猪流行性腹泻病毒G2c亚型HB2505株的分离鉴定及S基因序列分析OA

To obtain the G2c subtype strain of porcine epidemic diarrhea virus(PEDV),a method of incubating with high-concentration trypsin and supplementing with trypsin was adopted.Vero cells were used for the isolation of PEDV virus strains.The isolated strains were identified and analyzed by RT-PCR,indirect immunofluorescence assay(IFA),electron microscopy observation,animal reversion test,S gene homology analysis and phylogenetic tree analysis.The results of the virus strain isolation showed that one sample from Hubei Province caused cytopathic effects in Vero cells.Through RT-PCR,IFA and electron microscopy,it was identified as the PEDV strain,and was named PEDV HB2505 strain.The results of the animal reversion test showed that 2×105.0 TCID50 of HB2505 strain virus solution could cause diarrhea,dehydration and death in 5 out of 5 3～5-day-old piglets.The homology analysis of the S gene showed that PEDV HB2505 shared the highest nucleotide identity(99.9%)with the PEDV G2c strain CH-JX2301,while exhibiting the lowest identity(93.6%)with G1 strains Br1-87 and JS2008.The identities with PEDV G2b strain AJ1102 and G2a strain CH-HNLH-2015 were 97.3%and 98.8%respectively.The phylogenetic tree analysis further confirmed that it was located on the G2c branch.The analysis of the S protein amino acid sequence of HB2505 revealed that there were varying degrees of variations in the S1-NTD and COE domain,while the SS2,SS6 and 2C10 epitope regions were relatively conserved.The successful isolation of PEDV G2c strain HB2505 provides an essential foundation for further research on the basic biological characteristics and potential applications of PEDV G2c strains.