Isolation And Identification Of Porcine Epidemic Diarrhea Virus Strain CH/HLJ/18

Porcine epidemic diarrhea(Porcine epidemic diarrhea,PED)is a highly contagious disease caused by porcine epidemic diarrhea virus(Porcine epidemic diarrhea virus,PEDV).The virus can infect pigs of different ages through respiratory tract and digestive tract,among which newborn suckling piglets are the most sensitive and have obvious intestinal tropism,which can occur all the year round.So far,porcine epidemic diarrhea virus has been detected in 31provinces in China,and the fatality rate of newborn piglets is as high as 100%,which has caused huge economic losses to Chinese pig industry.According to the whole genome and S gene,PEDV can be divided into type 1 gene group(G1),type 2 gene group(G2)and type 3gene group(G3),and type G1 and G2 can be subdivided into G1a,G1b,G2a and G2b subtypes.In recent years,PEDV is prevalent in China.Most of the isolated PEDV strains are members of G2,and their genomes are obviously mutated,which makes it more difficult to control the disease in China.Therefore,the isolation and identification of PEDV is of great significance to master the epidemiology and control methods of the disease.In this study,the clinical piglet diarrhea materials were isolated,cultured and identified by PEDV,and the genetic variation and pathogenicity of the genomic sequence were analyzed.The specific results are as follows:1.Isolation and identification of PEDV strain CH/HLJ/18In this study,PEDV-positive piglet diarrhea materials detected by RT-PCR were inoculated on Vero cells after grinding with liquid nitrogen,and then subcultured in vitro;after blind passage to the fifth generation,obvious cytopathic changes were found,and the RT-PCR method was identified correctly.The results of IFA test showed that the infected cells had bright green fluorescence and obvious cell fusion,which was consistent with the typical pathological characteristics of PEDV.The virions with round shape,about 100 nm diameter,obvious envelope and spikes on the surface were clearly observed under transmission electron microscope,and the morphology was consistent with that of PEDV virions.The titer of the 5th generation virus was 10^5.00 TCID₅₀/0.1 m L,and the titer of the 30th generation virus was 10^5.75TCID₅₀/0.1 m L.The results of growth curve showed that the virus replicated rapidly from 6 h to 24 h after PEDV infection.At the 30th hour,the virus titer of the 5th generation virus reached10^5.82 TCID₅₀/0.1 m L,the virus titer of the 30th generation virus reached 10^6.25TCID₅₀/0.1m L.The above results showed that the PEDV strain CH/HLJ/18 was successfully isolated and had good adaptability to cells.2.Amplification and analysis of the whole genome sequence of PEDV strain CH/HLJ/18In order to analyze the genomic genetic variation of PEDV strain CH/HLJ/18,the whole genome was sequenced.The results showed that compared with the classical strain CV777,the5'UTR and S gene of the isolate had base insertion,deletion and point mutation,in which the S1 region of S gene included two insertions(58?59aa,138aa)and one deletion(161?162aa),and one insertion(1379aa)in S2 region.There are only base point mutations in other genes.The whole genome,S gene,M gene,N gene and ORF3 gene of strain CH/HLJ/18 were compared with the gene sequences of 34 representative strains of PEDV registered in Gen Bank.Among them,the nucleotide homology of the whole genome is 94.7%?98.1%?93.7%?99.6%,97.1%?100%?95.5%?99.9%?95%?99.4%.The results of phylogenetic evolution of whole genome and S gene showed that CH/HLJ/18 was far from G1 subtype virus,closer to G2a subtype epidemic strain in China and the United States,and closest to WHLL in China.Their S gene nucleotide homology was 99.6%,amino acid sequence homology was 98.6%,and nucleotide homology of ORF3 gene was 97.3%.There was no nucleotide deletion in ORF3gene.It is confirmed that the isolated strain CH/HLJ/18 obtained in this experiment is a G2a subtype virus strain currently prevalent in China.3.Animal experimentIn order to explore the pathogenicity of PEDV isolate CH/HLJ/18 to piglets,the fecal excretion,virus content in intestinal contents,clinical symptoms and dissection changes,gastrointestinal dissection changes and intestinal histopathology of 3-day-old newborn piglets were evaluated.The results showed that persistent diarrhea,vomiting,anorexia,weight loss and death occurred after each piglet was challenged with 5 m L10^5.0TCID₅₀ virus,and the mortality rate was 100%.The target virus could be detected in the feces and intestinal samples of piglets in the challenge group by RT-PCR method.After autopsy,different degrees of gastrointestinal lesions were observed in piglets,including enlargement of stomach,thinning of intestinal wall,thinning of contents and so on.The histopathological results showed that the pathological changes of jejunum and ileum were the most obvious,which showed high atrophy,necrosis,even disintegration and exfoliation of villi;atrophy,loss of outline,disintegration and exfoliation of apical villi in duodenum;and the least pathological changes in colon and cecum.Immunohistochemical examination of the ileal tissue with obvious pathological changes showed that a large number of PEDV virions could be detected in the villous epithelial cells of the ileum of infected piglets.The above results showed that the PEDV strain CH/HLJ/18 isolated in this study had strong pathogenicity to newborn piglets.In this study,the G2a subtype PEDV strain CH/HLJ/18 was successfully isolated in China,and it was confirmed that the isolate had strong pathogenicity to piglets,which laid a foundation for the pathogenic mechanism of PEDV and the prevention and treatment of porcine epidemic diarrhea.