The Study Of Viral Metagenomics In Rodents From Shandong

Objective:Rodents as a natural reservoir of viruses,especially rats are not only closely related to humans but also as an infection source of many viruses.In this study,we have mastered the composition of virus in intestine and tissues of rodentsfrom Shandong Province,the purpose is to understand potential pathogenic viruses and novel viruses,which will help to quickly respond to the sudden viral public health events caused by these viruses;Discovery novel viruses provide early warning for possible cross-host transmission,updating the host species and geographical range of many rodent-borne viruses,expanding the spectrum of viruses carried by rodents;The genomic structure analysis,phylogenetic analysis and epidemiological investigation of novel viruses provide theoretical support for preventingthat diseases caused by novel viruses.Method: The samples of rodents were collected from three regions(Huandao district of Qingdao city,Zichuan district of Zibo city and Jiaxiang county of Jining city)of Shandong province in China.The samples of 210 intestinal and 241 tissue were mixed into 10 intestinal pools and 10 tissue pools respectively,then filter,digested by DNase and extract nucleic acids;N ucleic acid were sequenced by Hiseq high throughput sequencing in Beijing Genomics Institute(BGI);the obtained mass sequences of intestinal and tissue were analyzed by our established biological information analysis platform for Viral metagenomics.The significant viruses were confirmed and screened by PCR.The whole genome sequence of novelviruses,including Wenzhou virus,Murine norovirus,sapovirusand bocavirus were amplified using the methods of general PCR,walking PCR,3' and 5' RACE in order to understanding the characteristic of genome and evolutionary relationship.Cell culture and animal effction experiments were conducted for further studing Wenzhou virus' infectious and pathogenic.Results: 5GB high-throughput data was obtained in every pool.1416731 sequences in intestinal pool S1-S10 and 57695 sequences in tissue pool T1-T10 were annotated to virus.By sequence analysis,these viruses in pool S1-S10 can be divided into 73 families and 237 genera.Vertebrate viruses contain 28 families and 114 genera;plant viruses contain 19 plant families and 66 genera;invertebrate viruses include 10 families and 17 genera and 16 families of phage.Viruses in pool T1-T10 can be divided into 35 families and 78 genera,vertebrate viruses contain 18 families and 44 genera;plant viruses contain 8 plant families and 22 genera;invertebrate viruses include 5 families and 7 genera(this virus can infect vertebrates and invertebrates at t he same time,these virus found in mice with less homologous)and 4 families of phage.Vertebrate virus is the largest,mainly including coronavirus(24%),astroviruses(18%),parvoviruses(11%)and picornaviruses(10%).Many of these viruses can cause animal diseases,such as coronavirus,astrovirus,rotavirus,norovirus,bocavirus.If there viruses can cross-species transmission to humans,it may be cause adverse impact on human life and health,and sometimeseven lead to public health incident.We found almost all the groups of rat rotavirus,especially group A and B,we have amplified the nearly whole genome sequence of 11 segments in group B.We choose Wenzhou virus(G107)?Murine noroviruses(SDHD46)?sapovirus(S4-82)and bocavirus(S9-108)for future research.The whole genome of L and S segment in Wenzhou virus is 7,131 nt and 3,322 nt,respectively,the indentity comparaed with other known strains is 84.7%~86.8%(L)and 84.2%~87.8%(S).DH82 cell culture and animal infection tests were negative,indicating it is no potential infection risk and pathogenicity to this cell and animals.MNV(SDHD46)is 7,382 nt in length,comparaed it's whole sequence with other MNVs showing about 76.8%~78.1% indentity.Additionally,there are 11 mutation sites in P2 region that known as high mutation area,5 mutation sites in P1 region and 1 mutation site in S region.Sapovirus S2-82 is the first complete genome of the rodent virus in Gen Bank,which is 6,841 in length.The bocavirus S9-108 is detected in host of Shrew and Hamster,it's nearly whole genome is 4,977 nt.The identity between S9-108 and other 6 rodent bocavirus strains(host is Rattus norvegicus)is just 50.1%.According to the International Committee on Taxonomy of Viruses(ICTV),S9-108 belongs to a novel species in bocaviruses of Parvoviridae.Conclusion:This study fully understood the viral spectrum existing in rodents from Shandong province with the method of Viral Metagenomic.There are a lot of viruses that may cuase disease in animals and humans,additionally,a large number of viruses that have lower homology with known virus,such as Wenzhou virus(a new arenavirus),astroviruses,bocaviruses,norovirus and sapovirus.These viruses greatly enrich the viral database of wild animals all over the world.