WO1999025838A1 - Compositions et procedes destines a prevenir l'enterite de la dinde - Google Patents

Compositions et procedes destines a prevenir l'enterite de la dinde Download PDF

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Publication number
WO1999025838A1
WO1999025838A1 PCT/US1998/024313 US9824313W WO9925838A1 WO 1999025838 A1 WO1999025838 A1 WO 1999025838A1 US 9824313 W US9824313 W US 9824313W WO 9925838 A1 WO9925838 A1 WO 9925838A1
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Prior art keywords
coronavirus
turkey
seq
turkeys
polypeptide
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PCT/US1998/024313
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English (en)
Inventor
Thomas P. Brown
Pedro Villegas
Anapatricia Contreras
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University Of Georgia Research Foundation, Inc.
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Application filed by University Of Georgia Research Foundation, Inc. filed Critical University Of Georgia Research Foundation, Inc.
Priority to AU14084/99A priority Critical patent/AU1408499A/en
Publication of WO1999025838A1 publication Critical patent/WO1999025838A1/fr

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Coronavirus, alphavirus, cryptosporidiosis, cochlasoma, hexamita, and reovirus have been considered as possible causes for SMT (Ficken et al., 1993), and other viral agents have been documented to produce milder enteritis in young turkey poults (Reynolds et al., 1987).
  • PEMS Poult Enteritis and Mortality Syndrome
  • PEMS/SMT Poult Enteritis and Mortality Syndrome/Spiking Mortality of Turkeys
  • PEMS/SMT is infectious and transmissible, but the specific cause(s) remains unidentified. It is spread by contact with litter or feces and is carried by recovered turkeys. Diligent decontamination efforts and biosecurity have failed to eliminate or to limit the slow spread of PEMS/SMT from contaminated premises.
  • Turkey enteric coronaviruses are one of the major causative agents of diarrhea in turkey poults (Dea et al., 1985; Dea et al., 1990b).
  • a coronavirus was identified as the primary etiologic agent of Bluecomb Disease or Transmissible Enteritis of Turkeys (Naqi et al., 1975; Pensaert et al., 1994; Pomeroy, 1980).
  • PEMS/SMT Transmissible Enteritis
  • infected material e.g., feces or organ homogenates
  • infected material was also clarified and passaged through cell culture.
  • a coronavirus, turkey enteritis coronavirus (TCV) was identified as an infectious agent that was associated with SMT.
  • TCV turkey enteritis coronavirus
  • TCV-MN Antibody to the reference Minnesota strain of turkey coronavirus
  • physiological samples e.g., tissues, blood sera or plasma, from an afflicted or exposed turkey or an organism suspected of being a carrier of TCV
  • coronavirus e.g., tissue, blood sera or plasma
  • viral-specific nucleic acid e.g., by an amplification reaction such as the polymerase chain reaction
  • antibodies specific for a virally encoded polypeptide or antibodies specific for the coronavirus e.g., antibodies specific for a virally encoded polypeptide or antibodies specific for the coronavirus.
  • the presence or amount of the virus, genomic viral nucleic acid, viral polypeptide or viral-specific antibodies in the sample can then be compared to a control sample, e.g., from a disease-free organism.
  • the present invention provides isolated preparations or compositions comprising a cell culture adapted TCV isolate obtained from a turkey having SMT, or a multicellular organism exposed to a turkey having SMT.
  • the preparations or compositions of the invention are substantially free from other infectious agents.
  • substantially free means below the level of detection for a particular infectious agent using standard detection methods for that agent.
  • the cell cultures infected with these TCV isolates produce titers of virus useful to prepare killed coronaviral vaccines or derive modified-live (attenuated) vaccines, to prepare antibodies useful for disease prevention (passive immunization), and for diagnostics.
  • killed virus can be administered to hens for vertical transmission.
  • TCV isolates include TCV-NC (also termed UGA-APN herein), TCV- GA (also referred to as UGA-APG herein), TCV-BOB (also termed UGA- APBOB), and TCV-BOA (also referred to as UGA-APBOA).
  • TCV-NC also termed UGA-APN herein
  • TCV- GA also referred to as UGA-APG herein
  • TCV-BOB also termed UGA- APBOB
  • TCV-BOA also referred to as UGA-APBOA
  • TCV-MN Classical Minnesota-type isolates of turkey intestinal coronaviruses
  • TCV-MN Classical Minnesota-type isolates of turkey intestinal coronaviruses
  • the claimed TCV isolates are genetically distinct from TCV-MN.
  • the claimed isolates unlike TCV-MN, produce severe thymic and bursal atrophy due to lymphocytolysis.
  • infection of turkeys with the claimed isolates resulted either in extremely high mortality, persistent infection without seroconversion, or recovery with stunting and immune system suppression. Recovered non- carrier individuals were susceptible to re-infection.
  • Bovine coronaviruses (BCV) and TCV are highly related. A 99% similarity exists between the nucleocapsid and membrane proteins of BCV and TCV-MN (Dea et al., 1990). Spike (“S 1 ") proteins of TCV-MN and BCV are also highly related, but, as described hereinbelow, the SI gene sequences of the TCV isolates of the invention are more closely related to BCV than to TCV-MN. Naturally occurring infection of cattle with the claimed pathogenic TCV strains occurs and cattle can serve as subclinical carriers or vectors. Further support for the relatedness of BCV and TCV is shown by the cross-reactivity of polyclonal antibody to BCV and TCV.
  • TCV is a diverse viral population composed of differing pathotypes including classical TCV-MN and the more recent highly pathogenic types, .and that these isolates vary in their relatedness to BCV.
  • the claimed preparation or composition comprising at least one cell culture adapted TCV isolate, e.g., TCV-NC, is also useful as an immunogenic composition or vaccine in cattle, preferably to provide neutralizing and/or protective antisera to BCV.
  • a preparation or composition comprising at least one cell culture adapted BCV isolate is useful to prepare immunogenic compositions or vaccines for turkeys so as to result in antibodies that bind to TCV.
  • a preparation or immunogenic composition comprising a plurality of enteric coronavirus isolates, e.g., a TCV isolate and a BCV isolate.
  • the preparation of the invention which comprises TCV or BCV, or a combination thereof, may optionally be combined with a physiologically acceptable carrier and/or optionally combined with one or more adjuvants, to yield an immunogenic composition or a vaccine.
  • the immunogenic composition or vaccine is injectable.
  • the .amount of isolated TCV and/or BCV in the immunogenic composition is preferably effective to actively immunize a susceptible bird, e.g., a turkey, or induce a humoral response in a mammal, e.g. a bovine.
  • Maternal antibodies obtained from the sera of a female vertebrate exposed to at least one of the preparations or immunogenic compositions of the invention, or obtained from an egg from an egg-laying female vertebrate exposed to at least one of the preparations or immunogenic compositions of the invention, may be isolated and administered to animals, e.g., in feed or by injection, to provide passive immunity.
  • the invention further provides an isolated and purified nucleic acid molecule comprising a preselected nucleic acid sequence encoding a coronavirus polypeptide, e.g., a SI polypeptide, a biologically active subunit, or a biologically active variant thereof, the presence of which is associated with SMT.
  • the preselected DNA sequence encodes a TCV SI polypeptide.
  • a more preferred embodiment of the invention includes a preselected DNA sequence comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO: 30, which encodes a polypeptide having an amino acid sequence corresponding to SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:31, respectively.
  • the nucleic acid molecules of the invention are double-stranded or single- str.anded.
  • the invention also provides an expression cassette comprising a preselected DNA sequence operably linked to a promoter functional in a host cell wherein said DNA sequence encodes a coronavirus polypeptide, a biologically active variant or subunit thereof, wherein the presence of said polypeptide in turkeys is associated with SMT.
  • a preferred DNA sequence encodes a TCV S 1 polypeptide.
  • Such expression cassettes can be placed into expression vectors which can then be employed to transform prokaryotic or eukaryotic host cells.
  • the expression of the preselected DNA sequence in the transformed cell results in the production of recombinant coronavirus polypeptide.
  • the resultant recombinant polypeptide can be isolated from transformed cells.
  • the invention also provides isolated, purified recombinant TCV SI polypeptide, a biologically active variant or a subunit thereof.
  • Recombinant coronavirus S 1 polypeptide is useful to prepare polyclonal or monoclonal antibodies.
  • the recombinant polypeptide and antibodies thereto are useful in assays to detect the presence of coronavirus-specific antisera, or coronavirus, respectively, in vertebrates, e.g., turkey, chicken, and cattle.
  • the polypeptide can be used as a "capture antigen" to bind to anti-polypeptide or anti-coronavirus antibodies in a sample of a physiological fluid or tissue obtained from an animal.
  • a physiological sample comprising antibodies is mixed with purified SI polypeptide, or a preparation of isolated coronavirus, so as to yield a binary complex.
  • the .antibodies which are bound to the polypeptide or the virus are separated from the antibodies which are not bound to the polypeptide or the virus.
  • the complex is detected or determined.
  • the complex is detected by antibodies.
  • Recombinant coronavirus S 1 polypeptide is also useful in a vaccine or immunogenic composition which, when administered to an animal, can elicit antibodies which can inhibit or block subsequent infection of the host by the coronavirus from which the SI gene was obtained, or a highly related coronavirus.
  • an isolated and purified "antisense” nucleic acid molecule which has at least about 80%, preferably at least about 90%, and more preferably at least about 98%, nucleotide sequence complementary to SEQ ID NO:2, SEQ NO:4 or SEQ ID NO:30. These molecules can be introduced into expression cassettes, which, when expressed in a host cell can provide "anti- sense" mRNA transcripts which can alter, e.g., inhibit, TCV expression.
  • nucleic acid sequences (molecules) of the present invention are useful to detect the replication or presence of the virus in infected samples, to detect related nucleic acid molecules and to amplify nucleic acid sequences, wherein said sequences fall within the scope of the present invention.
  • An oligonucleotide or primer of the invention preferably has at least about 80%), more preferably at least about 85%, and more preferably at least about 90%, sequence identity or homology, or sequence complementarity, to a nucleic acid molecule encoding an enteric coronavirus SI polypeptide, such as a BCV SI polypeptide or a TCV SI polypeptide which is associated with SMT, e.g., SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:30.
  • anti-sense oligonucleotides of the invention do not hybridize under stringent conditions to mouse hepatitis virus (MHV) nucleic acid sequences or other non-enteric avian coronavirus nucleic acid sequences.
  • An oligonucleotide or primer of the invention has at least about 7 to about 50, preferably at least about 10 to about 40, and more preferably at least about 15 to about 35, nucleotides.
  • the oligonucleotide primers of the invention comprise at least 7 nucleotides at the 3' of the oligonucleotide primer which have at least about 85%, more preferably at least about 90%, and more preferably at least about 95%, identity or complementarity, to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:30.
  • the oligonucleotides of the invention may also include sequences which are unrelated to nucleic acid sequences of a coronavirus SI gene, e.g., they may encode restriction endonuclease recognition sequences.
  • Preferred oligonucleotides include, but are not limited to, Sl-la (5' CCG ACG TAT ACC TAA TCT TCC 3'; SEQ ID NO:9), Sl-lb (5' TGC TCA CCT ATG CCA ACT 3'; SEQ ID NO: 10); SI -2a (5 'GAT AAG TCG GTG CCC TCT CCA 3'; SEQ ID NO:l 1), and SI -2b (5' ATG AAA GGC CGC TGA AAC AC 3'; SEQ ID NO: 12).
  • the oligonucleotides of the invention are useful in amplification reactions, and to detect the replication or presence of virus in a sample.
  • the method comprises contacting an amount of purified coronavirus SI polypeptide, or a clarified preparation of a cell cultured adapted isolate of coronavirus, with the sample which is suspected of comprising antibodies specific for the coronavirus, for a sufficient time to form binary complexes between at least a portion of the antibodies and a portion of the purified polypeptide or isolated virus. The presence or amount of the complexes is then determined or detected.
  • the detection of antibody responses specific for the polypeptide or specific for the isolated virus can be used in immunoassays, e.g., ELISA-based assays, for the serodiagnosis of SMT or TCV infection in animals.
  • immunoassays e.g., ELISA-based assays
  • a diagnostic kit for detecting or determining .antibodies that specifically react with a coronavirus which is associated with SMT comprises packaging, containing, separately packaged, a solid phase capable of binding a polypeptide or an isolated virus preparation, and a known amount of a purified coronavirus S 1 polypeptide or isolated cell culture adapted coronavirus.
  • the polypeptide has an amino acid sequence comprising SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:31, a variant or subunit thereof.
  • Preferred coronavirus isolates include TCV-NC, TCV-GA, TCV-BOB, and TCV-BOA.
  • the method comprises immunizing a turkey with (i) a recombinant coronavirus S 1 polypeptide, and/or (ii) an isolated cell culture adapted coronavirus preparation.
  • the SI polypeptide comprises SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:31, a variant or subunit thereof.
  • the cell culture adapted coronavirus is an isolate such as TCV-NC, TCV-GA, TCV-BOB or TCV-BOA.
  • the invention also provides an animal immunized with or exposed to (a) a recombinant coronavirus S 1 polypeptide, and/or (b) an isolated cell culture adapted coronavirus.
  • the immunized or exposed animal produces antibodies to the polypeptide and/or virus.
  • the invention provides a diagnostic method comprising contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample from a turkey at risk of, or afflicted with, SMT, or an animal exposed to said tukey, with .an amount of at least two complementary oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified DNA.
  • At least one oligonucleotide binds specifically to a nucleic acid sequence encoding a coronavirus polypeptide, wherein the coronavirus is associated with SMT. The presence of the amplified DNA is then detected or determined.
  • the presence of the amplified DNA is indicative of a turkey at risk of, or afflicted with, SMT, or an animal that is a carrier of the coronavirus associated with SMT.
  • the invention further provides a method for detecting DNA encoding an immunogenic polypeptide associated with SMT.
  • the method comprises preparing an amount of DNA from a physiological sample, wherein the DNA is obtained by reverse transcription of RNA from said sample.
  • the DNA is contacted with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified DNA. At least one oligonucleotide is specific for the DNA encoding the immunogenic polypeptide.
  • Yet another embodiment of the invention is a diagnostic kit for detecting a nucleic acid molecule that encodes at least a portion of a polypeptide which is specific for a turkey coronavirus associated with SMT in a physiological sample suspected of containing said nucleic acid molecule.
  • the kit comprises packaging containing, separately packaged, (a) a known amount of a first oligonucleotide, wherein the oligonucleotide consists of at least about 7 to about 50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:30, and (b) a known amount of a second oligonucleotide, wherein the oligonucleotide consists of at least about 7 to about 50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to a nucleotide sequence which is complementary to SEQ ID NO:2 , SEQ ID NO:4 or SEQ ID NO:30.
  • Figure 1 Codons for specified amino acids.
  • FIG. Immunofluorescent staining of turkey enteritis coronavirus- (TCV) infected HRT-18 cells.
  • FIG. 10 Nucleotide sequence relatedness of DNA encoding coronavirus SI polypeptide.
  • FEV feline enteric coronavirus (SEQ ID NO: 13).
  • HC-043 human coronavirus strain OC43 (Genbank Accession No. L14643; SEQ ID NO: 14).
  • TGEV porcine transmissible gastroenteritis virus (SEQ ID NO: 15).
  • Conn Connecticut strain of Infectious Bronchitis Virus (IBV; SEQ ID NO: 16).
  • Florida Florida strain of IBV (SEQ ID NO: 17).
  • Mass Massachusetts strain of IBV (SEQ ID NO: 18).
  • Beaudette Beaudette strain of IBV (SEQ ID NO: 19).
  • Gray Gray strain of IBV (SEQ ID NO.20).
  • JMK JMK isolate of IBV (SEQ ID NO:21 ).
  • GAV Georgia strain of IBV (SEQ ID NO:22).
  • CA California strain of IBV (SEQ ID NO:23).
  • BoCV BOA strain of turkey coronavirus (SEQ ID NO:30).
  • MN Minnesota strain of TCV (SEQ ID NO:3).
  • GA Georgia isolate of TCV (SEQ ID NO:2).
  • NC North Carolina isolate of TCV (SEQ ID NO:4).
  • BCV-L9 bovine coronavirus strain BCV-L9 (Genbank Accession No. M64667; SEQ ID NO:24).
  • BCV-LY138 bovine coronavirus strain BCV-LY138 (Genbank Accession No. M646669; SEQ ID NO:25).
  • BCV- VACCINE bovine coronavirus strain BCV- Vaccine (Genbank Accession No. M64668; SEQ ID NO:26).
  • BCV-QUEB bovine coronavirus Quebec strain (SEQ ID NO:27).
  • BCV-F15 bovine coronavirus strain F15 (SEQ ID NO:28).
  • MHV2 mouse hepatitis virus strain A59 (Genbank Accession No. Ml 8379; SEQ ID NO:29).
  • FIG. 11 Amino acid sequence relatedness of 21 coronavirus SI polypeptides.
  • FEV feline enteric coronavirus (Genbank Accession No. X80799; SEQ ID NO:32).
  • HC-043 human coronavirus strain OC43 (Genbank Accession No. L14643; SEQ ID NO:33).
  • TGEV porcine transmissible gastroenteritis virus (Genbank Accession No. X53128; SEQ ID NO:34).
  • Conn Connecticut strain of IBV (SEQ ID NO:35).
  • Florida Florida strain of IBV (SEQ ID NO:36).
  • Mass Massachusetts strain of IBV (SEQ ID NO:37).
  • Beaudette Beaudette strain of IBV (SEQ ID NO:38).
  • Gray Gray strain of IBV (SEQ ID NO:39).
  • JMK JMK isolate of IBV (SEQ ID NO :40).
  • GAV Georgia strain of IBV (SEQ ID NO:41 ).
  • CA California strain of IBV (SEQ ID NO:42).
  • BoCV BOA strain of turkey coronavirus (SEQ ID NO:31).
  • MN Minnesota strain of TCV (SEQ ID NO:7).
  • GA Georgia isolate of TCV (SEQ ID NO:5).
  • NC North Carolina isolate of TCV (SEQ ID NO:8).
  • BCV-L9 bovine coronavirus strain BCV-L9 (Genbank Accession No. M64667; SEQ ID NO:43).
  • BCV-LY138 bovine coronavirus strain BCV-LY138 (Genbank Accession No. M646669; SEQ ID NO:44).
  • BCV- VACCINE bovine coronavirus strain BCV-Vaccine (Genbank Accession No. M64668; SEQ ID NO:45).
  • BCV-QUEB bovine coronavirus Quebec strain (Genbank Accession No. D00662; SEQ ID NO:46).
  • BCV-F15 bovine coronavirus strain F15 (Genbank Accession No. P00731 ; SEQ ID NO:47).
  • MHV2 mouse hepatitis virus strain A59 (Genbank Accession No. Ml 8379; SEQ ID NO:48).
  • isolated and/or purified refer to in vitro preparation, isolation and/or purification of a nucleic acid molecule, polypeptide or peptide of the invention, so that it is not associated with in vivo substances.
  • isolated with respect to a viral isolate or strain of the invention refers to a virus preparation that was obtained by in vitro culture .and propagation, or alternatively, by in vivo passage and subsequent in vitro isolation.
  • an isolated virus preparation of the invention is substantially free of other infectious agents.
  • a coronavirus SI polypeptide or peptide is preferably a TCV polypeptide having an amino acid sequence comprising SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:31 , or a biologically active subunit thereof.
  • a "variant” coronavirus SI polypeptide is a polypeptide having an amino acid sequence which has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, but less than 100%, identity or homology to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, or a biologically active subunit thereof.
  • a variant polypeptide or peptide of the invention may include amino acid residues not present in the corresponding wild type SI polypeptide or peptide, as well as internal deletions relative to the corresponding wild type SI polypeptide or peptide.
  • the polypeptides, peptides and viral preparations of the invention are biologically active.
  • biologically active coronavirus SI polypeptides, peptides and variants thereof falling within the scope of the invention have at least about 1%, preferably at least about 10%, more preferably at least about 50%, and even more preferably at least about 90%, the activity of the polypeptide comprising SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:31.
  • the activity of a coronavirus polypeptide or peptide, or a viral isolate can be measured by methods well known to the .art including, but not limited to, the ability of the polypeptide, peptide or virus to be bound by antibodies specific for TCV, e.g., specific for the TCV SI protein, the ability of the polypeptide, peptide or virus to elicit a sequence-specific immunologic response when the polypeptide is administered to an animal such as a bird or a mammal, e.g., rabbit, goat, bovine, sheep, rat or mouse, or the correlation of the presence of the virus, or a virus having the polypeptide, and SMT.
  • TCV e.g., specific for the TCV SI protein
  • an animal such as a bird or a mammal, e.g., rabbit, goat, bovine, sheep, rat or mouse, or the correlation of the presence of the virus, or a virus having the polypeptide, and SMT.
  • the immunologic response is a humoral response, i.e, antibody response, directed to a particular epitope on the polypeptide, peptide or virus. More preferably, the presence of antibodies specific for that epitope correlates with the SMT infection status of the organism.
  • the biological activity of a viral preparation of the invention can be measured by methods known to the art, some of which are described hereinbelow.
  • a coronavirus isolate of the invention is propagated in culture, e.g., in HRT-18 cells.
  • the isolates of the invention induce cytopathic effect (cpe) in vitro in sensitive cells, .and/or have hemagluttination activity (Dea et al., 1991).
  • the isolate binds anti-TCV antibodies, as determined by assays such as IF A or a neutralization assay (Example 4).
  • the biological activity of a virus preparation of the invention in vivo can be determined by administering the virus preparation to turkeys so as to induce SMT, or to animals, e.g., turkeys or chickens, so as to elicit a specific immune response, e.g., a response which results in immunization or a virus-specific humoral response.
  • An isolated "vari.ant" nucleic acid molecule of the invention is a nucleic acid molecule which has at least 80%, preferably at least about 90%, and more preferably at least about 95%, but less than 100%, nucleotide sequence homology or identity to the nucleotide sequence of the corresponding wild type nucleic acid molecule, e.g., a DNA sequence comprising SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:30.
  • a variant nucleic acid molecule of the invention may include nucleotide bases not present in the corresponding wild type nucleic acid molecule, as well as internal deletions relative to the corresponding wild type nucleic acid molecule.
  • tissue sources of enteric coronaviruses include feces, duodenum, jejunum, ileum, bursa (for avian sources), lymph node (for non-avian sources, e.g., mammals), spleen, and thymus from these animals.
  • Further sources include turkeys or embryos thereof, chickens or embryos thereof, bovines and cell lines that have been experimentally infected with the viruses, as well as insects and wildlife which harbor the viruses (e.g., deer, birds, and rodents). "Carriers" of virus .are organisms in which the virus replicates but does not cause significant disease.
  • Coronaviruses falling within the scope of the invention may be adapted to grow in intestinal cultures from any species, e.g., rat, pig, dog and human.
  • a preferred cell culture is a permanent cell line, e.g. HRT-18 cells.
  • cultures which exhibit cytopathic effect upon infection are also preferred.
  • the viruses may also be maintained and propagated in vivo in adult turkeys, poults or embryos, chickens, cattle and wild life (see above).
  • HRT-18 cells are cultured in MEM with 3-10% fetal bovine sera at 37 °C in 5% C0 2 . Once a monolayer is established (approximately 24-48 hours after 8.12 x 10 5 cell/ml HRT-18 cells .are seeded onto a surface of 35 cm 2 or greater), the media is removed. Clarified and filtered (0.45 micron) inoculum is then added to the monolayer at 37 °C, 5% C0 2 for one hour. After incubation, MEM is added and the infected cells incubated for 5-7 days. The cells are then frozen at -70 °C and thawed.
  • Isolated coronaviruses falling within the scope of the invention include viruses which bind to anti-TCV antibodies and/or anti-BCV .antibodies, e.g., as determined by immunofluorescent assays (IF A) or neutralization assays, and/or have hemagluttination (HA) properties, and which induce SMT upon inoculation into susceptible animals. If the isolate has been propagated in vitro or in vivo in a species which is not susceptible, the isolate may require several back passages before pathogenicity is observed.
  • IF A immunofluorescent assays
  • HA hemagluttination
  • Sources of nucleotide sequences from which the present nucleic acid molecules, i.e., molecules which encode coronavirus polypeptides, can be derived include nucleic acid from any euk.aryotic source, preferably a farm animal, known or believed to be naturally or experimentally infected by a coronavirus of the invention, i.e., TCV associated with SMT or BCV useful to induce antibodies in turkeys which bind to TCV.
  • the source may be cellular or acellular (sera or plasma which contain the virus, or acellular egg components such as yolk and albumin) in origin.
  • the source is an in vitro source, i.e., cell cultures infected with the virus.
  • a nucleic acid molecule encoding a coronavirus SI polypeptide can be identified and isolated using standard methods, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor. NY (1989). For example, reverse transcriptase-polymerase chain reaction (RT-PCR) can be employed to isolate and clone coronavirus SI genes.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • PCR refers to a procedure or technique in which amounts of a preselected fragment of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195.
  • RNA sequence information from the ends of the region of interest or beyond is employed to identify and synthesize oligonucleotide primers comprising at least 7-8 nucleotides. These primers will be identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See generally Mullis et al., Cold Spring Harbor Svmp. Quant. Biol.. 51, 263 (1987); Erhlich, ed., PCR Technology. (Stockton Press, NY, 1989).
  • Primers are made to correspond to nucleic acid molecules encoding highly conserved regions of polypeptides or to nucleotide sequences which were identified and compared to generate the primers, e.g., by a sequence comparison of other coronavirus SI genes.
  • at least two primers are prepared, one of which is predicted to anneal to the antisense strand, and the other of which is predicted to anneal to the sense strand of a nucleic acid molecule which encodes the coronavirus SI polypeptide.
  • the products of each PCR reaction are separated via an agarose gel and all consistently amplified products can be gel- purified and cloned directly into a suitable vector, such as a plasmid vector.
  • the resultant plasmids are subjected to restriction endonuclease and dideoxy sequencing of double-stranded plasmid DNAs.
  • RNA is isolated from a cellular source believed to be infected with a coronavirus isolate of the invention, or supernatants from cells infected in vitro with the isolate.
  • the RNA is reverse transcribed to form a single strand cDNA.
  • the cDNA is then mixed with primers, as described above, and PCR is performed.
  • the resultant DNA fragments encode all or a portion of a gene encoding a coronavirus S 1 polypeptide.
  • These fragments can be further characterized by sequence analysis, or by expression in host cells and subsequent screening for binding to antibodies from an animal that has been infected by the same or a related coronavirus.
  • DNA fragments that have at least some sequence identity or homology to other coronavirus SI polypeptides, or which encode polypeptides that are immunoreactive with above-mentioned antibodies, can be subcloned into a suitable vector and used as probes to identify other nucleic acid sequences encoding all or a portion of a coronavirus SI polypeptide.
  • isolated and/or purified refer to in vitro isolation of a nucleic acid molecule or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or protein, so that it can be sequenced, replicated, and/or expressed.
  • isolated nucleic acid encoding coronavirus SI polypeptide is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more, sequential nucleotide bases.
  • the sequential nucleotide bases encode a biologically active coronavirus S 1 polypeptide, preferably a polypeptide associated with SMT, or a fragment thereof, or a biologically active variant coronavirus S 1 polypeptide, or a fragment thereof.
  • the DNA or RNA is complementary to the non-coding strand, or complementary to the coding strand, of RNA from the virus which causes, or is associated with, SMT and not complementary to the RNA or DNA from related organisms which do not cause, or are not associated with, SMT, or hybridizes to said RNA or DNA and remains stably bound under stringent conditions.
  • the RNA or DNA is "isolated” in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the RNA, and the isolated RNA or DNA is preferably substantially free of any other viral or eukaryotic RNA or DNA.
  • RNA or DNA encoding a polypeptide of the invention is RNA or DNA that encodes a biologically active, i.e., immunogenic, coronavirus S 1 polypeptide sharing at least about 80%, preferably at least about 90%, sequence identity with the S 1 polypeptide encoded by the nucleic acid molecules of Figure 12 ("NC", "GA” and "BO").
  • recombinant nucleic acid or "preselected nucleic acid,” e.g., “recombinant nucleic acid sequence or segment” or “preselected nucleic acid sequence or segment” refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from any appropriate cellular or acellular source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a non-recombinant viral genome.
  • RNA sequence that is obtained from said source by chemical means, e.g., by the use of reagents which remove viral capsids without affecting the integrity of viral nucleic acid, so that it can be further manipulated, e.g., reverse transcribed and amplified, for use in the invention by the methodology of genetic engineering.
  • RT-PCR of coronavirus nucleic acid may be employed to obtain amplified fragments having SI genes.
  • preselected DNA includes completely synthetic DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from biological sources, and DNA sequences derived from RNA, as well as mixtures thereof.
  • RNA molecule has complementary sequence identity to a particular DNA molecule.
  • Nucleic acid molecules encoding amino acid sequence variants of a coronavirus SI polypeptide can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non- variant version of a coronavirus SI polypeptide.
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing amino acid substitution variants of a coronavirus SI polypeptide.
  • This technique is well known in the art as described by Adelman et al., DNA. 2, 183 (1983). Briefly, coronavirus SI -specific DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the SI polypeptide. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the coronavirus SI DNA.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al., Proc. Natl. Acad. Sci. U.S.A.. 75, 5765 (1978).
  • the DNA template can be generated by those vectors that are either derived from bacteriophage Ml 3 vectors (the commercially available Ml 3mpl 8 and M13mpl9 vectors are suitable), or those vectors that contain a single- stranded phage origin of replication as described by Viera et al., Meth. Enzymol.. 153. 3 (1987). Thus, the DNA that is to be mutated may be inserted into one of these vectors to generate single-stranded template. Production of the single- stranded template is described in Sections 4.21-4.41 of Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, N.Y. 1989).
  • single-stranded DNA template may be generated by denaturing double-stranded plasmid (or other) DNA using standard techniques.
  • the oligonucleotide is hybridized to the single- stranded template under suitable hybridization conditions.
  • a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.
  • a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of the coronavirus SI DNA, and the other strand (the original template) encodes the native, unaltered sequence of the coronavirus SI DNA.
  • This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101. After the cells are grown, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabeled with 32-phosphate to identify the bacterial colonies that contain the mutated DNA. The mutated region is then removed and placed in an appropriate vector for peptide or polypeptide production, generally an expression vector of the type typically employed for transformation of an appropriate host.
  • the method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutations(s).
  • the modifications are as follows:
  • the single-stranded oligonucleotide is annealed to the single-stranded template as described above.
  • a mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP) is combined with a modified thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template- oligonucleotide complex.
  • this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.
  • the template strand of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme
  • the template strand can be digested with ⁇ xoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized.
  • the reaction is then stopped to leave a molecule that is only partially single-stranded.
  • a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E coli JM101.
  • a preferred embodiment of the invention is an isolated and purified DNA molecule comprising a preselected DNA segment encoding a coronavirus SI polypeptide, the expression of which in turkeys is associated with SMT, such as a preselected DNA encoding SEQ ID NO:6, e.g., a DNA comprising SEQ ID NO:2, or a DNA having nucleotide substitutions which are "silent" (see Table 1). That is, when silent nucleotide substitutions are present in a codon, the same amino acid is encoded by the codon with the nucleotide substitution as is encoded by the codon without the substitution. For example, valine is encoded by the codon GTT, GTC, GTA and GTG.
  • a variant of SEQ ID NO:2 at the fifteenth codon in a SI polypeptide includes the substitution of GTC, GTA or GTG for GTT.
  • Other "silent" nucleotide substitutions in SEQ ID NO:2 which can encode polypeptide having SEQ ID NO: 6 can be ascertained by reference to Table 1 and page DI in Appendix D in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989), as well as Table 1 hereinbelow. Nucleotide substitutions can be introduced into DNA segments by methods well known to the art, some of which are described above. See, also, Sambrook et al., supra.
  • nucleic acid molecules encoding other SI polypeptides may be modified in a similar manner.
  • nucleic acid molecules encoding at least a portion of SEQ ID NOJ or SEQ ID NO:8, or the complement thereto may be modified so as to yield nucleic acid molecules of the invention having silent nucleotide substitutions, or to yield nucleic acid molecules having nucleotide substitutions that result in amino acid substitutions (see polypeptide or peptide variants hereinbelow).
  • the recombinant or preselected nucleic acid sequence or segment may be circular or linear, double-stranded or single-stranded.
  • a preselected DNA sequence which encodes an RNA sequence that is substantially complementary to a mRNA sequence encoding a coronavirus SI polypeptide is typically a "sense" DNA sequence cloned into a cassette in the opposite orientation (i.e., 3 ' to 5 ' rather than 5' to 3').
  • the preselected nucleic acid sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the preselected DNA present in the resultant cell line.
  • chimeric means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species.
  • a portion of the preselected DNA may be untranscribed, serving a regulatory or a structural function.
  • the preselected DNA may itself comprise a promoter that is active in prokaryotic or eukaryotic cells, or may utilize a promoter already present in the genome that is the transformation target.
  • promoters in eukaryotes include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements), although many other promoter elements well known to the art may be employed in the practice of the invention.
  • elements functional in the host cells such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the preselected DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.
  • Control sequences is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotic cells include a promoter, and optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • "Operably linked” is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a peptide or polypeptide if it is expressed as a preprotein that participates in the secretion of the peptide or polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
  • the preselected DNA to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure.
  • Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956).
  • Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art.
  • a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity.
  • Preferred genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E. coli, .and the luciferase gene from firefly Photinus pyralis. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • vectors such as baculovirus vectors, E. coli or Salmonella vectors, or pox virus vectors are useful to express the polypeptide of the invention.
  • the recombinant DNA can be readily introduced into the host cells, e.g., mammalian, bacterial, e.g., E. coli or Salmonella, fungal, yeast or insect cells by transfection with an expression vector comprising a nucleic acid molecules of the invention by any procedure useful for the introduction into a particular cell, e.g., physical or biological methods, to yield a transformed cell having the recombinant DNA, preferably stably integrated into its genome, so that the nucleic acid molecules, sequences, or segments of the present invention are expressed by the host cell.
  • Physical methods to introduce a preselected DNA into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Biological methods to introduce the DNA of interest into a host cell include the use of DNA and RNA viral vectors.
  • the main advantage of physical methods is that they are not associated with pathological or oncogenic processes of viruses. However, they are less precise, and can result in multiple copy insertions, random integration, disruption of foreign and endogenous gene sequences, and unpredictable expression.
  • Viral vectors can be derived from poxviruses, herpes simplex virus I, retroviruses, adenoviruses and adeno-associated viruses, and the like.
  • Bacterial vectors, useful to express fusion proteins encoded by at least a portion of an SI gene operably linked to another gene in bacteria, e.g., in E. coli or Salmonella, can also be employed.
  • the transformed bacteria administered in vehicles such as water or feed, can populate the host organism's intestine where the recombinant S 1 polypeptide, preferably a fusion polypeptide comprising at least a portion of an SI polypeptide is expressed and excreted.
  • This method of administration can give rise to cellular and/or mucosal immunity.
  • cell line or "host cell” is intended to refer to well-characterized homogenous, biologically pure populations of cells. These cells may be eukaryotic cells that are neoplastic or which have been “immortalized” in vitro by methods known in the .art, as well as primary cells, or prokaryotic cells.
  • the cell line or host cell is preferably of mammalian origin, but cell lines or host cells of non-mammalian origin may be employed, including avian, plant, insect, yeast, fungal or bacterial sources. Preferred mammalian and avian cells are intestinal in origin.
  • Transfected or transformed is used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one preselected nucleic acid sequence, e.g., DNA, which DNA is also referred to in the art of genetic engineering as “heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “genetically engineered,” “non-native,” or “foreign DNA,” wherein said DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering.
  • the host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, a viral expression vector, or as an isolated linear DNA sequence.
  • the transfected DNA is a chromosomally integrated recombinant DNA sequence, which comprises a gene encoding a coronavirus SI polypeptide or its complement.
  • assays include, for example, "molecular biological” assays well known to those of skill in the .art, such as Southern and Northern blotting, RT-PCR and PCR; or "biochemical” assays, such as detecting the presence or absence of a SI polypeptide, e.g., by immunological means (ELISAs and Western blots).
  • “molecular biological” assays well known to those of skill in the .art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical such as detecting the presence or absence of a SI polypeptide, e.g., by immunological means (ELISAs and Western blots).
  • the present isolated, purified coronavirus SI polypeptides, peptides, or variants thereof can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant DNA approaches (see above).
  • a coronavirus SI polypeptide of the invention When a coronavirus SI polypeptide of the invention is expressed in a recombinant cell, it is necessary to purify the polypeptide from other recombinant cell proteins or polypeptides to obtain preparations that are substantially homogenous as to the SI polypeptide.
  • the culture medium or lysate can be centrifuged to remove particulate cell debris.
  • the membr.ane .and soluble protein fractions are then separated.
  • the SI polypeptide may then be purified from the soluble protein fraction.
  • the SI polypeptide may be purified from the insoluble fraction, i.e., retractile bodies (see, for example, U.S. Patent No. 4,518,526), if necessary.
  • SI polypeptide may be purified from contaminant soluble or membrane proteins and polypeptides by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography, and the like.
  • the fusion polypeptide may be purified by methods specific for the non-Si polypeptide portion of the polypeptide. For example, if the fusion polypeptide is a glutathione-S transferase (GST) fusion polypeptide, GST 4B beads may be employed to purify the fusion polypeptide.
  • GST glutathione-S transferase
  • SI polypeptide, variant SI polypeptide, or a biologically active subunit thereof can also be prepared by in vitro tr.anscription .and translation reactions.
  • a S 1 polypeptide expression cassette can be employed to generate S 1 gene-specific transcripts which are subsequently translated in vitro so as to result in a preparation of substantially homogenous SI polypeptide, variant SI polypeptide, or a biologically active subunit thereof.
  • the construction of vectors for use in vitro transcription/translation reactions, as well as the methodologies for such reactions, are well known to the art.
  • the solid phase peptide synthetic method is an established and widely used method to prepare peptides and polypeptides, which is described in the following references: Stewart et al., Solid Phase Peptide Synthesis. W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc. 85 2149 (1963); Meienhofer in "Hormonal Proteins and Peptides,” ed.; CH. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; and Bavaay and Merrifield, "The Peptides,” eds. E. Gross and F. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285.
  • polypeptides or peptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; eth.anol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
  • derivatives e.g., chemically derived derivatives, of a given SI polypeptide or peptide can be readily prepared.
  • amides of the SI polypeptide, peptide or variants thereof of the present invention may also be prepared by techniques well known in the .art for converting a carboxylic acid group or precursor, to an amide.
  • a preferred method for amide formation at the C-terminal carboxyl group is to cleave the peptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
  • Salts of carboxyl groups of a polypeptide, peptide or variant of the invention may be prepared in the usual manner by contacting the polypeptide or peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
  • a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
  • an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • N-acyl derivatives of an amino group of the polypeptide, peptide or variant of the invention may be prepared by utilizing an N-acyl protected amino acid for the final condensation
  • O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.
  • Formyl-methionine, pyroglutamine .and trimethyl-alanine may be substituted at the N-terminal residue of the polypeptide, peptide or variant.
  • Other amino-terminal modifications include aminooxypentane modifications (see Simmons et al., Science. 276. 276 (1997)).
  • amino acid sequence of the polypeptide or can be modified so as to result in a polypeptide or peptide variant.
  • the modification includes the substitution of at least one amino acid residue in the peptide for another amino acid residue, including substitutions which utilize the D rather than L form, as well as other well known amino acid analogs.
  • amino acid substitutions are preferred—that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • the invention also envisions polypeptide or peptide variants with non- conservative substitutions.
  • Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
  • Acid addition salts of the polypeptide, peptide or variant thereof or of amino residues of the polypeptide, peptide or variant may be prepared by contacting the polypeptide, peptide, variant or amine thereof with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid.
  • Esters of carboxyl groups of the polypeptides or peptides may also be prepared by any of the usual methods known in the art.
  • the present invention further relates to diagnostic assays for use in veterinary medicine.
  • diagnostic assays for use in veterinary medicine.
  • diagnosis of SMT the presence of antibodies to the SI polypeptide of TCV or to the corona virus associated with SMT in animal serum is determined.
  • Many types of test formats can be used. Such tests include, but are not limited to, IF A, RIA, RIST, ELISA, agglutination and hemagglutination.
  • the diagnostic assays can be performed using standard protocols such as those described by Magnarelli et al., J. Clin. Microbiol.. 20, 81 (1984); Craft et al., J. Infect. Pis.. 149. 789 (1984); Enguall et al., Immunochemistry. 8, 871 (1971); .and Russell et al., J. Infect. Dis.. 149, 465 (1984).
  • a diagnostic assay of the present invention can be constructed by coating on a surface (i.e., a solid support) for example, a plastic bead, a microtitration plate or a membrane (e.g., nitrocellulose membrane), all or a unique portion of the SI polypeptide (natural or synthetic), or an isolated virus preparation, and contacting it with the serum or other physiological fluid taken from an animal suspected of having a TCV infection or SMT.
  • any antibody bound to the immobilized SI polypeptide or virus preparation can be detected, preferably by reacting the binary antibody-antigen complexes with a "detection antibody", which detection antibody comprises a detectable label or a binding site for a detectable label.
  • Suitable detectable labels are enzymes, fluorescent labels or radiolabels.
  • all or a unique portion of the SI polypeptide or isolated virus preparation is bound to an inert particle of, for example, bentonite, polystyrene or latex.
  • the particles are mixed with serum from an animal in, for example, a well of a plastic agglutination tray.
  • the presence or absence of antibodies in the animal's serum is determined by observing the settling pattern of the particles in the well.
  • the presence or absence of TCV in a serum sample is detected.
  • Antibodies specific for the SI polypeptide or a unique antigenic portion thereof, or antibodies specific for the virus can be coated onto a solid surface such as a plastic and contacted with the serum sample. After washing, the presence or absence of TCV bound to the fixed .antibodies is detected by addition of a labeled (e.g., fluorescently labeled) antibody specific for TCV or the SI polypeptide.
  • virus preparations including cells infected with the coronavirus isolates of the invention
  • nucleic acid molecules including cells infected with the coronavirus isolates of the invention
  • polypeptides or peptides of the invention are preferably administered to an animal, e.g., a turkey or bovine, so as to result in .an immune response specific for the virus or a related virus (e.g., administration of TCV to cattle may protect cattle against BCV infection)
  • polypeptide including the polypeptide encoded by the nucleic acid molecules of the invention, or peptide.
  • the dosage of virus required for efficacy will range from about 0.1 cc containing about 10 3 virions to about 1.0 cc containing about 10 3 virions, preferably about 0.1 cc containing about 10 4 virions to about 1.0 cc containing about 10 4 virions, and more preferably about 0.1 cc containing 10 5 virions to about 1.0 cc containing 10 5 virions, although other dosages may provide beneficial effects.
  • Methods to determine the titer of a viral stock include the determination of TCID 50 and hemaglutinin titer (HA titer).
  • the dosage required is about 0.01 ⁇ g to about 300 mg, preferably about 0.1 ⁇ g to about 200 mg, and more preferably about 5 ⁇ g to about 100 mg, although other amounts may prove efficacious.
  • Dosages within these ranges can be administered via bolus doses or via a plurality of unit dosage forms, until the desired effects have been obtained.
  • the amount administered will vary depending on various factors including, but not limited to, the specific immunogen chosen, the age of the animal, live versus killed virus (for virus or infected cell inocula), and the route of inoculation.
  • the administration is to a turkey so as to result in an immune response which inhibits or prevents SMT, or to a chicken or bovine so as to result in the production of antibodies to the virus, polypeptide or peptide employed as an immunogen.
  • a turkey so as to result in an immune response which inhibits or prevents SMT
  • a chicken or bovine so as to result in the production of antibodies to the virus, polypeptide or peptide employed as an immunogen.
  • Both local and systemic administration is contemplated.
  • Systemic administration is preferred.
  • maternal antibody which antibody is obtained from a female animal exposed to a virus preparation, virus infected cells, a nucleic acid molecule or polypeptide of the invention.
  • a hen is vaccinated with at least one of the immunogenic compositions of the invention. The hen then provides passive immunity to progeny through the transfer of maternal antibody to the embryo.
  • an egg-laying animal e.g., a chicken, may be immunized and the eggs from that animal collected.
  • Antibody is recovered from the eggs and then administered to susceptible animals, e.g., turkeys, to provide passive protection.
  • the turkeys are subsequently exposed to live or killed virus, or other compositions of the invention, to provide active protection.
  • Administration of sense or antisense nucleic acid molecules may be accomplished through the introduction of cells transformed with an expression cassette comprising the nucleic acid molecule (see, for example, WO 93/02556) or the administration of the nucleic acid molecule (see, for example, Feigner et al., U.S. Patent No. 5,580,859, Pardoll et al., Immunity. 3, 165 (1995); Stevenson et al., Immunol. Rev.. 145. 211 (1995); Moiling, J. Mol. Med.. 75, 242 (1997); Donnelly et al., Ann. N.Y. Acad. Sci.. 772. 40 (1995); Yang et al., Mol. Med. Today. 2, 476 (1996); Abdallah et al., Biol. Cell. 85, 1 (1995)).
  • Pharmaceutical formulations, dosages and routes of administration for nucleic acids are generally disclosed, for example, in Feigner et al., supra.
  • the viral compositions may be administered as live, modified-live (attenuated) or inactivated virus, or optionally administered as a combination of attenuated, inactivated, and/or live virus, or in combination with a nucleic acid molecule of the invention, a polypeptide or peptide of the invention, or any combination thereof. Moreover, the administration of more than one immunogenic agent of the invention to an animal may occur simultaneously or at different times.
  • the virus may be inactivated by formalin, beta-propriolactone or ethylenimines.
  • immunogenic compositions are prepared for injection or infusion, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection or infusion may also be prepared.
  • the preparation may also be emulsified.
  • the active ingredient can be mixed with diluents, carriers or excipients which are physiologically acceptable and compatible with the active ingredient(s).
  • Suitable carriers can be positively or negatively charged or neutral avridine-containing liposomes, oil emulsions; live-in-oil; killed-in-oil, water-in-oil; Al(OH) 3 ; oil emulsion with terpene oils squalene or squalene; or aqueous.
  • Suitable diluents and excipients are, for example, water, saline, PBS, glycerol, or the like, and combinations thereof.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH- buffering agents, and the like.
  • compositions are conventionally administered parenterally, by injection, for ex.ample in birds, either intravenously, intramuscular injection to breast, lung or thigh, subcutaneous or via the beak, as well as by spraying the animals and their environment, e.g., their housing or yard.
  • the administration of maternal antibody is preferably in feed or water.
  • Nucleic acid, polypeptide and virus of the invention are preferably administered in feed, water or by spraying.
  • Formulations which are suitable for other modes of administration include suppositories, cloaca, insufflated powders or solutions, eye drops, nose drops, intranasal aerosols, and oral formulations, e.g., introduced into drinking water.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of alkylcelluloses, mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • these compositions can take the form of solutions, suspensions, tablets, pills, hard or soft gelatin capsules, sustained-release formulations such as liposomes, gels or hydrogels; or powders, and can contain about 10% to about 95% of active ingredient, preferably at about 25% to about 70%.
  • One or more suitable unit dosage forms comprising the virus preparations, nucleic acid molecules, polypeptides or peptides of the invention, which may optionally be formulated for sustained release.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the preparation may be administered at dosages of at least about 10 1 to about 10 2 virions, preferably about 10 2 to about 10 3 virions and more preferably about 10 3 to about 10 4 virions, although other dosages may provide beneficial results.
  • a unit dose of the vaccine is preferably administered parenterally, e.g., by subcutaneous or intramuscular injection.
  • an immunogenic composition comprising a coronavirus SI polypeptide, peptide or variant thereof
  • the purified SI polypeptide, peptide or variant can be isolated as described hereinabove, lyophilized and stabilized.
  • the polypeptide antigen may then be adjusted to an appropriate concentration, optionally combined with a suitable carrier and/or suitable vaccine adjuvant, and preferably packaged for use as a vaccine.
  • Suitable adjuvants include, but are not limited to, surfactants, e.g., hexadecylamine, octadecylamine, lysolecithin, di- methyldioctadecylammonium bromide, N,N-dioctadecyl-n'-N-bis(2- hydroxyethyl-propane di-amine), methoxyhexadecyl-glycerol, and pluronic polyols; pol.anions, e.g., pyran, dextran sulfate, poly IC, polyacrylicacid, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin, oil emulsions, alum, and mixtures thereof.
  • the immunogenic product may be incorporated into liposomes for use in a vaccine formulation, or may be conjugated to polysaccharides or
  • the amount administered may be at dosages of at least about 0.01 ⁇ g to about 300 mg, preferably about 0.1 ⁇ g to about 200 mg, more preferably about 5 ⁇ g to about 100 mg, and even more preferably at least about 10 ⁇ g to about 50 mg of polypeptide, although other dosages may provide beneficial results.
  • the absolute weight of the polypeptide, peptide or nucleic acid included in a given unit dosage form of vaccine can vary widely, and depends upon factors such as the species, age, weight and physical condition of the animal considered for vaccination, as well as the method of administration, e.g., feed, water or spray.
  • a unit dose of a polypeptide vaccine is preferably administered parenterally, e.g., by subcut.aneous or by intramuscular injection.
  • the polypeptides or peptides of the invention may also be conjugated or linked to an immunogenic protein, such as KLH or albumin, to enhance their immunogenicity.
  • Production animals may be individually exposed to an immunogenic composition of the invention, preferably virus, either live or killed, preferably at 1 day of age (doa) by ingestion, inhalation or injection.
  • a breeder animal may be exposed at 1 doa with killed virus as well as at multiple later points in time, preferably by interspersing live and killed virus inocula.
  • organ homogenate was also prepared from clinically normal poults from the same geographic production area (non-SMT organ homogenate).
  • Samples of small intestine, bursa, thymus, bone marrow, brain, lung, liver, spleen, kidney, sciatic nerve, heart, .and puboischiofemoralis muscle were collected at necropsy for histopathology and fixed by immersion in 10% neutral buffered formalin. Tissues were embedded in paraffin blocks, and 5 micron sections prepared. The resultant slides were stained with hematoxylin and eosin employing routine methods, and then examined using light microscopy.
  • 0.5 cc of SMT-organ homogenate was administered by oral gavage to each of 120 poults at 1 day-of-age.
  • An additional 120 poults were administered 0.5 cc of the non- SMT organ homogenate and served as vehicle controls.
  • Another 120 1 day-old poults were not gavaged, placed in identical isolation units, and served as negative controls.
  • Ambient air temperature in the isolators was 30°C on days 1 to 2, 35°C on days 4 to 7, 32°C on days 8 to 14, and 30°C on days 14 to 21. This experiment was repeated in the same isolation units with an equal number of poults. For both methods of transmission, the number of birds dying each day, totaled for each week, was recorded. Body weights and feed conversions were measured once weekly. Poults dying naturally were necropsied within 8 hours of death. All surviving poults were killed at 21 days of age by rapid cervical disarticulation and necropsied immediately. Samples of small intestine, bursa, and thymus were collected from each poult at necropsy and sections for histopathology prepared and examined as described above.
  • Follicular cortices were 2-4 lymphocytes thick, follicular epithelium was hyperplastic, and medullary portions of follicles were moderately depleted of lymphocytes. Thymic sections had severe cortical thinning with medullary tissue comprising over 90% of the thymic mass ( Figure 4). Direct electron microscopy of intestinal and bursal contents revealed a pleomorphic 70-140 nm diameter particle with short 5 to 10 nm surface projections over its profile ( Figure 5). Identical particles were adhered into large aggregates by mixing with polyclonal antisera containing anti-coronaviral antibody.
  • BW mean body weight
  • SMT organ homogenate from turkeys with SMT
  • Non-SMT Non-SMT organ homogenate
  • Severe enteritis consistent with SMT is positively correlated statistically with enteric coronavirus infection, but not with other enteric virus infections (Goodwin et al., 1995).
  • SMT has been experimentally reproduced (Brown et al., 1996b).
  • the histologic lesions described hereinabove are identical to those produced by turkey enteric coronavirus infection (Dea et al., 1991 ; Gonder et al, 1976), rotaviral infection in turkeys (Yason et al., 1987), and enteritis secondary to exposure to turkey litter contaminated with rotavirus, cryptosporidium, and other infectious agents (Perry et al., 1991).
  • the 70-140 nm particle in the small intestinal and bursal content of the naturally occurring and experimentally transmitted SMT cases is morphologically consistent with turkey enteric coronaviruses (Naqi et al., 1972; Dea et al, 1988; Dea et al., 1991; Ritchie et al, 1973).
  • the 70-140 nm particle has been isolated and antigenically confirmed as a coronavirus by fluorescent antibody analysis (Garcia et al., 1996). Major outbreaks of coronaviral enteritis in turkeys have occurred previously in Minnesota (Patel et al., 1977) and Quebec (Dea et al., 1988).
  • Coronavirus-containing organ homogenates were prepared as previously described (Example 1). Briefly, poults were experimentally inoculated with a coronavirus-containing SMT organ homogenate, killed, and samples of intestines, bursa, spleen, and thymus were collected aseptically, homogenized in a sterile blender with equal volumes of 4°C phosphate buffered saline, and divided into 7 equal aliquots.
  • Poults in each treatment were administered 1 ml of one of the 7 aliquots of inocula by oral gavage. Poults in one treatment were not gavaged and served as negative controls.
  • organ homogenates from infected poults were employed as inocula.
  • the ability of these inocula to produce disease and induce coronaviral fecal shedding was resistant to pH 12, heating to 57 °C, lyophilization, and NaCl exposure.
  • Treatment at pH 2 partially ameliorated mortality and negative effects on weight depression .and feed conversion to levels intermediate between the positive and negative controls.
  • MN-TCV Minnesota strain of turkey coronavirus
  • Virus propagation Isolation and propagation of viruses was performed by two methods. In one method, clarified clinical specimens of UGA-APN and UGA-APG isolates and the reference Minnesota strain of TCV were inoculated (0.2-0.3 ml) into the amniotic cavity of 22 to 24 day old embryonated turkey eggs (Adams et al., 1970; Dea et al., 1985; Deshmukh et al., 1973; Pomeroy, 1980) obtained from a source known to be free from common pathogens of turkeys .and with no history of coronaviral enteritis. Negative control turkey embryos were similarly inoculated with deionized distilled water.
  • Embryo intestines were harvested and homogenized in tryptose phosphate broth (TPB) (Dea et al., 1985). The homogenates were clarified by centrifugation at 5,000 x g for 10 minutes and by filtration using 0.45- ⁇ m filters. The resultant supernatants were used for subsequent embryo inoculations.
  • TPB tryptose phosphate broth
  • the other method employed to isolate and propagate viruses was the human rectal adenocarcinoma cell line HRT-18 (Dea et al., 1985), which was prepared in culture flasks and on Leighton coverslips (Dea et al., 1989e; Dea et al., 1985; Dea et al., 1989b; Dea et al., 1991).
  • the HRT-18 cell line was propagated in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), 50 ⁇ g of gentamicin/ml (Atlanta Biologicals, Atlanta, GA), and 10 U of bovine crystallized trypsin/ml (Sigma Chemical, St.
  • Confluent monolayers were inoculated with the clarified UGA-APN and UGA-APG clinical specimens or with the reference Minnesota strain of TCV. Five to six days postinoculation (PI), the monolayers were frozen and collected (Pomeroy, 1980) for electron microscopy (EM) analysis, hemagglutination (HA) activity and indirect immunofluorescence (IF A). Non-inoculated HRT-18 monolayers were used as negative controls and were processed similarly.
  • the samples were then centrifuged at 5,000 x g for 15 minutes (Lyerla et al., 1979).
  • the pellets were placed in glass slides using sterile loops, air dried and fixed in cold acetone (-29°C) for 15 minutes, rinsed three times in PBS and air dried.
  • the smears were processed for indirect staining using the hyperimmune rabbit anti-TCV serum (dilution 1 :5), and incubated at 37°C for 15 minutes, rinsed in phosphate buffered saline (PBS) and air dried.
  • a fluorescein-conjugated mouse .anti-rabbit IgG Sigma Immunochemicals, St.
  • Electron microscopy Specimens were processed in a similar fashion as mentioned above. Aliquots (0.5 ml) from clarified fecal specimens from poults with diarrhea and clarified infected-cell culture fluid were negatively stained with 2% sodium phosphotungstate (pH 7) for direct EM (Bozzola et al., 1991; Dea et al., 1989e; Dea et al., 1989d).
  • Confluent cell monolayers in Leighton tubes were inoculated with 0.2 to 0.5 ml of clarified viral suspensions of the Minnesota TCV (MN-TCV), UGA- APN or UGA-APG isolates. After incubation for 30 minutes at 37°C, the cell cultures were given maintenance medium (with 3% FBS, Atlanta Biologicals, Atlanta, GA) containing 10 U of bovine crystallized trypsin/ml (Sigma Chemical, St. Louis, MO). Leighton tubes were checked daily for CPE. The coverslips were fixed with 2% glutaraldehyde for 2 hours and washed in TBS (0.2 M Tris-buffered saline, Sigma, St. Louis, MO).
  • a second fixation was performed with 2% osmium tetroxide and then dehydrated in ethanol series of progressively increasing concentrations up to absolute ethanol, sectioned and stained with toluidine blue. Thick sections (1.0 ⁇ ) were checked for cellular morphology and CPE by light microscopy and affected monolayers were further processed.
  • tissues were sectioned (600 A thick), mounted on 200-mesh formvar carbon-coated nickel grids and stained with methanolic uranyl acetate (5% uranyl acetate, Ernest F. Fullam, Inc., Lathan, NY) for one minute, jet washed in deionized water, post-stained in Reynolds lead citrate (2.66% lead citrate, Baker, Phillipsburg, NJ; 3.52% sodium citrate, Baker, Phillipsburg, NJ; and 0.64%) sodium hydroxide, Fisher Scientific, Norcross, GA) for eight minutes and washed in deionized water (Bozzola et al., 1991).
  • the cell monolayers on the Leighton coverslips were examined by transmission electron microscopy (TEM) at 24 hours, 48 hours, 72 hours, and 96 hours PI.
  • CPE induced by the TCV isolates was first apparent on the fourth or fifth passage.
  • the first noticeable CPE in infected cell monolayers was the appearance of small, discrete syncytia and round, large, granular and refractile cells at 48 hours PI.
  • syncytia cells were more frequent and contained up to four to five nuclei.
  • there was detachment of the affected monolayer formation of syncytia cells containing up to six nuclei, and increased cytoplasmic granulation. Monolayers were not completely destroyed. All viral isolates produced similar CPE.
  • the negative control monolayers did not exhibit CPE.
  • Inoculated HRT-18 cell monolayers were mostly intact and in fair condition at 48 and 72 hours PI. In contrast, at 96 hours PI these monolayers were severely disrupted and cells were fragmented.
  • Typical coronaviral particles were detected using transmission electron microscopy (TEM). Viral particles were found from 48 hours to 96 hours PI. At 48 hours and 72 hours, the coronaviral particles were seen in a Golgi apparatus forming face or a smooth transitional region of the rough endoplasmic reticulum, previously characterized as a pre-Golgi compartment or vesicle (Avers, 1986; Holmes et al., 1995). Later, newly synthesized virions were also present in the lumina of the endoplasmic reticulum and Golgi apparatus (Holmes et al., 1995). Some particles were budding from the endoplasmic reticulum membrane (Figure 7). These particles were moderately pleomorphic but mostly spherical in shape, enveloped, and approximately 60 to 200 nm in diameter ( Figure 8).
  • TEM transmission electron microscopy
  • Typical particles had a central electron lucent or dense matrix 60 to 70 nm in diameter, surrounded by a double-layered band 17 to 30 nm in thickness (Figure 9).
  • the inner layer of this band consisted of darkly stained, knob-like long surface projections and a less obvious inner fringe of shorter surface projections (Dea et al., 1989e; Dea et al., 1989d; Dea et al., 1989c; Dea et al., 1990b; Holmes et al., 1995).
  • the light, more evenly stained outer layer had no distinguishing features. Surface projections, particularly the shorter fringe, were difficult to appreciate in the thin section preparations.
  • cytoplasmic vesicles were markedly enlarged, contained numerous coronaviral particles, and in many cases had ruptured onto the cellular surface. Discussion Previously, coronaviruses had been observed using electron microscopy in the feces of PEMS/SMT-infected turkeys, but propagation and isolation of these viruses had proven difficult. However, two PEMS/SMT-associated TCV strains were propagated in HRT-18 cells and in turkey-embryonated eggs. Evidence of viral replication in UGA-APN and UGA-APG TCV HRT-18 infected cells included the production of typical coronaviral particles as observed by electron microscopy, and the production of time-dependent increases in fluorescent staining for coronaviral antigens.
  • coronaviral particles have two different types of surface projections
  • mammalian hemagglutinating coronaviruses bovine coronavirus, porcine hemagglutinating encephalomyelitis virus
  • avian coronaviruses turkey enteric coronavirus
  • IFA confirmed that the reference Minnesota TCV is related to these two coronaviruses.
  • the strong positive reaction observed in the UGA-APN and UGA-APG TCV-infected cells and the absence of labeling in the negative controls confirms that the viruses that replicated in the HRT-18 cells shared antigenic determinants with the reference Minnesota strain.
  • Chickens were inoculated twice intramuscularly (0.5 cc in breast and 0.5 cc in thigh at 22 and 23 weeks of age) with killed UGA-APN (exposed to 0.01 % formaldehyde for 24 hours on a stir plate). Serum from these hyperimmunized chickens (collected at 24 weeks of age) neutralized UGA-APN.
  • Turkeys were inoculated three times intramuscularly (0.5 cc in the breast at 6, 7, and 8 weeks of age) with killed UGA-APN (exposed to 0.01% formaldehyde for 24 hours on a stir plate). Serum from these turkeys neutralized UGA-APN (endpoint of 8-1024). Serum from turkeys inoculated subcutaneously (i.e., in the neck at 11 and 12 weeks of age) had a virus neutralization endpoint of ⁇ 2-16.
  • Hygromycin B (5 mM in low pH media) had no TCV neutralizing activity in vitro.
  • TCV UGA-APN isolate of turkey intestinal coronavirus grown in HRT-18 cells. Mortality in all groups was less than 6%.
  • TCV UGA-APN isolate of turkey intestinal coronavirus grown in HRT-18 cells.
  • bovine-origin intestinal coronaviruses (Nebraska BCV, and bovine-origin TCV "A” , i.e., TCV-BOA, and "B", i.e., TCV-BOB) were inoculated orally (1 cc of 10 6 TCID 50 /ml) into 1 day-old turkey poults and backpassed ten times. For each passage, effects on clinical signs, body weight (Table 8), feed conversion (Table 9) and pathologic changes were determined. Three to five days postinoculation, the inoculated birds were depressed, dehydrated and many had a moderate to severe diarrhea, but had no significant gross lesions. Poor body weight gain and feed conversion was noted in inoculated turkeys.
  • Gross pathology included emaciation and diarrhea, small intestinal dilatation, distension of the ceca, mild bursal atrophy and mild thymic atrophy. Histopathological examination of thymus, bursa of Fabricius, small and large intestine revealed moderate to severe bursa atrophy and villus atrophy of the duodenum, jejunum, and ileum. Moderate lymphocytic enteritis and moderate to severe atrophy of cortex and medulla of the thymus was also observed. Marked differences were found in the body weight .and feed conversion between control and inoculated birds.
  • HRT- 18 cells were grown in MEM with 10% FBS, 5%> C0 2 at 37°C, and seeded in plates or flasks at a concentration of 8.12 x l0 5 cells/ml.
  • Neutralization assay The neutralization assay was performed essentially as described above (Example 4).
  • TCV isolates were titrated by serial dilution of virus in HRT-18 cells to endpoints as visualized by cytopathic effect (cpe).
  • a virus neutralization (VN) assay was employed to determine the neutralizing titer of sera taken from infected turkeys, vaccinated turkeys, and suspected carriers of TCV.
  • VN virus neutralization
  • virus neutralization assay described above detected virus neutralizing antibodies to the homologous TCV. Moreover, while anti-BCV antibodies neutralized TCV, there was little cross-reactivity of TCV isolates with IBV antibodies.
  • Example 7 Sequence comparison of the SI gene of turkey enteric coronavirus with bovine enteric coronavirus and avian and mammalian coronaviruses
  • Coronaviruses are enveloped viruses and the envelope contains the viral glycoproteins M, S ("spike protein"), and HE.
  • the spike protein (“SI”) binds to a specific host cell receptor and may induce fusion of viral envelope with the cell membrane.
  • Neutralizing antibodies are produced against S 1.
  • coronaviruses there is diversity in nucleotide sequence and molecular mass of SI.
  • there are several hypervariable regions in SI which include deletions and insertions.
  • RNA isolation was performed essentially according to Stern and Kennedy (1980) using the Rnaid Kit (BIO 101, Inc., Vista, CA).
  • Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) was used in a reverse transcriptase reaction (1 hour at 37°C) to obtain single-stranded cDNA from TCV RNAs using protocols such as those described in Sambrook et al. (1989).
  • a primer pair (e.g., SEQ ID NO:9 and SEQ ID NO: 10 or SEQ ID NO.l 1 and SEQ ID NO: 12), Taq DNA polymerase (5 ⁇ l/reaction), and PCR buffer (Promega Corp., Madison, WI; 50 mm KCl, 10 mM Tris HCl, pH 9.0 at 25°C), 0.1% Triton X-100 and 3 mm MgCl 2 ) were then added to individual samples. The mixture was then subjected to 30 cycles at 95 °C for 1 minute, 50 °C for 2 minutes, and 72 °C for 3 minutes.
  • the PCR products were separated by gel electrophoresis on 1% agarose gels, stained with ethidium bromide and visualized under UV light.
  • the primer pair SEQ ID NO:9 and SEQ ID NO: 10 yielded a PCR product of 717 bp while SEQ ID NO:l 1 and SEQ ID NO: 12 yielded a PCR product of 1070 bp.
  • the amplified products were sequenced using an automated sequencer.
  • the SI nucleotide sequence and corresponding amino acid sequence of the amplified SI genes were compared to other avian, bovine and mammalian coronavirus SI sequences using Dnasis ( Figure 11).
  • the TCV SI gene is about 700 bases longer than the avian IBV SI gene.
  • Phylogenetic analysis of the deduced amino acid sequence reveals a 98% similarity with most of the BCV isolates analyzed, and ⁇ 20% similarity with avian bronchitis isolates ( Figures 9 and 10). Sequence analysis indicated that UGA-APBOA and UGA-APG are the same virus. References
  • Deshmukh, D. R., C. T. Larsen, and B. S. Pomeroy Survival of bluecomb agent in embryonating turkey eggs and cell cultures. Am. J. Vet. Res.. 34:673-675. 1973. Deshmukh, D. R., and B. S. Pomeroy. Physiochemical characterization of a bluecomb coronavirus of turkeys. Am. J. Vet. Res.. 35:1549-1552. 1974.

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Abstract

L'invention concerne un segment d'ADN codant le polypeptide S1 du coronavirus entérique de la dinde. L'invention concerne également des procédés destinés à inhiber ou prévenir la mortalité foudroyante des dindes. L'invention a aussi pour objet des compositions immunogènes renfermant des coronavirus conçus pour la culture cellulaire. L'invention concerne enfin un procédé permettant de détecter la présence du coronavirus entérique de la dinde ou de ses anticorps, dans un échantillon prélevé sur un oiseau ou un mammifère.
PCT/US1998/024313 1997-11-14 1998-11-13 Compositions et procedes destines a prevenir l'enterite de la dinde WO1999025838A1 (fr)

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WO2005005596A3 (fr) * 2003-06-18 2005-05-12 Chinese Nat Human Genome Ct Sh Caracterisation des stades les plus precoces du virus du syndrome respiratoire aigu severe (sras), et applications
US8329194B2 (en) 2003-07-01 2012-12-11 The Royal Veterinary College Vaccine composition for vaccinating dogs against canine infectious respiratory disease (CIRD)
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004011651A1 (fr) * 2002-07-27 2004-02-05 The Royal Veterinary College Proteine a pointes du coronavirus respiratoire canin (crcv), polymerase et hemagglutinine/esterase correspondantes
EP2182066A3 (fr) * 2002-07-27 2010-08-11 The Royal Veterinary College Protéine de spicule, polymérase et hémaggllutininestérase de coronavirus respiratoire canin (crcv)
US7776340B2 (en) 2002-07-27 2010-08-17 The Royal Veterinary College Canine respiratory coronavirus (CRCV) spike protein, polymerase and hemagglutinin/esterase
US7981427B2 (en) 2002-07-27 2011-07-19 The Royal Veterinary College Canine respiratory coronavirus (CRCV) spike protein
WO2005005596A3 (fr) * 2003-06-18 2005-05-12 Chinese Nat Human Genome Ct Sh Caracterisation des stades les plus precoces du virus du syndrome respiratoire aigu severe (sras), et applications
US8329194B2 (en) 2003-07-01 2012-12-11 The Royal Veterinary College Vaccine composition for vaccinating dogs against canine infectious respiratory disease (CIRD)
US9023366B2 (en) 2003-07-01 2015-05-05 The Royal Veterinary College Vaccine composition for vaccinating dogs against canine infectious respiratory disease (CIRD)

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