PORCINE ENTERIC CALICIVIRUS: MOLECULAR ANALYSIS AND SYNTHETIC RNA TRANSCRIPTS

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4030	1101	40%
311	3510	1090	10%
712	4030	1101	40%
712	3510	1090	10%

Progress 10/01/99 to 09/30/04

Outputs
Enteric calicivirus (ECV) are emerging pathogens of humans and animals and comprise 2 genera: Sapovirus (SaV) and Norovirus (NoV). Data on the prevalence and genetic similarities of PEC to human SaV is lacking. The SaV porcine enteric calicivirus (PEC)/Cowden is the only cultivable ECV, but grows only with intestinal contents (IC) from uninfected gnotobiotic pigs in the medium. Our goals were to develop diagnostic tests for SaV PEC, survey their prevalence and genetic diversity, identify the factors in IC promoting growth of PEC and develop an infectious clone of the cell-adapted (TC) PEC/Cowden. Using RT-PCR and newly designed SaV primers, we found SaV PEC are prevalent in nursing, post-weaning and finisher swine from OH and two other states. We also detected a high seroprevalence of antibodies to SaV PEC among OH swine using recombinant PEC virus-like-particles in an antibody ELISA. The SaV PEC were genetically diverse with three genetic clusters. Some strains were closely related to human SaV suggesting the possibility of swine as reservoirs for human SaV. Sequence analysis of the attenuated TC and virulent wild type (WT) PEC/Cowden revealed only 6 amino acid differences encoded by their genomes. The potential role of these mutations in the cell adaptation and virulence of PEC will be explored in future studies by use of the infectious cDNA clone of TC PEC/Cowden successfully generated in this project. This is the first report of a reverse genetics system for any ECV. We further found that an initial cAMP cell signaling pathway induced by IC is essential for PEC replication in vitro. Bile acids were identified as the active factor in IC that induced the increased cAMP and down-regulated interferon-mediated STAT 1 activation, a key element in innate immunity to viruses. We propose a novel mechanism for the targeted ECV growth in the proximal intestine dependent on bile acids at this site and involving the PKA cell signaling pathway with down-regulation of innate immunity.

Impacts
Enteric caliciviruses are the leading cause of food-borne viral gastroenteritis in humans. However, no information was available on the prevalence or diversity of PEC and their relationship to human caliciviruses. We developed sensitive assays for viral detection (RT-PCR) and antibody detection using PEC VLP. Using these assays we found a high SaV PEC prevalence (42-86%) in OH, NC and MI farms tested and high antibody prevalence in OH swine sera. After sequence analysis of the full length genomes of WT PEC and TC PEC/Cowden, we identified only a few mutations between them, which should aid in determining the molecular basis for cell culture adaptation and virulence of PEC. We analyzed the sequences of field strains of SaV PEC and found 3 distinct genetic clusters, indicating genetic diversity among SaV PEC. Our data revealed that PEC virus replication is dependent on a cAMP signaling pathway induced by IC, a novel mechanism whereby virus growth is dependent on a cell signal transduction pathway induced by the surrounding intestinal microenvironment (IC). Moreover bile acids which induced increased cAMP concentrations and down-regulated STAT1 were identified as an active factor in IC required for PEC growth. Finally, we successfully generated synthetic TC PEC/Cowden viral RNA transcripts infectious for LLC-PK cells. Development of this first reported reverse genetics system for an enteric calicivirus is important to investigate the molecular basis for in vitro replication and in vivo pathogenesis of enteric caliciviruses and to serve as a model system for uncultivable HECV.

Publications

Guo, M, K.O. Chang, M.E. Hardy, Q. Zhang and L.J. Saif. 1999. Cloning and sequencing of a porcine enteric calicivirus genetically related to Sapporo like viruses. J. Virol. 73:9625-31.
Guo M, Qian Y, Chang KO, Saif LJ. 2001. Expression and self-assembly in baculovirus of porcine enteric calicivirus capsids into virus-like particles and their use in an enzyme-linked immunosorbent assay for antibody detection in swine. J Clin Microbiol 39:1487-93.
Guo M, Hayes J, Cho KO, Parwani AV, Lucas LM, Saif LJ. 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 75:9239-51.
Chang, K.O., Y. Kim, K.Y., Green, and L.J. Saif. 2002. Cell culture propagation of porcine enteric caliciviruses mediated by intestinal contents is dependent on cyclic AMP signalling pathway. Virology 304:302-310.
Guo, M., J. Vinje and L.J. Saif. 2003. Caliciviruses and other potential foodborne viruses. In Microbial. Food Safety in Animal Agriculture: Current Topics. (R. Isaacson and M.E. Torrence, eds.), Iowa State Press, Ames, Iowa, pp. 333-350.
Guo, M, K.O. Chang, M.E. Hardy, Q. Zhang and L.J. Saif. 1999. Cloning and sequencing of a porcine enteric calicivirus genetically related to Sapporo like viruses. Presented at the International Workshop on Human Caliciviruses. Atlanta, Georgia, March 29-31, 1999, Abst.
Guo, M, K.O. Chang, M.E. Hardy, Q. Zhang and L.J. Saif. 1999. The complete nucleotide sequence of the RNA genome of a porcine enteric calicivirus genetically related to Sapporo like viruses. Presented at the18th annual meeting of American Society for Virology. Amherst, MA, July10-14, 1999, Abst.
Guo, M. A.V. Parwani, K-O. Cho, L.M. Lucas and L.J. Saif. Pathogenesis of tissue culture adapted porcine enteric calicivirus in gnotobiotic pigs. Conference of Research Workers in Animal Disease, Abst. #106, Chicago, IL, Nov. 8-9, 1999.
Guo M, Qian Y, Chang KO, and Saif LJ. 2000. Expression and self-assembly in baculovirus and immunogenicity of the porcine enteric calicivirus/Cowden capsid virus-like particles. American Society of Virology, Abst, Ft. Collins, CO, 2000.
Wang, Q.H., M.G. Han, A.E. Hoet, and L.J. Saif. Hemagglutination by Cowden porcine enteric calicivirus and development of hemagglutination-inhibition test. Conference for Research Workers in Animal Disease, Abst. #102, St. Louis, MO November 10-12, 2002.
Chang, K.O., S. Sosnovtsev, G. Belliot, L.J. Saif and K.Y. Green. Generation and characterization of infectious RNA transcripts or porcine enteric calicivirus in cell culture. Am. Soc. Virol., Abst. Davis, CA, July 12-16, 2003.
Wang, Q.H., M. Guo and L.J. Saif. 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). Am. Soc. Virol., Abst., Davis, CA, July 12-16, 2003.
Wang, Q., S. Cheetham, M. Souza, M.G. Han, W. Zhang, J. Funk and L.J. Saif. Prevalence and molecular characterization of porcine noroviruses and sapoviruses. Am. Soc. Virol., Abst., Montreal, Canada, July 10-14, 2004.
Wang, Q.H., M.G. Han, J. Funk, and L.J. Saif. Prevalence and molecular characterization of porcine noroviruses and sapoviruses. 2nd Int. Calicivirus Conf, Abst. #190, Dijon, France, November 6-9, 2004.
Chang, K.O., S.V. Sosnovtsev, G. Belliot, Y. Kim, L.J. Saif, and K.Y. Green. 2004. Bile acids are essential for porcine enteric calicivirus replication in association with down-regulation of signal transducer and activator of transcription 1. Proc. Natl. Acad. Sci. USA 101:8733-8.
Chang, K.O., S.V. Sosnovtsev, G. Belliot, Q. Wang, L.J. Saif and K.Y. Green. 2005. A reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae. J. Virol. (in press).
Guo, M.and L.J. Saif. 2002. Pathogenesis of enteric calcivirus infections. In Viral Gastroenteritis, (U. Desselberg and J. Gray, eds.) Elsevier Science, Amsterdam, The Netherlands, pp. 489-504.
Han, M.G., Q.H. Wang, J. Smiley, C. Thomas, K. Chang, and L.J. Saif. Antigenic and genetic relationships of bovine noroviruses to human noroviruses. 2nd International Calicivirus Conf., Abst # 180, Dijon, France, November 6-9, 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 Int. Calicivirus Conf, Abst, Dijon, France, November 6-9, 2004.

Progress 01/01/03 to 12/31/03

Outputs
Enteric caliciviruses cause diarrhea in pigs and are the leading cause of foodborne viral gastroenteritis in humans. We previously found that growth of porcine enteric calicivirus (PEC) Cowden strain (Sapovirus, GIII) in cell culture requires the presence of intestinal contents (IC) in the cell culture medium. Recently we cloned the full-length cDNA of the tissue-culture adapted (TC) PEC Cowden strain into the vector pCDNA3.1 directly downstream from the T7 RNA polymerase promoter. Capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells, but only in the presence of IC in the medium, as determined by immunofluorescence, antigen-ELISA, and electron microscopy (EM) detection of viral particles. The infectious cDNA clone has 10 nucleotide (4 amino acids) point mutations throughout its genome compared to the parent TC PEC/Cowden. The recovered progeny virus was plaque purified 3 times and sequence analysis of the RT-PCR fragments confirmed the presence of the previously identified nucleotide (nt) differences with wild type PEC/Cowden. Further studies are in progress to define the infectivity of this clone in pigs. In additional studies, porcine norovirus RNA was recently detected in 4 healthy finisher pigs by RT-PCR using the broadly reactive primer pair p290 and p110 targeting the RNA dependent RNA polymerase gene (RdRp Screening of 280 pig field fecal samples collected from 5 Ohio and 2 NC swine farms revealed only 4 norovirus positives or only 1.4% (4/280) of the samples. After cloning and sequencing or direct sequencing of the RT-PCR products, 274 nt sequences for each sample, excluding the primer sequences, were obtained and compared. The 4 samples shared 71-99% nt identity. They shared the highest nt identity (71-93%) with the porcine norovirus Sw43 strain (Norovirus GII/9) detected in the cecum of healthy pigs in Japan.

Impacts
The lack of an in vitro cell propagation system remains a major limitation for the detailed study of human enteric caliciviruses (HuCV). The TC PEC/Cowden provides a cell culture model to study receptors and replication strategies, for verification of infectious clones, and to assay neutralizing antibodies to determine viral serotypes. Because PEC/Cowden has been shown to cause disease and induce similar intestinal lesions as HuCV infections, it is possible to use PEC/Cowden as a model to investigate the pathogenesis of enteric caliciviruses. Our infectious clone of TC-PEC/Cowden provides a useful tool to study the molecular basis for replication, growth restriction, and pathogenicity of enteric caliciviruses. Porcine norovirus RNA has been detected after screening of 280 pig fecal samples collected from Ohio farms. Norovirus RNA was previously detected in pigs in Japan and Europe, but not in the U.S. Detection of porcine norovirus RNA in pigs raises questions about its zoonotic potential and the pig as a possible animal reservoir for human noroviruses.

Publications

Chang K-O, Sosnovtsev S, Belliot G, Saif LJ, and Green KY. 2003. Generation and characterization of infectious RNA transcripts of porcine enteric calicivirus in cell culture. 22nd Annual Meeting of the American Society for Virology, July 12-16,2003, Davis, CA.
Chang, K.O., Y.Kim, K.Y. Green and L.J. Saif. 2002. Cell culture propagation of porcine enteric calicivirus mediated by intestinal contents is dependent on teh cyclic AMP signaling pathway. Virology 304:302-310.

Progress 01/01/02 to 12/31/02

Outputs
Enteric caliciviruses cause diarrhea in pigs and are the leading cause of food-borne viral gastroenteritis in humans. We found that porcine enteric calicivirus (PEC/Cowden strain) growth in cell culture requires the presence of an intestinal contents preparation (ICP) in the cell culture medium, and the effective factor for virus growth is a protein (or peptide) < 50 kDa. Recently we found that the tissue culture-adapted PEC/Cowden (TC-PEC) hemagglutinates both young and adult pig red blood cells (RBC) with high hemagglutination (HA) titers (1,024), but does not agglutinate RBCs of rats, mice, guinea pigs, chickens, calves and sheep. The HA of PEC was inhibited by hyperimmune antiserum to PEC or PEC virus-like particles (VLP) in a hemagglutination-inhibition (HI) test, and these antisera had high HI titers (20,480-81,920). The histo-blood group antigens are present on intestinal epithelial cells of pigs, so these RBC antigens may function as PEC receptors during infection. At present, we are treating pig RBCs with different enzymes to identify whether the receptors on pig RBCs are proteins or carbohydrates. We compared use of human enteric calicivirus (HuCV) primer sets in RT-PCR for detection of PEC in swine farms. We then sequenced the PEC strains detected to determine their genetic relatedness to one another and to HuCV. Finally based on the PEC sequences obtained, we designed new primer sets specific for these PEC strains to monitor their prevalence among swine. Of the HuCV primer sets tested, the p289/290 set was the most sensitive for detecting PEC. Using 15 PEC positive fecal samples, a newly designed primer set PEC66/65 (15/15, 100%) was more sensitive than p289/290 (7/15, 47%). Using the PEC66/65 set, PEC was detected in 51% (80/156) of fecal samples collected from 3 swine herds in Ohio. The PEC was shed mainly by post-weaning and nursing pigs, but not adults in one of the three farms. Sequence analyses indicated that the PEC detected in swine fecal samples shared the highest nucleotide (71-93%) and amino acid (94-99%) sequence identities in the partial RNA dependent RNA polymerase (RdRp) region with PEC/Cowden, indicating that they also belong to the Sapovirus genus containing the HuCV. These PEC strains shared higher amino acid sequence identity with the human sapoviruses (55-66%) than with human noroviruses. The PEC field strains and PEC/Cowden fell into 3 clusters (genotypes), suggesting genetic diversity exists among PEC. To monitor the existence of RT-PCR inhibitors, we designed and produced one internal RNA control based on PEC66/65 and PEC46/45 primer sets, which can be amplified by these two sets of primers, but the product sizes can be differentiated from those of PEC products on agarose gel electrophoresis. Clones containing the full-length TC-PEC genome were generated and work continues toward development of a TC-PEC infectious clone.

Impacts
The lack of an in vitro cell culture system remains a major limitation for the detailed study of HuCV. The cultivable TC-PEC, a sapovirus, is a good model to study enteric calicivirus receptors and replication strategies, for verification of infectious clones and for assay of neutralizing antibodies to determine viral serotypes and immune responses. Because the histo-blood group antigens are present on intestinal epithelial cells of pigs, PEC hemagglutination of pig RBCs may provide insight into the cell receptors for PEC infection. Viremia occurs during PEC infection of gnotobiotic pigs; whether the RBCs bind to PEC and transport virus to other tissues, requires further investigation. The development and improvement of molecular diagnosis of PEC infections of pigs will provide tools to study the epidemiology of PEC infections of swine.

Publications

Wang, Q. H., M. G. Han, A. E. Hoet, and L. J. Saif.Hemagglutination by Cowden porcine enteric calicivirus and development of a hemagglutination-inhibition test. Abs # 102. Proc. 83rd Conference of Research Workers in Animal Diseases, St. Louis, Missouri, Nov. 10-12, 2002.
Chang, K.O., Y.Kim, K.Y. Green, and L.J. Saif. 2002. Cell culture propagation of porcine enteric calicivirus mediated by intestinal contents is dependent on the cyclic AMP signaling pathway. Virology 304:302-310.

Progress 01/01/01 to 12/31/01

Outputs
Enteric caliciviruses cause diarrhea in pigs and are the leading cause of food-borne viral gastroenteritis in humans. Our goal is the molecular analysis of the attenuated, tissue culture-adapted [TC] porcine enteric calicivirus (PEC/Cowden strain) and generation of synthetic RNA transcripts. Because this strain grows in pig kidney (LLC-PK) cells, but only with intestinal contents (IC) from uninfected gnotobiotic pigs in the medium, we investigated the relationship between IC and growth of Cowden PEC. Pretreatment of cells or the virus with IC or transfection of viral RNA into cells did not promote virus growth unless medium was supplemented with IC. After various pretreatments of IC and size exclusion fractionation of IC, we found that the IC effective factor(s) for virus growth was protein(s) (<50,000 MW). Several inhibitors related to modulation of cell signal transduction inhibited the IC effects on PEC virus growth in a dose dependent manner for up to 72 hrs. Although the cAMP level did not differ between IC and mock treated cells, it was significantly decreased by treatment of cells with the inhibitors for up to 36 hrs. The viral replicase complex was isolated from virus infected cells only in the presence of IC at 30 hrs, and actively synthesized genomic and subgenomic RNA of PEC in vitro. The viral RNA was infectious after transfection into LLC-PK cells and infectious virus particles were recovered in the cell culture supernatants. Thus it is feasible to generate synthetic infectious RNA. Several additional full length PEC clones were generated after amplifying full length RT-PCR products. The clones contain a full-length Cowden TC PEC genome including the poly-A tail, directly downstream of the T7 promoter. The TNT (in vitro transcription and translation) assay of the clones showed that they could produce mature viral proteins. However, synthetic RNA of the clones was not infectious after transfection into LLC-PK cells. Sequence analysis of the clones showed that they contained at least 10 point mutations throughout the genome of each clone. At present, we are trying to generate consensus full length PEC clones for the infectious RNA transcripts.

Impacts
The lack of an in vitro cell propagation system remains a major limitation for the detailed study of enteric caliciviruses including production of infectious virus, studies of receptors and replication strategies, verification of infectious clones and assay of neutralizing antibodies to determine viral serotypes and immune responses. Thus, studies of the potentially novel growth-promoting factors in IC for Cowden PEC and the generation of infectious RNA transcripts would be a breakthrough for research on enteric caliciviruses and potentially other uncultivable viruses.

Publications

Guo M, Qian Y, Chang KO, Saif LJ. 2001. Expression and self-assembly in baculovirus of porcine enteric calicivirus capsids into virus-like particles and their use in an enzyme-linked immunosorbent assay for antibody detection in swine.J Clin Microbiol 39(4):1487-93.

Progress 01/01/00 to 12/31/00

Outputs
Enteric caliciviruses are emerging pathogens associated with diarrhea in swine and food-borne gastroenteritis in humans. Our goal is to characterize porcine enteric caliciviruses (PEC), to study their epidemiology in swine and to create synthetic RNA transcripts of PEC. By using RT-PCR with broadly reactive human caliciviral primers, PEC were detected in fecal samples from diarrheic pigs in 4 swine farms examined. Further studies of one of the 4 swine farms indicated that PEC were detected by RT-PCR in pigs of several age groups: nursery (19/31), post-weaning (39/43), and finisher (3/10), but not in the adult breeders (0/10). Serologic studies using PEC virus-like particles (VLP) in ELISA demonstrated that there was a high seroprevalence to PEC/Cowden in nursery (10/10, 1:800-6400), finisher (10/10, 1:800-12800), and adult breeder (10/10, 1:400-12800) swine. Three different strategies were used for constructing a full-length PEC clone; 1) by ligating 3 partial length clones (F1 to F3) covering the enteric PEC genome into a full length clone; 2) by generating full-length RT-PCR products; and 3) by constructing cDNA libraries. Several full length PEC clones were generated after ligating the 3 partial clones and by amplifying full length RT-PCR products. The clones contain a full-length Cowden [cell culture adapted (TC)] PEC genome including the poly-A tail, directly downstream of the T7 promoter. An in vitro transcription and translation assay of the RT-PCR generated clones showed that these clones produced mature viral proteins. After synthesizing RNA by in vitro transcription of the clones using the T7 polymerase, transfection of the RNA into LLC-PK cells is being performed. Sequencing of the clones is also being done to identify and generate consensus full length PEC clones. In future work we will continue to investigate if any of the full length PEC cones are infectious for LLC-PK cells. Once we produce infectious clones, we will generate several mutant PEC viruses by site-directed mutagenesis to study the molecular basis of viral replication in the LLC-PK cell culture system and the pathogenesis of PEC in pigs.

Impacts
The human enteric caliciviruses (HuCV) are the leading cause of foodborne viral gastroenteritis worldwide. A porcine enteric calicivirus (PEC) is the only cell culture adapted enteric calicivirus and is genetically similar to HuCV. Thus studies of the epidemiology of PEC infections, genetic analysis of PEC strains and creation of infectious PEC clones may lead to an increased understanding of the prevalence of PEC in swine, their similarity to human caliciviruses and the molecular basis for the pathogenesis of human and animal enteric caliciviruses.

Publications

Guo, M., J.F. Evermann and L. J. Saif. 2000. Detection and molecular characterization of enteric caliciviruses associated with diarrhea in mink and cultivable caliciviruses from clinically normal mink. Conference of Research Workers in Animal Disease, Abst. #79, Chicago, IL, Nov. 13-14.
Guo, M., Y. Qian, K.O. Chang and L. J. Saif. 2000. Expression and self-assembly in baculovirus and immunogenicity of the porcine enteric calicivirus/Cowden capsid virus-like particles. American Society of Virology, Ft. Collins, CO, July 8-12.
Guo, M. (L.J. Saif, advisor) 2000. Molecular studies of porcine enteric caliciviruses: relationship with human caliciviruses, pathogenesis in gnotobiotic pigs, and capsid protein expression in baculovirus. The Ohio State University.

Progress 01/01/99 to 12/31/99

Outputs
Enteric caliciviruses cause diarrhea in pigs and are the leading cause of food-borne viral gastroenteritis in humans. Our goal is the molecular analysis of the attenuated, tissue culture-adapted [TC] porcine enteric calicivirus (PEC/Cowden strain) and generation of synthetic RNA transcripts. Sequence analysis showed that the TC PEC/Cowden has an RNA genome of 7,320 bp, excluding its 3' poly(A)(+) tail. The genome is organized in two open reading frames (ORFs), similar to the organization of the human Sapporo-like viruses (SLVs) and rabbit caliciviruses, the lagoviruses. 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 has 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 PEC is genetically most closely related to human SLVs. The complete RNA genome of virulent wild-type (WT) PEC/Cowden was also sequenced. Sequence comparisons revealed that the WT and TC PEC/Cowden have 100% nucleotide sequence identities in the 5' terminus, 2C helicase, ORF2, and the 3' nontranslated region. TC PEC/Cowden has one silent mutation in its protease, two amino acid changes and a silent mutation in its RNA polymerase, and five nucleotide substitutions in its capsid that result in one distant and three clustered amino acid changes and a silent mutation. These substitutions may be associated with adaptation of TC PEC/Cowden to cell culture or reduced virulence for pigs. Our goal is to study the genetic basis for cell culture adaptation and virulence of the PEC.

Impacts
The human enteric caliciviruses (HuCV) are the leading cause of foodborne viral gastroenteritis worldwide. A porcine enteric calicivirus (PEC) is the only cell culture adapted enteric calicivirus and is genetically similar to HuCV. Thus the genetic analysis and infectivity studies of PEC may lead to an increased understanding of the molecular basis for the cell culture adaptation and pathogenesis of human and animal enteric caliciviruses.

Publications

Guo, M., Chang, K.O., Hardy, M.E., Zhang, Q., Parwani, A.V., Saif, L.J. 1999. Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human caliciviruses. J. Virol. 73:9625-9631.
Guo, M, Chang, K.O., Hardy, M.E, Zhang, Q., Saif, L.J. 1999. Cloning and sequencing of a porcine enteric calicivirus genetically related to Sapporo-like viruses. Presented at the International Workshop on Human Caliciviruses. Atlanta, Georgia, Abstract #S2-7.
Guo, M, Chang, K.O., Hardy, M.E, Zhang, Q., Saif, L.J. 1999. The complete nucleotide sequence of the RNA genome of a porcine enteric calicivirus genetically related to Sapporo-like viruses. Proc. American Society for Virology, abstract W8-9. Amherst, MA, July10-14.
Guo, M, Parwani, A.V., Cho, K.O., Lucas, L.M., Saif, L.J. 1999. Pathogenesis of tissue culture adapted porcine enteric calicivirus in gnotobiotic pigs. Abst. #106, Proc. CRWAD, Chicago, IL.
Chang, K.O., Parwani, A.V., Cho, K.O., Guo, M, Nielsen, P.R., Saif, L.J. 1999. Pathogenesis of bovine enteric calicivirus in gnotobiotic calves. Abst. #107, Proc. CRWAD, Chicago, IL.