Progress 09/15/00 to 06/30/05
Outputs
A NoV strain (GII/4) of human origin was successfully serially passaged in Gn piglets. Viral shedding was demonstrated by RT-PCR and antigen ELISA, infected cells were identified by IF and viral particles could be observed by TEM. Also, specific immune responses demonstrated seroconversion, by antibody ELISA and B and T cell ELISPOTs. The characterization of the immune responses of pigs provides preliminary data on the pattern of cytokine responses in Gn pigs after human GII NoV infection followed by homologous challenge, and also on intestinal immune (cytokine) responses to HuNoV which are difficult to assess in human volunteers. Also, we reported the first detection of NoVs in US swine, similar to HuNoVs both genetically and antigenically, suggesting that pigs could be potential reservoirs of NoVs strains. Reagents and techniques were developed to continue the study of these HuNoVs and the new porcine NoV strains. Also, studies between two distinct strains of BEC (NB
and NoV CV186-OH) revealed a lack of cross-protection between BEC of 2 different genogroup/genera, a new genogroup and potential new genus of enteric caliciviruses represented by BEC NB strain. Further characterization of the pathogenesis of the NoV BEC (CV186-OH) belonging to NoV GIII showed that, based on our preliminary results, age and inoculation route could be an important factor for BEC pathogenesis. BEC infected calves developed a transient viremia as previously shown for Gn piglets infected with PEC. The development of new reagents (VLPs and hyperimmune sera) and techniques permitted an increased sensitivity and specificity for the detection of these strains, the assessment of their prevalence in the cattle population and the evaluation of antigenic cross-reactivity between human and bovine strains, besides analysis of their genetic relationships. No antigenic cross-reactivity was evident between GI or GII HuNoVs and the GIII BoNoV (CV186-OH). Vaccine studies using BoNoV VLPs
have great significance because they not only provide information about immune responses, but they also permit evaluation of protection in contrast to the NoV VLP studies performed in human volunteers and mice. Also the prevalence of SaVs in the swine population was determined. The PoSaV strains identified are more closely related to HuSaVs and potential pig recombinant strains were found as described for human strains. The basis of the requirement of intestinal contents for SaV PEC/Cowden growth in cell culture was identified. Within the intestinal contents, bile acids were shown through an increase of cAMP, to down-regulate STAT1 phosphorylation and a subsequent signaling pathway that plays a major role in innate immunity therefore providing a possible explanation for PEC pathogenesis in the proximal intestine. In addition drugs that interfere with viral replication were tested in this system offering possibilities of studying antiviral strategies in vitro. The development of an
infectious clone of the SaV PEC/Cowden permits future studies of the genetic basis for virulence and cell culture adaptation of enteric caliciviruses using PEC/Cowden as a model.
Impacts
The replication of a HuNoV in Gn pigs is not only of great significance because it is now possible to study the pathogenesis of this disease in vivo and the hosts immune response, but also because it further supports the possibility that pigs may be reservoirs of human strains. It is also possible that pigs have their own NoVs strains but the fact that they get infected with a human isolate could imply that recombination between pig and human strains could emerge. NoV and SaV have a high prevalence in US swine. The bovine vaccine study could assess protection rates that have not been described for human or mice vaccine studies. In vitro studies are revealing the replication mechanism of these viruses and providing ground for the development of antiviral drugs.
Publications
- Han, M.G., Wang, Q., Smiley JR, Chang KO and LJ., S. 2005b. Self-Assembly of Recombinant Capsid Protein of a Bovine Norovirus (BoNV) into Virus-Like Particles and Evaluation of Cross-Reactivity of BoNV with Human Noroviruses. Journal of Clinical Microbiology 43(2).
- Souza M., Cheetham S., Saif L.J. (2005) Case Report: Evaluation of Norovirus shedding in stool samples collected during a twelve-hour period from an acute case of human gastroenteritis. American Society for Virology Annual Meeting. Pennsylvania State University. June 18-22, 2005.
- Wang, Q., K. O. Chang, M. K. Han, S. Sreevatsan, and L. J. Saif. 2005a. Development of a new microwell hybridization assay and an internal control RNA for the detection of porcine noroviruses and sapoviruses by reverse transcription-PCR. J Virol Methods (in press).
- Wang, Q., M. G. Han, S. Cheetham, M. Souza, J. A. Funk, and L. J. Saif. 2005b. Porcine noroviruses: genetic and antigenic relationships to human noroviruses. Emerg Infect Dis (in press).
- Wang, Q., M. K. Han, J. A. Funk, G. Bowman, and L. J. Saif. 2005. Genetic diversity and recombination of porcine sapoviruses. J Clin Microbiol (in press).
- Chang, K.O., Sosnovtsev, S.S., Belliot, G., Wang, Q., Saif, L.J. and Green, K.Y. 2005. Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae. J Virol 79(3), 1409-16.
- Cheetham S, Souza M, Grimes S, Han M, Wang Q, Meulia T, K, J. and L.J, Saif. 2005. Pathogenesis of a human norovirus in the gnotobiotic pig model. American Society for Virology Annual Meeting. Pennsylvania State University. June 18-22, 2005.
- Han, M.G., Cheetham S., Azevedo M., Thomas C. and Saif L.J. 2005a. Immune responses to bovine norovirus like particles with various adjuvants and analysis of protection in gnotobiotic calves. Vaccine (in press).
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Progress 01/01/04 to 12/31/04
Outputs
A human norovirus (NoV) genogroup II cluster 4 (GII-4) strain HS66 belongs to the most common genotype circulating worldwide and has the widest range of binding activity to the histo-blood group antigens, binding to all ABOH phenotypes (Harrignton, 2003). We have typed pigs for A/H phenotype (pigs do not possess the B antigen) by ELISA, immunofluorescence and immunohistochemistry (IHC) to match human NoV strains with their corresponding ABOH binding activity. When inoculated into gnotobiotic (Gn) piglets, the HS66 strain caused mild diarrhea and viral RNA was detected by RT-PCR from rectal swab fluids and intestinal contents at post-inoculation days (PID) 1-4. Some intestinal contents were NoV positive by antigen-ELISA. We have engineered virus-like particles (VLPs) of the HS66 strain and produced hyperimmune antisera to this strain in a pig and guinea pigs. We are optimizing an antibody-ELISA for testing seroconversion to human GII-4 NoVs in the exposed pigs and to
monitor serologic cross-reactivity with antiserum to porcine NoVs. We are also testing pigs intestinal tissues by IHC using a monoclonal antibody to GII NoVs. We previously reported the detection of porcine NoV RNA from fecal samples of normal adult US swine. Recently, the 3-end 3 kb of 5 porcine NoV strains were sequenced to investigate their genetic diversity and relationships to human, bovine and murine NoVs. Phylogenetic analysis of the complete major capsid protein sequences showed that the 5 strains belong to 3 genetic clusters within GII. One cluster includes the prototype porcine NoV Sw43/98 and Sw918/98 strains from Japan and the other 2 represent new clusters. One of the new clusters is located between human and porcine GII NoVs. Further analysis of the partial RNA polymerase (RdRp) and the RdRp-capsid junction regions also identified 2 strains that were likely recombinants. One strain, closely related to human NoVs, replicated in Gn pigs. Viral shedding detected by RT-PCR,
started on PID 2 and lasted for 4 days or until the pig was euthanized. NoV particles were observed in intestinal contents by immune electron microscopy using convalescent antiserum from an infected pig. The pig convalescent antiserum also cross-reacted with moderate ELISA titers (200-800) to GII-3, GII-4, and GII-6 human NoV VLPs, but with low titers (10) to GII-1 and GI-3 human NoV VLPs. In conclusion, porcine NoVs are genetically and antigenically closely related to human NoVs and certain strains may infect Gn pigs. These findings raise questions of whether pigs may be reservoirs for emergence of human NoVs.
Impacts
An antibody-ELISA using baculovirus-expressed VLPs of a human NoV GII-4 strain has been developed and will be used for detecting seroconversion of human NoV-exposed pigs and serologically related NoV infections in pigs. Because a close genetic and antigenic relationship exits between porcine and human NoVs, porcine NoV infection of pigs should provide an infection or disease model for the study of human NoV infections.
Publications
- Han, M. G., J. R. Smiley, C. Thomas, and L. J. Saif. 2004. Genetic Recombination between Two Genotypes of Genogroup III Bovine Noroviruses (BoNVs) and Capsid Sequence Diversity among BoNVs and Nebraska-Like Bovine Enteric Caliciviruses. J Clin Microbiol 42:5214-24.
- Wang Q.H., S. Cheetham, M. Souza, M.G. Han, W. Zhang, J. Funk, and L.J. Saif. Prevalence and molecular characterization of porcine noroviruses and sapoviruses. 23rd Annual Meeting of American Society for Virology, W38-9, Quebec, Canada, July 10-14, 2004.
- Wang Q.H., M.G. Han, L.J. Saif. Prevalence and molecular characterization of porcine noroviruses and sapoviruses. 2nd International Calicivirus Conference, Abstract 0190, Dijon, France, Nov. 6-10, 2004.
- Han M.G., Q. Wang, J. Smiley, C. Thomas, K. Chang, L.J. Saif. Antigenic and genetic relationships of bovine noroviruses to human noroviruses. 2nd International Calicivirus Conference, Abstract 0180, Dijon, France, Nov. 6-10, 2004.
- Chang, K.O., S. Sosnovtsev, G. Belliot, Y. Kim, Q. Wang, L. J. Saif and K. Y. Green. A Reverse Genetics System for Porcine Enteric Calicivirus is Dependent on Bile Acids for Recovery of Virus in Cultured Cells. 2nd International Calicivirus Conference, Abstract, Dijon, France, Nov. 6-10, 2004.
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Progress 01/01/03 to 12/31/03
Outputs
Gnotobiotic (Gn) pigs were evaluated as a model for the pathogenesis of Human enteric caliciviruses (HuCV). We attempted to adapt human strains to pigs and examined pig strains genetically related to HuCV. The Porcine enteric calicivirus (PEC) Cowden belongs to a different genogroup (GIII) than the human sapoviruses. We recently detected noroviruses in the same genogroup (GII) as HuCV in pigs from Ohio. We also inoculated 4 Gn pigs with or without immunosuppressive treatment, with norovirus GII/4 stools collected from a nursing home outbreak in Ohio. The animals did not develop diarrhea and rectal swabs were negative by RT-PCR. Attempts are in progress to produce human norovirus-like particles (VLPs) and use these in an ELISA to evaluate seroconversion in the HuCV-exposed pigs. Serum virus neutralizing (VN) and serum and intestinal isotype antibody (IgM, IgA or IgG) responses were compared after oral inoculation of Gn pigs with wild type (WT) and tissue culture
adapted (TC) PEC Cowden and challenge with WT PEC at 21 post-inoculation day (PID). The PEC VN antibody was first detected at PID 14, peaked at PID21 and increased again at PID 28/PCD 7 in the TC and WT PEC pigs. However, there were no significant differences in the antibody titers between the TC and WT PEC pigs at the same PID/PCD. After challenge with WT PEC, no diarrhea occurred in the TC or WT PEC inoculated pigs. These results indicate that infection of Gn pigs with TC or WT PEC induced similar serum VN and serum or intestinal IgM, IgA and IgG antibody responses and protection against diarrhea. We continued to screen cattle to identify bovine enteric caliciviruses (BECV) that may represent zoonotic strains similar to HuCV and for pathogenesis studies in Gn calves. To characterize the BECV circulating in two veal calf farms in Ohio and the genetic relationships between HuCV and BECV, we determined the complete capsid gene sequence of 19 BECV. Based on phylogenetic analyses, 13
BECV belonged to norovirus GIII distinct from human norovirus GI and GII. Nucleotide and amino acid (aa) identities among the 13 bovine noroviruses were 81 to 98% and 84 to 99%, respectively. These viruses were more closely related to NA-2 strain and other BECV from Europe, which share 87 to 97% aa identities. The aa identities to the bovine norovirus Jena strain were 59 to 69%. The CV521-OH bovine norovirus had high aa identity (94%) to the capsid gene of NA-2, whereas the RNA polymerase gene sequence was closely related to Jena BECV suggesting that CV521-OH may be a recombinant. Six BECV shared 85 to 98% nucleotide and 92 to 99% aa identities with the NB BECV (undefined genus). Our analysis of the BECV capsid gene sequences suggests that BECV with capsid gene diversity and belonging to different genera and genogroups circulate in calves on the same farm.
Impacts
There are multiple swine strains circulating that are closely related to human noroviruses. These may represent an animal reservoir and they also allow us to study the pathogenesis of noroviruses in the Gn pig model. The BECV sequenced belonged to a distinct genotype (GIII/2) from HuCV, suggesting that bovine noroviruses are unlikely to be of risk for human outbreaks. The HuCV strain from the nursing home (norovirus GII/4) did not cause diarrhea or shedding in 4 Gn pigs, in contrast to other HuCV strains described by us previously. Studies of seroconversion to norovirus in the HuCV exposed pigs will permit us to monitor subclinical infections.
Publications
- Guo M., and L.J. Saif. 2002. Pathogenesis of enteric calicivirus infections. In: Viral gastroenteritis 1st ed (Desselberger U and Gray J, eds.) Elsevier Science, Amsterdam, The Netherlands, pp.489-504.
- Guo M., Vinje J. and L.J. Saif. 2003. Caliciviruses and other potential foodborne viruses. In: Current Topics in Food Safety in Animal Agriculture, (R. Isaacson and M.E. Torrence, eds.) Iowa State Univ. Press, Ames, IOWA, pp.333-350.
- Wang Q.H., M. Guo and L. J. Saif. 2003. Serum neutralizing and isotype antibody responses in gnotobiotic (Gn) pigs inoculated orally with tissue culture-adapted (TC) or wild type (WT) porcine enteric calicivirus (PEC). In: 22nd Annual Meeting of American Society for Virology; July 12-16, 2003; Davis, California.
- Han M.G., Thomas C., Smiley J.R. and L. J. Saif. 2003. Capsid sequence diversity among bovine noroviruses and NB-like bovine enteric caliciviruses. In: 84th Annual Meeting of Conference of Research Workers in Animal Diseases; November 9-11, 2003 Chicago, Illinois.
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Progress 01/01/02 to 12/31/02
Outputs
Human enteric caliciviruses (HECV) are the leading cause of acute epidemic gastroenteritis. The relatedness of animal enteric caliciviruses (ECV) to HECV including their detection using primers to HECV in RT-PCR raises public health concerns about their zoonotic potential. We identified BECVs with similar genomic organization to Sapoviruses, but with Norovirus-like morphology. Our goals were to investigate the pathogenesis of HECV in gnotobiotic (Gn) animals and characterize bovine (BECV) and porcine (PECV) ECVs to determine their relatedness to HECV. We conducted Gn pig exposure trials using HECV to identify strains that may be adapted to this model. A human Sapovirus GI, was tested in 3 Gn pigs, 2 inoculated orally (PO) and one intravenously (IV). Rectal swabs, serum and intestinal contents, were tested by RT-PCR for viral RNA. Sera were tested for antibodies to Sapovirus by ELISA. Three human norovirus strains (1 GI and 2 GII) have also been similarly examined in
Gn pigs. In trials completed to date for all HECV strains, similar results were observed: viral shedding in most pigs at PID 1-4 detected by RT-PCR, mild diarrhea, mild histopathologic lesions in the proximal small intestine and enlarged mesenteric lymph nodes. Seroconversion occurred at post-inoculation day (PID) 29 in the Sapovirus IV-inoculated pig. We also analyzed the complete genomic sequence of the BECV NB strain and studied the pathogenesis of BECV NB in gnotobiotic calves. The 5' UTR of the NB strain which consisted of 7,453 bp and 2 ORFs was 74 bp in length. Sequence identity of the non-structural (NS) proteins of NB strain with caliciviruses of other genera was low and varied from 12.9% to 41.0% identity, Sequence identity of the NB strain with other caliciviruses in the complete ORF-1 NS protein sequence varied between 13.8%-22.6% and was lowest with viruses of the Norovirus genus. Like the ORF-1 comparison, the NB virus shared the lowest overall identity with viruses of
the Norovirus genus. A 3,205 bp sequence corresponding to the 3' terminus of the BECV CV-23 strain recently isolated from a veal calf in Ohio was determined. By sequence comparison to the NB strain and ORF analysis, the CV-23 strain had a similar genomic organization to the NB strain. A nucleotide identity of 89.1% was observed over the entire aligned sequence with the the NB strain. We also studied the pathogenesis of the NB strain in Gn calves. In calves examined for histologic evaluation, changes in small intestinal tissues were similar to those described previously for calicivirus infections in humans, calves, and pigs. Each NB strain-inoculated Gn-calf developed diarrhea on post-inoculation days 3 or 4 and no major differences occurred between oral and IV exposed calves in virus shedding or histopathologic lesions. Histopathologic lesions were limited to the small intestine and followed general patterns in which pathologic changes were most severe in duodenum. Tissues of major
organs including the lung, liver, kidney and spleen had no visible microscopic lesions, even in calves exposed to BECV NB via the IV route.
Impacts
At least limited replication of human Noroviruses and Sapoviruses occurred in Gn pigs, but confirmation by additional serial pig passage is needed. We identified a potentially new genogroup or genus of enteric calicivirus, the BECV NB strain. Whether similar NB-like counterpart calicivirus strains exist in humans or other species is unknown. Studies of the antigenic relationships of BECV and PECV to human enteric caliciviruses are needed to clarify the existence of possible animal reservoirs for human caliciviruses.
Publications
- Smiley, J.R., K.O. Chang, J. Hayes, J. Vinje and L.J. Saif. 2002. Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus. J. Virol. 76:10089-10098.
- Smiley, J., K.O. Chang, J. Vinje and L.J. Saif. Genomic characterization of the enteropathogenic bovine caliciviruses: CV95/OH, CV186/OH and NB strain-A prototype virus of a new calicivirus genogroup. 21st Annual Meeting of the Conference of American Society for Virology, Lexington, KT, Abst., July 20-24, 2002.
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Progress 01/01/01 to 12/31/01
Outputs
Human enteric caliciviruses (HuCV) are a leading cause of foodborne illness and acute gastroenteritis. The lack of an animal disease model or in vitro culture system impedes studies of the pathogenesis and host immunity to human caliciviruses (HuCV). We discovered a porcine enteric calicivirus (PEC) Cowden strain that is genetically most closely related to the HuCV Sapporo-like viruses (SLV). Similarly other researchers described bovine enteric caliciviruses (BEC) from calves and PEC from pigs that were most closely related to the Norwalk-like virus (NLV) genus of HuCV. These reports raise concerns about the zoonotic potential of animal enteric caliciviruses. Our goals were to detect and characterize additional strains of calf and pig enteric caliciviruses and compare them genetically to HuCV. We also studied the pathogenesis of PEC and BEC in gnotobiotic (Gn) pigs and calves as animal models of disease. Using the RT/PCR assay, with primer sets designed for HuCV
detection, and immune electron microscopy (IEM) we identified BECs in 2 Ohio veal herds. The 3.5 kb 3' genomic sequences of 2 US BEC strains designated BEC CV-95 OH and CV-186 OH were determined and were found to be related to European BEC strains, Jena and NA-2 and in a distinct genogroup (GIII) from current HuCV-NLVs. A BEC pathogenesis study in Gn calves using the NLV CV-186 strain was initiated. Using degenerate primers, we determined the complete (7,453 bp, NB strain) and partial (3,205 bp, CV-23 OH) genomic sequences of two other US BEC strains that may represent a new calicivirus genus. Unlike NLV related BECs, the genomic organization of the NB and CV-23 OH viruses is similar to lagoviruses and SLVs, but morphologically these viruses resemble NLV. To develop an animal model for enteric caliciviruses, we studied the pathogenesis of wild-type (WT) PEC/Cowden and tissue culture adapted (TC) PEC/Cowden in Gn pigs. We found differences in clinical signs, histopathologic lesions,
and virus-shedding patterns between the TC/PEC and WT/PEC following oral or IV inoculation. These studies confirmed attenuation of TC/PEC as compared to WT/PEC. Significantly, viremia accompanied WT/PEC infection following either oral or IV inoculation. This result was confirmed by induction of clinical PEC infections in additional Gn pigs following oral or IV inoculation with PEC positive acute sera. We previously reported only 6 amino acid changes with 3 clustered in the capsid region, between WT and TC PEC strains. Thus we are conducting studies to investigate the genetic basis for the reduced virulence and cell culture adaptation of the attenuated TC/PEC by creation of an infectious clone of the TC/PEC strain. Purified genomic TC/PEC RNA was used to transfect LLC-PK cells. Virus growth in this system was dependent on the presence of an intestinal content preparation in the cell culture medium as is required for passage of TC/PEC in LLC-PK cells. Several full-length TC/PEC clones
were constructed and in vitro transcription-translation assays using some of these clones yielded mature viral proteins. In the future we will investigate if any of the full-length PEC clones or transcripts are infectious.
Impacts
Enteric caliciviruses are the major cause of foodborne viral gastroenteritis in humans resulting in significant economic losses due to medical care and lost productivity. The lack of a susceptible animal disease model or in vitro culture system for enteric caliciviruses hinders our understanding of the pathogenesis of disease caused by these viruses in infected hosts, as well as our knowledge of cellular or molecular determinants in the virus life cycle. The research objectives addressed in this study will advance our understanding of the pathogenesis and distribution of disease caused by these viruses in calves and pigs, using either characterized PEC or BEC strains, or NLV or SLV HuCV strains to establish an animal model for HuCV. Major findings include: 1) the identification of a potentially new genus of enteric caliciviruses represented by the BEC/NB and CV23 OH strain and the development of new primer sets for their detection to examine the possible existence of
a similar counterpart HuCV; 2) the first report of an attenuated enteric calicivirus; 3) induction of diarrhea and intestinal lesions in gnotobiotic pigs following intravenous inoculation with WT/PEC and the presence of viremia following PEC infection; and 4) demonstration that genomic RNA of TC/PEC is infectious in LLC-PK cells with intestinal contents in the medium.
Publications
- Guo, M., J. Hayes, K.O. Cho, A.V. Parwani, L.M. Lucas and L. J. Saif. 2001. Comparative pathogenesis of tissue culture adapted and wild type Cowden porcine enteric calicivirus (PEC) in gnotobiotic pigs and induction of diarrhea by intravenous inoculation of wild type PEC.J. Virol. 75:9239-9251.
- Guo, M., J.F. Evermann, and L.J. Saif. 2001. Detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink. Arch. Virol. 146:479-493.
- Smiley, J.R., K.O. Chang, and L.J. Saif. Complete genome sequence of a bovine enteric calicivirus genogroup. Conference of Research Workers in Animal Disease, Abst. #117, St. Louis, MO, Nov. 12-13, 2001.
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