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

Porcine enteric calicivirus (PEC/Cowden) causes diarrhea in pigs, grows in cell culture, and is morphologically and genetically similar to the Sapporo -like human caliciviruses. Genetic analysis revealed that the tissue cultur e-adapted (TC) Cowden PEC has one distant and three clustered amino acid su bstitutions in the capsid region and 2 amino acid changes in the RNA polyme rase region compared to wild-type (WT) PEC (M. Guo, K.-O. Chang, M. E. Hard y, Q. Zhang, A. V. Parwani, and L. J. Saif, J. Virol. 73:9625-9631, 1999). In this study, the TC PEC, passaged in a porcine kidney cell line, and the WT PEC, passaged in gnotobiotic (Gn) pigs, were used to orally inoculate 13 4- to 6-day-old Gn pigs. No diarrhea developed in the TC-PEC-exposed pigs, whereas moderate diarrhea developed in the WT-PEC orally inoculated pigs, persisting for 2 to 5 days. Fecal virus shedding persisting for at least 7 days was detected by both reverse transcription (RT)-PCR and antigen-enzyme -linked immunosorbent assay (antigen-ELISA) in both TC-PEC and WT-PEC orall y inoculated pigs but not in mock-inoculated pigs. The PEC particles were d etected by immunoelectron microscopy (IEM) in intestinal contents from all the WT-PEC-inoculated pigs, but not from the TC-PEC-inoculated pigs. Mild ( duodenum and jejunum) or no (ileum) villous atrophy was observed in histolo gic sections of the small intestines of TC-PEC-inoculated pigs, whereas WT PEC caused mild to severe (duodenum and jejunum) villous atrophy and fusion . Scanning electron microscopy confirmed mild shortening and blunting of vi lli in the duodenum and jejunum of the TC-PEC-inoculated pigs, in contrast to moderate to severe villous shortening and blunting in the duodenum and j ejunum of WT-PEC-inoculated pigs. Higher numbers of PEC antigen-positive vi llous enterocytes were detected by immunofluorescent (IF) staining in the p roximal small intestine of the WT-PEC-inoculated pigs, in contrast to low n umbers of PEC antigen-positive enterocytes in only one of four TC-PEC-inocu lated pigs. No PEC antigen-positive cells were observed in the colon or ext raintestinal tissues of all inoculated pigs or in the small intestine of on e mock-inoculated pig. Thus, the TC PEC was at least partially attenuated ( no diarrhea, mild lesions) after serial passage in cell culture. In further experiments, three 4- to 6-day-old Gn pigs were intravenously (i.v.) inocu lated with WT PEC, and all pigs developed diarrhea and villous atrophy in t he small intestines resembling that observed in the orally inoculated pigs. Fecal viral shedding persisting for 8 days was detected by both RT-PCR and antigen-ELISA, and PEC was detected by IEM in feces or intestinal contents . The PEC RNA and antigens (at low titers) were detected in acute-phase ser a from all the WT-PEC i.v.-inoculated pigs and also from seven of nine of t he WT-PEC orally inoculated pigs. Oral or i.v. inoculation of four addition al pigs with the PEC-positive acute-phase sera induced diarrhea, small inte stinal lesions, PEC shedding in feces, and seroconversion to PEC, confirmin g the occurrence of viremia during PEC infection, with infectious PEC prese nt in acute-phase sera. No diarrhea, histopathologic changes, or IF stainin g in the small intestine or fecal or serum detection of PEC was evident in two pigs i.v. mock-inoculated or a pig inoculated i.v. with inactivated WT PEC. To our knowledge, this is the first report of an attenuated enteric ca licivirus, the induction of diarrhea, and intestinal lesions in Gn pigs cau sed by i.v. inoculation of WT PEC and the presence of viremia following PEC infection.