Viral Diversity And Epidemiology Study In Neophocaena Asiaeorientalis Asiaeorientalis

The Yangtze finless porpoise（Neophocaena asiaeorientalis asiaeorientalis,YFP）is a critically endangered freshwater cetacean with 1,249 individuals in existence.Although the population of Yangtze finless porpoise has recovered from before,its endangered status remains unchanged and there are still gaps in factors such as viral diseases.Therefore,this paper investigates the viral epidemiology of Yangtze finless porpoise in the Swansea Baiji National Nature Reserve using viral macrogenomics and other methods to investigate the diversity of viruses contained in the anal swab samples of Yangtze finless porpoise.We also established a detection method for the newly identified papillomaviruses（NaPVs）and constructed a prokaryotic expression plasmid containing the major capsid protein L1 gene using the papillomavirus E1 and L1 genes,respectively.The main results are as follows:1.A total of 19 viruses belonging to 9 families were identified by macrogenome sequencing,including Papillomaviridae,Herpesviridae,Picornaviridae,Picobirnaviridae,Caliciviridae,Retroviridae.Parvoviridae,Virgaviridae,Narnaviridae and other unclassified viruses.Among the above-mentioned viruses,based on the potential pathogenicity of the newly identified YFPs herpesvirus（Na HV）,crestovirus（Na KV1-2）and papillomavirus（NaPV1-6）,the carriage of viruses in the samples was detected by molecular biology,and the results showed that most YFPs carried one or more viruses（52/58,89.7%）.And crestoviruses and herpesviruses showed a clear trend of age distribution,while PVs showed a clear gender difference characteristic in YFPs.Then,two new PVs species（referring to Omikronpapillomavirus 2 and 3）and four newly isolated PVs were identified in YFPs according to the species delimitation criteria of each genus of Papillomaviridae.Further evolutionary analysis showed that NaPVs occupied mucosal ecological niche and that the co-evolution,replication,recombination and host transformation events together drove the evolution of cetacean PVs.Finally,the estimated divergence times of PVs in YFP and other cetaceans reflected the status of YFPs as incipient species.In conclusion,the results revealed the potential virus species,virus prevalence,and evolutionary history in YFPs,and provided a direction for research on the effects of virus infection on the health and reproduction of YFPs.2.The whole genome sequences of six NaPVs were compared by multiple sequences,and the most conserved E1 gene was selected as the target sequence.The simplified primers E1 gene were designed,and the NaPVs detection method was established by amplification system optimization.Using the above six viral sequences as templates,PCR amplification was followed by sequencing,and the optimal primer annealing temperature was determined to be 50°C.The correctly sequenced NaPV4gene sequences were ligated into the T-vector,and the constructed plasmids were diluted at 10-fold ratio to make the copy number between 1×10¹⁰-1×10⁰,and the sensitivity of the check method was 10.6 copies/μL.3.The whole genome sequence of the newly discovered YFPs papillomavirus type2（NaPV2）was used as a template to amplify its region encoding the L1 gene and ligated into the prokaryotic vector p GEX-4T-1-L1.The recombinant plasmid was double digested,the target gene sequenced and then transferred into E.coli BL21（DE3）,and the expression was induced by IPTG to explore the expression form of the fusion protein at different temperatures.The result of SDS-PAGE showed that NaPV2 L1-GST fusion protein was efficiently expressed as soluble and inclusion bodies at 20℃with a relative molecular mass of about 82 000,which laid the foundation for the preparation of NaPV2 L1 vaccine.