Application Of High-throughput Sequencing Technology In Diagnosis Of Viral Diseases In Wild Animals

Infectious diseases are spread among wild animals,humans and domestic animals,posing a serious threat to human health and animal protection.Due to the variety of wild animals,complex habitats and unclear origin of the Infectious diseases,they are often used in the routine detection of human and domestic animals,most of which are no longer suitable for the diagnosis of wild animal diseases,which brings difficulties to pathogen diagnosis.In this study,we explore the application of high-throughput sequencing technology to the diagnostic confirmation of wildlife viral diseases.1.Establishment of a rapid diagnostic high-throughput sequencing method for viral diseases in wild animals.In order to meet the requirements of rapid detection of wild animal viral diseases,the four links of nucleic acid extraction,library construction,computer sequencing and data analysis were optimized in terms of methods.In terms of nucleic acid extraction,the combination of Trizol method and QIAamp kit not only increases the content of RNA extraction but also shortens the detection time.In terms of library construction,only RNA library construction is carried out,including the construction of RNA virus library and DNA virus m RNA library during the propagation period in the host,which simplifies the experimental process.When performing sequencing on the machine,select the Next Seq ?1000/2000 P2 Reagent Cartridge kit with a small throughput of 50 double-ends according to the characteristics of the case,which can shorten the time for sequencing on the machine.In the data analysis process,the virus database is directly used for annotation comparison,which improves the data analysis speed and shortens the analysis time.In order to evaluate the availability and accuracy of the detection method,the rapid diagnostic method was validated using the tissue samples of dead brown-headed gulls infected with H5N1 as the verification object.Nucleic acid extraction,library establishment,and computer sequencing can be completed within 36 hours,and the comparison and analysis of sequencing data can be completed within 8 hours.Compared with the viral sequence obtained by first-generation sequencing,the base matching rate is 94.75%,which greatly reduces the detection time.And at the same time,most of the sequence can be directly used for analysis.The verification results confirm that the application of this method effectively shortens the detection and analysis time and can be used for rapid diagnosis of wild animal viral diseases.2.High-throughput sequencing technology for the confirmation of LSDV.For a case of a giraffe that died suddenly without typical clinical symptoms,some organ tissue samples were obtained from the dead giraffe,and the established rapid diagnostic method was used to complete high-throughput sequencing and rapid analysis of pathogenic data within 5 days.Through data analysis,4922 reads were screened,which can be classified into 3 viridae families.Poxviridae has a total of 4886 reads,accounting for 95.8%,and Baculoviridae has a total of 5 reads,accounting for 95.8%.The ratio was 0.1%,and Herpesviridae had a total of 31 reads,accounting for 0.6%.Among them,Poxviridae had the highest abundance,with a matching degree of 100%.The sequences of Poxviridae were analyzed and compared,and the result was LSDV of Poxviridae.The validation and sequence alignment analysis using firstgeneration sequencing technology showed that the GPCR gene sequence of LSDV of giraffe was highly homologous with 100% nucleotide identity in Vietnamese giraffe from China,Thailand and Russia.It is different from wild-type strains and vaccine-type strains,and it is far related to GTPV and SPPV strains.The giraffe tissue samples were ground and inoculated with bovine testicular interstitial cell line,which produced typical lumpy skin disease virus CPE.The harvested cell cultures were determined by sequencing and electron microscopy to prove the successful acquisition of a giraffe-derived LSDV virus strain and the feasibility of high-throughput sequencing methods for rapid detection.3.High-throughput sequencing technology was used to confirm Feline astrovirus 3 in wild Amur leopard samples.For a wild Amur leopard rescued in the wild,the wild Amur leopard throat,anal swab,feces,eye and nose secretions and blood samples were obtained.Using this detection method,high-throughput sequencing and rapid analysis of pathogenic data were completed within 5 days.Through analysis and screening,5 virus reads were obtained,and the virus sequence information was all classified into Astroviridae,with a matching degree of 80%,among which the complete 2464 bp ORF2 gene was spliced.The viral sequence information of the Astroviridae family was compared by BLASTn at NCBI,and the astrovirus was the Feline astrovirus 3 from cats.Then,the first-generation sequencing technology was used to further verify the viral disease pathogen,and the results showed that the ORF1 b and ORF2 genes of type 3 astrovirus in the Far Eastern leopard had high homology with type 3 astrovirus from cats.The ORF1 b gene is 91.09% and 98.46% and the ORF2 gene is 90.33% and amino acid identity 98.47%.Using electron microscopic morphological detection,virions were found with astral-like structures.In this study,the high-throughput sequencing technology had been established successfully and were used in rapid diagnosis of two unknown wildlife diseases.By using the method,the giraffe LSDV infection and Amur leopard feline astrovirus 3 infection were confirmed.The research results provide a reference for the subsequent application of highthroughput sequencing technology in the diagnosis of wild animal viral diseases.