likely that numerous swine diseases either require the presence of more than one virus, or that the primary effect of certain "primary" pathogens is potentiated by TTV infection. As stated, the possibility exists that numerous diseases of swine can be treated or lessened by administering anti-TTV agents to affected or potentially affected animals.
Notwithstanding the well established interest in TTV, effective vaccines have not emerged.
TTV is a small, non-enveloped virus comprised of negative polarity, single- strand circularized DNA. The genome includes three major open reading frames, ORF1 , ORF2 and ORF3, which overlap, and ORF1 encodes the capsid protein. (Biagini et al., supra). For a detailed discussion thereof, please see the following references, which are incorporated by referenece: Kakkola et al., Virology, vol. 382 (2008), pp. 182-189; Mushahwar et al., Proc. Natl. Acad. Sci, USA, vol 96, (1999) pp. 3177-3182; and T. Kekarainen and J. Segales, "Torque teno virus infection in the pig and its potential role as a model of human infection", The Veterinary Journal, accepted December 13, 2007 for 2008.
Despite the relatively simple genome, it has been generally very difficult to propagate the virus in cell culture or by other in vitro methods. The present invention is directed to recombinant constructs whereby TTV can be propagated in vitro, including via infectious clones. More particularly, the invention is directed to the discovery that effective vaccines can in fact be made from TTV, most particularly when the TTV antigen is the expression product of a single ORF, or a fragment thereof. In a preferred embodiment, the invention provides for ORFI protein vaccines.
Summary of the Invention
The present invention provides a method of treating or preventing a disease or disorder in an animal caused by infection with torque teno virus (TTV), including disease states that are directly caused by TTV, and disease states contributed to or potentiated by TTV. In a preferred example, the animal treated is a swine. Disease states in swine that may be potentiated by TTV, and which may also be treated or prevented according to the practice of the invention, include those caused by or associated with porcine circovirus (PCV), and porcine reproductive and respiratory syndrome virus (PRRS).
The present invention also includes the option to administer a combination vaccine, that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-TTV pathogen, with appropriate choice of adjuvant.
Based in part upon the unique TTV amino acid sequences as disclosed herein, the present invention also provides a diagnostic kit for differentiating between porcine animals vaccinated with the above described TTV vaccines and porcine animals infected with field strains of TTV.
Representative embodiments of the invention include an isolated polynucleotide sequence that comprises a polynucleotide selected from the group consisting of:
(ai ) the DNA of genotype 2 sequence TTV13 (SEQ ID NO: 1 ); the DNA genotype 2 sequence TTV10 (SEQ ID NO: 2); or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
(32) the DNA of a genotype 1 sequence selected from the group consisting of ttvg1 -7 (SEQ ID NO: 4), ttvGT1 -17 (SEQ ID NO: 5), ttvGT1 -21 (SEQ ID NO: 6), ttvgtl -27 (SEQ ID NO: 3), ttvgt1 -178 (SEQ ID NO: 7) or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
(b) the complement of any sequence in (a);
(c) a polynucleotide that hybridizes with a sequence of (a) or (b) under stringent conditions defined as hybriding to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 xSSC/0.1 % SDS at 68°C;
(d) a polynucleotide that is at least 70% identical to the polynucleotide of (a) or (b);
(e) a polynucleotide that is at least 80% identical to the polynucleotide of
(a) or (b); (f) a polynucleotide that is at least 90% identical to the polynucleotide of (a) or (b); and
(g) a polynucleotide that is at least 95% identical to the polynucleotide of (a) or (b).
The invention further provides RNA polynucleotide molecules that are the complement of any such DNA polynucleotide sequence, and vectors and plasmids for the expression of any such RNA or DNA polynucleotides, and for TTV virus that is expressed from such nucleotide sequences, wherein said virus is live, or fully or partially attenuated.
The invention also provides a DNA vaccine that comprises a
polynucleotide sequence as aforementioned, and corresponding nucleotide sequences that function as infectious clones.
The invention provides a polypeptide encoded by any of the open reading frames of the genotype 2 TTV 13 (SEQ ID NO: 1 ) or genotype 2 TTV10 (SEQ ID NO: 2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
The invention also provides a polypeptide encoded by any of the open reading frames of the (all sertotype 1 ) ttvg1 -7 (SEQ ID NO:10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), ttvgt1 -27 (SEQ ID NO: 13), and ttvgtl - 178 (SEQ ID NO:9) ORF1 polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
Despite continued failures as reported in the art, to provide effective vaccines against TTV (or to limit the ability of TTV to potentiate other diseases), the present invention provides for such effective vaccines, which preferably comprise a polypeptide resultant from expression of a single TTV open reading frame, or a mixture thereof. In a preferred embodiment, the polypeptide is expressed from ORF1 , and preferred mixtures include a combination of the polypeptides of ORF1 and ORF2, and ORF1 and ORF3. In a further preferred embodiment, and taking advantage of the substantial polypeptide sequence information disclosed herein, there are further provided polypeptide vaccines wherein the antigen is defined by (a) the first 100 N- terminal amino acids of the capsid protein of TTV13 (SEQ NO:1 ) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto; or (c) an arginine rich region thereof.
Brief Description of the Drawings Figure 1 (panels A and B) shows detection of ORF1 protein by immunological methods.
Figure 2 evidences successful expression of codon-optimized TTVgl ORF1 protein in E. coli, with a 6X His tag for affinity purification
Figure 3 provides a vector map for the Chromos construct pcTV-TTV1 -7 ORF1 (plus yeast invertase) expression plasmid from which is expressed (following integration into an artificial chromosome in CHO cells) vaccinating ORF1 protein. Figure 4 provides a phylogenetic tree for various TTV strains including a compilation of percent identities.
Figure 5 (panels A, B and C) provides identification of in-common arginine rich regions of ORF1 proteins as expressed from various TTV isolates.
Figure 6 provides a vector map for TTVgl -178 as assembled.
Figure 7 demonstrates that Chromos-expressed gITTV ORF1 significantly reduced lung lesions compared to the challenge controls and reduces the magitude and duration of gITTV viremia, again compared to the challenge controls.
Figure 8 provides a vector map for the pCR2.1 +TTVg1 -178 construct that contains a ttvg1 -178 strain full length infectious clone.
Brief Description of the Sequence Listings SEQ ID NO:1 provides the genotype gt2 TTV 10 DNA sequence.
SEQ ID NO:2 provides the genotype 2 gt2 TTV 13 DNA sequence. SEQ ID NO:3 provides the genotype 1 ttvgt1 -27 DNA sequence. SEQ ID NO:4 provides the genotype 1 ttvgtl -7 DNA sequence. SEQ ID NO:5 provides the genrotype 1 ttvgtl -17 DNA sequence.
SEQ ID NO:6 provides the genotype 1 ttvgtl -21 DNA sequence.
SEQ ID NO:7 provides the genotype 1 ttvg1 -178 DNA sequence SEQ ID NO:8 provides the amino acid sequence of TTV strain AY823991 ORF1 .
SEQ ID NO:9 provides the amino acid sequence of TTV strain ttvgtl -178 ORF1 (TTV genotype 1 ). SEQ ID NO: 10 provides the amino acid sequence of TTV strain ttvgtl -7 ORF1 . SEQ ID NO: 1 1 provides the amino acid sequence of TTV strain ttvgtl -17 ORF1 . SEQ ID NO: 12 provides the amino acid sequence of TTV strain ttvgtl -21 ORF1 .
SEQ ID NO: 13 provides the amino acid sequence of TTV strain ttvgtl -27 ORF1 .
SEQ ID NO: 14 provides the amino acid sequence of TTV strain gt2 TTV10 ORF1 (genotype 2).
SEQ ID NO: 15 provides the amino acid sequence of TTV strain gt2 TTV13 ORF1
SEQ ID NO: 16 provides the DNA sequence of known strain AY823991
(genotype 2).
SEQ ID NO: 17 provides the DNA sequence of known strain AY823990
(genotype 1 ).
SEQ ID NO:18 provides the 76057-3 TTV capsid encoding sequence, codon optimized for E. coli. as cloned into the pUC57 GenScript® vector.
SEQ ID NO:19 provides the 76057-4 TTV capsid encoding sequence, codon optimized for E. coli. as cloned into the Invitrogen pET101/D-TOPO® expression plasmid.
SEQ ID NO:20 provides the 76057-5 TTV capsid encoding sequence, codon optimized for Saccharomyces cerevisiae as cloned into the pUC57 GenScript® vector. SEQ ID NO:21 provides the DNA sequence for a construct that encodes ttvgtl -7 ORF1 with a yeast invertase expression tag (Yl). SEQ ID NO:22 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
corresponding to residues 167-185, which is used with the C-terminal AA in amidated form.
SEQ ID NO:23 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
corresponding to residues 459-479.
SEQ ID NO:24 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
corresponding to residues 612-637. SEQ ID NO:25 provides the amino acid sequence of TTV strain AY823990 ORF1 .
SEQ ID NOS:26-29 define primer sequences. In connection with the descriptors for the sequences, those familiar with the art will recognize that numerous slightly different abbreviations are commonly used interchangeably for specific serotypes, for example, gITTV, TTVgl , genotype 1 TTV, serotype 1 TTV, gtl TTV, and the like. A similar situation exists for genotype 2.
Detailed Description of the Invention
The following definitions and introductory matters are applicable in the
specification.
The terms "porcine" and "swine" are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig. "Mammals" include any warm-blooded vertebrates of the Mammalia class, including humans.
An "infectious DNA molecule", for purposes of the present invention, is a DNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell. Likewise, an "isolated polynucleotide molecule" refers to a composition of matter comprising a polynucleotide molecule of the present invention purified to any detectable degree from its naturally occurring state, if any.
For purposes of the present invention, the nucleotide sequence of a second polynucleotide molecule (either RNA or DNA) is "homologous" to the nucleotide sequence of a first polynucleotide molecule , or has "identity" to said first polynucleotide molecule, where the nucleotide sequence of the second polynucleotide molecule encodes the same polyaminoacid as the nucleotide sequence of the first polynucleotide molecule as based on the degeneracy of the genetic code, or when it encodes a polyaminoacid that is sufficiently similar to the polyaminoacid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in practicing the present invention. Homologous polynucleotide sequences also refers to sense and anti-sense strands, and in all cases to the complement of any such strands. For purposes of the present invention, a polynucleotide molecule is useful in practicing the present invention, and is therefore homologous or has identity, where it can be used as a diagnostic probe to detect the presence of TTV virus or viral polynucleotide in a fluid or tissue sample of an infected pig, e.g. by standard hybridization or amplification techniques. Generally, the nucleotide sequence of a second polynucleotide molecule is homologous to the nucleotide sequence of a first polynucleotide molecule if it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the
BLASTN algorithm (National Center for Biotechnology Information, otherwise known as NCBI, (Bethesda, Maryland, USA) of the United States National Institute of Health). In a specific example for calculations according to the practice of the present invention, reference is made to BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences- a new tool for comparing protein and nucleotide sequences." (1999) FEMS Microbiol Lett. 174:247-250.]. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1 , and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 89:10915-10919. 1992). The percent identity is then calculated as: Total number of identical matches x 100/ divided by the length of the longer sequence+number of gaps introduced into the longer sequence to align the two sequences.
Preferably, a homologous nucleotide sequence has at least about 75% nucleotide sequence identity, even more preferably at least about 80%, 85%, 90% and 95% nucleotide sequence identity. Since the genetic code is
degenerate, a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.
A homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention. In this regard, certain conservative amino acid substitutions may be made which are generally recognized not to inactivate overall protein function: such as in regard of positively charged amino acids (and vice versa), lysine, arginine and histidine; in regard of negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and in regard of certain groups of neutrally charged amino acids (and in all cases, also vice versa), (1 ) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and
phenylalanine.
Homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above. Alternatively, homologous nucleotide sequences can be determined by hybridization under selected conditions. For example, the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO: 1 (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO: 1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2xSSC/0.1 % SDS at 42°C (see Ausubel et al editors, Protocols in Molecular Biology, Wiley and Sons, 1994, pp. 6.0.3 to 6.4.10), or conditions which will otherwise result in hybridization of sequences that encode a TTV virus as defined below. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51 .
In another embodiment, a second nucleotide sequence is homologous to SEQ ID NO: 1 (or any other sequence of the invention) if it hybridizes to the complement of SEQ ID NO: 1 under highly stringent conditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 xSSC/0.1 % SDS at 68°C, as is known in the art.
It is furthermore to be understood that the isolated polynucleotide molecules and the isolated RNA molecules of the present invention include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.
Polvpetides and Polynucleotides of the Invention
Representative embodiments of the invention include an isolated polynucleotide sequence that comprises a polynucleotide selected from the group consisting of:
(ai ) the DNA of genotype 2 sequence TTV13 (SEQ ID NO: 1 ); the DNA genotype 2 sequence TTV10 (SEQ ID NO: 2); or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
(32) the DNA of a genotype 1 sequence selected from the group consisting of ttvg1 -7 (SEQ ID NO: 4), ttvGT1 -17 (SEQ ID NO: 5), ttvGT1 -21 (SEQ ID NO: 6), ttvgtl -27 (SEQ ID NO: 3), ttvgt1 -178 (SEQ ID NO: 7) or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
(b) the complement of any sequence in (a);
(c) a polynucleotide that hybridizes with a sequence of (a) or (b) under stringent conditions defined as hybriding to filter bound DNA in 0.5M NaHP04,
7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 xSSC/0.1 % SDS at 68°C;
(d) a polynucleotide that is at least 70% identical to the polynucleotide of (a) or (b);
(e) a polynucleotide that is at least 80% identical to the polynucleotide of (a) or (b);
(f) a polynucleotide that is at least 90% identical to the polynucleotide of (a) or (b); and
(g) a polynucleotide that is at least 95% identical to the polynucleotide of (a) or (b).
The invention also provides a polypeptide encoded by any of the open reading frames of the genotype 2 TTV13 (SEQ ID NO: 1 ) or genotype 2 TTV10 (SEQ ID NO:2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
The invention also provides a polypeptide encoded by any of the open reading frames of the (all sertotype 1 ) ttvg 1 -7 (SEQ ID NO: 10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), ttvgt1 -27 (SEQ ID NO: 13), and ttvgtl - 178 (SEQ ID NO:9) ORF1 polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
In a preferred embodiment, the polypeptide is expressed from ORF1 , and preferred mixtures include a combination of the polypeptides of ORF1 and ORF2, and ORF1 and ORF3.
In a further preferred embodiment, there are further provided TTV polypeptide-based vaccines wherein the antigen is defined by: (a) the first 300 N-terminal amino acids of the ORF1 capsid protein of TTV13 (SEQ NO:1) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto;
(b) the first 200 N-terminal amino acids of the ORF1 capsid protein of TTV13 (SEQ NO:1) orTTVIO (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto;
(c) the first 100 N-terminal amino acids of the ORF1 capsid protein of TTV13 (SEQ NO:1) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto;
(d) the first 300 N-terminal amino acids of the ORF1 capsid protein of any of (all sertotype 1)ttvg1-7 (SEQ ID NO:10), ttvGT1-17 (SEQ ID NO:11), ttvGT1-21 (SEQ ID NO:12), ttvgt1-27 (SEQ ID NO:13), and ttvgt1-178 (SEQ ID NO:9) or a polypeptide that is at least 90% identical thereto;
(e) the first 200 N-terminal amino acids of the ORF1 capsid protein of any of (all sertotype 1)ttvg1-7 (SEQ ID NO:10), ttvGT1-17 (SEQ ID NO:11), ttvGT1-21
(SEQ ID NO:12), ttvgt1-27 (SEQ ID NO:13), and ttvgt1-178 (SEQ ID NO:9) or a polypeptide that is at least 90% identical thereto; and
(f) the first 100 N-terminal amino acids of the ORF1 capsid protein of any of (all sertotype 1)ttvg1-7 (SEQ ID NO:10), ttvGT1-17 (SEQ ID NO:11), ttvGT1-21 (SEQ ID NO:12), ttvgt1-27 (SEQ ID NO:13), and ttvgt1-178 (SEQ ID NO:9) or a polypeptide that is at least 90% identical thereto.
Further Genetic Manipulations
The DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of the viral genes and their encoded gene products. Knowledge of a polynucleotide encoding a viral gene product of the invention also makes available anti-sense polynucleotides which recognize and hybridize to
polynucleotides encoding a polypeptide of the invention, or a fragment thereof. Full length and fragment anti-sense polynucleotides are useful in this respect. The worker of ordinary skill will appreciate that fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to a specific RNA (as determined by sequence comparison of DNA encoding a viral polypeptide of the invention as well as (ii) those which recognize and hybridize to RNA encoding variants of the encoded proteins. Antisense polynucleotides that hybridize to RNA DNA encoding other TTV peptides are also identifiable through sequence comparison to identify characteristic, or signature sequences for the family of molecules. Such techniques (see Example 8) are further of use in the study of antigenic domains in TTV polypeptides, and may also be used to distinguish between infection of a host animal with remotely related non-TTV members of the Circoviridae.
Example 4 provides guidance as to effective codon optimization for enhanced expression in yeast and E. coli for the constructs of the invention.
Vaccine formulations
Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
Vaccines of the present invention may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, MT), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta GA), QS-21 (Cambridge Biotech Inc., Cambridge MA), SAF-M (Chiron, Emeryville CA), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant. Non-limiting examples of oil- in-water emulsions useful in the vaccine of the invention include modified
SEAM 62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1 % (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1 % (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, with may be additionally lipidated, such as those described in
WO 2006/084319, WO2004/014957, and WO2004/014956.
In a preferred embodiment of the present invention, ORFI TTV protein, or other TTV proteins or fragments thereof, is formulated with 5% AMPHIGEN®.
Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention. Examples of such sustained release formulations include virus, infectious DNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in texts known in the art, for example M. Chasin and R. Langer (eds), 1990, "Biodegradable Polymers as Drug Delivery Systems" in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY, which is also incorporated herein by reference. Alternatively, or additionally, the virus, plasmid, or viral vector can be microencapsulated to improve
administration and efficacy. Methods for microencapsulating antigens are well- known in the art, and include techniques described, e.g., in U.S. Patent
3, 137,631 ; U.S. Patent 3,959,457; U.S. Patent 4,205,060; U.S. Patent 4,606,940; U.S. Patent 4,744,933; U.S. Patent 5,132, 1 17; and International Patent
Publication WO 95/28227, all of which are incorporated herein by reference.
Liposomes can also be used to provide for the sustained release of virus, plasmid, viral protein, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Patent
4,016, 100; U.S. Patent 4,452,747; U.S. Patent 4,921 ,706; U.S. Patent 4,927,637; U.S. Patent 4,944,948; U.S. Patent 5,008,050; and U.S. Patent 5,009,956, all of which are incorporated herein by reference.
An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, viral protein plasmid or viral vector, and then increasing the dosage while monitoring the effects. An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine. Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies (see, for example, Examples 2 and 3 below).
One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above. The effective dose amount of virus, protein, infectious DNA molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated. The dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 101 to about 109 pfu (plaque forming units), more preferably from about 102 to about 108 pfu, and most preferably from about 103 to about 107 pfu. The dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about O.^g to about 100mg, more preferably from about ^g to about 10mg, even more preferably from about 1 (^g to about 1 mg. The dose amount of an infectious DNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 μg to about 100mg, more preferably from about ^g to about 10mg, even more preferably from about 10μg to about 1 mg. The dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 101 pfu to about 109 pfu, more preferably from about 102 pfu to about 108 pfu, and even more preferably from about 103 to about 107 pfu. A suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
Suitable doses for viral protein or peptide vaccines according to the practice of the present invention range generally from 1 to 50 micrograms per dose, or higher amounts as may be determined by standard methods, with the amount of adjuvant to be determined by recognized methods in regard of each such substance. In a preferred example of the invention relating to vaccination of swine, an optimum age target for the animals is between about 1 and 21 days, which at pre-weening, may also correspond with other scheduled vaccinations such as against Mycoplasma hyopneumoniae. Additionally, a preferred schedule of vaccination for breeding sows would include similar doses, with an annual revaccination schedule. Antibodies
Also contemplated by the present invention are anti-TTV antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, porcine, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a TTV polypeptide of the invention. The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind a TTV polypeptide exclusively (i.e., are able to distinguish a single TTV polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) to interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA
techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the Ab molecule.
Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the TTV polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, a TTV polypeptide of the invention from which the fragment was derived.
For the purposes of clarity, "antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions. Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab')2, as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences. Diagnostic Kits
The present invention also provides diagnostic kits. The kit can be valuable for differentiating between porcine animals naturally infected with a field strain of a TTV virus and porcine animals vaccinated with any of the TTV vaccines described herein. The kits can also be of value because animals potentially infected with field strains of TTV virus can be detected prior to the existence of clinical symptoms and removed from the herd, or kept in isolation away from naive or vaccinated animals. The kits include reagents for analyzing a sample from a porcine animal for the presence of antibodies to a particular component of a specified TTV virus. Diagnostic kits of the present invention can include as a component a peptide or peptides from ORF1 , 2, or 3 which is present in a field strain but not in a vaccine of interest, or vice versa, and selection of such suitable peptide domains is made possible by the extensive amino acid sequencing as provided for in Examples 1 and 2 of the Specification. As is known in the art, kits of the present invention can alternatively include as a component a peptide which is provided via a fusion protein. The term "fusion peptide" or "fusion protein" for purposes of the present invention means a single polypeptide chain consisting of at least a portion of a TTV virus protein, preferably of ORF1 , and a heterologous peptide or protein.
Examples
Example 1 Cloning of swine TTV complete genome A. TTV genotype 2.
DNA was purified from porcine serum using a DNA blood mini kit (Qiagen) per manufacturer's protocol. DNA was eluted from the columns in 50 μΙ_ Tris- EDTA buffer. DNA was then amplified via random primed rolling circle
amplification. Briefly, 5 uL of purified DNA and 100 ng random hexamers
(Invitrogen) were then added to 71 μΙ water and heated at 95C for 3 min and cooled on ice. One mM dNTP's, 100 ng random hexamers (Invitrogen), 1X phi29 polymerase buffer and 1 μΙ_ of phi29 polymerase were then added and the reaction was incubated overnight at 30C.
One-fifth total volume was digested with EcoRI and electrophoresed on 0.8% E-gel (Invitrogen) to detect presence of 2.7 kB fragment. EcoRI digested material was purified using a Qiagen PCR purification kit following
manufacturer's protocol, and ligated into an EcoRI digested/shrimp alkaline phosphatase-treated pGem3zf(+) vector (Promega). Ligated DNA was used to transform chemically competent E. coli DH5a. Transformed E. coli was selected on LB/amp agar plates.
Plasmid DNA was isolated from transformed colonies and digested with EcoRI to confirm presence of an approximately 2.7 kB insert. Four clones (4, 7, 10 and 13) were selected and submitted to ACGT, Inc. for sequencing.
Alignment of sequence data indicated that clones 10 and 13 demonstrated homology to TTV published sequence and aligned more closely to TTV genotype 2 than genotype 1 . These clones were subsequently named TTV10 and TTV13.
Analyses of sequencing data for PAH TTV genotype 2.
Nucleotide Alignment of TTV13 (SEQ ID NO:2) and TTV10 (SEQ ID NO: 1 ) to published TTV genotype 2 AY823991 DNA sequence (SEQ ID NO: 16).
AY823991 (1) TCATGACAGGGTTCACCGGAAGGGCTGCAAAA-TTACAGCTAAAACCACA
TTV13 (1) TAATGACAGGGTTCACCGGAAGGGCTGCAAAA-TTACAGCTAAAACCACA
TTV10 (1) TAATGACAGGGTTC-CAGGAAGTGCTGCAAAAATTACAGCTAAAACCACA
AY823991 (50) AGT-CTAACACAATAAACCACAAAGTATTACAGGAAACTGCAATAAATTT
TTV13 (50) AAT-CTAACACAATAAACCACAAAATATTACAGGAAACTGCAATAAATTT
TTV10 (50) ACTACTTACACAT--AACCACAAAATATTTCAGGAAACTGCAATAATTTT
AY823991 (99) AGAAATAAGTTACACATAACCACCA AACCACAGGAAAC
TTV13 (99) AGAAATAAATTACACATAACCACCA AACCACAGGAAAC
TTV10 (98) CAACACACATTGCACAAAACCACAAGATATCAACATAAACCACAGGAAAC
AY823991 (137) TGTGCAAAAAAGAGGAAATAAATTTCATTGGCTGGGCCTGAAGTCCTCAT
TTV13 (137) TCTGCAAAAAAGAGGAAATAAATTTCATTGGCTGGTCCATAAGTCCTCAT
TTV10 (148) TCTGCAAAAAAGAGGAAGTAAATGCTATTGGCTAAATCTGAAGTCTTCAT AY823991 (187) TAGAATAATAAAAGAACCAATCAGAAGAACTTCCTCTTTTAGAGTATATA
TTV13 (187) TAGAATACAAAAAGAACCAATCAGAAACACTTCCTCTTTTAGAGTATATA
TTV10 (198) TAGCATACACAACCAACCAATCAGAAACACTTCCTCATTTGAAGTATATA
AY823991 (237) AGTAAGTGCGCAGACGAATGGCTGAGTTTATGCCGCTGGTGGTAGACACG
TTV13 (237) AGTAAGTGCGCAGACGAATGGCTGAGTTTATGCCGCTGGTGGTAGACACG
TTV10 (248) AGTAAATGCGCAGACGAATGGCTGAGTTTATGCCGCTGGTGGTAGACACG
AY823991 (287) AACAGAGCTGAGTGTCTAACCGCCTGGGCGGGTGCCGGAGCTCCTGAGAG
TTV13 (287) AACAGAGCTGAGTGTCTAACCGCCTGGGCGGGTGCCGGAGCTCCTGAGAG
TTV10 (298) AACAGAGCTGAGTGTCTAACCGCCTGGGCGGGTGCCGGAGCTCCAGAGAG
AY823991 (337) CGGAGTCAAGGGGCCTATCGGGCAGGCGGTAATCCAGCGGAACCGGGCCC
TTV13 (337) CGGAGTCAAGGGGCCTATCGGGCAGGCGGTAATCCAGCGGAACCGGGCCC
TTV10 (348) CGGAGTCAAGGGGCCTATCGGGCGGGCGGTAATCCAGCGGAACCGGGCCC
AY823991 (387) CCC-TCGATGGAAGAAAGATGGCTGACGGTAGCGTACTGCGCACACGGAT
TTV13 (387) CCCCTCCATGGAAGAAAGATGGCTGACGGTAGCGTACTGCGCCCACGGAT
TTV10 (398) CCC-TCCATGGAGGAGAGATGGCTGACGGTAGCGTACGCCGCCCACGGAT
AY823991 (436) TATTCTGCAGCTGTAAAGACCCGAAAAAACATCTTGAAAAATGCCTTACA
TTV13 (437) TATTCTGCGACTGTAAAGACCCGAAAAAACATCTTGAAAAATGCCTTACA
TTV10 (447) TATTCTGCGCCTGCAGTAAGCCCAAAGACCACCTTGAAAAATGCCTTTCC
AY823991 (486) GACGCTATCGCAGACGCCGAAGAAGACCGACACGGAGATGGAGGCACCGG
TTV13 (487) GACGCTATCGCAGACGCCGAAGGAGACCGACAAGAAGATGGAGGCACCGG
TTV10 (497) ACCGCTATCGCCGACGCCGAAGGAGACCCACCAGGAGATGGAGGAGAAGG
AY823991 (536) AGGTGGAGACGCTACTTTCGATATCGGTATCGACGCGCTCCTCGCCGCCG
TTV13 (537) AGGTGGAGACGCTACTTTCGATATCGGTATCGACGCGCTCCTCGCCGCCG
TTV10 (547) AGGTTCCAGCGCTACTTTCGATATCGGTATAGACGCGCTCCTCGCCGCCG
AY823991 (586) CCG CACAAAGGTAAGGAGACGGAGG AAAAAAGCTCCGGTCATAC
TTV13 (587) CCG CACAAAGGTAAGGAGACGGAGG AGGAAAGCTCCGGTCATAC
TTV10 (597) CCGACGCTACAAGGTAAGGAGACGGAGGGTTAAAAAGGCTCCGGTCATTC
AY823991 (630) AATGGTTCCCTCCTAGCCGGAGAACCTGCCTCATAGAGGGATTTTGGCCG
TTV13 (631) AATGGAACCCTCCTAGCCGGAGGACCTGCCTCATAGAGGGGTTCTGGCCG
TTV10 (647) AATGGTTCCCCCCAACAGTCAGAAACTGTTTTATCAAGGGAATCTGGCCG
( 680) TTGAGCTACGGACACTGGTTCCGTACCTGTCTCCCCTTTAGGCGGTTAAA
( 681) TTGAGCTACGGACACTGGTTCCGTACCTGTCTCCCCTTTAGAAGAAAAAA ( 697 ) TTGAGCTACGGACACTGGCTCCGTACCTGTCTCCCTATGAGAAAAGAAAA
(730) TGGACTAGTATTCCCGGGTGGAGGTTGTGACTGGAGCCAGTGGAGTTTAC
(731) TGGACTAATATTTACGGGAGGAGGTTGTGACTGGACTCAGTGGAGCTTAC (747) CGGACTCATATTCCTAGGAGGTGGCATAGACTGGACTGTCTGGAGTTTAC
(780) AAAACCTTTACAATGAAAAACTTAACTGGAGAAATATATGGACAGCTAGT
(781) AAAACCTTTATCATGAAAAACTAAACTGGAGAAATATATGGACAGCTAGT (797) AGAATCTATACCATGAAAAACTAAACTGGAGGAATGTGTGGACTTCTTCA
(830) AATGTTGGAATGGAATTCGCTAGATTTTTAAAAGGAAAGTTTTACTTTTT
(831) AACGTGGGAATGGAATTCGCTAGATTTTTAAAAGGAAAATTCTACTTTTT (847) AATGATGGCATGGAGTTCGCTAGATTCAGATATGCAAAGTTTAAATTTTT AY823991 (880 CAGACATCCATGGAGAAATTATATAATAACTTGGGATCAAGATATACCAT
TTV13 (881 TAGACATCCTTGGAGAAACTATATAGTGACTTGGGATCAGGACATTCCTT
TTV10 (897 TAGACACACAACCAGATCCTACGTAGTAACATGGGACCAAGACATACCAT
AY823991 (930 GCAGGCCACTACCTTATCAAAACCTGCATCCACTCCTAATGCTACTAAAA
TTV13 (931 GTAAACCTTTACCATATCAGAACTTACACCCATTATTAATGCTATTAAAA
TTV10 (947 GTAAACCTTTACCATACACAAATTTACATCCATTTGTAATGCTTCTAAAA
AY823991 (980 AAACAGCACAAAATTGTACTTTCACAGCAAAACTGTAACCCAAACAGAAA
TTV13 (981 AAACAACACAAATTAGTACTCTCACAACAAAACTGTAACCCTAACAGAAA
TTV10 (997 AAACATCATAAAGTAGTTCTAAGCAAACAAGACTGTAATCCTAGAAAAAT
AY823991 (1030 ACAAAAACCTGTCACATTAAAATTCAAACCTCCGCCAAAACTAACATCAC
TTV13 (1031 ACAAAAACCTGTAACTTTAAAATTCAGACCGCCACCAAAACTAACTTCAC
TTV10 (1047 GGACAAACCAGTCACCTTAAAAATAAAGCCACCACCAAAACTCACATCAC
AY823991 (1080 AATGGAGACTAAGTAGAGAATTAGCAAAGATGCCACTAATAAGACTTGGA
TTV13 (1081 AATGGAGACTAAGTAGAGAATTAGCAAAAATGCCACTCATTAGACTAGGA
TTV10 (1097 AGTGGAGACTAAGCAGAGAATTATCAAAAATACCGCTCTTAAGACTAGGA
AY823991 (1130 GTAAGCTTTATAGACCTAACAGAACCATGGGTAGAAGGGTGGGGAAATGC
TTV13 (1131 GTTAGTTTTATAGACTTAACAGAACCGTGGCTAGAAGGTTGGGGAAATGC
TTV10 (1147 GTTTCTTTAATAGACTTCAGAGAACCATGGGTTGAAGGTTTTGGAAATGC
AY823991 (1180 ATTTTATTCCGTGCTAGGATATGAAGCAGTAAAAGACCAAGGACACTGGT
TTV13 (1181 ATTTTACTCAGTACTAGGATATGAAGCCATAAAAGAACAAGGACACTGGT
TTV10 (1197 ATTCTTTAGTACTTTAGGATATGAAGCAGATAAAAGCAATTTAAAAACAA
AY823991 (1230 CAAACTGGACACAAATAAAATACTATTGGATCTATGACACGGGAGTAGGA
TTV13 (1231 CAAATTGGTCACAAATTAAATATTACTGGATATATGATACAGGAGTAGGA
TTV10 (1247 GCGCTTGGTGCCAATGTAAATACTTCTGGATATATGATACCGGAGTAAAT
AY823991 (1280 AATGCAGTATATGTTATACTATTAAAAAAAGACGTTACTGATAATCCAGG
TTV13 (1281 AATGCTGTATATGTAGTTATGCTAAAACAAGATGTAGACGACAACCCAGG
TTV10 (1297 AATCATGTATATGTAGTCATGTTAAACAAAGACGCAGGAGATAATGCAGG
AY823991 (1330 AAACATGGCAACAACCTTTAAAGCATCAGGAGGACAGCATCCAGATGCAA
TTV13 (1331 AAAAATGGCATCAACATTTAAAACAACTCAGGGACAACATCCCAATGCTA
TTV10 (1347 AGACCTAATAACAA ATCAAAACTCAA
AY823991 (1380 TAGATCACATTGAATTGATAAACCAAGGATGGCCTTACTGGTTATACTTT
TTV13 (1381 TAGATCACATAGAATTAATAAATGAAGGATGGCCGTACTGGTTATACTTT
TTV10 (1373 TAGCACACATAGAACAGATAGGAGAAGGTTATCCATACTGGTTATATTTT
AY823991 (1430 TATGGTAAAAGTGAACAAGACATTAAAAAAGAGGCACAC AGCGCAGA
TTV13 (1431 TTTGGTAAAAGTGAACAAGACATAAAAAAGGAAGCACAT AGCGCTGA
TTV10 (1423 TTTGGAAGATCTGAAAGAGACTTAAAAGCACTAGCAACTTCAAACACAAA
AY823991 (1477 AATATCAAGAGAATATACTAGAGACCCAAAATCTAAAAAACTAAAAATAG
TTV13 (1478 AATAGCAAGAGAATATGCTACAAATCCAAAATCAAAAAAACTAAAAATAG
TTV10 (1473 CATAAGAAACGAATTCAATACTAATCCTAACAGCAAAAAATTAAAAATAG
AY823991 (1527 GAATAGTAGGATGGGCATCTTCAAACTACACAACAACAGGCAGTGATCAA
TTV13 (1528 GAATAGTAGGATGGGCATCCTCTAACTTCACAACACCAGGCAGTTCACAA
TTV10 (1523 CTGTAATAGGATGGGCTAGCAGTAACAACACAGCACAAGATAGTACACAA AY823991 (1577) AACAGTGGTGGATCAACATCAGCTATACAAGGTGGATATGTAG CA
TTV13 (1578) AACTCAGGGGGAAATATAGCAGCAATACAAGGAGGATACGTAG CA
TTV10 (1573) GGAGCGAATACTCCAATAGAAGGAACATATTTAATATCACA
AY823991 (1622) TATGC-AGG-GTCCGGGGTCA TAGGAGCAGGGTCAATAGGAA
TTV13 (1623) TGGGC-AGGAGGACAAGGAAAACTAAATCTAGGAGCAGGATCAATAGGAA
TTV10 (1614) TGTGCTACAAACATCAGGACATACAG CAGGAGCAGCACAAATAAATA
AY823991 (1662) ATTTATATCAACAAGGATGGCCATCTAATCAAAACTGGCCTAATACAAAC
TTV13 (1672) ATTTGTACCAACAAGGATGGCCATCAAATCAAAACTGGCCAAATACAAAC
TTV10 (1661) ACCTATTCGCCTCTGGATGGCCTAACTCTCAAAACTATCCACCTTTAAAT
AY823991 (1712) AGAGACAAAACAAACTTTGACTGGGGAATACGAGGACTATGTATACTCAG
TTV13 (1722) AGAGACGAAACTAACTTTGATTGGGGACTCAGATCACTTTGTATACTAAG
TTV10 (1711) CTAGACAAAAACAACTTTGACTGGGGAAAAAGAGCGCTATGTATACTAAG
AY823991 (1762) AGATAACATGCACTTAGGAAGCCAAGAATTAGATGATGAATGCACAATGC
TTV13 (1772) AGATAACATGCAATTAGGAAATCAAGAATTAGATGATGAATGTACCATGC
TTV10 (1761) AAACAACATGAAAATTGGAAACCAAAATTTAGATGATGAGACCACTATGT
AY823991 (1812) TCACATTGTTCGGACCCTTTGTAGAAAAAGCAAATCCAATATTTGCAACA
TTV13 (1822) TCTCACTCTTTGGACCTTTTGTAGAAAAAGCAAATCCAATATTTGCAACA
TTV10 (1811) TTGCCCTCTTCGGACCCTTGGTAGAAAAAGCAAA-CTGGGAAGGCCTAGA
AY823991 (1862) ACAGACCCTAAATTCTTTAAACCTGAACTCAAAGACTATAATATAATCAT
TTV13 (1872) ACAGACCCTAAATACTTTAAACCAGAACTAAAAGACTATAATTTAATCAT
TTV10 (1860) AAAAATACCAGAA--CTAAAACCAGAACTCAAAGACTATAATATCTTAAT
AY823991 (1912) GAAATATGCCTTTAAATTTCAGTGGGGAGGACATGGCACAGAAAGATTTA
TTV13 (1922) GAAATATGCCTTTAAATTCCAGTGGGGAGGACATGGCACAGAAAGATTTA
TTV10 (1908) GAGATATAACTTTCGCTTTCAGTGGGGCGGACACGGAACAGAGACCTTCA
AY823991 (1962) AAACCAACATCGGAGACCCCAGCACCATACCCTGCCCCTTCGAACCCGGG
TTV13 (1972) AAACAACCATCGGAGACCCCAGCACCATACCCTGCCCCTTCGAACCCGGG
TTV10 (1958) AAACAAGTATTGGAGACCCCAGCCAAATACCCTGTCCCTACGGACCAGGT
AY823991 (2012) GACCGCTTCCA-CAGCGGGATACAAGACCCCTCCAAGGTACAAAACACCG
TTV13 (2022) GACCGCTTCCA-CAGCGGGATACAAGACCCCTCCAAGGTACAAAACACCG
TTV10 (2008) GAAGCCCCCCAACACCTTGTCAGGA-ACCCCTCCAAGGTACACGAGGGGG
AY823991 (2061) TCCTCAACCCCTGGGACTATGACTGTGATGGGATTGTTAGAAAAGATACT
TTV13 (2071) TCCTCAACCCCTGGGACTATGACTGTGATGGGATTGTTAGAAAAGATACT
TTV10 (2057) TCCTCAATGCGTGGGATTATGACTATGATGGAATTGTTAGAAAAGACACT
AY823991 (2111) CTCAAAAGACTTCTCGAACTCCCCACAGAGACAGAGGAGGAGGAGAAGGC
TTV13 (2121) CTCAAAAGACTTCTCGAACTCCCCACAGAGACAGAGGAGGAGGAGAAGGC
TTV10 (2107) CTCAAAAGACTGCTTGCCATCCCCACAGACTC GGAGGAGGAGAAAGC
AY823991 (2161) GTACCCACTCCTTGGACAAAAAACAGAGAAAGAGCCATTATCAGACTCCG
TTV13 (2171) GTACCCACTCCTTGGACAAAAAACAGAGAAAGAGCCATTATCAGACTCCG
TTV10 (2154) GTACCCGCTCGCTGGACCCAAAACAGAGAAATTGCCCTCCTCAGACGAAG
AY823991 (2211) ACGAAGAGAGCGTTATCTCAAGCACGAGCAGTGGATCCTCTCAAGAA
TTV13 (2221) ACGAAGAGAGCGTTATCTCAAGCACGAGCAGTGGATCCGATCAAGAA
TTV10 (2204) AAGGAGAGAGCGATATCAGTTCTTCGAGCGACTCATCGACGCAAGAAAGC AY823991 (2258 GAAGAAACGCAGAGAC GAAGACACCACAAGCCAAGCAAGCGACGACT
TTV13 (2268 GAAGAGACGCAGAGAC GAAAGCACCACAAGCCAAGCAAGCGACGACT
TTV10 (2254 GAAGAAGAGAAGAGATACAGAAGACGACACAAGCCCTCAAAGCGAAGACT
AY823991 (2305 CCTCAAGCACCTCCAGCGGGTGGTAAAGAGGATGAAAACACTGTGATAGA
TTV13 (2315 CCTCAAGCACCTCCAGCGGGTGGTAAAGAGGATGAAAACACTGTGATAGA
TTV10 (2304 CCTCCAGCATGTCCAGCGACTGGTGAAGAGATTCAGGACCCT ATAGA
AY823991 (2355 TAAATATAGAAACCTAGCAGACCCCTCACTCAATGTCACAGGACACATGG
TTV13 (2365 TAAATACAGAAACCTAGCAGACCCCTCACTCAATGTCACAGGACACATGG
TTTV10 (2351 CAAATACAGAAACTTAGCAGACCCCTCATTAAATGTCACAGGACATTTTG
AY823991 (2405 AAAAATTCATGCAGTTACATATTCAAAACGTACAAGAAATAAGAGCTAAA
TTV13 (2415 AAAAATTCATGCAACTACATATCCAAAACATACAAGAAATAAGAGCTAAA
TTV10 (2401 AACACTTCTGCCGCTTACACTATAAAAACATAGCAGAAATCAGAGCTAGA
AY823991 (2455 AATGCTAAAAAATCCCTCAATAAACTTTACTTTTCTGATTAATAGCGGCC
TTV13 (2465 AATGCTAAAAAATCCCTCAATAAACTTTACTTTTCTGATTAATAGCGGCC
TTV10 (2451 AATGCCAAAAAAAACCTCAATAAACTATACTTTTCAGACTAAAAGAAG--
AY823991 (2505 TCCTGTGTCCAACCTATTTTTCCTAAACCCCTTCAAAATGGCGGGCGGGA
TTV13 (2515 TCCTGTGTCCAATCTATTTTTTTAAACACCCTTCAAAATGGCGGGAGGGA
TTV10 (2499 TTT ATTTCTTTATTTAAAACACC
AY823991 (2555 CACAAAATGGCGGAGGGACTAAGGGGGGGGCAAGCCCCCCT N
TTV13 (2565 CACAAAATGGCGGAGGGACTAAGGG TG N
TTV10 (2522 ACTA GAGGGCG
AY823991 (2605 NGGGGGGCGACCCCCCCGCACCCCCCCCTGCGG
TTV13 (2598 NNNNNNNNNTAGGCTCTTCG CCCCCGCACCCCCCC-TGCGG
TTV10 (2533 TAGCGGGGGGGGGACC CCCCTGCACCCCCCCATGCGG
AY823991 (2655 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
TTV13 (2638 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
TTV10 (2570 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
AY823991 (2705 CCCGCGGGGGG-CAAGCCCCCCTGCACCCCCC-
TTV13 (2688 CCCGCGGGGGG-CAAGCCCCCCTGCACCCCCC-
TTV10 (2620 CCCGCGGGGGGGCAAGCCCCCCTGCACCCCCCC
Figure imgf000026_0001
Nucleotide Identity
Figure imgf000026_0002
TTV 13 shows 92% identity when compared with previously published AY823991 sequence. However, TTV10 only show 76% similarity between either AY823991 or TTV13 and may be considered a separate genotype. Amino Acid Alignment of PAH TTV genotype ORF1 for TTVI O (SEQ ID NO: 14) and TTV 13 (SEQ ID NQ:15) with AY823991 ORF1 (SEQ ID NQ:8).
AY823991 Orfl (1 MPYRRYRRRRRRPTRRWRHRRWRRYFRYRYRRAPRRRR-TKVRRR-RKKA
TTVlOOrfl (1 MPFHRYRRRRRRPTRRWRRRRFQRYFRYRYRRAPRRRRRYKVRRRRVKKA
TTV130RF1 (1 MPYRRYRRRRRRPTRRWRHRRWRRYFRYRYRRAPRRRR-TKVRRR-RRKA
AY823991 Orfl (49 PVIQWFPPSRRTCLIEGFWPLSYGHWFRTCLPFRRLNGLVFPGGGCDWSQ
TTVlOOrfl (51 PVIQWFPPTVRNCFIKGIWPLSYGHWLRTCLPMRKENGLIFLGGGIDWTV
TTV130RF1 (49 PVIQWNPPSRRTCLIEGFWPLSYGHWFRTCLPFRRKNGLIFTGGGCDWTQ
AY823991 Orfl (99 WSLQNLYNEKL WRNIWTAS VGMEFARFLKGKFYFFRHPWRNYIITWDQ
TTVlOOrfl (101 WSLQNLYHEKL WR VWTSSNDGMEFARFRYAKFKFFRHTTRSYWTWDQ
TTV130RF1 (99 WSLQNLYHEKL WRNIWTAS VGMEFARFLKGKFYFFRHPWRNYIVTWDQ
AY823991 Orfl (149 DIPCRPLPYQNLHPLLMLLKKQHKIVLSQQNCNPNRKQKPVTLKFKPPPK
TTVlOOrfl (151 DIPCKPLPYTNLHPFVMLLKKHHKVVLSKQDCNPRKMDKPVTLKIKPPPK
TTV130RF1 (149 DIPCKPLPYQNLHPLLMLLKKQHKLVLSQQNCNPNRKQKPVTLKFRPPPK
AY823991 Orfl (199 LTSQWRLSRELAKMPLIRLGVSFIDLTEPWVEGWGNAFYSVLGYEAVKDQ
TTVlOOrfl (201 LTSQWRLSRELSKIPLLRLGVSLIDFREPWVEGFGNAFFSTLGYEADKSN
TTV130RF1 (199 LTSQWRLSRELAKMPLIRLGVSFIDLTEPWLEGWGNAFYSVLGYEAIKEQ
AY823991 Orfl (249 GHWS WTQIKYYWIYDTGVGNAVYVILLKKDVTDNPG MATTFKASGGQH
TTVlOOrfl (251 LKTSAWCQCKYFWIYDTGV NHVYWMLNKDAGDNAGDLITNQNS
TTV130RF1 (249 GHWS WSQIKYYWIYDTGVGNAVYWMLKQDVDDNPGKMASTFKTTQGQH
AY823991 Orfl (299 PDAIDHIELINQGWPYWLYFYGKSEQDIKKEAHS-AEISREYTRDPKSKK
TTVlOOrfl (296 IAHIEQIGEGYPYWLYFFGRSERDLKALATSNTNIRNEFNTNPNSKK
TTV130RF1 (299 PNAIDHIELINEGWPYWLYFFGKSEQDIKKEAHS-AEIAREYATNPKSKK
AY823991 Orfl (348 LKIGIVGWASSNYTTTGSDQNSGG-STSAIQGGYVAYAGSG VIGAGS
TTVlOOrfl (343 LKIAVIGWASS NTAQDSTQGANTPIEGTYLISHVLQTSGH TAGAAQ
TTV130RF1 (348 LKIGIVGWASSNFTTPGSSQNSGG-NIAAIQGGYVAWAGGQGKLNLGAGS
AY823991 Orfl (394 IGNLYQQGWPSNQ WPNTNRDKTNFDWGIRGLCILRD MHLGSQELDDEC
TTVlOOrfl (390 I NLFASGWPNSQNYPPLNLDK NFDWGKRALCILR MKIGNQNLDDET
TTV130RF1 (397 IGNLYQQGWPSNQ WPNTNRDETNFDWGLRSLCILRD MQLGNQELDDEC
AY823991 Orfl (444 TMLTLFGPFVEKANPIFATTDPKFFKPELKDYNIIMKYAFKFQWGGHGTE
TTVlOOrfl (440 TMFALFGPLVEKAN-WEGLEKIPELKPELKDYNILMRYNFRFQWGGHGTE
TTV130RF1 (447 TMLSLFGPFVEKANPIFATTDPKYFKPELKDYNLIMKYAFKFQWGGHGTE
AY823991 Orfl (494 RFKTNIGDPSTIPCPFEPGDRFHSGIQDPSKVQNTVLNPWDYDCDGIVRK
TTVlOOrfl (489 TFKTSIGDPSQIPCPYGPGEAPQHLVRNPSKVHEGVLNAWDYDYDGIVRK
TTV130RF1 (497 RFKTTIGDPSTIPCPFEPGDRFHSGIQDPSKVQNTVLNPWDYDCDGIVRK
AY823991 Orfl (544 DTLKRLLELPTETEEEEKAYPLLGQKTEKEPLSDSDEESVISSTSSGSSQ
TTVlOOrfl (539 DTLKRLLAIPTDSEEE-KAYPLAGPKTEKLPSSDEEGESDISSSSDSSTQ
TTV130RF1 (547 DTLKRLLELPTETEEEEKAYPLLGQKTEKEPLSDSDEESVISSTSSGSDQ
AY823991 Orfl (594 EEETQR--RRHHKPSKRRLLKHLQRWKRMKTL--
TTVlOOrfl (588 ESEEEKRYRRRHKPSKRRLLQHVQRLVKRFRTL--
TTV130RF1 (597 EEETQR--RKHHKPSKRRLLKHLQRWKRMKTL-- Amino Acid Alignment of TTV10 TTV13 ORF with Published Sequence
On the amino acid level, TTV10 ORF demonstrates only 65% homology to the published sequence and may represent a unique phenotype of TTV
Cloning of TTV genotype 2 ORF1 for baculovirus expression
Based on sequence data derived above, primers were designed to clone the ORF from TTV10 and TTV13 for expression in baculovirus using the
Invitrogen Gateway® system. Primer sequences were:
For TTV13 ORF: Ttv13Rev121 1 : 5' cgt act cga gtc aca gtg ttt tea tec (SEQ ID NO:26); TTV13For121 1 : 5' eta ggt acc atg cct tac aga cgc tat (SEQ ID NO:27)
For TTV10 ORF: tt10for1207: 5' eta ggt acc atg cct ttc cac cgc tat (SEQ ID NO:28) and ttvrev1207: cgt act cga get ata ggg tec tga at (SEQ ID NO:29)
Since the EcoRI cloning into pGem resulted in interrupting the reading frame of the ORF1 , the TTV insert in pGem was isolated by EcoRI digestion, gel- purified and re-circularized using standard ligation conditions. Following an overnight ligation at 4°C, ligase was inactivated at 65°C, and the reaction was purified using QuiQuick purification kit (Qiagen) following the manufacturer's protocol.
TTVORF13 was the PCR amplified using re-circularized TTV13 genomic DNA with Expand Hi-Fidelity® enzyme (Roche) using the above described TTV13 forward and reverse primers (0.15μΜ each), 0.2 mM dNTP's in 1 X Hi Fidelity enzyme buffer. PCR conditions were: 1 cycle at 4 min, 95°C; 35 cycles with 94°C, 15 sec denaturation, 55°C, 30 sec anneal, and 68°C 1 .5 min extension; and 1 cycle of 72°C, 7 min extension.
Similarly, TTVORF10 was PCR amplified using re-circularized TTV10 genomic DNA with Expand Hi-Fidelity® enzyme (Roche) using the above described TTV10 forward and reverse primers (0.15μΜ each) 0.2 mM dNTP's in 1 X Hi Fidelity enzyme buffer. PCR conditions were: 1 cycle at 4 min, 95®°C; 35 cycles with 94°C, 15 sec denaturation, 56°C, 30 sec anneal, and 68°C 1 .5 min extension; and 1 cycle of 72°C, 7 min extension.
PCR products were purified using QiaQuick PCR purification kit (Qiagen) following the manufacturer's protocol. Both PCR TTV10Orf1 anfd TTV130rf1 products and the Gateway entry plasmid, pENTR3C, were digested with Kpnl. Digested DNA was purified using QIAquick PCR amplification kit and
subsequently digested with Xhol . Following QIAquick purifications, the TTV10 ORF1 or the TTV130RF1 DNAs were ligated into pENTR3C using standard ligation procedures. Following a 2 hour ligation at room temperature, ligated DNA was used to transform chemically competent E. coli DH5a. Transformed colonies were selected using Kanamycin. Plasmid was purified from transformed E.coli and ORF1 DNA insertion was verified by restriction fragment analysis. pENTR3C plasmids containing TTV10 ORF1 or TTV13 ORF1 were then inserted into Invitrogen destination vectors pDESTI O or pDEST 20 encoding a His6X or a GST protein N-terminal to the TTV Orf1 reading frame. Recombinant pDEST vectors containing the open reading frame of TTV Orf1 were used to transform DHI OBac E. coli. Recombinant bacmid DNA was isolated and used for transfection of SF9 cells following standard protocol. Recombinant baculovirus containing the native Orf1 were isolated by plaque purification.
Confirmation of recombinant baculovirus was performed using PCR. Native TTVOrfl construction for baculovirus expression.
Standard PCR was used to incorporate a BamH1 restriction site upstream from the initiation codon in TTV10 Orf1 or an Xbal restriction site upstream from the initiation codon in TTV Orf13. These constructs were cloned into pFastBac transfer vector and used to transform E. coli DHI OBac. The resultant
recombinant bacmids were subsequently used to transfect SF9 cells.
Recombinant baculovirus containing the native Orf1 were isolated by plaque purification. Confirmation of recombinant baculovirus was performed using PCR.
Cloning of TTV genotype 2 ORF1 for E. coli expression.
Full-length TTVOrfl 0 was also cloned into a PGex-6p-1 vector for expression of a GST-fusion protein in a bacterial system. The TTV ORFs contain an arginine rich amino terminus. To determine if protein production could be increased in a bacterial expression system, the arginine rich segment was removed from TTVOrfl 3 at a convenient restriction site (EcoR1 ) located at nucleotide 368 of the Orf1 open reading frame and was in frame with the GST coding region of pGex-6p-1 . This clone resulted in the removal of 100 amino- terminal amino acids containing a highly enriched arginine segment.
B. TTV genotype 1 .
Total cellular DNA from porcine bone marrow was amplified by rolling circle amplification following procedures described above, except that single- stranded binding protein was added to improve the efficiency of the amplification reaction. Amplification products were digested with EcoR1 , purified using a
QIAquick PCR purification kit (Qiagen), and ligated into pGem3zf(+) vector which had been previously treated with shrimp alkaline phosphatase. Recombinant vector containing putative TTV genomic DNA was selected based on restriction digests with EcoR1 and/or BamH1 . Plasmids containing approximately 2.7 kB inserts were purified and submitted to ACGT, Inc. for sequencing of the ORF1 sequences to confirm genotype. The complete genome, i.e. the region containing the high G/C rich region, was not sequenced to entirety.
Analyses of sequencing data for PAH TTV genotype 1
Nucleotide alignment of PAH TTV7 (SEQ ID NO:4), TTV 17 (SEQ ID NO:5), TTV21 (SEQ ID NO:6), and TTV27 (SEQ ID NO:3) with published sequence, AY823990 (SEQ ID NO: 17).
AY823990 (1 TACACTTTGGGGTTCAGGAGGCTCAATTTGGCTCGCTTCGCTCGCACCAC ttvgl-7 (1 TACACTTCCGGGTTCAGGAGGCTCAATTTGGCTCGCTTCGCTCGCACCAC ttvGTl-17 (1 TACACTTCCGGGTTCAGGAGGCTCAATTTGGCTCGCTTCGCTCGCACCAC ttvGTl-21 (1 TACACTTCCGGGTTCAGGAGGCTCAATTTGGCTCGCTTCGCTCGCACCAC ttvgtl-27 (1 TACACTTCCGGGTTCAGAGGGCTCAATTTGGCTCGCTTCGCTCGCACCAC
AY823990 (51 GTTTGCTGCCAGGCGGACCTGATTGAAGACTGAAAACCGTTAAATTCAAA ttvgl-7 (51 GTTTGCTGCCAGGCGGACCTGTTTGAAGACTGAAAACCGTTAAATTCAAA ttvGTl-17 (51 GTTTGCTGCCAAGCGGACCTGATTGAAGACTGAAAACCGTTACATTCAAA ttvGTl-21 (51 GTTTGCTGCCAGGCGGACCTGATTGAAGACTGAAAACCGTTAAATTCAAA ttvgtl-27 (51 GTTTGCTGCCAGGCGGACCTGATTGAAGACTGAAAACCGTTAAGTTCAAA
AY823990 (101 ATTGAAAAGGGCGGGCAAA-ATGGCGGACAGGGGGCGGAGTTTATGCAAA ttvgl-7 (101 TTTGAAATTGGCGGT-AAACATGGCGGAAGGGGGGCGGAGTATATGCAAA ttvGTl-17 (101 TTTGAAAATGGCGCCCAAACATGGCGGATGTGGG-CGGAGTATATGCAAA ttvGTl-21 (101 TTTGAAATTGGCGGT-AAATATGGCGGAAGGGGGGCGGAGTATATGCAAA ttvgtl-27 (101 TTTGAAAATGGCGCCCAAACATGGCGGAG-GGGGGCGGAGTTTATGCAAA
AY823990 (150 TTAATTTATGCAAAGTAGGAGGAGCTCGATTTTAATTTATGCAAAGTAGG ttvgl-7 ... (150 TTAATTTATGCAAAGTAGGAGGAGCTCGATTTTAATTTATGCAAAGTAGG ttvGTl-17... (150 TTAATTTATGCAAAGTAGGAGGAGCTCGATTTTAATTTATGCAAAGTAGG ttvGTl-21... (150 TTAATTTATGCAAAGTAGGAGGAGCTCGATTTTAATTTATGCAAAGTAGG ttvgtl-27... (150 TTAATTTATGCAAAGTAGGAGGAGCTCCATTTTAATTTATGCAAAGTAGG
AY823990 (200 AGGAGTCAAATCTGATTGGTCGGGAGCTCAAGTCCTCATTTGCATAGGGT ttvgl-7 ... (200 AGGAGTCAAATCTGATTGGTCGGGAGCTCAAGTCCTCATTTGCATAGGGT ttvGTl-17... (200 AGGAGTCACTTCTGATTGGTCGGGAACTCAAGCCCTCATTTGCATAGGGT ttvGTl-21... (200 AGGAGTCAAATCTGATTGGTCGGGAGCTCAAGTCCTCATTTGCATAGGGT ttvgtl-27... (200 AGGAGTCACTTCTGATTGGTCGGGAGCTCAAGTCCTCATTTGCATAGGGT
AY823990 (250 GTAACCAATCAGAATTAAGGCGTTCCCACGAAAGCGAATATAAGTAGGTG ttvgl-7 ... (250 GTAACCAATCAGAATTAAGGCGTGCCCACTAAAGTGAATATAAGTAAGTG ttvGTl-17... (250 GTAACCAATCAGAATTAAGGCGTTCCCCGTGAAGTGAATATAAGTAAGTA ttvGTl-21... (250 GTAACCAATCAGAATTAAGGCGTGCCCACTAAAGTGAATATAAGTGAGTG ttvgtl-27... (250 GTAACCAATCAAACTTAAGGCGTTCCCACTAAAGTGAATATAAGTAAGTG
AY823990 (300 AGGTTCCGAATGGCTGAGTTTATGCCGCCAGCGGTAGACAGAACTGTCTA ttvgl-7 ... (300 CAGTTCCGAATGGCTGAGTTTATGCCGCCAGCGGTAGACAGAACTGTCTA ttvGTl-17... (300 AAGTTCCGAATGGCTGAGTTTATGCCGCCAGCGGTAGACAGAACTGTCTA ttvGTl-21... (300 CAGTTCCGAATGGCTGAGTTTATGCCGCCAGCGGTAGACAGAACTGTCTA ttvgtl-27... (300 CGGTTCCGAATGGCTGAGTTTATGCCGCCAGCGGTAGACAGAACTGTCTA AY823990 350 GCGACTGGGCGGGTGCCGGAGGATCCCTGATCCGGAGTCAAGGGGCCTAT ttvgl-7 ... 350 GCGACTGGGCGGGTGCCGGAGGATCCCAGATCCGGAGTCAAGGGGCCTAT ttvGTl-17... 350 GCGACTGGGCGGGTGCCGAAGGATCCCAGATCCGGAGTCAAGGGGCCTAT ttvGTl-21... 350 GCGACTGGGCGGGTGCCGGAGGATCCCAGATCCGGAGTCAAGGGGCCTAT ttvgtl-27... 350 GCGACTGGGCGGGTGCCGGAGGATCCCTGATCCGGAGTCAAGGGGCCTAT
AY823990 400 CGGGCAGGAGCAGCTAGGCGGAGGGCCTATGCCGGAACACTGGGAGGAAG ttvgl-7 ... 400 CGGGCAGGAGCAGCTGAGCGGAGGGCCTATGCCGGAACACTGGGAGGAGG ttvGTl-17... 400 CGGGCAGGAGCAGCTGAGCGGAGGGCCTATGCCGGAACACTGGGAGGAGG ttvGTl-21... 400 CGGGCAGGAGCAGCTGAGCGGAGGGCCTATGCCGGAACACTGGGAAGAGG ttvgtl-27... 400 CGGGCAGGAGCAGCTGAGCGGAGGGCCTATGCCGGAACACTGGGAAGAAG
AY823990 450 CCTGGTTGGAAGCTACCAAGGGCTGGCACGATCTCGACTGCCGCTGCGGT ttvgl-7 ... 450 CCTGGTTGGAAGCTACCAAGGGCTGGCACGACCTTGACTGCCGCTGCGGT ttvGTl-17... 450 CCTGGTTGGAAGCTACCAAGGGCTGGCACGACCTCGACTGCCGCTGCGGT ttvGTl-21... 450 CCTGGTTGGAAGCTACCAAGGGCTGGCACGACCTTGACTGCCGCTGCGGT ttvgtl-27... 450 CCTGGTTGGAAGCTACCAAGGGCTGGCACGACTTAGACTGCCGCTGCGGT
AY823990 500 AACTGGCAGGACCACCTATGGCTCCTACTCGCCGATGGAGACGCCGCTTT ttvgl-7 ... 500 AATTGGCAAGACCACCTATGGCTTTTGCTCGCCGATGGAGACGCCGCTTT ttvGTl-17... 500 AACTGGCAAGACCACCTATGGCTCCTGCTCGCCGATGGAGACGCGGCTTT ttvGTl-21... 500 AATTGGCAAGACCACCTATGGCTTTTGCTCGCCGATGGAGACGCCGCTTT ttvgtl-27... 500 AACTGGCAGGACCACCTATGGCTCCTACTCGGCGATGGAGACGCCGCTTT
AY823990 550 GGCCGCCGCCGTAGACGCTATAGAAAGAGACGCTATGGCTGGAGACGACG ttvgl-7 ... 550 GGCCGCCGCCGTAGACGCTATAGAAAGAGACGCTATGGATGGAGGAGACG ttvGTl-17... 550 GGCCGCCGCCGTAGACGCTATAGAAAGAGACGCTGGGGCTGGAGAAGGCG ttvGTl-21... 550 GGCCGCCGCCGTAGACGCTATAGAAAGAGACGCTATGGATGGAGGAGACG ttvgtl-27... 550 GGCCGCCGCCGTAGACGCTATAGAAAGAGACGCTATGGCTGGAGAAGACG
AY823990 600 CTACTACCGCTACAGGCCGCGTGACTATCGGCGACGATGGCTGGTAAGGA ttvgl-7 ... 600 CTACTACCGCTACAGACCGCGTTACTATCGGAGACGATGGCTGGTAAGGA ttvGTl-17... 600 CTACTGGAGATACCGACCGCGTTACCGTCGGCGCAGATGGCTGGTAAGGA ttvGTl-21... 600 CTACTACCGCTACAGACCGCGTTACTATCGGAGACGATGGCTGGTAAGGA ttvgtl-27... 600 CTACTACCGCTACAGACCGCGTTACTATCGGAGACGATGGCTGGTAAGGA
AY823990 650 GAAGGCGGCGTTCCGTCTACCGTAGAGGTGGACGTAGAGCGCGCCCCTAC ttvgl-7 ... 650 GAAGGCGGCGTTCCGTCTACCGACGAGGTGGACGTAGAGCGCGCCCCTAC ttvGTl-17... 650 GAAGGCGGCGTTCCGTCTACCGAAGAGGTGGACGTAGAGCGCGCCCCTAC ttvGTl-21... 650 GAAGGCGGCGTTCCGTCTACCGACGAGGTGGACGTAGAGCGCGCCCCTAC ttvgtl-27... 650 GAAGGCGGCGTTCCGTCTACCGTAGAGGTGGACGTAGAGCGCGCCCCTAC
AY823990 700 CGA CTG--TTTAATCCAAAAGTAATGCGGAGAGTAGTAATTAGGGG ttvgl-7 ... 700 CGCATTTCTGCCTTTAATCCGAAAGTAATGCGTAGAGTAGTGATTAGAGG ttvGTl-17... 700 CGTATTTCTGCTTTTAATCCAAAAATAATGCGGAGAGTAGTAATAAGGGG ttvGTl-21... 700 CGCATTTCTGCCTTTAATCCGAAAGTAATGCGTAGAGTAGTGATTAGAGG ttvgtl-27... 700 CGGGTATCTGCCTTTAACCCCAAAGTAATGCGGAGAGTAGTAATAAGGGG
AY823990 744 GTGGTGGCCTATTTTACAATGCTTAAAAGGACAGGAGGCACTAAGATATA ttvgl-7 ... 750 GTGGTGGCCAATACTGCAGTGCCTAAAAGGTCAGGAATCACTAAGATACA ttvGTl-17... 750 ATGGTGGCCAATCCTACAATGTCTAAGAGGACAGGAATCACTAAGATATA ttvGTl-21... 750 GTGGTGGCCAATACTGCAGTGCCTAAAAGGTCAGGAATCACTAAGATACA ttvgtl-27... 750 GTGGTGGCCAATACTACAGTGCTTAAAAGGACAGGAATCGCTGAGATATA AY823990 (794 GACCTCTACAGTGGGACACAGAGAGACAGTGGAGAGTGAGATCAGACTTC ttvgl-7 ... (800 GACCACTTCAGTGGGACGTAGAGAAAAGCTGGAGAATAAACACAACTCTT ttvGTl-17... (800 GACCGTTACAGTGGGACGTAGAAAAAAGCTGGAGAATAAAGACAGACTTA ttvGTl-21... (800 GACCACTTCAGTGGGACGTAGAGAAAAGCTGGAGAATAAACACAACTCTT ttvgtl-27... (800 GACCACTACAGTGGGACACAGAAAGACAGTGGAGAGTGAGACAAGACTTC
AY823990 (844 GAAGACCAGTACGGATACCTCGTACAATACGGGGGAGGTTGGGGAAGTGG ttvgl-7 ... (850 GAGGACAACTATGGATACTTAGTACAGTATGGAGGTGGTTGGGGTAGCGG ttvGTl-17... (850 GAAGACAACTACGGCTACTTAGTACAGTACGGAGGAGGTTGGGGGAGCGG ttvGTl-21... (850 GAGGACAACTATGGATACTTAGTACAGTATGGAGGTGGTTGGGGTAGCGG ttvgtl-27... (850 GAGGATCAATACGGATACCTGGTGCAATACGGTGGAGGTTGGGGAAGTGG
AY823990 (894 TGATGTGACACTTGAAGGTCTCTACCAAGAGCACTTATTGTGGAGAAACT ttvgl-7 ... (900 AGAGGTAACACTGGAGGGGCTGTATCAGGAGCACCTACTATGGAGAAACT ttvGTl-17... (900 AGAGGTGACTCTAGAAGGACTGTACCAGGAACACCTACTATGGAGAAATT ttvGTl-21... (900 AGAGGTAACACTGGAGGGGCTGTATCAGGAGCACCTACTATGGAGAAACT ttvgtl-27... (900 TGATGTGACACTAGAGGGACTATACCAGGAACACTTACTATGGAGAAATT
AY823990 (944 CTTGGTCTAAAGGAAACGATGGAATGGACCTAGTAAGATACTTTGGATGT ttvgl-7 ... (950 CTTGGTCAAAAGGAAACGATGGGATGGACTTAGTGAGATACTTCGGCTGC ttvGTl-17... (950 CATGGTCAAAAGGAAATGATGGAATGGATCTAGTAAGATACTTCGGCTGC ttvGTl-21... (950 CTTGGTCAAAAGGAAACGATGGGATGGACTTAGTGAGATACTTCGGCTGC ttvgtl-27... (950 CCTGGTCAAAAGGAAATGATGGCATGGACTTAGTGAGATACTTTGGCTGT
AY823990 (994 GTAGTATACCTATATCCACTAAAGGACCAGGACTATTGGTTCTGGTGGGA ttvgl-7 ... (1000 ATAGTATATCTATATCCGTTAAAAGATCAAGACTACTGGTTTTGGTGGGA ttvGTl-17... (1000 ATAGTATACCTGTACCCACTGAAAGATCAGGACTACTGGTTTTGGTGGGA ttvGTl-21... (1000 ATAGTATATCTATATCCGTTAAAAGATCAGGACTACTGGTTTTGGTGGGA ttvgtl-27... (1000 GTGGTATACCTCTACCCACTTAAAGATCAGGACTATTGGTTCTGGTGGGA
AY823990 (1044 CACGGACTTCAAAGAATTATATGCAGAAAACATAAAGGAATACAGCCAAC ttvgl-7 ... (1050 CACAGATTTTAAAGAATTATATGCAGAGAGTATCAAAGAATACTCACAGC ttvGTl-17... (1050 CACAGACTTTAAGGAACTCTATGCAGAAAGTATTAAGGAGTACTCACAAC ttvGTl-21... (1050 CACAGATTTTAAGGAATTATATGCAGAGAGTATCAAAGAATACTCACAGC ttvgtl-27... (1050 CACTGACTTTAAAGAGCTATACGCAGAAAACATAAAAGAATACAGCCAAC
AY823990 (1094 CATCAGTAATGATGATGGCAAAAAGAACAAGAATAGTAATAGCCAGAGAA ttvgl-7 ... (1100 CATCTGTAATGATGATGGCAAAAAGAACAAAAATAGTGATCGCAAGAAGT ttvGTl-17... (1100 CATCAGTAATGATGATGGCAAAAAAAACAAAAATTGTAATAGCGAGAAGT ttvGTl-21... (1100 CATCTGTAATGATGATGGCAAAAAGAACAAAAATAGTGATCGCAAGAAGT ttvgtl-27... (1100 CATCAGTAATGATGATGGCAAAAAGAACTAGAATAGTAATAGCGAGAGAC
AY823990 (1144 AGGGCACCACATAGAAGAAAAGTAAGAAAAATATTTATTCCGCCACCTTC ttvgl-7 ... (1150 AGAGCCCCACATAGAAGGAAGGTACGCAGAATTTTCATACCGCCTCCAAG ttvGTl-17... (1150 AGGGCACCACACAGACGAAAAGTAAGAAAAATATTCATACCGCCACCAAG ttvGTl-21... (1150 AGAGCCCCACATAGAAGGAAGGTACGCAGAATTTTCATACCGCCTCCAAG ttvgtl-27... (1150 AGAGCTCCACATAGAAGAAAAGTGAGAAAAATATTCATCCCACCACCATC
AY823990 (1194 GAGAGACACAACACAGTGGCAGTTTCAGACAGATTTCTGCAATAGAAAGT ttvgl-7 ... (1200 TAGAGACACGACACAGTGGCAATTTCAAACTGACTTTTGCAATAGACCAC ttvGTl-17... (1200 TAGAGACACTACACAATGGCAATTTCAAACAGAGTTCTGCAACAAACCAC ttvGTl-21... (1200 TAGAGACACGACACAGTGGCAATTTCAAACTGACTTTTGCAATAGACCAC ttvgtl-27... (1200 AAGAGACACTACGCAGTGGCAGTTTCAGACAGACTTCTGTAATAGGAAGC AY823990 1244 TATTTACGTGGGCAGCTGGTCTAATAGACATGCAAAAACCGTTCGATGCT ttvgl-7 ... 1250 TATTCACATGGGCTGCAGGACTCATAGACCTCCAAAAACCATTTGACGCA ttvGTl-17... 1250 TATTCACTTGGGCTGCAGGACTAATAGACCTCCAAAAGCCATTTGACGCA ttvGTl-21... 1250 TATTCACATGGGCTGCAGGACTCATAGACCTCCAAAAACCATTTGACGCA ttvgtl-27... 1250 TATTTACCTGGGCGGCAGGACTAATAGACATGCAAAAACCCTTTGATGCC
AY823990 1294 AATGGAGCCTTTAGAAATGCTTGGTGGCTGGAACAGAGAAATGATCAGGG ttvgl-7 ... 1300 AACGGTGCGTTCAGAAATGCCTGGTGGTTAGAACAGAGAAACGAGGCAGG ttvGTl-17... 1300 AACGGAGCTTTTAGAAATGCGTGGTGGTTAGAACAGAGAAATGAGGCAGG ttvGTl-21... 1300 AACGGTGCGTTCAGAAATGCCTGGTGGTTAGAACAGAGAAACGAGGCAGG ttvgtl-27... 1300 AACGGAGCTTTTAGAAATGCGTGGTGGCTGGAGCAGAGAACGGAACAGGG
AY823990 1344 AGAAATGAAATACATAGAACTGTGGGGAAGAGTACCCCCACAAGGAGATT ttvgl-7 ... 1350 AGAAATGAAATACATAGAGCTATGGGGTAGAGTACCACCCCAGGGGGACA ttvGTl-17... 1350 AGAGATGAAATACATAGAATTATGGGGGAGAGTCCCACCGCAAGGAGACA ttvGTl-21... 1350 AGAAATGAAATACATAGAGCTATGGGGTAGAGTACCACCCCAGGGGGACA ttvgtl-27... 1350 TGAAATGAAGTACATAGAACTGTGGGGAAGAGTGCCCCCACAAGGAGACT
AY823990 1394 CAGAGCTGCCCAAAAAAAAAGAATTCTCCACAGGAACAG ATAACCCA ttvgl-7 ... 1400 CGGAATTACCCGTTCAAACAGAATTCCAAAAACCCTCGGGATATAACCCA ttvGTl-17... 1400 CAGAATTGCCGGCCCAAAAAGAATTCCAGAAACCAGACGGGTATAACCCA ttvGTl-21... 1400 CGGAATTACCCCTTCAAACAGAATTCCAAAAACCCTCGGGATATAACCCA ttvgtl-27... 1400 CAGAACTACCCAAGAAAAGTGAATTCACAACAGCTACAG ACAATAAA
AY823990 1441 AACTACAATGTTCAGGACAATGAGGAGAAAAACATATACCCCATTATAAT ttvgl-7 ... 1450 AAATACTACGTAAACCCGGGGGAGGAAAAACCAATCTACCCAGTAATAAT ttvGTl-17... 1450 AAATACTATGTGCAGGCAGGAGAGGAAAAACCTATATATCCAATAATAAT ttvGTl-21... 1450 AAATACTACGTAAACCCGGGGGAGGAAAAACCAATCTACCCAGTAATAAT ttvgtl-27... 1447 AACTACAATGTGAATGACGGTGAGGAAAAACCTATATACCCCATAATTAT
AY823990 1491 ATACGTAGACCAAAAAGATCAAAAACCAAGAAAAAAGTACTGCGTATGTT ttvgl-7 ... 1500 ATACGTAGACATGAAAGACCAAAAACCAAGAAAAAAGTACTGCGTCTGCT ttvGTl-17... 1500 TTACGTAGACAAAAAAGATCAGAAAGCAAGAAAGAAATACTGTGTCTGTT ttvGTl-21... 1500 ATACGTAGACATGAAAGACCAAAAACCAAGAAAAAAGTACTGCGTCTGCT ttvgtl-27... 1497 ATACGTAGACCAAAAAGACCAAAAACCAAGGAAAAAGTACTGTGTATGTT
AY823990 1541 ATAATAAGACCCTCAACAGATGGAGACTAGGACAGGCAAGTACTCTAAAG ttvgl-7 ... 1550 ACAACAAGACGCTTAACAGGTGGCGCAGCGCTCAAGCAAGCACATTAAAA ttvGTl-17... 1550 ACAATAAGACACTAAACAGATGGAGAGCAGCACAAGCAAGTACCCTAAAA ttvGTl-21... 1550 ACAACAAGACGCTTAACAGGTGGCGCAGCGCTCAGGCAAGCACATTAAAA ttvgtl-27... 1547 ACAACAAAACTCTGAACAGGTGGAGATTAGGACAAGCGAGTACTCTAAAA
AY823990 1591 ATAGGAAACCTGAAAGGACTAGTACTAAGACAGCTGATGAATCAAGAAAT ttvgl-7 ... 1600 ATTGGTGACTTGCAGGGGCTAGTATTGAGACAGCTAATGAACCAAGAAAT ttvGTl-17... 1600 ATAGGAGACCTGCAAGGACTAGTACTAAGACAATTAATGAACCAGGAAAT ttvGTl-21... 1600 ATTGGTGACTTGCAGGGGCTAGTATTGAGACAGCTAATGAACCAAGAAAT ttvgtl-27... 1597 ATAGGAAACCTGAAAGGACTAGTGCTAAGACAGTTGATGAACCAAGAGAT
AY823990 1641 GACGTATATATGGAAAGAAGGAGAATACAGTGCCCCCTTTGTACAAAGGT ttvgl-7 ... 1650 GACATACACATGGAAAGAAGGAGAATTTACCAATGTATTCCTGCAGAGGT ttvGTl-17... 1650 GACATATATTTGGAAAGAGGGAGAGTTCACAAACGTATTCCTGCAAAGGT ttvGTl-21... 1650 GACATACACATGGAAAGAAGGAGAATTTACAAATGTATTCCTGCAAAGGT ttvgtl-27... 1647 GACTTACATATGGAAGGAAGGAGAGTACAGCTCACCATTTGTACAAAGGT AY823990 (1691) GGAAAGGCAGCAGATTCGCTGTGATAGACGCAAGAAAGGCAGACCAAGAA ttvgl-7 ... (1700) GGAGAGGTTTCAGATTAGCAGTAATAGACGCAAGAAAGGCAGACACAGAA ttvGTl-17... (1700) GGAAAGGCTTCAGACTAGCAGTCATAGACGCCAGAAAGGGAGACACAGAA ttvGTl-21... (1700) GGAGAGGTTTCAGATTAGCAGTAATAGACGCTAGAAAGGCAGACACAGAA ttvgtl-27... (1697) GGAAAGGAAGCAGATTTGTTGTGATAGACGCAAGAAAGGCTGACCAGGAA
AY823990 (1741 AACCCGAAAGTATCAACATGGCCAATTGAGGGAACGTGGAACACACAGGA ttvgl-7 ... (1750 AACCCGACAGTCCAAACTTGGAAGGTGGACGGACAGTGGAACACACAAGG ttvGTl-17... (1750 AATCCAACAGTACAAACATGGAAAGTAGACGGAAACTGGAACACTAGTGG ttvGTl-21... (1750 AACCCGACAGTCCAAACTTGGAAGGTGGACGGACAGTGGAACACACAAGG ttvgtl-27... (1747 AATCCCAAAGTATCTACATGGCCAATAGAGGGAGTGTGGAACACACAGGG
AY823990 (1791 CACAGTACTGAAGGATGTATTCGGTATTAACTTGCAAAATCAACAATTTA ttvgl-7 ... (1800 GACAGTGCTTAAAGAGGTTTTCAATATAAACCTGAATAATGAACAGATGA ttvGTl-17... (1800 AACAGTACTACAAGAAGTGTTCGGCATAAACCTCACCCAACAACAAATGA ttvGTl-21... (1800 GACAGTTCTTAAAGAGGTTTTCAATATAAACCTGAATAATGAACAGATGA ttvgtl-27... (1797 TACAGTACTTAAGGATGTATTCCAGATTGACTTAAACAGTACTAATTTCA
AY823990 (1841 GGGCGGCGGACTTTGGTAAACTCACACTACCAAAATCACCGCATGACTTA ttvgl-7 ... (1850 GACAGGCAGACTTTGGAAAACTAAACTTACCAAAATCCCCGCACGACATT ttvGTl-17... (1850 GGGCATCGGACTTTGCTAAGCTAACACTACCAAAATCGCCACATGACATT ttvGTl-21... (1850 GACAGGCAGACTTTGGAAAACTAAACTTACCAAAATCCCCGCACGACATT ttvgtl-27... (1847 GAGCGGCAGACTTTGGAAAACTAACACTACCAAAATCACCGCACGACTTA
AY823990 (1891 GACTTCGGTCACCACAGCAGATTTGGGCCATTTTGTGTGAAAAATGAACC ttvgl-7 ... (1900 GACTTTGGACACCACAGTAGATTTGGACCTTTCTGTGTAAAAAACGAACC ttvGTl-17... (1900 GACTTTGGACACCACAGTAGATTTGGGCCATTTTGTGTCAAAAACGAACC ttvGTl-21... (1900 GACTTTGGACACCACAGTAGATTTGGACCTTTCTGTGTAAAAAACGAACC ttvgtl-27... (1897 GACTTCGGACATCACAGTAGATTCGGACCATTCTGTGTGAAAAATGAACC
AY823990 (1941 ACTGGAGTTTCAGGTATACCCTCCAGAACCAACTAACTTGTGGTTTCAGT ttvgl-7 ... (1950 ACTGGAGTTTCAACTAACAGCCCCAGAGCCAACTAACCTGTGGTTTCAGT ttvGTl-17... (1950 GCTGGAGTTTCAACTAACCGCTCCAGAACCTATTAATCTTTGGTTTCAGT ttvGTl-21... (1950 ACTGGAGTTTCAACTAACAGCCCCAGAGCCAACTAACCTGTGGTTTCAGT ttvgtl-27... (1947 ACTGGAATTTCAGGTATACCCGCCAGAACCCACTAACCTGTGGTTTCAGT
AY823990 (1991 ACAGATTTTTCTTTCAGTTTGGAGGTGAATACCAACCCCCCACAGGAATC ttvgl-7 ... (2000 ACAAATTTCTGTTTCAGTTTGGAGGTGAATACCAACCACCAACAGGCATC ttvGTl-17... (2000 ACAAATTTCTCTTTCAGTTTGGAGGTGAATACCAACCACCAACAGGCATC ttvGTl-21... (2000 ACAAATTTCTGTTTCAGTTTGGAGGTGAATACCAACCACCAACAGGCATC ttvgtl-27... (1997 ACAGATTTTTCTTTCAGTTTGGAGGTGAATACCAACCCCCCACAGGAATC
AY823990 (2041 CGGGATCCATGCGTTGATACACCAGCCTATCCTGTGCCGCAGTCAGGAAG ttvgl-7 ... (2050 CGCGATCCCTGCGCTGATAACCCAGCCTATCCTGTGCCGCAGTCAGGAAG ttvGTl-17... (2050 CGCGATCCCTGCGCTGATAACCAACCCTATCCTGTGCCGCAGTCAGGAAG ttvGTl-21... (2050 CGCGATCCCTGCGCTGATAACCCAGCCTATCCTGTGCCGCAGTCAGGAAG ttvgtl-27... (2047 CGCGATCCATGCGTTGATACACCAGCCTATCCTGTGCCGCAGTCAGGAAG
AY823990 (2091 TATTACACACCCCAAATTCGCCGGAAAAGGAGGAATGCTCACGGAAACAG ttvgl-7 ... (2100 TATTACACACCCCAAATTCGCCGGAAAAGGCGGCATGCTCACGGAAACAG ttvGTl-17... (2100 TATTACACACCCAAAATTCGCCGGGAAAGGAGGAATGCTCACGGAAACAG ttvGTl-21... (2100 TATTACACACCCCAAATTCGCCGGAAAAGGCGGCATGCTCACGGAAACAG ttvgtl-27... (2097 TATTACACACCCCAAATTCGCCGGAAAAGGCGGAATGCTCACGGAAACAG AY823990 (2141 ACCGTTGGGGTATCACTGCTGCCTCTTCCAGAGCCCTCAGTGCAGATACA ttvgl-7 ... (2150 ACCGTTGGGGTATCACTGCTGCCTCTTCCCGAACCCTCAGTGCAGATACA ttvGTl-17... (2150 ACCGTTGGGGTATCACTGCTGCCTCTTCCAGAGCCCTCAGTGCAGATACA ttvGTl-21... (2150 ACCGTTGGGGTATCACTGCTGCCTCTTCCCGAGCCCTCAGTGCAGATACA ttvgtl-27... (2147 ACCGTTGGGGTATCACTCCTGCCTCTACCAGAGCCCTCTGTGCAGATACA
AY823990 (2191 CCCACAGAGGCAGCGCAAAGTGCACTTCTCCGAGGGGACTCGGAAGCGAA ttvgl-7 ... (2200 CCCACGGAAGCAACGCAAAGTGCACTTCTCCGAGGGGACTCGGAAAAGAA ttvGTl-17... (2200 CCCACGGAGGCAGCGCAAAGTGCACTTCTCCGAGGGGACTCGGAAAAGAA ttvGTl-21... (2200 CCCACGGAAGCAACGCAAAGTGCACTTCTCCGAGGGGACTCGGAAAAGAA ttvgtl-27... (2197 CCCACAGAAGCAACGCAGAGTGCACTTCTCCGAGGGGACTCGGAAAAGAA
AY823990 (2241 AGGAGAGGAAACCGAGGAAACCGCGTCATCGTCCAGTATCACGAGTGCCG ttvgl-7 ... (2250 AGGAGAGGAAACCGAGGAAACCTCGTCATCGTCCAGTATCACGAGTGCCG ttvGTl-17... (2250 AGGAGAGGAAACCGAGGAAACCACGTCATCGTCCAGTATCACGAGTGCCG ttvGTl-21... (2250 AGGAGAGGAAACCGAGGAAACCTCGTCATCGTCCAGTATCACGAGTGCCG ttvgtl-27... (2247 AGGAGAGGAAACCGAGGAAACCACGTCATCGTCCAGTATCACGAGTGCCG
AY823990 (2291 AAAGCTCTACTGAGGGAGATGGATCGTCTGATGATGAAGAGACAATCAGA ttvgl-7 ... (2300 AAAGCTCTACTGAAGGAGATGGATCGTCTGATGATGAAGAGACAATCAGA ttvGTl-17... (2300 AAAGCTCTACTGAAGGAGATGGATCGTCTGATGATGAAGAGACAATCCGA ttvGTl-21... (2300 AAAGCTCTACTGAAGGAGATGGATCGTCTGATGATGAAGAGACAATCAGA ttvgtl-27... (2297 AAAGCTCTACTGAGGGAGATGGATCGTCTGATGATGAAGAGACAGTCAGA
AY823990 (2341 CGCAGAAGGAGGACCTGGAAGCGACTCAGACGAATGGTCAGAGAGCAGCT ttvgl-7 ... (2350 CGCCGAAGGAGGACCTGGAAGCGACTCAGACGGATGGTCCGAGAGCAGCT ttvGTl-17... (2350 CGCAGAAGGAGGACCTGGAAGCGACTCCGACGAATGGTCAGAGAGCAGCT ttvGTl-21... (2350 CGCCGAAGGAGGACCTGGAAGCGACTCAGACGGATGGTCCGAGAGCAGCT ttvgtl-27... (2347 CGCCGAAGGAGGACCTGGAAGCGACTCAGACGAATGGTCCGAGAGCAGCT
AY823990 (2391 TGACCGACGAATGGACCACAAGCGACAGCGACTTCATTGACACCCCCATA ttvgl-7 ... (2400 TGACCGACGAATGGACCACAAGCGACAGCGACTTCATTGACACCCCCATT ttvGTl-17... (2400 TGACCGACGAATGGACCACAAGCGACAGCGACTTCATTGACACCCCCATA ttvGTl-21... (2400 TGACCGACGAATGGACCACAAGCGACAGCGACTTCATTGACACCCCCATT ttvgtl-27... (2397 TGACCGACGAATGGACCACAAGCGACAGCGACTTCATTGACACCCCCATT
AY823990 (2441 AGAGAAAGATGCCTCAATAAAAAACAAAAGAAACGCTAAACAGTGTCCGA ttvgl-7 ... (2450 AAACAGAGATGCCTCAATAAAAAACAAAAGAAACGCTAAGCAGTGTCC-C ttvGTl-17... (2450 AGAGAACGATGCCTGAATAAAAAACAAAAAAAACGCTACACAGTGTCCGC ttvGTl-21... (2450 AGACAGAGATGCCTCAATAAAAAGCAAAAGAAACGCTAAACAGTGTCC-C ttvgtl-27... (2447 AGAGACAGATGCCTCAATAAAAAGCAAAAGAAACGCTAAACTGCCTCCGC
AY823990 (2491 TTACTAATGGGGGGGGGTCCGGGGGGGGCTTGCCCCCCCGCAAGCTGGGT ttvgl-7 ... (2499 TATTATTTTGGGGGG--TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGGGT ttvGTl-17... (2500 TTATTTGTAGGGGGGG-TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGGGT ttvGTl-21... (2499 TATTACTTTGGGGGGG-TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGTGT ttvgtl-27... (2497 TTATTTTTTGGGGGG--TCCGGGGGGGGCTTGCCCCCCCGAAAGCTGGGT
AY823990 (2541 TACCGCACTAACTCCCTGCCAAGTGAAACTCGGGGACGAGTGAGTGCGGG ttvgl-7 ... (2547 TACCGCACTAACTCCCTGCCAAGTGAAACTCGGGGACGAGTGAGTGCGGG ttvGTl-17... (2549 TGCCGCACTAACTCCCTGCCAAGTGAAACTCGGGGACGAGTGAGTGCGGG ttvGTl-21... (2548 TACCGCACTAACTCCCTGCCAAGTGAAACTCGGGGACGAGTGAGTGCGGG ttvgtl-27... (2545 TACCGCACTAACTCCCTGCCAAGTGAAACTCGGGGACGAGTGAGTGCGGG AY823990 (2591 ACATCCCGTGTAATGGCTACATAACTACCCGGCTTTGCTTCGACAGTGGC ttvgl-7 ... (2597 ACATCCCGTGTAATGGCTACATAACTACCCGGCTTTGCTTCGACAGTGGC ttvGTl-17... (2599 ACATCCCGTGTAATGGCTACATAACTACCCGGCTTTGCTTCGACAGTGGC ttvGTl-21... (2598 ACATCCCGTGTAATGGCTACATAACTACCCGGCTTTGCTTCCACAGTGGC ttvgtl-27... (2595 ACATCCCGTGTAATGGCTACATAACTACCCGGCTTTGCTTCGACAGTGGC
AY823990 (2641 CGTGGCTCGACCCTCACACAACACTGCAGGTAGGGGGCGCAATTGGGATC ttvgl-7 ... (2647 CGTGGCTCGACCCTCACACAACACTGCAGGTAGGGGGCGCAATTGTGATC ttvGTl-17... (2649 CGTGGCTCGACCCTCACACAACAATGCAGGTAGGGGGCGCAATTGGGATC ttvGTl-21... (2648 CGTGGCTCGACCCTCACACAACACTGCAGGTAGGGGGCGCAATTGGGATC ttvgtl-27... (2645 CGTGGCTCGACCCTCACACAACACTGCAGATAGGGGGCGCAATTGGGATC
AY823990 (2691 GTTAGAAAACTATGGCC--GAGCATGGGGG NCCAACC ttvgl-7 ... (2697 GTTAGAAAACTATGGCCCGGAGCATGG-CCCCCCAAAC CCCCCC ttvGTl-17... (2699 GTTAGAAAACTATGGCCCG-AGCATGGGCCCCCCAAAA CCCCCC ttvGTl-21... (2698 GTTAGAAAACTATGGCCCCAAGCATGG-CCCA--AAAC CCCCCC ttvgtl-27... (2695 GTTAGAAAACTATGGCC--GAGCATGGGCCCCCACAAA CCCCCCC
AY823990 (2739 CCCCCGGTGGGGGGGCCAAGGCCCCCCCTACACCCCCCCATGGGGGGCTG ttvgl-7 ... (2740 TTGCCCGGGGCTGTGCCCCGGACCCCC
ttvGTl-17... (2742 TTGCCCGGGGCTGTGCCCCGGACCCCC
ttvGTl-21... (2739 TT-CCCGGGGCTGTGCCCCGGACCCCC
ttvgtl-27... (2738 CTGCCCGGGGCTGTGCCCCGGACCCCCC
AY823990 (2789 CCGCCCCCCAAACCCCCCGCGTCGGATGGGGGGGGCTGCGCCCCCCCCAA ttvgl-7 ... (2767
ttvGTl-17... (2769
ttvGTl-21... (2765
ttvgtl-27... (2766
AY823990 (2839 ACCCCCCTTGCCCGGGGCTGTGCCCCGGACCCCC
ttvgl-7 ... (2767
ttvGTl-17... (2769
ttvGTl-21... (2765
ttvgtl-27... (2766
Nucleotide Identity among PAH TTV's and Published Sequence
Figure imgf000037_0001
TTVgt1 -27 demonstrates the greatest homology with published sequence, AY823990, demonstrating 91 % identity. TTVgt1 -7,17, and 21 demonstrate 85- 87% identity. TTVgt1-7 and TTVgt1 -21 share 99% nucleotide identity Orf1 Amino Acid Alignment
The following provides a comparison of the published AY823990 sequence (SEQ ID NO:25) to the corresponding amino acid sequences for TTV7 (SEQ ID NO: 10), TTV17 (SEQ ID NO: 1 1 ), TTV21 (SEQ ID NO: 12), and TTV27
(SEQ ID NO: 13)
AY823990 (1) MAPTRRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRDYRRRWLVRRRRRSV
Ttvgl-70rfl (1) MAFARRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV
Ttgl-170rfl (1) MAPARRWRRGFGRRRRRYRKRRWGWRRRYWRYRPRYRRRRWWRRRRRSV
Ttgl-270rfl (1) MAPTRRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV ttgl-210rfl (1) MAFARRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV
AY823990 (51) YRRGGRRARPYRL--FNPKVMRRWIRGWWPILQCLKGQEALRYRPLQWD
Ttvgl-70rfl (51) YRRGGRRARPYRISAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD
Ttgl-170rfl (51) YRRGGRRARPYRISAFNPKIMRRWIRGWWPILQCLRGQESLRYRPLQWD
Ttgl-270rfl (51) YRRGGRRARPYRVSAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD ttgl-210rfl (51) YRRGGRRARPYRISAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD
AY823990 (99) TERQWRVRSDFEDQYGYLVQYGGGWGSGDVTLEGLYQEHLLWRNSWSKGN
Ttvgl-70rfl (101) VEKSWRINTTLEDNYGYLVQYGGGWGSGEVTLEGLYQEHLLWRNSWSKGN
Ttgl-170rfl (101) VEKSWRIKTDLEDNYGYLVQYGGGWGSGEVTLEGLYQEHLLWRNSWSKGN
Ttgl-270rfl (101) TERQWRVRQDFEDQYGYLVQYGGGWGSGDVTLEGLYQEHLLWRNSWSKGN ttgl-210rfl (101) VEKSWRINTTLEDNYGYLVQYGGGWGSGEVTLEGLYQEHLLWRNSWSKGN
AY823990 (149) DGMDLVRYFGCWYLYPLKDQDYWFWWDTDFKELYAENIKEYSQPSVMMM
Ttvgl-70rfl (151) DGMDLVRYFGCIVYLYPLKDQDYWFWWDTDFKELYAESIKEYSQPSVMMM
Ttgl-170rfl (151) DGMDLVRYFGCIVYLYPLKDQDYWFWWDTDFKELYAESIKEYSQPSVMMM
Ttgl-270rfl (151) DGMDLVRYFGCWYLYPLKDQDYWFWWDTDFKELYAENIKEYSQPSVMMM ttgl-210rfl (151) DGMDLVRYFGCIVYLYPLKDQDYWFWWDTDFKELYAESIKEYSQPSVMMM
AY823990 (199) AKRTRIVIARERAPHRRKVRKIFIPPPSRDTTQWQFQTDFCNRKLFTWAA
Ttvgl-70rfl (201) AKRTKIVIARSRAPHRRKVRRIFIPPPSRDTTQWQFQTDFCNRPLFTWAA
Ttgl-170rfl (201) AKKTKIVIARSRAPHRRKVRKIFIPPPSRDTTQWQFQTEFCNKPLFTWAA
Ttgl-270rfl (201) AKRTRIVIARDRAPHRRKVRKIFIPPPSRDTTQWQFQTDFCNRKLFTWAA ttgl-210rfl (201) AKRTKIVIARSRAPHRRKVRRIFIPPPSRDTTQWQFQTDFCNRPLFTWAA
AY823990 (249) GLIDMQKPFDANGAFRNAWWLEQRNDQGEMKYIELWGRVPPQGDSELPKK
Ttvgl-70rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPVQ
Ttgl-170rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPAQ tgl-270rfl (251) GLIDMQKPFDANGAFRNAWWLEQRTEQGEMKYIELWGRVPPQGDSELPKK ttgl-210rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPLQ
AY823990 (299) KEFSTGT-DNPNYNVQDNEEKNIYPIIIYVDQKDQKPRKKYCVCYNKTLN
Ttvgl-70rfl (301) TEFQKPSGYNPKYYVNPGEEKPIYPVIIYVDMKDQKPRKKYCVCYNKTLN
Ttgl-170rfl (301) KEFQKPDGYNPKYYVQAGEEKPIYPIIIYVDKKDQKARKKYCVCYNKTLN
Ttgl-270rfl (301) SEFTTAT-DNKNYNVNDGEEKPIYPIIIYVDQKDQKPRKKYCVCYNKTLN ttgl-210rfl (301) TEFQKPSGYNPKYYVNPGEEKPIYPVIIYVDMKDQKPRKKYCVCYNKTLN
AY823990 (348) RWRLGQASTLKIGNLKGLVLRQLMNQEMTYIWKEGEYSAPFVQRWKGSRF
Ttvgl-70rfl (351) RWRSAQASTLKIGDLQGLVLRQLMNQEMTYTWKEGEFTNVFLQRWRGFRL
Ttgl-170rfl (351) RWRAAQASTLKIGDLQGLVLRQLMNQEMTYIWKEGEFTNVFLQRWKGFRL
Ttgl-270rfl (350) RWRLGQASTLKIGNLKGLVLRQLMNQEMTYIWKEGEYSSPFVQRWKGSRF ttgl-210rfl (351) RWRSAQASTLKIGDLQGLVLRQLMNQEMTYTWKEGEFTNVFLQRWRGFRL AY823990 (398) AVIDARKADQENPKVSTWPIEGTWNTQDTVLKDVFGINLQNQQFRAADFG
Ttvgl-70rfl (401) AVIDARKADTENPTVQTWKVDGQWNTQGTVLKEVFNINL NEQMRQADFG
Ttgl-170rfl (401) AVIDARKGDTENPTVQTWKVDG WNTSGTVLQEVFGINLTQQQMRASDFA
Ttgl-270rfl (400) WIDARKADQENPKVSTWPIEGVWNTQGTVLKDVFQIDLNSTNFRAADFG ttgl-210rfl (401) AVIDARKADTENPTVQTWKVDGQWNTQGTVLKEVFNINL NEQMRQADFG
AY823990 (448) KLTLPKSPHDLDFGHHSRFGPFCVKNEPLEFQVYPPEPTNLWFQYRFFFQ
Ttvgl-70rfl (451) KLNLPKSPHDIDFGHHSRFGPFCVKNEPLEFQLTAPEPTNLWFQYKFLFQ
Ttgl-170rfl (451) KLTLPKSPHDIDFGHHSRFGPFCVKNEPLEFQLTAPEPINLWFQYKFLFQ
Ttgl-270rfl (450) KLTLPKSPHDLDFGHHSRFGPFCVKNEPLEFQVYPPEPTNLWFQYRFFFQ ttgl-210rfl (451) KLNLPKSPHDIDFGHHSRFGPFCVKNEPLEFQLTAPEPTNLWFQYKFLFQ
AY823990 (498) FGGEYQPPTGIRDPCVDTPAYPVPQSGSITHPKFAGKGGMLTETDRWGIT
Ttvgl-70rfl (501) FGGEYQPPTGIRDPCADNPAYPVPQSGSITHPKFAGKGGMLTETDRWGIT
Ttgl-170rfl (501) FGGEYQPPTGIRDPCADNQPYPVPQSGSITHPKFAGKGGMLTETDRWGIT
Ttgl-270rfl (500) FGGEYQPPTGIRDPCVDTPAYPVPQSGSITHPKFAGKGGMLTETDRWGIT ttgl-210rfl (501) FGGEYQPPTGIRDPCADNPAYPVPQSGSITHPKFAGKGGMLTETDRWGIT
AY823990 (548) AASSRALSADTPTEAAQSALLRGDSEAKGEETEETASSSSITSAESSTEG
Ttvgl-70rfl (551) AASSRTLSADTPTEATQSALLRGDSEKKGEETEETSSSSSITSAESSTEG
Ttgl-170rfl (551) AASSRALSADTPTEAAQSALLRGDSEKKGEETEETTSSSSITSAESSTEG
Ttgl-270rfl (550) PASTRALCADTPTEATQSALLRGDSEKKGEETEETTSSSSITSAESSTEG ttgl-210rfl (551) AASSRALSADTPTEATQSALLRGDSEKKGEETEETSSSSSITSAESSTEG
AY823990 (598) DGSSDDEETIRRRRRTWKRLRRMVREQLDRRMDHKRQRLH-
Ttvgl-70rfl (601) DGSSDDEETIRRRRRTWKRLRRMVREQLDRRMDHKRQRLH-
Ttgl-170rfl (601) DGSSDDEETIRRRRRTWKRLRRMVREQLDRRMDHKRQRLH-
Ttgl-270rfl (600) DGSSDDEETVRRRRRTWKRLRRMVREQLDRRMDHKRQRLH- ttgl-210rfl (601) DGSSDDEETIRRRRRTWKRLRRMVREQLDRRMDHKRQRLH-
Figure imgf000039_0001
Hydrophobicity plots of the proteins demonstrate 5 areas of hydrophilicity, which may indicate surface-exposed regions that are potentially antigenic. Two of these regions are at the amino terminus and at the carboxy terminus, and are both arginine-rich and highly conserved. A highly conserved hydrophilic region between amino acids 190 and 232 was observed and may potentially serve as antigenic site. The remaining hydrophilic regions between amino acids 295 and 316, and between amino acids 415 and 470 are also be antigenic. Additionally, it has been determined that the putative start codons for ORF1 and coding region are as follows: ttvgtl -27 nt 517-2435; ttvg1 -7 nt 517- 2435; ttvgtl -17 nt 517—2436; ttvgtl -21 nt 517-2439; ttv10 nt 487-2346; and ttv13 nt 477-2363. The putative start codons for ORF 2 and coding region are as follows: ttvgtl -27 nt 428-646; ttvg1 -7 nt 428-643; ttvgtl -17 nt 428-643; ttvgtl - 21 nt 428-646; ttv10 nt 404-610; and ttv13 nt 394-597.
TTV ORF1 protein expression utilizing recombinant baculovirus
A series of experiments was then undertaken to express the genotype 2 TTV ORF1 protein utilizing insect cells and recombinant baculovirus.
Optimization of protein expression was conducted with three cell lines (SF9, SF21 and Hi Five), multiple media configurations (ExCell 420, SF900 I I I SFM, Express Five SFM), various cell densities (5e5, 1 e6, 2e6 and 4e6 cells/ml), and various multiplicities of infection (0.005, 0.1 , 0.5, 2.0), and the resultant cultures were monitored daily over a seven day post infection period.
The processes were monitored for cell density and viability, and infection was monitored through monitoring of cell size and virus titration. Protein expression was monitored through SDS-PAGE, Coomassie gel analysis and Western blotting. To ensure proper control, negative and positive controls were maintained throughout all experiments. Although all experiments were able to confirm expression of the target protein, optimal conditions were found when utilizing SF9 cells maintained in ExCell 420 media (Sigma, SAFC) with a cell density of 2x106 cells/ml and an MOI of 0.1 , with the process terminated following a three day infection. The majority of the recombinant expressed protein can be located within the cell pellet although some resides in the resultant supernatant.
Confirmation of protein expression with Western Blotting (GST-tag)
As the Invitrogen destination vectors (pDESTI O) contained a GST protein
N-terminal to the TTV Orf1 reading frame, a resultant GST-ORF1 fusion protein of approximately 95kD was generated, which was detected using a commercially available rabbit anti-GST (CALBIOCHEM) antibody. Of the 95kD fusion protein, approximately 68kD is considered to be ORF1 and 25kD to be the GST protein. No commercial antibody was available for standardized detection of TTV ORF1 protein, which necessitated the use of the anti-GST antibody.
Production of rabbit anti-TTV ORF1 antibody
Due to the initial lack of availability of known TTV reagents, efforts were undertaken to produce anti-TTV ORF1 antibodies. Following the optimized expression protocol for preparing the TTV ORF1 recombinant protein, the resultant material was further purified utilizing the commercially available
Baculogold GST purification kit. Purified TTV10 and TTV13 ORF1 protein was then utilized to hyperimmunize rabbits for the subsequent production of antibodies against the ORF1 recombinant protein.
In regard of protein detection, Figure 1A sample lanes were as follows (from right to left)
Samples:
1 SeeBlue Plus 2
2 ORF1 TTV 13 d.3 1 e6 cell/ml (GST purified pellet)
3 ORF1 TTV 13 d.3 1 e6 cell/ml (GST unbound)
4 ORF1 TTV 13 d.3 2e6 cell/ml (GST purified pellet)
5 ORF1 TTV 13 d.3 2e6 cell/ml (GST unbound)
6 ORF1 TTV 13 d.3 4e6 cell/ml (GST purified pellet)
7 ORF1 TTV 13 d.3 4e6 cell/ml (GST unbound)
8 ORF1 TTV 13 d.3 4e6 cell/ml (GST purified supe)
9 ORF1 TTV 13 d.3 4e6 cell/ml (GST unbound)
10 ORF1 TTV 13 d.3 4e6 cell/ml untreated supe.)
1 1 ORF1 TTV 13 d.3 4e6 cell/ml untreated cell pellet
12 SF9 Negative Control d.3 pellet 1 e6 cell/ml Lanes 2, 4 and 6 demonstrate the purified 95kD TTV13 ORF1 fusion protein which was later utilized for the rabbit immunization, see Figure 1A. Detection of Native TTV ORF1 utilizing the Rabbit anti-ORF1 protein
Additional expression experiments were conducted with the native TTV ORF1 recombinant baculovirus. This recombinant baculovirus was constructed without a 6xHis or GST fusion tag and hence requires a specific anti-TTV ORF1 antibody. Consequently, post expression Western blot analysis was conducted utilizing the rabbit anti-TTV ORF1 antibody to confirm expression of the native protein, and to confirm the reagent reactivity. Western blot analysis
demonstrated a faint reaction at approximately 69kD, which is approximately the predicted size of TTV ORF1 as well as reaction to an additional band at approximately 49kD (see Figure 1 B). The 49kD protein band is unknown. The faint banding at 69kD is assumed to be a function of either low protein
expression in the native TTV ORF1 construct or poor antibody yield from the rabbit immunization. It should be noted that no purification of the antigen or antibody was conducted in this particular analysis. Lane 5 (see the arrows in Figure 1 B) demonstrates a unique reaction to a ~69kD and 49kD protein in the native TTV ORF1 expression utilizing anti-TTV ORF1 rabbit polyclonal antibody.
Accordingly, there was demonstrated binding of antibody to capsid protein as antigen, herein the antigen provided only TTV sequence and was not tagged.
Figure 6B sample lanes were as follows (from right to left)
Figure imgf000043_0001
Example 2 Backpassaging
A liver was collected aseptically from a caesarean-derived, colostrum deprived (CDCD) pig. The liver tissue was tested for g1 and g2 TTV in unique qPCR assays and confirmed to be positive for only gITTV. A 10% (wt/vol) liver homogenate was then prepared in media containing antibiotics and antimycotics. Finally, the homogenate was clarified by centrifugation, designated as gITTVpO and frozen at -70C. The resulting gITTV homogenate was tested to be free of extraneous viruses, bacteria and mycoplasma via routine testing. Following satisfactory testing, two milliliters of freshly thawed gITTVpO was IP inoculated into each of six 11 -day old gnotobiotic piglets. At approximately 12 days post- inoculation the pigs were euthanized and the bone marrow, spleen and livers were aseptically collected. Each of the resulting livers were confirmed by qPCR to be rich in gITTV and negative for g2TTV. Liver homogenates were then prepared from each of the resulting livers as aforementioned, labeled and aliquoted as gI TTVpl and placed at -70C. A further second passage (g1 TTVp2) was created from gI TTVpl .
Example 3 Evaluation of the Efficacy of Three Torque Teno Virus (TTV)
Vaccines in Young Pigs
The present study was conducted to evaluate the efficacy of three TTV vaccine candidates administered at ~7 days of age, and again at weaning (-21 days of age) followed by a challenge at ~5 weeks of age.
This study provided a preliminary immunogenicity evaluation in pigs injected intramuscularly with formulations for TTV. As previously mentioned, TTV is a small, non-enveloped virus with a single-stranded circular DNA genome of negative polarity. The genome includes an untranslated region and at least three major overlapping open reading frames. Porcine TTV is ubiquitous and PCR-detection of the virus in serum samples collected from various geographical regions shows prevalence in pigs ranging from 33 to 100%. McKeown et al., Vet. Microbiol. (2004) 104: 1 13-1 17. Krakowka et al., AJVR (2008) 69: 1623-1629, reported that g1 -TTV inoculated pigs had no clinical signs but developed interstitial pneumonia, transient thymic atrophy, membranous
glomerulonephropathy and modest lymphocytic to histiocytic infiltrates in the liver after inoculation. The present study provided a comparison of three different formulations of TTV vaccines, and evaluated if any of these prototype
formulations can be numerically or statistically differentiated when compared to challenge control groups.
Materials and Methods
Animals: Six clinically healthy, crossbred pregnant, PRRSV and M hyo
seronegative females without a history of disease caused by PRRSV or M hyo (or vaccination against the same organisms) were sourced from Lincoln Trail / Puregenic Pork, Alton, IL, and transported to the Pfizer Animal Health Research Farm in Richland, Ml at approximately 3 weeks pre-partum. If necessary, sows were induced to farrow within a 2 or 3 day period using injectable prostaglandin (Lutalyse®). Normal piglets from these sows were allotted to study according to the allotment design. Pigs were randomized to treatment by litter and each litter had at least one piglet assigned to each treatment. Housing: During the vaccination phase, pigs were housed with their mother with no cross-fostering, in BL-2 isolation facilities. Pigs remained housed by litter until the time of 2nd vaccination. Post-second vaccination pigs were moved to a further facility and housed in two rooms (one room contains NTX (non-vaccinated and non-challenge controls) animals, the second room vaccinates), and each room contains 4 or 8 pigs per pen.
Feed: Following farrowing, sows were fed a lactating sow diet as appropriate. Piglets accessed creep feed and milk replacer prior to weaning. Once weaned, piglets were feed an age-appropriate diet offering free choice. Water was available to all animals ad libitum. Allotment/Randomization: Pigs were randomized to treatment by litter. Each litter had at least one piglet assigned to each treatment.
Study Design
Figure imgf000045_0001
* Minimum of 1 NTX pig from each litter Masking: Vaccine was masked using a numeric code prior to vaccination. The investigator, vaccine administrator and study personnel were masked to treatment and did not have access to the masking code unless treatment information was required for the welfare of an animal. Investigational Veterinary Products
Table 1. IVP Formulation
Figure imgf000046_0001
Challenge Material Preparation: gITTV pass 1 was derived from liver homogenate tested positive (7.6x10e8 to 1.6x10e9 DNA copies/2ml_) for gITTV and negative for g2TTV by qPCR. An appropriate number of bottles were removed from the freezer and thawed shortly before challenge. An aliquot was then removed from one of the bottles, and held for retitration at a later time.
Challenge stock was transported on ice to the research facility and maintained on ice during the challenge procedure. A challenge dose equals 2.0ml_ of stock solution (2.0 mL intraperitoneal). The dose was delivered to each pig is therefore expected to be 7.6x10e8 to 1 .6x10e9 DNA copies/2mL. Following challenge, an aliquot of challenge stock was kept for titration to confirm challenge dose.
General Health Observations: Animals were observed daily by a qualified individual and general health observations were recorded.
Body Weights: All pigs were weighed Day 0, the day of challenge (Day 28) and at necropsy. All weights were recorded.
Vaccination: At approximately 7 days of age (Day 0), -10 randomly allotted pigs per treatment group (Groups T01 thru T04) were vaccinated as described in Table 1 . Pigs were injected in the right neck with a single dose syringe (2.0 mL intramuscular (IM) dose) of IVP, or a 2 mL IM dose of control according to allotment. A second dose of the same IVP or control was administered in the left neck at the time of weaning (-21 days of age).
Blood Sampling: Prior to Day 0, Day 14 (prior to vaccination) and Day 28 prior to challenge (as well as Day 31 , 34, 37, and 40), a blood sample was collected, using 5 mL or 9 mL serum separator tubes (dependant on body weight), from all pigs for gITTV status (qPCR-Pfizer-VMRD Laboratory Sciences). Serum samples were aliquoted by site personnel to at least three separate tubes and were stored at -80 C. Table 2: gITTV qPCR analysis to be performed on sera by time point
Study Day DO D14 D28 D31 D34 D37 D40 qPCR gI TTV X X X X X X X Challenge: At ~5 weeks of age, piglets were inoculated with a 2.0 mL (IP or IN) dose of a TTV isolate according to allotment. Challenge material was shipped to the facility identified by a treatment code for masking purposes.
Rectal Temperatures post challenge were recorded once per day on Day 28 prior to challenge as well as Day 31 , 34, 37, and 40.
Necropsies: On Day 40 all animals were euthanized and necropsied. Upon necropsy, lung lesions were scored using the following methods: 1 ) a numeric score (0, 1 , 2, 3) and 2) the percentage of consolidation for each lobe (left cranial, left middle, left caudal, right cranial, right middle, right caudal, and accessory) was scored and recorded as percent of lobe observed with lesions. Liver, kidney, thymus and lymph nodes were also scored. A blood sample was also taken prior to euthanasia. Tissues were collected as indicated in the following table:
Figure imgf000048_0001
In regard of assessment of safety and/or efficacy, no confounding secondary disease conditions were detected. Animals were vaccinated and challenged according to protocol. In regard of outcome criteria, reduction in any or all of the following were used: decreased gross or microscopic lesions;
decreased viremia by qPCR; and decreased incidence of fever, weight loss or death, two-sided tests. Method of Analysis
Upon necropsy, lung lesions were scored using the following methods: 1 ) a numeric score (0=no lesions, 1 =mild lesions, 2=moderate lesions, 3=severe lesions) and 2) the percentage of consolidation for each lobe (left cranial, left middle, left caudal, right cranial, right middle, right caudal, and accessory) was scored and recorded as percent of lobe observed with lesions.
The percentage of total lung with lesions was transformed and analyzed with a general linear mixed model with fixed effects, treatment, and random effect litter. Linear combinations of the parameter estimates were used in a priori contrasts after testing for a significant (P<0.10) treatment effect. The 10% level of significance (P<0.10) was used to assess statistical differences. qPCR data will be transformed prior to analysis with an appropriate log transformation. The transformed titers will be analyzed using a general linear repeated measures mixed model analysis. Pairwise treatment comparisons will be made at each time point if the treatment or treatment by time point interaction effect is significant (P≤0.10). Treatment least squares means, 90% confidence intervals, the minimum and maximum will be calculated and back-transformed for each time point. Descriptive statistics, means, standard deviations, and ranges, will be calculated for each treatment and day of study, pre-challenge. Study Results and Discussion
Lung Lesions
Although the overall percent lung lesions observed was low throughout all treatment groups, significant differences were found. T01 (Chromos expressed gI TTV ORF1 ) yielded significantly lower lung lesions when compared to both the T02 (Baculovirus expressed g2TTV ORF1 ) and T04 (Challenge controls). Since the challenge virus was comprised of infectious gITTV, it may not be surprising that the genotype 2 ORF1 from Baculovirus did not provide very substantially lower lung lesions as compared to the challenge controls. It is however interesting to note that while not substantial, it did offer numerically lower lung lesion scores compared to the challenge controls, thereby indicating that some level of cross protection is possible between different TTV genotypes upon optimization of dose and adjuvant selection. It was surprising that the inactivated challenge virus (T03, g1TTVp1 Killed Virus) did not offer cross-protection against the live gITTV challenge virus as evidenced by the lack of any statistical difference between T03 and T04. This surprising lack of cross protection further enhances the veterinary importance of novel vaccines of the invention, such as gITTV ORF1 (T01 Chromos).
Figure imgf000050_0001
Contrast 2-tailed p-value (1 ) significance of 2-tailed p-value T01 vs T02 0.2167 N.S. T01 vs T02 0.0472 T01 vs. T04 0.0389 T02 vs T03 0.5394 N.S. T02 vs T04 0.4955 N.S. T02 vs. T04 0.9454 N.S.
(1) P-Values > 0.10 are designated as "N.S." (Not Significant) and P-Values < or = 0.10 are designated as "*" (Signficant). glTTV qPCR
Analysis of the TTV qPCR viremia data (Figure 7) reveals that T01
(Chromos glTTV ORF1 ) has numerically lower TTV qPCR values as compared to T04 (Challenge controls). There exists a decrease in viremia magnitude and duration, which along with a reduction in lung lesions are indicators of efficacy. In addition, T02 (Baculovirus g2TTV ORF1 ) demonstrates a numerical reduction in viremia magnitude and duration compared to T04 (Challenge controls) but for a shorter period of time. This combined with the numerically lower lung lesions indicates that some genotypic cross protection (g2TTV ORF1 vaccine vs glTTV challenge virus) was observed. One can suggest that with an optimized dose and adjuvant that broad genotypic cross protection can be realized. It is also interesting to note that (T03) gITTVpl KV offered no reduction in TTV qPCR viremia when compared to the challenge controls. This observation in
conjunction with the lung lesion data further illustrate the novel finding that the recombinantly expressed glTTV ORF1 (T01 ) provides efficacy as a vaccine.
Example 4 Codon optimization and recombinant expression glTTV ORF1 as a full length protein with a 6His tag, and detection thereof by an antibody.
The TTVgl nucleotide sequence was submitted to GenScript (Piscataway, New Jersey, USA) for codon optimization and gene synthesis for both E. coli and Saccharomyces cerevisiae. In both cases, the codon optimized gene was cloned into the GenScript pUC57 vector, as product. The GenScript OptimumGene™ codon optimization analysis involves analysis of numerous parameters including codon usage bias,GC content, CpG dinucleotide content, mRNA secondary structure, identification of possible cryptic splicing sites, presence of premature polyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motifs (ARE), inhibition sites (INS), repeat sequences of various kinds (including direct, reverse and dyad), and also restriction sites that may interfere with cloning. Translational performance may be additionally improved via translational initiation Kozak sequences, Shine-Dalgarno sequences, and to increase efficiency of translational termination via stop codons.
SEQ ID NO: NOS 18-20 provide TTV capsid gene that were codon optimized for both Escherichia coli (NOS: 18-19) and Saccharomyces cerevisiae (NO: 20). The sequences for E. coli are very similar, however, to clone the gene into the commercial pET101/D-TOPO expression vector (Invitrogen) to create 76057-4 (SEQ ID NO:19), additional CA nucleotides had to be added at the N- terminus. The pET101/D-TOPO expression vector also has a C-terminal V5 tag and 6X-His for purification, although the sequences for 76057-3 (SEQ ID NO: 18) and 76057-4 are otherwise identical. The expressed codon-optimized TTVgl protein is approximately 68 kD in size, relative to the 63kD protein, due to the addition of a 10 amino acid protective peptide at the amino terminus, and 32 amino acids corresponding to the V5 epitope and a 6X His tag at the carboxy terminus (Figure 2).
The sequence for 76057-5 (SEQ ID NO: 20) has been codon optimized for S. cerevisiae, and it thus differs slightly from the E. coli sequences. In addition, this sequence lacks a 10 amino acid protective peptide at the N-terminus (which was added to the E. coli sequence), and it also has flanking restriction
endonuclease sites, Not\ at the N-terminus and 4afl l at the C-terminus, for subcloning of the gene into yeast vectors.
Aditionally, it should be noted that the protective peptide of ten amino acids was added to N-terminus of the TTVgl sequence for expression in E. coli. since this has been shown to increase protein stablility when fused to the amino terminus. Restriction sites have been engineered such that the peptide can be removed for evaluation of the full length protein. Expression of the codon optimized TTVgl was evaluated in the pET101/D-TOPO vector with and without the protective peptide N-terminal fusion. The TTVgl sequence codon optimized for S. cerevisiae was also subcloned into a pESC-Trp vector with the potential for producing suface-expressed protein in yeast that can be used to elicit an antibody response in vivo.
Example 5. TTV Peptide Conjugation and Antibody Production (Polyclonal and Monoclonal)
Rabbit polyclonal antibodies were raised against Baculovirus expressed g2 TTV GST-ORF1 protein prepared in Example 2. Two rabbits were
hyperimmunized, but only one rabbit responded. The rabbit antiserum cross- reacts to various preparations of g1 TTV whole virus that was propagated in pigs and also reacts against the immunizing antigen, Baculovirus expressed g2TTV ORF1 . The rabbit antibody did not, however, respond to the E.coli expressed g2TTV ORF1 that had the 100 A.A. N-terminal arginine-rich region removed from the amino terminus as described in Example 2. This may suggest that a major antigenic epitope may be in the 100 amino acid region that was missing in the truncated g2 TTV ORF1 , and that there is homology between g1 and g2 TTV in this region.
Monoclonal antibodies can be generated against full-length g1 TTV ORF1 , or other g1 TTV antigens. Other potential immunizing antigens include g1 TTV whole virus, g2 TTV GST-ORF1 (Baculo), g1 TTV GST-truncated ORF1 (E coli), and g2 TTV GST-truncated ORF1 (E coli). A peptide library can be generated to identify linear epitopes that are antigenic. For example, 18mer peptides, with a 10AA overlap, can be utilized to cover the TTV genome. The peptides can then be utilized in Western blots or ELISA's to determine their overall reactivity to the gI TTV ORF1 or g2TTV ORF1 monoclonal and/or polyclonal antibodies so that immunogenic domains can further be identified.
Rabbit polyclonal antibodies may also be raised against three g1 TTV ORF1 peptides cross-linked to KLH, and subsequently screened using peptide- ovalbumin conjugates. The peptide-KLH conjugates can also be used to produce monoclonal antibodies. In this respect, in one embodiment, multiple g1 TTV ORF1 peptides copies may be conjugated together, including from different strains.
In particular examples, once peptides were generated (CPC Scientific), they were then conjugated to KLH or ovalbumin (by the Proteos Co). The KLH- conjugated peptides were used for immunization of rabbits, while the Ovalbumin conjugated peptides are used for screening the serum (i.e., to detect antibodies to the peptides and not the carrier protein).
Example 6 Peptide Sequences for Polyclonal Antibody Generation
The following peptide sequences were chosen from TTVgl (numbering based on AY823990) for polyclonal antibody generation, and represent SEQ ID NOS; 22-24 respectively. 1 . [L167C]TTV(167-185)-NH2 : CKDQDYWFWWDTDFKELYA-NH2 (19 aa. pl 4)
2. TTV(459-479): DFGHHSRFGPFCVKNEPLEFQ (21 aa, pi 6.9)
3. [Cys612]-TTV( 612-637):
CTWKRLRRMVREQLDRRMDHKRQRLH (26 aa, pi 13)
Each of the three peptides has a single cysteine residue present in the sequence to enable selective peptide coupling to a carrier protein. In
[L167C]TTV(167-185)-NH2 and [Cys612]-TTV( 612-637), an extra cysteine residue was added at the N-terminus, while in TTV(459-479) there is a native cys present at position 470. Additionally, [L167C]TTV(167-185)-NH2 has an amidated C-terminus to yield a less acidic peptide. The peptides were selected based on sequence identity for different TTV isolates. Additionally, the C-terminal fragment [Cys612]-TTV( 612-637) appears to be surface exposed. The peptides were custom made by solid phase peptide synthesis at CPC Scientific and obtained with >95% purity.
A further and highly preferred peptide is constructed by using the peptide sequence corresponding to residues 601 -620 of SEQ ID NO:9 (20AA, pi 13) except that a cysteine residue is used at the N-terminus in replacement for Asn 601 . This peptide is also likely surface exposed in the native protein.
Example 7 TTV g1 ORF1 protein expression using the Chromos system
The Chromos ACE system is a protein expression platform that consists of three main components. The first component is a neutral, functional mammalian artificial chromosome called the Platform ACE, which resides in the genetic material of a modified Chinese Hamster Ovary (CHO) cell line. The second component is the ACE targeting vector, which is a plasmid used for loading target genes onto the Platform ACE. The third element is a site-specific, unidirectional integrase, which catalyzes the direct and specific loading of the target gene onto the Platform ACE. Additional information concerning the ACE System can be found of the website of Chromos Molecular Systems, Inc. of Canada, or by contacting the company directly at 604-415-7100 where the technology is available for license.
The Chromos ACE system has a number of significant advantages over traditional protein production platforms. The first of these is speed. The
Chromos ACE system allows for the rapid, efficient and reproducible insertion of selected genes. The second advantage is expression. High level constituitive protein expression is achieved over time. A third advantage is stability. The Chromos ACE system allows selective and controlled protein expression. Briefly, restriction sites were added to both ends of the TTV7 ORF1 gI DNA using PCR. Additionally, the sequence for yeast invertase was added to the 5' end of a separate PCR preparation. The amplified sequences were then treated with restriction enzymes and sub-cloned into the plasmid pCTV927. The DNA sequence was verified by ACGT Inc. CHk2 (Chinese Hamster Ovary) cells were then transfected with the plasmids using Lipofectamine 2000 (Invitrogen), and selective pressure was added using hygromycin B. Ten single-cell clones were analyzed for TTV protein production using SDS PAGE and Western Blotting. More specifically, the ACE Targeting Vector pCTV-TTV7ORF1 +YI was generated as follows (see Figure 3). The gene TTV7ORF1 was obtained as a PCR product. A primer was designed to contain the yeast invertase secretion signal and the restriction site EcoRV at the 5' end of the gene. A second primer was designed to contain the restriction site Kpnl at the 3' end of the gene. These sequences were added to the gene TTV7ORF1 using the polymerase chain reaction. The modified gene was then subcloned into the ACE Targeting Vector ATVCHS4Hyg, which contained a hygromycin resistance marker suitable for downstream antibiotic selection. The new plasmid was named pCTV- TTV7ORF1 +YI .
The plasmids pCTV-TTV7ORF1 +YI and pSIO343, which coded for TTV7ORF1 /yeast invertase and the unidirectional lambda integrase, respectively, were transfected into the Chk2 cell line, which contained the Platform ACE. The transfected cells were named Chk2-TTV7ORF1 +YI . These cells were seeded in 96-well plates and monitored for the formation of single-cell clones. Media containing Hygromycin was added to each 96-well plate to select for cell clones that contained the ACE targeting vector. Once single-cell clones were identified, twelve of them were expanded into 24-well plates, and then to 6-well plates. Finally, the clones were expanded into suspension cell culture. Culture Chk2- TTV7ORF1 +YI #75 was used to generate cell-free supernatant for subsequent experimental vaccine preparation. Figure 7 demonstrates that Chromos-expressed gI TTV ORF1 significantly reduced lung lesions compared to the challenge controls, and reduced the numerical magitude and duration of gI TTV viremia, again compared to the challenge controls. Vaccination was at Day 0 and 14, with challenge at Day 28. The geometric mean of detected gI TTV copies was reported exponentially, i.e. 1 .00 E+00 is 1 , 4.25E+00 is 4.25, and 4.42E+01 is 44.2. Example 8 Nuclear Localization Signals
Figures 4 and 5 provides a 7-way amino acid alignment of ORF1 (capsid proteins) from 5 TTV gt1 viruses of the present invention and two TTV gt2 (or gt2-like) viruses of the invention. There are, of course, many gaps and mismatches because the gt1 capsids are only about 22.3 to 23.2% identical to the gt2 capsids. The five gt1 capsids are 85.6 to 99.7% identical, however, among themselves. The two gt2 capsids (TTV10 and TTV13) are similarly 66.8% identical.
Two known types of NLS signals (Pat7 and Pat4, see US Patent
7,544,362, for example) were identified by inspection. In Figure 5, the The NLS signals are underlined. Note the all seven capsids contain multiple NLS of both pat7 and pat4 type. Some are conserved between genotypes, some within a genotype, and some are not conserved. Most are near the N-terminus, where they tend to form overlapping poly-NLS regions. Numerous of these arginine - rich motifs are substantially immunogenic in mammals, and peptides containing them are useful in the generation of anti-TTV antibodies. Example 9 Clone Fragments for infectious clone construction
The following provides a basis for the construction from overlapping clones of TTV genotype 1 strain ttvgtl -178 (see SEQ ID NO:7) for which the amino acid sequence is shown as SEQ ID NO:9.
In summary, two TTV fragments (1900bp and 2200bp), which together span the entire TTV circular genome, were separately cloned into separate pCR 2.1 TA (Invitrogen) cloning vectors. The clone fragments were as follows: Clone 1 : 680s to 2608a=~1900bp, and Clone 2: 1340s to 764a= ~2200bp. In order to accomplish this, PCR primers were designed using the consensus sequence that was generated from strains of the present invention (ttvgt1 -27, -7, -17 and -21 ), and also from published sequences (AY823990(g1 ) and AB076001 -(Sd-TTV31 )). Primer pairs that correspond to the sequence at 680s and 2608a or 1340s and 764a were used to amplify PCR products from DNA that was extracted from liver homogenate samples of pigs infected with TTV challenge strain. These PCR fragments were cloned into Invitrogen's pCR2.1 - TOPO TA vector using directions that were supplied with the kit. Clones were subsequently used to generate DNA sequences across the entire 2880 base genome and the sequence was found to be 86% homologous to published sequences GQ120664.1 and AY823990.1 .
The fully correct sequences will now be combined for construction of a full length infectious clone.
Example 10 Infectious clone for gI TTV
Cloning of ql TTV dsDNA fragments. gI TTV is a single-stranded DNA (ssDNA) virus. Fragments of gI TTV are converted to double-stranded DNA (dsDNA) using polymerase chain reaction (PCR). The dsDNA fragments of gI TTV are then cloned into pUC-based plasmid cloning vectors and transformed into E. coli. The fragments of gITTV are less than 1 full-length dsDNA equivalent of the gI TTV genome. Amplification of gI TTV dsDNA concatemers. Concatemers of full-length g1 TTV dsDNA genome equivalents are generated using φ29 polymerase amplification kits (e.g., illustra TempliPhi). Full-length gITTV dsDNA fragments are generated by digestion of the concatemers at appropriate restriction endonucleases (RE) sites. These full-length gI TTV dsDNA fragments can be cloned into plasmid vectors. Alternatively, the concatemers or the uncloned fragments (resulting from RE digestion) can be used without immediate cloning in subsequent molecular biology constructions (see below).
Tandem duplications of the gI TTV genome. Plasmid constructs encoding tandem duplications of the gI TTV genome are next generated. The tandem duplications in the constructs are approximately greater than 1 .2 copies of full- length dsDNA equivalents of the the gI TTV genome. The tandem duplications in plasmids are generated using (1 ) subcloning employing appropriate RE sites, (2) PCR assembly of tandem duplications, or (3) other molecular biology methods. The templates for the generation of the tandem duplications are the gI TTV dsDNA fragments and/or the full-length gI TTV dsDNA clones (yielded by φ29 polymerase amplification).
In vivo recombination and generation of gITTV virus. The tandem duplication plasmid constructs are not identical to the gITTV virus. The tandem duplication constructs are dsDNA while the virus is ssDNA, the constructs encode >1 .2 full- length dsDNA equivalents of the gI TTV genome while the virus has only one full- length equivalent, the construct contains interrupting plasmid sequences while the virus has only viral sequences. To generate the bona fide gI TTV virus, the tandem duplication plasmid constructs are introduced into pigs (by inoculation, injection, electroporation, or other methods of introduction) or introduced into tissue culture cells (by transfection, electroporation, or other methods of introduction) where the plasmid construct recombines at homologous sequences to regenerate a unit-length dsDNA equivalent of the gI TTV genome. The dsDNA equivalent of the gITTV genome is a presumed replicative intermediate of the gI TTV viral life cycle. The presence of this presumed dsDNA replicative intermediate will lead to the production of the bona fide ssDNA gI TTV.
Enabling in vivo generation of gI TTV virus by co-transfection of gI TTV ORF- expressing constructs. It is expected that a circular dsDNA gI TTV genome would be capable of yielding virus production. In the unexpected event that the dsDNA form of gITTV is not replication-competent, the immediate expression of a gITTV ORF may be required for the initiation of gITTV replication from the dsDNA replicative intermediate. Plasmid constructs directing in vivo transcription of gITTV ORFs can be made, such as the fusion of transcriptional promoters (e.g., CMV) to gITTV ORFs. Alternatively, plasmid constructs directing the in vitro generation of gITTV ORF transcripts can be made, such as the fusion of transcriptional promoters (e.g., T7) to gITTV ORFs followed by use of in vitro transcription kits. Either gITTV ORF-expressing plasmids or gITTV ORF- expressing RNA transcripts can be co-injected into pigs or co-transfected into cells along with the tandem duplication plasmid constructs to yield gITTV virus.
Detection of gITTV virus production. To date, whole gITTV virus cannot be propagated in tissue culture cells. The generation of gITTV virus is detected by immune reagents (e.g., a-g1TTV antibody) or by molecular methods (e.g., qPCR).
Example 1 1 Provision of TTV1-178 Clone in pCR2.1 Vector
Total DNA was isolated (DNEasy Blood and Tissue Kit, Qiagen, Valencia, CA ) from a frozen liver homogenate sample (200 microliters) derived from a prior TTV challenge study. The DNA was then PCR-amplified using forward and reverse primers selected to overlap at the unique EcoRI site of the swine TTV 1 - 178 genome. The forward primer: for TTVg1 -178 was selected as positions 1399 to1428 (ACGG... CCAA) from SEQ ID NO:7, and the reverse primer:was selected to correspond to base positions 1443 (5')-to 1416 (3') ATAT... TTGT (opposite strand) from SEQ ID NO:7. PCR conditions were as follows: 1 cycle of denaturation at 94°C for 1 minute; 35 cycles of 94°C, 30 seconds; 55°C, 30 sec; 72°C, 3 minutes; followed by a final 10 minute extension at 72°C. The resultant ~2.8kb fragment was cloned into pCR2.1 vector using a TOPO TA cloning kit. (Invitrogen, Carlsbad, CA). Upon sequence verification, this plasmid (named pCR2.1 +TTV_178) was found to contain the entire TTV 1 -178 genome sequence. pCR2.1 +TTV_178 vector was then linearized with EcoRI in order to release the full length TTV genome, which was then transfected into human embryonic kidney (293), baby hamster kidney (BHK-21 ), swine testicular (ST) and porcine kidney (PK) cells lines using Lipofectin (Invitrogen, Carlsbad, CA). The transfection was allowed to proceed for a total of 5 days at which time the cells were fixed, and then used for IFA staining to determine if the TTV DNA provided expression of ORF 1 protein. IFA staining was accomplished with rabbit polyclonal sera that was raised against a peptide corresponding to a C- terminal region of the capsid protein (residues 601 -620 in SEQ ID NO:9), except that the N terminal residue thereof (Asn 601 ) was replaced with a cysteine residue. The results indicate that the TTV-transfected DNA successfully expressed the ORF-1 protein in at least 293, BHK-21 , ST and PK cells lines.

Claims

Claims
1 . An isolated polynucleotide sequence that comprises a polynucleotide selected from the group consisting of:
(ai ) the DNA of genotype 2 sequence TTV13 (SEQ ID NO: 1 ); the DNA genotype 2 sequence TTV10 (SEQ ID NO: 2); or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
(32) the DNA of a genotype 1 sequence selected from the group consisting of ttvg1 -7 (SEQ ID NO: 4), ttvGT1 -17 (SEQ ID NO: 5), ttvGT1 -21 (SEQ ID NO: 6), ttvgtl -27 (SEQ ID NO: 3), and ttvgt1 -178 (SEQ ID NO: 7) or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
(b) the complement of any sequence in (a);
(c) a polynucleotide that hybridizes with a sequence of (a) or (b) under stringent conditions defined as hybriding to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 xSSC/0.1 % SDS at 68°C;
(d) a polynucleotide that is at least 70% identical to the polynucleotide of (a) or (b);
(e) a polynucleotide that is at least 80% identical to the polynucleotide of (a) or (b);
(f) a polynucleotide that is at least 90% identical to the polynucleotide of
(a) or (b); and
(g) a polynucleotide that is at least 95% identical to the polynucleotide of (a) or (b).
2. An RNA polynucleotide molecule that is complement of any DNA
polynucleotide sequence according to claim 1 .
3. A vector or plasmid that comprises a polynucleotide according to Claim 1 or Claim 2.
4. A host cell that comprises a vector or plasmid according to Claim 3.
5. A virus that be expressed from a nucleotide sequence according to claim 1 , wherein said virus is live, or fully or partially attenuated.
6. A vaccine comprising a virus according to Claim 5.
7. A DNA vaccine that comprises a polynucleotide sequence of Claim 1 .
8. A polypeptide encoded by any of the open reading frames of the TTV13 (SEQ ID NO: 1 ) or TTVI O (SEQ ID NO: 2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof.
9. A polypeptide encoded by any of the open reading frames of the ttvg1 -7 (SEQ ID NO: 10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), and ttvgtl - 27 (SEQ ID NO: 13) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof.
10. A vaccine that comprises a polypeptide according to Claim 8 or Claim 9.
1 1 . A peptide that comprises: (a) the first 100 N-terminal amino acids of the capsid protein of TTV13 (SEQ NO: 1 ) or TTV10 (SEQ ID NO:2); (b) an amino acid sequence that is at least 90 percent identical theret; or (c) an arginine rich region thereof.
12. A vaccine composition comprising a peptide of Claim 1 1 .
13. A monoclonal or polyclonal antibody composition that binds specifically to a protein or peptide of Claims 8, 9 or 1 1 .
14. A vaccine that comprises one or more polypeptides encoded by an open reading frame selected from the group consisting of ORF1 , ORF2 or ORF3.
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