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Molecular Studies of Porcine Enteric Caliciviruses: Relationship with Human Calciviruses, Pathogenesis in Gnotobiotic Pigs, and Capsid Protein Expression in Baculovirus

Guo, Mingzhang
http://rave.ohiolink.edu/etdc/view?acc_num=osu1391676674

Abstract Details

2000, Doctor of Philosophy, Ohio State University, Veterinary Preventive Medicine.
Human caliciviruses (HuCVs) are the leading cause of food- and waterborne epidemic viral gastroenteritis worldwide. Enteric caliciviruses of animals are emerging pathogens causing diarrhea in their respective hosts. Porcine enteric caliciviruses (PEC) are associated with diarrhea in pigs and to date the PEC/Cowden is the only cultivable enteric calicivirus (TC PEC). By using RT-PCR and DNA sequencing, the complete genomes of both the wild type (WT) and TC PEC/Cowden were sequenced. The PEC/Cowden has an RNA genome of 7,320 nucleotides (excluding its 3' poly(A)+ tail), which is composed of two ORFs, similar to those of the human “Sapporo-like viruses" (SLVs) and the lagoviruses. The ORF1 encodes the polyprotein that is fused to and contiguous with the capsid protein. ORF2 at the 3' end encodes a small basic protein of 164 amino acids. Among caliciviruses, PEC shares the highest amino acid sequence identities in the putative RNA polymerase (66%), 2C helicase (49.6%), 3C-like protease (43.7%) and capsid (39%) regions with the SLVs, indicating that it is most closely related genetically to the SLVs. The WT and TC PEC/Cowden share 100% nucleotide sequence identities in the 5' terminus, 2C helicase, ORF2 and the 3' NTR. The TC PEC/Cowden has 2 aa substitutions in the RNA polymerase, and 1 distant and 3 clustered aa changes in the predicted capsid region. These substitutions may be associated with adaptation of TC PEC/Cowden to cell culture. Both the TC and WT PEC/Cowden were used to orally inoculate 14 neonatal gnotobiotic pigs. No diarrhea developed in the TC PEC exposed pigs, whereas moderate to severe diarrhea developed in the WT PEC exposed pigs by postinoculation day (PID) 2 to 4, persisting for 2 to 5 days. Fecal virus shedding was detected by both the RT-PCR and ELISA (by PID1 or 2) and persisted for at least 7 days (or until euthanasia) in both the TC and WT PEC orally inoculated pigs, but not in mock- inoculated pigs. Mostly mild (duodenum, jejunum) or no villous atrophy was observed in histologic sections of the small intestines of TC PEC-inoculated pigs, whereas the WT PEC caused mild to severe villous atrophy, villous fusion and vacuolization. Scanning electron microscopy confirmed mild shortening and blunting of villi in the duodenum and jejunum of the TC PEC-inoculated pigs, in contrast to the moderate to severe villous shortening and blunting in the duodenum and jejunum of WT PEC-inoculated pigs. Thus the TC PEC was at least partially attenuated after serial passage in cell culture. Furthermore, the neonatal gnotobiotic pigs that were intravenously (IV) inoculated with WT PEC also developed diarrhea and histopathologic changes (villous atrophy and fusion) in the small intestines, resembling those observed in the WT PEC-orally- inoculated pigs. Fecal viral shedding was detected as described above. No diarrhea and small intestinal lesions were observed in a formalin-inactivated WT PEC-IV-inoculated pig or 2 mock-IV-inoculated pigs. Interestingly, PEC RNA and antigens (in low titers) were detected sporadically in sera only from the WT PEC-IV-inoculated pigs. To our knowledge, this is the first report of an attenuated enteric calicivirus and the use of the IV inoculation route to induce diarrhea and intestinal lesions in gnotobiotic pigs with WT PEC. Infection of pigs with PEC should be a useful model for studies of the pathogenesis, replication and possible rescue of uncultivable human enteric caliciviruses. So far, no serologic assays are available for animal enteric caliciviruses. To develop diagnostic assays for PEC antigens and antibodies, the complete PEC capsid gene was cloned and used to generate the recombinant baculoviruses for expression of the PEC capsid protein in insect (Sf9) cells. The recombinant capsids produced in baculovirus self-assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs reacted with pig hyperimmune and convalescent sera to PEC/Cowden in ELISA and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400-204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP- ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP-ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific and reproducible, and might be used for large-scale serological investigations of PEC antibodies in swine.
Linda J. Saif (Advisor)
Kenneth W. Theil (Committee Member)
Daral J. Jackwood (Committee Member)
John H. Hughes (Committee Member)
210 p.
Veterinary Services

Recommended Citations

Citations

  • Guo, M. (2000). Molecular Studies of Porcine Enteric Caliciviruses: Relationship with Human Calciviruses, Pathogenesis in Gnotobiotic Pigs, and Capsid Protein Expression in Baculovirus [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1391676674

    APA Style (7th edition)

  • Guo, Mingzhang. Molecular Studies of Porcine Enteric Caliciviruses: Relationship with Human Calciviruses, Pathogenesis in Gnotobiotic Pigs, and Capsid Protein Expression in Baculovirus. 2000. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1391676674.

    MLA Style (8th edition)

  • Guo, Mingzhang. "Molecular Studies of Porcine Enteric Caliciviruses: Relationship with Human Calciviruses, Pathogenesis in Gnotobiotic Pigs, and Capsid Protein Expression in Baculovirus." Doctoral dissertation, Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1391676674

    Chicago Manual of Style (17th edition)

osu1391676674
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This open access ETD is published by The Ohio State University and OhioLINK.