Epidemiological Investigation Of PEDV In Hubei Province From 2018 To 2020 And The Isolation And Identification Of HB2018 Strain

Porcine epidemic diarrhea virus（PEDV）can cause acute,highly contagious infectious diseases characterized by vomiting,watery diarrhea and dehydration.Pigs of all ages are susceptible to the virus,and suckling piglets within 7 days of age have the most severe symptoms,with an infection fatality rate of 90%-100%,which has caused great economic losses to the pig industry.In 2010,a large-scale PEDV epidemic broke out in my country and even in the world.Studies have shown that compared with the traditional strain CV777,the epidemic strain has undergone larger mutations,especially the S gene.After China has used the attenuated vaccine of the GIIb subbranch,the PEDV epidemic has been effectively controlled,but the epidemiological survey results show that the strain is still mutating,so it is necessary to continue to track the mutation of the pathogen.In this study,by isolating PEDV epidemic strains,analyzing their genetic evolution,and studying the characteristics of the isolated strains based on the results,it will provide technology and material reserves for the prevention and control of PEDV epidemics.The main research contents are as follows:1.Epidemiological survey of PEDV in Hubei Province from 2018 to 2020In this study,the collection,isolation and identification of porcine epidemic diarrhea virus was carried out in Hubei Province,and an epidemiological investigation was carried out.The RT-PCR diagnostic method was used to detect 3,027 piglet diarrhea samples collected from 7 regions in Hubei Province from 2018 to 2020.Among them,PEDV was detected.The rate is 74.33%,and the detection rates from 2018 to2020 are 68.09%,70.80%,and 79.02%,which have a tendency to increase year by year.Among the positive samples,the detection rates of spring,summer,autumn and winter were 82.77%,36.08%,51.46%,and 83.46%,respectively.The detection rate of PEDV in spring and winter was higher than that in summer and autumn,and PEDV showed seasonal prevalence.Five PEDV strains of different years were selected and named HB2018,DY2018,HB2019,HB2020 and DY2020,respectively.Based on the conservative sequence of the PEDV S gene included in Genbank as a template,specific primers for the S gene were designed.The S gene sequences of five strains of virus were cloned by RT-PCR method,and the T vector was connected for testing.The S gene sequences of five strains were spliced together,and the genetic analysis of the S genes showed that the five strains belonged to the GIIa subgroup,which was comparable to SC-YB73,YC2014,CH/HNLH/2015 and AHCZ-2 The homology is relatively close,and the homology is between 98.8%-99.3%.The nucleotide and amino acid comparisons of the five strains showed that the variation of the PEDV S gene was mainly concentrated in the S1 gene.2.Isolation and identification of PEDV HB2018 strainFive positive disease materials from different years were inoculated with Vero cells,and through spot-selection purification and RT-PCR identification,one strain of PEDV virus was obtained,named HB2018.By the Reed-Muench method,the drug value was determined to be 10^6.7TCID50/m L.Using the PEDV conserved sequence included in Genbank as a template,21 pairs of specific primers were designed.The whole genome sequence of HB2018 was cloned by RT-PCR method,and it was connected to T vector for testing.The whole genome sequence of the HB2018 isolate was spliced.The genetic analysis of the whole gene of the strain showed that it belongs to the GII group and has high homology with the YC2014 strain and the KT199103.1strain,which are 99.28%and 97.00%,respectively.The genome-wide homology with vaccine strains CV777,DR13,AJ1102 and LW/L are 96.6%,97.4%,98.5%and 97.9%,respectively;the nucleotide homology of the S gene is 93.7%,93.5%,97.4%and 97.0%,the amino acid homology is 93.9%,93.3%,98.2%and 97.1%,and the amino acid has11 key position mutations.The ORF3 gene homology between the HB2018 strain and the vaccine strains CV777,DR13,AJ1102 and LW/L are 96.4%,92.0%,95.9%and95.3%,and the amino acid homology is 96.0%,90.1%,96.9%and 95.5%,respectively%,the amino acid has 3 key position mutations.HB2018 strain F20generation(10^6.7TCID50/m L)virus solution 4 m L/head was inoculated with 5 weaned piglets,and the negative control group was orally inoculated with 4 m L of DMEM medium/head to inoculate 5 weaned piglets.Observe the diarrhea and stool consistency of the piglets after inoculation,and collect stool samples for RT-PCR detection.After 7days,the piglets are euthanized.The results showed that all weaned piglets vaccinated with HB2018 strain showed diarrhea,and the weaned piglets of the negative control did not show diarrhea or other clinical symptoms.RT-PCR testing showed that viral RNA was detected in the weaned piglets of the inoculation group,and no viral RNA was detected in the feces of the negative control pigs.The histological examination of the weaned piglets in the inoculation group showed that the intestinal wall of the small intestine became thin and transparent,and a large amount of fluid accumulated in the small intestine lumen.There was no obvious histological damage in the negative control group.