Isolation,Identification And Pathogenicity Of Porcine Epidemic Diarrhea Virus In Guangxi

Porcine epidemic diarrhea（PED）infection of newborn piglets causes anorexia,vomiting,severe watery diarrhea and dehydration,and the morbidity and mortality rate are extremely high.The pathogen is Porcine epidemic diarrhea virus（PEDV）.Since October 2010,a large-scale PED epidemic has occurred in China.Once the disease occurs,the morbidity and mortality of piglets are both up to 100%.This study mainly focused on the PEDV-positive disease samples from January 2017 to May 2019 in Guangxi,to isolate and identify PEDV strains,and conduct pathogenicity studies.To understand the mutations and pathogenicity of PEDV strains in Guangxi,and provide effective scientific basis and support for the prevention and control of PEDV and the development of vaccines in Guangxi.1.Establishment and primary application of fluorescence quantitative PCR for detection of M gene of PEDV.In this study,the PEDV quantitative PCR method was successfully established,it has no cross-reaction with other common porcine viruses.The correlation coefficient of standard curve of plasmid concentration between3.2×102 copies/μL～3.2×109 copies/μL was 0.997,showing a good linear relationship.The variation coefficient of intra-group test was less than 1%.The minimum limit of detected samples was 3.2×102 copies/μL.The established fluorescence quantitative PCR was used for quantitative analysis of the PEDV strain isolated in this experiment,which can be used as a reference for screening high-copy number strains.2.Isolation,identification and pathogenicity of porcine epidemic diarrhea virus strains in Guangxi.In this study,a total of 116 PEDV-positive samples collected in various regions of Guangxi from January 2017 to May 2019 were used for virus isolation,and 12 PEDV strains were successfully isolated.When the virus solution was inoculated in Vero cells,the cells began to appear cytopathic,and the diseased cells became large and rounded,vacuolated,and formed multinuclear aggregates on 24 hpi post infection.The S gene sequences of all the isolated strains were amplified,it showed that the 12 PEDV strains were all belong to G2a subtype;one strain named CH/GXNN-1/2018 was purified by plaque assay,with the titer of 10^6.15PFU/m L;the growth curve showed that the virus began to multiply from 6 hpi,it reached the peak of virus replication and proliferation at 48 hpi;the results of the immunofluorescence test showed that the virus produced specific green fluorescence,indicating that the virus can be recognized by PEDV-specific monoclonal antibodies;transmission electron microscopy results showed the virus particles are round,about 100 nm in diameter,with petal-shaped fibrils radially distributed around,it has the typical morphological characteristics of coronavirus particles.Eight 7-day-old piglets with both negative for PEDV antigen and antibodies were selected for animal pathogenicity experiments,and the challenge group was orally inoculated with 2 m L of 10⁶ PFU/m L CH/GXNN-1/2018 strain.Piglets in the challenge group all showed typical clinical symptoms of PED,with weight loss,diarrhea,and messy hair stained with yellow stool.The virus shedding reached the maximum at 24 hpi,and then showed a stable detoxification trend;the results of viral load measurement in different tissues showed that the presence of PEDV was detected in the stomach,intestinal tissue,and mesenteric lymph nodes of piglets in the infected group,and the viral load in the jejunum was the highest;It showed that the virus mainly infected the intestinal tissues of piglets,especially the duodenum,jejunum and ileum.It was worth not that throughout the experiment,the symptoms of the challenge group gradually reduced with time,and the overall weight of the piglets in the challenge group was lower than that of the control group,and there was no lethality.Combined with the above experimental results,it is speculated that the CH/GXNN-1/2018 strain is a less pathogenic strain and has the potential to become an attenuated vaccine candidate.In summary,this study successfully established a quantitative detection PEDV fluorescent quantitative PCR method,which provided effective support for subsequent detection experiments;this study successfully obtained 12 PEDV strains,increasing the number of PEDV isolates in Guangxi,rich the pathogenic data of PEDV in our country is shown;the pathogenicity of CH/GXNN-1/2018strain showed that PEDV infection had a non-negligible effect on piglets.These studies lay a foundation for further understanding the strain variation,pathogenic mechanism of PEDV strains in Guangxi and the development of attenuated vaccines.