Isolation,Identification And Pathogenicity Research Of Porcine Epidemic Diarrhea Virus Prevalent Strains

Porcine epidemic diarrhea?PED?is an acute,highly contagious enteric disease caused by porcine epidemic diarrhea virus?PEDV?,which characterised by watery diarrhea,vomiting,dehydration and high mortality of piglets.All ages of pig can be infected by PEDV,especially piglets with the rate of mortality and morbidity nearly reaching 100%.The disease was first detected in United Kingdom in1971 and then spread to Europe and Asia.PEDV was first reported in United States in 2013 and spread to the whole country and caused serious economic losses.After that,PEDV has gotten more attention of many countries.The severe outbreaks of PED were occurred in China in later 2010.In recent years,the prevalence of PED has become more and more widespread and novel variants have constantly emerged.There are differences in the antigenicity of commercial vaccines and epidemic strains,resulting in poor immune effect,which seriously affects the healthy development of the pig industry in China.Because the genome characteristics and epidemiology of the new PEDV epidemic strains are not yet clear,the research has developed the following work to provide basis for further elucidating molecular characteristics and pathogenicity and selecting of candidate vaccine strain of Chinese PEDV epidemic strains.1.This study carried out an epidemiological investigation of piglet diarrhea from 6 provinces?Gansu,Xinjiang,Shaanxi,Hunan,Hainan and Henan?in China between 2016 and 2017 and 134diarrhea stool samples were collected.Meanwhile,the PEDV TaqMan probe real-time fluorescence quantitative RT-PCR method was successfully established.The minimum detectable amount was up to240 copies/?L and PEDV can only be detected,which showed its sensitivity and specificity were good.The variable coefficient of the method was less than 1%indicating its repeatability was good.The above samples were detected using the qPCR assay set up in this study and conventional PCR.and the results showed that the positive rates of PEDV and porcine kobuvirus?PKV?were 54.4%?73/134?and17.2%?23/134?,respectively.In addition,transmissible gastroenteritis virus?TGEV?and porcine deltacoronavirus?PDCo V?were not detected in those samples.It is noteworthy that PEDV infection with a positive rate of 59.5%?25/42?was first detected in Hainan samples in this study.2.11 PEDV strains were separated from 73 PEDV-positive samples and gene sequences of major antigen gene S of them were sequenced.Compared with classical strain CV777,the identities of nucleotide and amino acid of these strains varied from 93-94%and 92-93%,respectively and these strains existed one insertion(A¹³³),one deletion(G¹⁵⁵)and four continuous amino acid insertions(⁵⁶NNTN⁵⁹)in the S1 region.Phylogenetic tree analysis showed that these strains collected from 2016 to2017 are mainly G2 genotypes,and there are major differences between the S gene regions compared with classical vaccine strain.The results of this study provide the data for understanding of prevalence and genetic diversity of PEDV in China during 2016-2017.3.The 11 PEDV-positive strains detected in this study were cultured in vitro,and a cell-adapted strain of CH/HNPJ/2017?Genbank Accession:MF152604?was successfully isolated.The strain showed a typical cytopathic effect?CPE?in the P1 generation.With the increase of culture passages,the virus titer increased gradually and stabilized above 10⁷ TCID₅₀/mL.Fluorescent green PEDV particles can be detected by immunofluorescence.Typical coronavirus particles about 100 nm in diameter were observed by transmission electron microscop,which was surrounded by coronal spike formed by the S protein.The whole genome sequence of the CH/HNPJ/2017 strain was amplified and sequenced using specific primers.Phylogenetic analysis showed that the strain was located in the G2b genotype subgroup and was closely related to the AJ1102 and CHGD-01 strains.However,the phylogenetic relationship with vaccine strain CV777 is relatively far.Of note,the sequencing results showed that the CH/HNPJ/2017 strain occurred a 12 nucleotide insertion in S gene region after P6 passage.Compared with the strains from different genotypes or regions,only CH/HNPJ/2017 strain has the insertion mutation in that position.4.The results of animal experiment showed that piglets can be infected by P4 cell-adapted strain of CH/HNPJ/2017,and caused watery diarrhea and dehydration,which indicated it had strong pathogenicity for piglets.