Isolation,Identification And Molecular Characterization Analysis Of Porcine Epidemic Diarrhea Virus Variant Strain

Porcine epidemic diarrhea(PED)is an acute and highly contagious enteric infectious disease intended for pigs,caused by porcine epidemic diarrhea virus(PEDV),which is most harmful to piglets under 1 week of age.Their infection rate and death rate are as high as100%.Clinically,vomiting,diarrhea,and dehydration death are determined as main features for this disease.Since the outbreak of PED in China in 2010,the epidemic isolates have varied,affecting the effectiveness of existing vaccines,which fails to provide adequate protection for the herd.This brings about huge economic losses to the Chinese swine industry.Research has shown that compared with classic isolates CV777,the variation of the epidemic isolates is mainly reflected in S-gene.Therefore,comprehending the main epidemic trends of PEDV and conducting isolation and identification for epidemic isolates will provide a theoretical basis for vaccine development and application as well as development of new antiviral drugs.One pair of detection primers were designed for the highly conservative M gene.The RT-PCR test was conducted for the 180 samples collected from partial piggeries of Shandong province,like Jinnan,Jining,Liaocheng,Dezhou,Binzhou,and 47 samples were tested PEDV positive,with positive rate reaching 26.1%(47/180).Then specific primers for S1 and S2 genes were designed to amplify S gene,and thus the 2145bp,1752bp specific segments were obtained respectively.In order to look into the variation of PEDV in the current epidemic isolates,14 PEDV positive isolates of different time periods were adopted as representatives to conduct the homology analysis with domestic and overseas epidemic isolates for S gene.Compared with classic isolates CV777,the homology was ranging from91.5%to 92.6%;the homology with Chinese isolates SD-M was the lowest,91.0%-91.8%,while the homology with USA/Colorado/2013 isolates was the highest,97.9%-98.4%.The results of phylogeny trees for S-gene indicated that all the isolates had been divided into two groups:G1 and G2.The 14 isolates in the study all belonged to G2 group,different from the classic isolates CV777 and Chinese isolates SD-M in G1 group.They were not closely related.According to the sequence analysis,the difference of the 14 isolates in the study from the classic isolates CV777 lied in the 4 insertions(164-166nt,177-185nt,480-485nt,415-417nt),1 base deletion(114nt)and multiple mutations in S1 area.Positive samples should be selected from the submitted samples to isolate PEDV on Vero cells.The final concentration of trypsin in cell maintenance medium was 10?g/m L.Blind passage for 3 generations,the RT-PCR test should be conducted to analyze the virus isolation.Thereinto,one sample was submitted to the 2^ndgeneration,Vero cells formed syncytium and test results of PT-PCR came to be PEDV positive.The results were further observed by Indirect immunefluorescent testing and Transmission electron microscope(TEM).It proved to be successfully isolated to PEDV,which was named ZY2020.The TCID₅₀of the isolates was determined as 10^-5.1/0.1m L and the growth curve was made accordingly.The whole genome of the isolates was amplified and spliced with 37 pairs of primer segments.The total length reached 28035bp.The homology comparison on whole genome between the isolates ZY2020and 10 domestic and overseas reference isolates and the construction of phylogeny trees were conducted to further analyze the sequence characteristics of the isolates.These results suggested that the homology between isolates ZY2020 and classic isolates CV777 was the lowest,reaching 96.7%.The isolates ZY2020 was not closely related with the classic isolates CV777 since they belonged to different groups.According to the analysis on the S-gene of ZY2020 for its molecular characteristics,the homology of ZY2020 S-gene with that of classic isolates CV777 and Chinese isolates LZC was 93.9%and 93.3%respectively,sharing the lowest relation;while for the homology with American isolates USA/Colorado/2013,it reached 98.4%,sharing the highest relation.The S-gene of the isolates ZY2020 was divided into G2 subsets,different from the group of classic isolates CV777 and early epidemic isolates in China.There were multiple amino acid mutations on S-gene of ZY2020:two insertions of amino acid(4 amino acid inserted to QGVN at 59-62nt and insertion of amino acid N at position 140)and one deletion of amino acid(163-164nt),as well as the deletion of amino acid at position 1198.In conclusion,47 PEDV positive samples were obtained from the PT-PCR test of the submitted 180 samples in the study.Sequence characteristics of S-gene were analyzed by comparing with domestic and overseas reference isolates.One isolates ZY2020 was obtained by the isolation and identification of PEDV on Vero cells.It turned out to be far with classic isolates CV777 in accordance with genomic sequence analysis and there were characteristic mutations on S gene,which would provide clues to vaccine development and comprehension on the mechanism of PEDV self-multiplication and reduplication in the near future.