| Viruses are one of the most abundant species on our planet,the safety of humans and animals are seriously threatened by many virus. Emerging viral diseases become more and more common in recent years,which can easily cause social panic and instability, given its characteristics of heavily unknown,emergency, high mortality and great harm. The development of viral metagenomics in recent years does not rely on traditional virus isolation methods, which breaks through the limitations of traditional virology research methods and focus on all of the viral nucleic acids of one environment. It is able to identify the virus in one environmental community quickly and has become a powerful tool to discovery new viruses and monitor the virus dynamics.Tiger as a large predator, standing at the top of the food chain, have been infected by many virus,such as canine distemper virus, feline parvovirus, canine parainfluenza virus, feline calicivirus, canine adenovirus, and even bird flu virus. These virus are not only a threat to the survival of tigers, but also can threaten human health, especially in modern society, the chance of human contacting with tigers is becoming more and more common, the study of viral diseases of tigers becomes more and more necessary. While the systematic study about tiger viruses communitie have not been reported.Here, viral metagenomics technology was used to explore gastrointestinal virus of the siberian tigers. Firstly, faecal samples of Siberian tigers was collected,and diluted by PBS, then high-speed centrifugation was applied to remove the unrelated components and ultracentrifugation method was applied to concentrate the virus particles. Next, nucleic acids of polluted was digested by nuclease digestion method. After the extraction of nucleic acid, sequence independent single primer amplification was used to amplify the virus nucleic acids. Then the PCR amplication product was concentrated and purified,it was sequenced by Illumina Hi Seq2000 high-throughput sequencing technology through gene sequencing company. 3200,000,000 reads were obtained after Illumina Hi Seq2000 high-throughput sequencing, and 6766 Contigs were spliced out, the total length of the contigs were 8,451,561 bp. After bioinformatic analysis,these sequences were annotated to many virus,including feline parvovirus, canine parvovirus, feline calicivirus, porcine bocavirus, mammalian reovirus, bluetongue virus, influenza virus, mink enteritis virus, chicken anemia virus and so on.Feline parvovirus HRB T2014 and feline calicivirus TFHLJ-8 were isolated,from which the metagenomic results was verified. The homology analysis of VP2 gene of HRB T2014 showed that its similarity with other reference strains was 98.1%~99.9%, the highest similarity is the feline parvovirus FJ405225 strain and HT-374 strain,which were originated from tiger, and the lowest similarity is canine parvovirus cpv-nj01-06, HRB T2014 was in the tiger feline parvovirus branch from phylogenetic analysis. the homology analysis of whole gene of HRB T2014 showed that its similarity with other reference strains was 98.6%-99.3%. the homology analysis of whole gene of TFHLJ-8 showed that its similarity with other reference strains was 76.3%-92%, highest similarity is URB isolate and lowest similarity is GD isolate.In this study, viral metagenomics was used to analysis the gastrointestinal virus of siberian tigers, the results included sequence of feline parvovirus, feline calicivirus, canine parvovirus, bluetongue virus, mammalian reovirus, influenza virus and so on, and feline parvovirus and feline calicivirus were successfully verified; a strain of feline parvovirus, named HRB T2014, was isolated, and the virus may be a recombinant parvovirus of canine parvovirus and feline parvovirus, besides this, a strain of feline calicivirus, named TFHLJ-8, was isolated. |
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