Guo M, Hayes J, Cho K O, Parwani A V, Lucas L M, Saif L J
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691, USA.
J Virol. 2001 Oct;75(19):9239-51. doi: 10.1128/JVI.75.19.9239-9251.2001.
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 culture-adapted (TC) Cowden PEC has one distant and three clustered amino acid substitutions in the capsid region and 2 amino acid changes in the RNA polymerase region compared to wild-type (WT) PEC (M. Guo, K.-O. Chang, M. E. Hardy, 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 orally inoculated pigs but not in mock-inoculated pigs. The PEC particles were detected 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 histologic 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 villi 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 jejunum of WT-PEC-inoculated pigs. Higher numbers of PEC antigen-positive villous enterocytes were detected by immunofluorescent (IF) staining in the proximal small intestine of the WT-PEC-inoculated pigs, in contrast to low numbers of PEC antigen-positive enterocytes in only one of four TC-PEC-inoculated pigs. No PEC antigen-positive cells were observed in the colon or extraintestinal tissues of all inoculated pigs or in the small intestine of one 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.) inoculated with WT PEC, and all pigs developed diarrhea and villous atrophy in the 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 sera from all the WT-PEC i.v.-inoculated pigs and also from seven of nine of the WT-PEC orally inoculated pigs. Oral or i.v. inoculation of four additional pigs with the PEC-positive acute-phase sera induced diarrhea, small intestinal lesions, PEC shedding in feces, and seroconversion to PEC, confirming the occurrence of viremia during PEC infection, with infectious PEC present in acute-phase sera. No diarrhea, histopathologic changes, or IF staining 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 calicivirus, the induction of diarrhea, and intestinal lesions in Gn pigs caused by i.v. inoculation of WT PEC and the presence of viremia following PEC infection.
猪肠道杯状病毒（PEC/Cowden）可引起猪腹泻，能在细胞培养中生长，在形态和基因上与札幌样人杯状病毒相似。遗传分析表明，与野生型（WT）PEC相比，适应组织培养（TC）的考登PEC在衣壳区域有1个远距离和3个成簇的氨基酸替换，在RNA聚合酶区域有2个氨基酸变化（M. Guo、K.-O. Chang、M. E. Hardy、Q. Zhang、A. V. Parwani和L. J. Saif，《病毒学杂志》73:9625 - 9631，1999年）。在本研究中，在猪肾细胞系中传代的TC PEC和在无菌（Gn）猪中传代的WT PEC用于口服接种13头4至6日龄的Gn猪。接种TC - PEC的猪未出现腹泻，而口服接种WT - PEC的猪出现中度腹泻，持续2至5天。通过逆转录（RT）-PCR和抗原酶联免疫吸附测定（抗原-ELISA）在口服接种TC - PEC和WT - PEC的猪中均检测到粪便病毒排出持续至少7天，但在 mock 接种的猪中未检测到。通过免疫电子显微镜（IEM）在所有接种WT - PEC的猪的肠道内容物中检测到PEC颗粒，但在接种TC - PEC的猪中未检测到。在接种TC - PEC的猪的小肠组织切片中观察到轻度（十二指肠和空肠）或无（回肠）绒毛萎缩，而WT PEC导致轻度至重度（十二指肠和空肠）绒毛萎缩和融合。扫描电子显微镜证实接种TC - PEC的猪的十二指肠和空肠绒毛轻度缩短和变钝，相比之下，接种WT - PEC的猪的十二指肠和空肠绒毛出现中度至重度缩短和变钝。通过免疫荧光（IF）染色在接种WT - PEC的猪的近端小肠中检测到更多的PEC抗原阳性绒毛肠上皮细胞，相比之下，在接种TC - PEC的4头猪中只有1头猪中有少量PEC抗原阳性肠上皮细胞。在所有接种猪的结肠或肠外组织以及1头mock接种猪的小肠中均未观察到PEC抗原阳性细胞。因此，TC PEC在细胞培养中连续传代后至少部分减毒（无腹泻，轻度病变）。在进一步的实验中，3头4至6日龄的Gn猪静脉内（i.v.）接种WT PEC，所有猪均出现腹泻和小肠绒毛萎缩，类似于口服接种猪中观察到的情况。通过RT - PCR和抗原-ELISA检测到粪便病毒排出持续8天，并且通过IEM在粪便或肠道内容物中检测到PEC。在所有静脉内接种WT - PEC的猪的急性期血清中以及9头口服接种WT - PEC的猪中的7头猪的急性期血清中检测到PEC RNA和抗原（低滴度）。另外4头猪口服或静脉内接种PEC阳性急性期血清后出现腹泻、小肠病变、粪便中PEC排出以及对PEC的血清转化，证实PEC感染期间发生病毒血症，急性期血清中存在感染性PEC。在2头静脉内mock接种的猪或1头静脉内接种灭活WT PEC的猪中，未观察到腹泻、组织病理学变化或小肠中的IF染色，也未在粪便或血清中检测到PEC。据我们所知，这是关于减毒肠道杯状病毒、静脉内接种WT PEC导致Gn猪腹泻和肠道病变以及PEC感染后病毒血症存在的首次报告。
