Isolation,Identification And Molecular Epidemiological Investigation Of Porcine Epidemic Diarrhea Virus In Henan Province

Porcine epidemic diarhea(PED)is an acute,highly contagious and devastating disease,characterized by enteritis and fatal watery diarrhea.Since2010,a large-scale outbreak of PED caused by porcine epidemic diarrhea virus(PEDV)variants has occurred in China,leading to huge economic losses.Therefore,a comprehensive understanding of the genetic variation and interrelationship among different strains of PEDV will help to find out the reasons for the continuous outbreak of PEDV and develop new strategies for the control and prevention of PEDV infection.In order to understand the current epidemic trend and genetic variation of PEDV in Henan Province,the diarrhea samples collected from 2020 to 2022 were detected.On this basis,virus isolation,identification,pathogenicity evaluation and molecular epidemiological investigation were carried out.1.Molecular epidemiology of PEDV in Henan ProvinceIn this study,65 clinical samples collected from pig farms in some areas of Henan Province from 2020 to 2022 were tested for PEDV by RT-PCR,and the positive rate of PEDV was 29.23%.Among them,11 S and ORF3 genes of PEDV-positive samples were cloned and sequenced.The sequences were aligned and analyzed by biological software.The nucleotide homology of S gene and ORF3 gene between PEDV strains isolated in this study and other representative reference strains were 93.6-99.1% and 93.1-99.7%,respectively.Phylogenetic analysis based on S gene showed that 4 PEDV strains sequenced in this study were located in clade G2 a,the remaining 7 PEDV strains were clustered into an independent subclade G2 c between the G1 and G2 clades.The results of recombination analysis showed that the strains within the G2 c branch might be derived from natural recombination of two different subtypes of PEDV mutants.2.Isolation and identification of PEDV epidemic strainsThe identified PEDV positive samples were inoculated into Vero cells for serial passage.Cytopathic effect was observed in passage 3,which showed swelling and rounding,increased diopter,and the presence of vacuoles and syncytia.The isolate was continuously propagated in Vero cell culture,and identified as PEDV by RT-PCR,transmission electron microscopy and immunofluorescence,and named HN2021.The proliferation curve of HN2021 on Vero cells showed that the virus proliferation reached the peak at 48 h after inoculation,the virus titer was 1×104.6 TCID50/0.1 ml.3.Genetic evolution molecules and pathogenicity of PEDV isolatesThe sequences of PEDV strains published in Gen Bank were used for homology analysis and genetic evolution analysis.The results showed that the nucleotide homology between the strains obtained in this study and the references belonged to the G2 a variant strain was 95.6%-98.7%.Notably,this isolate was further confirmed by RT-PCR to have a large natural deletion in ORF3 gene at 207-373 nt,which has not been reported previously.To determine whether this deletion pattern was associated with the pathogenicity,the HN2021 isolate was selected for subsequent animal studies.In terms of pathogenicity evaluation,piglets challenged with PEDV HN2021 exhibited severe diarrhea and high mortality,confirming that PEDV HN2021 was a virulent strain.This study aims to understand the current prevalence of PEDV in swine populations in some regions of Henan Province,clarify the epidemic characteristics of the disease,further analyze the molecular characteristics and genetic variations PEDV isolates,and provide an important foundation for subsequent related vaccine preparation and disease control,with important clinical implications and potential application values.