Molecular Epidemiological Investigation In Some Regions Of China And Establishment Of Absolute Quantitative Detection Method Of Chicken Astrovirus

In recent years,the impact of avian astrovirus on poultry industry has received worldwide attention.Among them,chicken astrovirus can cause chicken nephrtis,diarrhea enteritis and slow development,and it is one of the important pathogens which causing diseases such as diarrhea and gout in chick flocks.Untilll now,the results of molecular epidemiological investigation of chicken astrovirus in China have not been reported publicly.In order to understand the prevalence of CAstV in China,this study carried out a molecular epidemiological investigation of chicken astrovirus in some areas of China and isolated first Chinese strain of CAstV.In addition,an absolute fluorescence quantification method for the detection of type ?chicken astrovirus was established,which provided technical means and biological materials for the further study of the biological function of CAstV.1.Epidemiological investigation of chicken astrovirus in some regions of ChinaIn this study,we collected 195 samples from 7 regions of Jiangsu,Anhui,Shandong and Guangdong provinces from 2017 to 2018 for detection chicken astrovirus infection by PCR.The results showed that only 1 of the 41(2.44%)samples of adult chickens were detected as positive,while the positive rate of 154 samples of 1 day old chicks was as high as 82.47%(127/154).The comprehensive positive rate of all samples was 65.64%.The positive samples contained 7 strains,all of which belonged to chicken astrovirus.The results of genetic analysis indicated that both type ? and type ? chicken astrovirus existed in chick flocks in China.According to the results,similar to previous reports,low-age chicks are more likely to detect the virus,and the detection rate of adult chickens is much lower.2.Establishment of absolute quantitative qRT-PCR detection method for chicken type ?astrovirusSo far,there is no relevant research about quantitative detection method of CAstV.In this study,a method for the detection of type ? chicken astrovirus by absolute quantitative real-time fluorescent PCR was established based on the chicken astrovius RNase-dependent RNA polymerase(RdRp)gene expression.By designing CAstV-RdRp-specific fluorescent quantitative PCR primers and preparing plasmid standards,a absolute quantitative real-time PCR method for detecting viral RdRp gene was established.And this method was used to detect the proliferation of virus in LMH cells.The results showed that although there was no obvious pathological change after infection of LMH cells by CAstV,the virus could grow and reproduce in LMH cells.The absolute quantitative PCR method for detecting CAstV-RdRp gene established in this study pvides a detection technique for studying the pathogenicity and biological function of viruses.3.Isolation and identification of chicken astrovirus and whole genome sequencing analysisIn order to further deep study of the CAstV epidemic in China,this study used the chicken liver cancer cell line(LMH)to isolate and identify the virus of CAstV NJ1701 strain.The results demonstrated that the CAstV could replicate in LMH cells,and after 4 passages LMH cells showed obvious cytopathic effect.A nearly full-length genome of a type ? chicken astrovirus CAstV NJ1701 strain was amplified and sequenced.The length of the genome was 7492nt.Its structural features were similar to those of the published CAstV genomic DNA genome,which contains three open reading frames,including ORFla,ORF1b and ORF2.Three open reading frames were used for genetic and evolutionary analysis.The results revealed that the isolated CAstV NJ1701 strain had higher homology with CAstV FP3 strain,and could be divided into CAstV B1 population by ORF2 capsid protein classification.The ORP1a and ORF1b gene sequences of CAstV NJ1701 strain had low homology with other ORF1a and ORF1b sequences of CAstV,and were in a relatively independent branch.4.Experiments on the pathogenicity of chicken embryos inoculated with CAstVThe isolated virus was inoculated into the 6-day-old chicken embryo through the yolk sac.After 4 passages of blind transmission,the chicken embryos were found to be weak and lethal.The qRT-PCR assay confirmed that the virus could proliferate in chicken embryos as well.The hatch rate of chicken embryo inoculated with virus showed that only 11/30 were successfully hatched with a hatch rate of 36.67%.There were obvious problems in the growth and development of the dead chicken embryo,which showed dwarfism embryo,indicating that the infection of CAstV could lead to the decrease of the hatching rate in the chick flock.The RdRp gene distribution and expression in different tissues of 1-day-old hatched chicks were detected by absolute quantitative qRT-PCR.The results showed that the CAstV gene had the highest expression level in the caecum tissue and the lowest expression level in the heart tissue,which provided a reference for the collection of clinical samples in the future.