Analysis Of Virome In The Plasma Of Cattle As Weii As Human From Forest Region And Identification Of Novel Genomes

As we all know,the virus is a kind of micro-organism that can spread among other human organisms and infect organisms.In recent years,resurgence and new epidemic diseases caused by viruses have frequently occurred,seriously threatening human health and the safety of life and property.The relationship between cattle and production and life of human is particularly close,especially in the northeastern forestry areas where animal husbandry is well-developed.Cows are vulnerable to be bited by mosquitoes that carrying pathogenic viruses,as well as ingestion of water and feed contaminated with related pathogenic viruses.If cow-borne pathogenic viruses threaten the health of cattle and even humans,cattle can become a source of pollution.Therefore,it is of crucial importance to be familiar with and to ascertain the viruses that have been or have not yet been identified in the blood of the cattle and people who are in close contact with in the northeastern forestry areas of China.Traditional virus detection methods have greater limitations in the detection of unknown viruses and new virus.The emergence of Viral Metagenomics based on second-generation sequencing technology solves this problem.This research method directly uses all virus nucleic acid sequences present in the sample as the research object,and has better detection of known and unknown viruses in the sample.The emergence and rise of this technology provides powerful technical support for the excavation of new viruses and early warning of new infectious diseases.In order to explore and control the composition of the virus community in the blood of cattle and the people who are closely contact with in the northeastern forest region,as well as to explore new types of potential pathogenic viruses,this study used the method of virus metagenomics to study the blood samples of cattle breed in the northeast forest region as well as the human.Results: 1.The composition of virus community of blood from cattleThe composition of the virus community in the bovine blood of the northeastern forest region includes: unclassified virus family,Phycodnaviridae 12.64%,Mimiviridae 4.44%,Parvoviridae 3.22%,Circoviridae 2.22%,Poxviridae 1.88%,Herpesviridae 1.11%,Iridoviridae 1.01%,Marseilleviridae virus 0.86%,Baculoviridae 0.55 %,0.29% for Geminiviridae,0.23% for Alloherpesviridae,0.22% for Bicaudaviridae,0.18% for Retroviridae,0.13% for Sphaerolipoviridae,0.13% for Adenoviridae,and others The Others 1.25%.The analysis of bovine blood virus community showed that:(1)The composition of virus community in bovine blood showed diversity of virus species;(2)Among all the viruses,unclassified viruses accounted for the largest proportion,reaching 69.65%,constituting the main body of the bovine blood virus community in the Northeast Forest Region.Followed by 12.46% of Phycodnaviridae,4.44% of Mimiviridae virus,3.22% of Parvoviridae,and 2.22% of Circoviridae;(3)The five virus libraries contain a variety of mammals-infecting viruses such as Picornaviridae,Parvoviridae,Circoviridae and Caliciviridae.2.The composition of virus community from human bloodThe results showed that the composition of the virus community in the blood of people who were in close contact with cattle in the northeast forest area included: Anelloviridae,Hepadnaviridae,and Human Endogenous Retrovirus.Human blood virus community analysis suggested that:(1)There are relatively few species in the virus community of human blood;(2)The viral DNA library of Forest05 contains three different species of human endogenous retrovirus(HERV)nucleic acid sequences;(3)A complete genome of Hepatitis B virus was found in the viral DNA library of Forest06.3.Comparison of Blood Virus communities between human and cattleThe composition of the blood virus community of cattle and their close contacts in the northeastern forest area is quite different.There is no correlation between the two virus communities.The viruses presented in the bovine blood virus community is almost absent in human blood.4.Identification of the new viruses from the blood of cattle(1)novel CircovirusesThree novel complete Circovirus genomes were found in the blood of cattle in the northeastern forest region of china.Named Bvch001,Bvch002,Bvch005.This type of virus is currently a circular single-stranded DNA virus that can only infect birds and pigs while the ability to infect cows and replicate in cattle is not yet reported.In this study,the complete genome sequences of the two viruses,Bvch001 and Bvch002,were obtained from library 5 and library 4.The full-length genes were 1881 and 2926 nt,respectively;Bvch003 has its complete genome in the library.Analysis of the entire genomes of these three viruses revealed that Bvch002 contains the Rep and Cap genes in the same orientation;the two genes(Rep)and(Cap)in Bvch002 and Bvch005 have opposite orientations.Using the virus' s conserved replication protein(REP)for the comparison by the BLASTp with the Gen Bank gene database,it was found that Bvch001 and the two small circoviruses(KP233191 and KP233192)from the gorilla shared similarity of 55%;Bvch002 shares the highest similarity with the circovirus-like virus(KM598397)from dragonfly,reaching 38%;Bvch005 has up to 38% similarity with a fecal-associated circular virus which was isolated from cattle.In order to further verify that the two Circoviruses do exist in the blood of cows in Northeastern China,the nucleic acid sequences of the conserved REP genes of the two Circoviruses found in bovine blood were designed to target these three viruses,respectively.The PCR primers were used to test blood samples of 80 cattle from northeastern forest area of china.The results showed that: Bvch001 detected nucleic acid sequences in three samples and sequenced and found that the similarity of the three sequencing results was greater than 99.3%;Both Bvch002 and Bvch005 were detected in a sample of the corresponding nucleic acid sequence,and then the detected nucleic acid sequence was sequenced.The sequencing results showed that the nucleic acid sequence of the two original circovirusREP genes was completely consistent.Subsequently,the phylogenetic analysis of the conserved Rep protein(Rep)amino acid sequences of the three strains showed that they all clustered into the Circovirus.The results above show that these two viruses belong to the novel circular virus Circovirus.(2)Novel GemycircularvirusesThe virus is a new single-stranded circular DNA virus that can exist in humans and other animals,even in environmental samples.Two new gemycircular viruses were found in the blood of cattle,named "BGmv001" and "BGmv002" respectively,and the full-length genomes were 2192 nt and 2926 nt,respectively.Gene sequence analysis revealed that the genomes of the two viruses contained two genes that were opposite in orientation,encoding the two proteins,Rep and Cap,and did not contain intron regions.Based on the alignment analysis of the BLASTp protein sequence based on the relatively conserved replication protein(Rep)of the virus,the two virus strains showed 56% and 36% similarity with the gemycircular virus derived from cockroaches and the gemycircular virus derived from bird feces,respectively.In this study,primers were designed based on the nucleic acid sequences of the two gemycircularvirusREP genes found in bovine blood samples and 80 cow blood samples were tested.The detection results showed that both BGmv001 and BGmv002 had their gene sequences found in the two samples.The two nucleic acid sequences of BGmv001 and BGmv002 were sequenced.The similarity of the two 290 bp long nucleic acid sequences of BGmv001 was 99%,based on BGmv002.The similarity of the sequencing results of the two 280 bp long nucleic acids was 98%.Phylogenetic analysis using the conserved protein replication protein(Rep)amino acid sequences of both viruses,both of which are clustered together with the gemycircularvirus family,showed that the two viruses belong to a new gemycircularvirus.(3)Novel Bovine parvovirusBovine pavovirus is a small class of linear DNA viruses that can cause cow diarrhea and adult cattle respiratory and reproductive system diseases.In the study,two novel bovine parvoviruses,Bovine parvovirus 2 and Bovine parvovirus 3,were identified in bovine blood and designated BPV2 and BPV3.Bovine parvovirus contains two genes with the same orientation and encodes nonstructural protein(NS)and structural proteins(VP1).The BLASTp and phylogenetic analysis of non-structural protein NS and structural protein VP1 proteins of the BPV2 and BPV3 revealed that the amino acid sequence of the NS protein of BPV2 shared 96%-98% similarity with three NS protein of the BPV2 in the Gen Bank database,but the VP protein amino acid sequence shared only 84%-86% similarity with BPV3 from GenBank database.Conclusions:(1)The use of virus metagenomics method mastered the composition of blood virus community in bovine blood and related blood of people;(2)The composition of bovine blood virus community presents diversity;the human blood virus community is relatively single;(3)Two new types of gemycircularviruses,three novel circoviruses and a novel parvovirus were revealed.