Isolation And Identification Of CAstV In Huai’an Area Of Jiangsu Province And Establishment Of Antibody Detection Method For CAstV

Astrovirus(Astroviruses,AstV)is a potential zoonotic pathogen,which often causes acute gastrointestinal diseases.It is a widespread pathogen that can infect multiple hosts.The whole genome of AstV consists of three open reading frames(ORF1a,ORF1b and ORF2).Chicken astrovirus(ChickenAstrovirus,CAstV),which belongs to the genus Avian astrovirus,is a new intestinal pathogen found in chicken flocks with stunted growth,and widely exists in different farms.It can be transmitted not only horizontally through fecal mouth,but also vertically to chicks through hens,resulting in clinical symptoms such as growth retardation syndrome,white chicken syndrome and renal gout.In recent years,the hatching characteristics of white chicks have appeared in Europe,North America and other regions,which are characterized by the decrease of hatching rate,pale and weak feathers and increased mortality.Now CAstV infection is on the rise in China,and CAstV infection can be detected in most provinces.In this study,a strain of chicken astrovirus CAstV/HA2101 was successfully isolated and purified from 1-day-old chickens in Huai’an,Jiangsu Province,and its genetic evolution and bioinformatics analysis were carried out,and the prepared monoclonal antibody against chicken astrovirus capsid protein was identified.At the same time,using LMH cells infected with chicken astrovirus as coating antigen,a Cell-ELISA antibody detection method for type Ⅱ chicken astrovirus was established.In addition,the recombinant protein GST-NJ1701-ORF2 with GST tag was successfully expressed and purified,which provides an effective and reliable detection technique for the rapid diagnosis of CAstV antibody.1.Isolation,purification and preparation of specific Monoclonal of CAstVAntibodies in Huai’an area in Jiangsu Province in order to investigate the prevalence of chicken astrovirus in Huai’an area of Jiangsu Province,66 cecal disease samples were collected from farms in Huai’an area of Jiangsu Province and detected by RT-PCR.Some positive samples were randomly selected and sequenced,and it was found that all of them were type II chicken astrovirus.A virus named CAstV/HA2101 was successfully isolated by inoculating positive materials into chicken embryos for three generations and then passing through LMH cells for three generations.Then the plaque was purified to improve the toxicity and purity of the virus.At the same time,the RNA of the virus was isolated and purified,and the whole gene was sequenced,and the sequencing results were analyzed by genetic evolution and bioinformatics analysis.The CAstV/HA2101 isolated and purified strain has high homology with other isolates in China,between 96.4%and 98.0%,belonging to an independent branch.Through genetic evolution and bioinformatics analysis of the whole gene and structural gene of the isolated strain,this study enriched the epidemiological data of CAstV and provided a reference for the prevention and control of CAstV in Jiangsu Province.At the same time,the monoclonal antibody 6C7 was prepared from Sf9 cells infected by recombinant baculovirus Bacmid-CAstV-Cap.The specificity of the monoclonal antibodies 3E12,5A11,6H3 and the monoclonal antibody 6C7 obtained in this study were identified.The results showed that the four monoclonal antibodies had no significant specific reaction with ALV-J,AIV,IBV and MDV,Preliminary identification of its epitopes showed that 3E12,5A11,6H3 and 6C7 all recognized Cap protein 297～505aa.The results of neutralization test showed that none of the four McAbs could neutralize the Cap protein of chicken astrovirus by Westernblot.The results showed that all the four McAbs could specifically recognize the recombinant Cap protein and the Cap protein in LMH cells infected by CAstV/NJ1701 strain.The results show that the prepared monoclonal antibody has high potential for the diagnosis and vaccine development of chicken astrovirus.2.Establishment of a method for detection of antibodies to chicken astrovirus.2.1 Establishment of Cell-ELISA method for detection of chicken astrovirus antibodyIn order to develop a rapid and accurate method for detection of CAstV antibody.In this study,using LMH cells infected with chicken astrovirus as coating antigen,a Cell-ELISA method for detection of CAstV antibody was established.The experimental conditions were optimized by chessboard titration,and the optimum reaction conditions were determined as follows:the optimum cell density was 3×105,the best amount of virus infection was MOI=3,the best serum dilution was 1purl 400,the best sealing solution was 3%FBS/PBS,the best sealing time was 2h,the best first antibody reaction time was 1h,the best second antibody reaction time was 1h,and the best chromogenic time was 15min.44 sera of SPF chickens were detected by this method,and their OD450nm readings were determined,and the critical value of negative and positive was 0.232.The cross-reactivity test was carried out with the established Cell-ELISA method:the Cell-ELISA method established in this study was only positive for the positive serum of chicken astrovirus,but no cross-reaction to other common avian viruses,and the sensitivity of the ELISA test was 112800,which was more sensitive than the conventional IF A method.And the coefficient of variation within interbatch is less than 10%,and the repeatability is good.It shows that the kit of the invention has good sensitivity,specificity and stability.Therefore,the Cell-ELISA method established in this study provides a high-throughput method for the detection of CAstV antibodies in chicken flocks.2.2 A method for detection of antibody against chicken astrovirus was established by eukaryotic expression of virus capsid protein.The capsid protein gene of chicken astrovirus was cloned into pAcG2T vector.Using flashBACTM baculovirus expression system,the constructed plasmid was transfected into Sf9 cells,and the recombinant protein GSTNJ1701-ORF2 with GST tag was successfully expressed.Through IF A and Westernblot tests,it was found that the recombinant protein had a good specific reaction with GST antibody and monoclonal antibody against chicken astrovirus capsid protein,and the expression of recombinant protein GST-NJ1701-ORF2 in the supernatant was up to 50%.The expressed protein was purified by GST protein affinity purification column as a coating antigen for detecting antibody,and an ELISA method for detecting CAstV antibody was established.