Identification And Phylogenetic Analysis Of A Novel Adenovirus Isolated Fromtupaia (Tree Shrew) With Severe Respiratory Disease

Tree shrew adenovirus is linear double-stranded DNA viruses.It is classified as a virus of the genus Mastadenovirus,family Adenoviridae.These viruses can cause a variety of illnesses,including acute respiratory tract infections,acute conjunctivitis,hemorrhagic cystitis,and diarrhea.Tupaia belangeri is a novel model animal closely related to primate,and it has high genetic homology both with human and primate.It is used as human disease models,especially in infectious disease and preclinical drug development.These animals play an indispensable role in the study of human diseases,especially infectious diseases,and have gradually applied in biomedical research.A previous study indicated that wild tree shrews have showed a high prevalence of adenovirus.Previous studies showed that the detection rate of adenoviral nucleic acid is high in wild-type tree shrews.Therefore,it has been listed as one of the detection indicators for the microbiological quality control of the animal.Prior to this study,there were no previous reports of TAV separation published in China.It suggests that the respiratory disease and diarrhea may be caused by a viral infection.Tree shrew adenovirus was isolated and identified from tree shrew using a virus isolation method.In order to reveal the evolutionary status,genetic information,and potential effect of TAV in tree shrew,the whole genomic sequence analysis of TAV was performed in this study.Firstly,TAV was obtained by cell culture of tree shrew kidney cells(TKC),and the pathological symptoms of interstitial pneumonia was made.The viruses were identified as adenovirus by molecular method,immunofluorescence,and electron microscopy detection,ultimately named as TAV-KM(Tree shrew adenovirus Kunming strain).Secondly,establish efficient and specific molecular detection methods.Based on the TAV genome sequence published,a 3' conserved sequence was used to design specific probe primers.A standard curve was prepared using a recombinant plasmid containing the target gene fragment.A real-time fluorescence quantitative PCR method was established for detecting TAV based on TaqMan probe.The detection method was specific and there was not cross-reactive with other common pathogens.The detection limit of the method was 3.7 copies/?L,which was sensitive,the correlation coefficient was 0.998,and the efficiency was 95.7%;the amplification result had a fine linear relationship,and the repeatability test effect was goodFinally,TAV was reproduced by TKC.It was purified and concentrated using a virus purification kit,and the complete genome sequence of TAV-KM was obtained through second-generation sequencing.The major encoded protein was then verified by one-generation sequencing.To further clarify the evolutionary taxonomic status of TAV-KM,a phylogenetic tree was subsequently constructed.It was found that the genetic relationship was close between TAV-KM and German isolated TAV1.The TAV-KM genome was found to be 33,487 bp,which is shorter than the genome of tree shrew adenovirus 1 as 14 bases.The mapping rate of the two genomes was 74.97%and the G+C content of TAV-KM was 50.07%,which is similar to that of tree shrew adenovirus 1(49.96%).We also found that there was a 161 bp reverse repeat sequence in the TAV-KM genome.Analyzing the coding capacity of the complete TAV-KM genome at least 40 codons in length,we identified the 110 open reading frames(ORF)of,of which 32 were confirmed as viral genes.To further characterize the molecular structure of the TAV-KM genome and determine how it correlates with tree shrew adenovirus 1,we compared the nucleotide and amino acid sequences of the two genomes.In our comparative study of the fiber proteins of TAV-KM and tree shrew adenovirus 1,a 1.43%difference between the amino acid sequences was detected.Nine amino acid differences in the fiber protein sequences of TAV-KM and tree shrew adenovirus 1 were identified,including two synonymous and seven non-synonymous differences.Presumably,these mutations lead to changes in the fiber protein structure,thereby increasing the infectivity of TAV-KM.A detailed study of the amino acid differences and fiber protein structure would be conducted in the future in order to understand the characteristics of this isolate preferably.The results of this dissertation enriched the animal virus database,provided experimental materials for the establishment of the tree shrew adenovirus infection model in the future,and laid the foundation for the pathogenic mechanism and other related research.At the same time,the mutation of the adenoviral fiber protein gene enhances its susceptibility,and these problems will be further clarified in future work.