Application Of Viral Metagenomics In Diagnosis Of Broiler Chicken Diseases

In recent years,the emergence of new infectious diseases occurring on poultry has brought serious losses to the poultry industry.Achieving rapid diagnosis of diseases is of great significance to the development of poultry industry.At present,there are many atypical symptoms in clinic,which can not be confirmed by traditional detection methods and bring certain difficulties to the prevention and control of epidemic diseases.The purpose of this study is to use viral metagenomics technology to detect sick chicken samples that are difficult to diagnose clinically and provid a basis for clinical diagnosis.Moreover,it would assist in the discovery of new viruses and their virological studies.A total of 2821 contig sequences were obtained from broilers with obvious respiratory symptoms by using metagenomics technology.After bioinformatics analysis,the measured sequences corresponded to several viruses,including Chicken parvovirus,Sicinivirus,Picornavirus,Avastrovirus and Nephritisvirus accounted for 3.5%(101/2821),2.5%(73/2821),0.8%(23/2821),0.1%(3/2821)and 0.1%(3/2821),respectively.In addition,483 contig sequences were obtained from the same batch of healthy chicken samples,which were analyzed by BLAST and did not match the virus.The sequences of Ch PV and Scinivirus were analyzed.In the sequence of Ch PV,two fragments matched the NS gene,the amino acid homology between the two fragments and the reference strain was 73.4%-76.6%,94.3%-99.4%,and the amino acid homology between the two fragments was 74.7%;one fragment matched the NP gene,the amino acid homology with other reference strains was 69.3%-99.0%;one fragment matched the VP gene,and the amino acid homology with other reference strains ranged from 74.0% to 96.6%.The results of genetic evolution of each segment of Ch PV showed that most segments had close genetic distances from the parvovirus in the United States,and one of the NS gene segments had a large degree of sequence variation and was in a large branch alone.Sequences of Sicinivirus included some or all of the nucleotide sequences of 10 genome segments: L(1379 nt),VP1(537 nt),VP0(1022 nt),VP3(617 nt),2A(587 nt),2B(587 nt),2C(1019 nt),3A(444 nt),3C(509 nt),3D(1415 nt),and no 3B segment sequence was measured.The results of homology analysis showed that the homology of each segment with domestic and abroad reference strains was below 91%,represented a high variability.The ChPV and Sicinivirus in the original disease material were verified by ordinary PCR,and the verification result was positive.In summary,the sample contained more ChPV and Sinivirus genome sequences,suggesting that the mixed ifection of the two viruses may be one of the reasons that causes this respiratory disease.In addition,the samples contained multiple ChPV sequences,one may be a new variant,and the other may derived from US strains.The measured Scinivirus sequences were more variable than the known sequences,suggesting that they may be new variants.A chicken embryo culture sample was tested and 94 virus-related contig sequences were obtained,including 78 avian leukosis virus(ALV)contig sequences,13 avian reovirus(ARV)contig sequences,and 3 Pandoravirus contig sequences.The sequencing results of ALV were compared to the complete capsule glycoprotein gene(gp85),and the homology analysis based on this gene showed that the ALV belonged to E group.In addition,the homology of 10 partial nucleotide sequences of ARV with domestic and abroad reference strains was below 91%,and the degree of variation was large.In summary,there were ALV,ARV infections in this sample,and there was a high degree of variation in the ARV gene,suggesting that it might be a new variant.In conclusion,two groups of broiler clinical samples were analyzed by viral metagenomics,and viral sequences such as Chicken parvovirus,Sicinivirus,Picornavirus,Avianastrovirus,Nephritisvirus,Avian leukemia virus,Avian reovirus were obtained from the sequencing results,which provided a certain basis for diagnosis and laid a foundation for the application of metagenomics technology in pathogen detection in broiler diseases.