Isolation And Identification Of Chicken Astrovirus And Development Of Monoclonal Antibodies Against Chicken Astrovirus

In recent years,the infection of chicken astrovirus(CAstV)is very common at home and abroad,and its diseases such as enteritis,glomerulonephritis and growth retardation have caused harm to the poultry industry.In the 1990s,virions similar to astrovirus were first found in chicken feces,and then CAstV infection was found in South Africa,the United States,Nigeria and other countries.Although there have been some investigations on the prevalence of chicken astrovirus in China,there are still few studies on the epidemic strains of CAstV in China.In this study,epidemiological investigation and analysis of chicken astrovirus infection in some areas of Jiangsu Province were carried out to understand the main domestic epidemic strains and their distribution characteristics.A chicken astrovirus strain was successfully isolated using chicken embryos and LMH cells,named CAstV/CZ1801,and its whole genome was sequenced,and its genetic evolution and biological information characteristics were analyzed.In order to understand the pathogenicity of CAstV isolated strain,the animal challenge experiment of CAstV was carried out.And the capsid protein of chicken astrovirus was expressed by baculovirus expression system,and the monoclonal antibody against the capsid protein of chicken astrovirus was generated,which made the foundation for the further study of the biological characteristics of capsid protein and the detection method of chicken astrovirus.1.Epidemiological investigation,isolation and identification of chicken astrovirusIn this study,98 intestinal samples of chicks were collected from some farms in Jiangsu Province.The CAstV infection was investigated by molecular biology methods.The results showed that the total positive rate was as high as 69%,and the sequencing results showed that all the samples belonged to type ? CAstV.A strain of chicken astrovirus was successfully isolated using chicken embryos and LMH cells,named CAstV/CZ1801,Its whole genome was sequenced,and the genetic and evolutionary relationship and biological information characteristics of the virus were analyzed.The results showed that this isolate had the structural characteristics of typical astrovirus genome and had high homology with CAstV/NJ1701 isolated in our laboratory asa well as other Chinese astrovirus isolates CAstV/GDYHTJ and CAstV/HBLP(97.5%-98.7%).Theybelonged to the same branch.Compared with other published sequences of GenBank,the amino acid sequence homology of ORFla and ORF1b of CAstV/CZ1801 strain was 86.4%-87.0%and 77.9%-86.9%,respectively.The difference of amino acid homology of ORF2 was obvious,between 38.8%and 95.9%,and it belonged to Bi group CAstV.2.Pathogenicity study of chicken astrovirusTwo strains of chicken astrovirus CAstV/CZ1801 and CAstV/NJ1701 isolated and identified in the laboratory were inoculated into 1-day-old SPF chicks by oral and intraperitoneal injection.The pathogenicity of chicken astrovirus was evaluated by observing its growth and development,evaluating its serum antibody level,detecting its detoxification and the replication level of the virus in different organs in the body.The results showed that CAstV/NJ1701 challenge group could significantly inhibit the growth of chicks,while the other group had no abnormal growth state,the antibody began to produce at 14th day post infection(P.I.),and reached to the higher level at 21st day P.I.,and the detoxification amount was the highest at the first dayP.I.,and then showed a fluctuating downward trend.The results of virus load detection in different organs of different ages showed that the virus first showed a high level of distribution in intestinal tissue,while the highest distribution level was found in kidney tissue 3-7 days P.I.,and 14 days P.I.,the virus mainly remained in the glandular stomach,but could not be detected in other tissues.After dissection,there were no visible pathological changes in organs by the naked eye in two groups.3.Development of Monoclonal Antibodies against Chicken AstrovirusIn this study,the capsid protein gene of chicken astrovirus was cloned into the eukaryotic expression plasmid of baculovirus.After transposition,the recombinant baculovirus Bacmid-CAstV-Cap was transfected into Sf9 cells and the recombinant baculovirus rBacmid-CAstV-Cap was successfully expressed.Mouse polyclonal serum was used as the first antibody,and the expressed protein had good reactivity verified by indirect immunofluorescence and Western Blot assay.Four hybridoma cell lines which can stably secrete monoclonal antibodies against CAstV capsid protein were screened by indirect immunofluorescence(IFA).All of them could detectnatural virus.The monoclonal antibodies named CAstV-Cap-6H3,CAstV-Cap-5A11,CAstV-Cap-3A2 and CAstV-Cap-3E12.The specific experimental results showed that the hybridoma supernatant of the four McAbs had no cross reaction with other avian viruses such as ALV-J,AIV,IBV,MDV and so on.According to the subclass identification detection,two McAbs were identified as IgGl subclass,one was IgG2b subclass,and the other one was IgM subclass.And the light chains were all Kappa chains.