| Caused by porcine epidemic diarrhea virus, PED is a highly contagious intestinal infectious disease, which was characterized by severe diarrhea and even death in neonates. To better understand its etiological characteristics, in this study we selected a PEDV strain FJzz1/2011 isolated from a PED plagued pig farm in Fujian Province as our subject. FJzz1/2011 was continuously passaged and plaque-purified though Vero cell and its biological features were worked out from etiological perspective. The results showed that all the parent F1 and its plaque-purified progenies F5, F10 and F20 viruses could induce obvious CPE characterized by fusion of cells and formation of syncytia and be identified as only PEDV positive by RT-PCR, and they could also be recognized by specific PEDV m Ab and p Ab through IFA, which excluded the possibility of contamination with other kinds of viruses. EM displayed FJzz1/2011 was crown-like particles with spikes protruding from the virion surface. Growth curves showed that all viruses proliferated at high speed within the first 24 hours and reached the replication peak at 24 hours after inoculation. Then the whole genome of FJzz1/2011 was sequenced and it exhibited prominent mutation in S gene, including 6 insertions(59QGVN62, 140 N and 157H) and 2 deletions(163NI164), compared with the prototype CV777. And sequence analysis with other related isolates suggested the new Chinese emerging PEDV strains might come from South Korea.To better understand the pathogenesis of FJzz1/2011, fifteen 3-day-old fit piglets were selected and assigned to orally inoculated group A and intramuscularly inoculated group B. Each of them was infected with 1×104.0TCID50. While piglets in control group behaved normally, all infected piglets in Group A and B got serious clinical sighs, including: diarrhea, vomiting, anorexia and emaciation, resulting in a high mortality of 80% and 100%, respectively. The intestinal wall of piglets died form infection became thin with massive faint yellow fluids in it. The villus became shortened and partially exfoliated. RT-PCR result showed that all anus swab samples of different time and intestinal contents of piglets from Group A and B exhibit PEDV positive and TGEV and PRV-A negative. We also isolated PEDV strain from the samples of Group A and B, and its homology with FJzz1/2011 reached 100% in S gene. The study results showed that FJzz1/2011 was a variant PEDV strain with high pathology to neonates.The N protein has good diagnostic value. To investigate its antigenicity, fusion N protein and its segmented proteins expressed based on the sequence of FJzz1/2011 were served as antigen to apply ELISA with m Abs PN-D6 and PN-D4. The result showed that PN-D4 and PN-D6 could specifically recognize segmented N1 and N2, respectively. Then we mapped the exact epitopes of N protein and found that one epitope bound with PN-D6 was N252RSDSSGKNTPK262, and the other epitope that PN-D4 recognized specifically range from N18 aa to N133 aa. And the consensus alignment results based on the amino acid sequences of these two epitopes revealed that they all exhibited high specificity among various coronaviruses and relative conservatism among different PEDV strains, suggesting PN-D4 and PN-D6 could be used as specific diagnosis tool of PEDV. In this study, we confirmed the variation and high piglets-pathogenicity of FJzz1/2011, as well as two epitopes on N protein of PEDV. These could all provide indispensable basis for the further study on effect of variation to pathogenesis, antigenic features and specific diagnosis at epitope level of emerging PEDV strains. |
PDF Full Text Request