| Microbiome is an integral part of the human body,which as we all know is composed of bacteria,fungi,and viruses.Viruses are kind of microorganism that can infect organisms and easily spread among animals and human beings.Recently,disease caused by viruses and new epidemic viral infection have frequently occurred,seriously threatening human health and the safety of life and property.Animals are always in close relation with humans.Rodents represent around 43% of all mammalian species and are widely distributed.Many rodents have mixed diets,but some eat mostly seeds or green vegetation.Rodents are known to vector more than 60 known human infectious diseases.Some rodents live in close association with humans offering numerous opportunities for cross-species viral transmission through their urine,feces,or their arthropod ectoparasites such as ticks,mites,and fleas.Traditional virus detection methods have greater limitations in the detection of new virus and unknown viruses.Development 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 emerging infectious diseases.In order to explore and control the composition of the virus community in wild rats as well as to explore new types of potentially pathogenic viruses,this study used the method of viral metagenomics to investigate the viral community in feces,blood,oral swab,Skin swab and in brain,lungs and liver tissues.ResultsComposition of virus communities in different samples of wild ratsThe composition of the virus community in the wild rats blood samples from Zhenjiang is so divergent and about 40 viruses from different families were detected,including Anelloviridae45.29%,Parvoviridae 29.63%,Secoviridae 7.78%,Retroviridae 4.35%,Flaviviridae 3.53%,Virgaviridae 1.32%,Microviridae 0.90%,and others viruses which makes wild rat blood virome.The analysis of wild rat brain virus community showed that the composition of the virus community in wild rat brain showed diversity of virus species.Among all the viruses,Retroviruses accounted for the largest proportion,reaching 45.60%,constituting the main body of the wild rats' virus community.Followed by 9.69% of Reoviridae,9.45% of Anelloviridae,7.15% of Microviridae,and 6.11% of Poxviridae.Results showed that the composition of the virus community in the wild rat feces included viruses from about 80 different families there are also viruses which can cause severe infection in animals and humans.It also includes insects and plants viruses.Variation has been found in wild rat liver samples there are viruses from different families which include: Parvoviridae,Retroviridae,Anelloviridae,Phycodnaviridae,Hepeviridae,Herpesviridae,Mimiviridae,Poxviridae,Asfaviridae,Baculoviridae and Iridoviridae.Wild rat Lungs samples includes viruses from about 55 different viral families.63.40 % of viruses in wild rat lungs sample belong to Retroviruses s 6.32% Poxviridae and3.60% Anelloviridae respectively.Diversity has been found in viral communities of oral and skin swab of wild rats there were viruses from family Picobirnaviridae and Parvoviridae;some reads were also detected from Bunyaviridae and Hepeviridae.Mostly insects and plants viruses in Skin swab.In wild rat Skin and oral swab,we found some novel viral reads from PapillomaviridaeIdentification of novel viruses from wild rat samplesA total of 10,329,088 unique reads were generated and sequenced from the extracted nucleic acid of 140 samples in this study using Illumina Mi Seq sequencing run with 250 bases paired ends.Sequence reads from each sample were assembled de novo into different length of contigs.Both contigs and singlets were then classified and compared to the Gen Bank non-redundant database using BLASTx(E score,10-5).Family Parvoviridae consist of small,resilient,non-enveloped viruses encoded by linear singlestranded DNA(ss DNA)genome of 4-6kb in length with two ORFs.Parvoviridae species can infect rodents,or domesticated animals species revealed the potential for vertical transmission.Canine minute virus,feline parvovirus and porcine parvovirus can induce intrauterine or placental infection in naturally infected host species.We characterize abundant sequences from different samples of wild rats showing similarities to Parvoviridae.These sequences were named Wr Pv.About 7 Fecal,3 Oral Swab,2,Blood and 2 Liver samples were positive for Parvoviruses like sequences.Sequences were de novo assembled and aligned.Phylogenetic tree constructed with best BLASTx matches in Gen Bank.Based on the phylogenetic tree result and genetic distance calculations,sequences from our data are significantly linked to other recognized species of Parvoviridae.We detected several sequence reads belong to Adenoviruses(AdVs).AdVs from different samples of wild rats.We named these sequences as Wr Ad Vs.The smallest nucleotide 1220 bp and the largest was 1590 bp.Sequence was the sequence reads shared a high nucleotide similarity of 76.18% to their best match Murine Ad V2 in Gen Bank after BLASTx.Phylogenetic tree were construct successfully based on hexon proteins.However,results showed that Wr Ad Vs sequences from our library make clade between Murine Ad Vs and Mouse adenovirus 2.In the present study,almost every sample of wild rat feces was positive for Picobirnaviruses(PBV)related sequences,the highest prevalence and number of mammalian virus-like sequences in fecal samples of wild rats.Sequences from libraries named as Wr PBVs.Along with that samples from wild rats also contained sequence reads that assembled and whose amino acid sequence showed identity to the viruses from Astroviridae.Alignment of sequences allowed the construction of a phylogenetic tree using four representative best BLASTx matches.Phylogenetic tree were constructed.Results showed that viral sequences from wild rats' samples are making separate branches.Circoviridae classified into two genera Cyclovirus and Circovirus,viruses in this family are round,non-enveloped with a single-stranded circular DNA(ss DNA).Circovirus infections have spread geographically and are highly prevalent,these viruses have been associated with various infections particularly in swine including dermatitis,reproductive disease,gastroenteritis and respiratory problems as well as in avian infections are psittacine beak and feather disease,chicken anemia Distribution of Circoviridae in different samples were found in recent study.Circoviridae in our samples were named Wr CVs.Among the 2 circovirus related genomes both were from fecal samples.Phylogenetic analysis included the Circoviruses identified in the present study and their best BLASTx matches based on Rep in Gen Bank.Cardiovirus is a genus of viruses that belongs to the order Picornavirales and family Picornaviridae.Human and animals serve as natural hosts and can cause severe disease.Cardiovirus has three species.Currently using viral metagenomic techniques,a novel cardiovirus is discovered.Using viral metagenomics,Samples were prepared,and nucleic acid was then isolated.Libraries were then constructed(Illumina)and sequenced(Mi Seq Illumina).The unique reads were de novo assembled and compared to the Gen Bank non-redundant protein database using BLASTx.PCR was performed for the confirmation of novel viruses in different samples.Inverse PCR was used to create the complete genome of the novel viruses in samples.A novel cardiovirus from feces of a wild rat was identified,named amzj-2018.The complete genome of amzj-2018 is 8,186 bp and has only one ORF which encodes a 2,292-aa polyproteins.Genomic organization of amzj-2018 shared similar features with the other cardioviruses.Phylogenetic analysis based on the complete amino acid sequence of polyprotein revealed that amzj-2018 formed a separate branch located between clusters of Saffold virus(SAFV)and Rat Theilovirus1(RTV-1).Phylogenetic analysis based on different regions of the polyproteins,including P1,P2,P3,and P2+P3,respectively,showed discordant trees,where the tree based on P3 region indicated that amzj-2018 clustered separately between Theiler's Murine Encephalomyelitis Virus(TMEV)and RTV-1.PCR screening results showed this cardiovirus strain was only present in one out of 20 fecal samples collected from wild rats.Conclusions(1)Using of virus metagenomics,we mastered the composition of viral community in wild rats(2)The composition of blood,brain,feces,liver,lung,oral swab and skin swab virus community in wild rats presents diversity.(3)Novel cardiovirus was discovered in the feces of wild rats. |
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