Molecular Epidemiology Of Rodent Pegivirus Infection In Sera Samples From Wild Rodents

Background:Pegivirus is a positive-sense,single-strand RNA virus of approximately 10kb in length,which contains only one open reading frame(ORF).Its genomic structure resembles that of hepacivirus.Intriguingly,unlike hepacivirus,pegivirus is primarily lymphotropic,asymptomatic,even benign in humans and other animals.A comprehensive understanding of the natural reservoirs for emerging pathogens could provide valuable insights into viral transmission and phylogeographic distribution.Until now,pegivirus has been found in human,horse,pig,monkeys,bats and rodents.Numerous rodent species have been considered as wild and synanthropic reservoirs of various pathogens.However,little epidemiological researches have been conducted in rodent pegivirus.Kapoor has reported detection of rodent pegivirus in Neotoma albigula.Another report of rodent pegivirus was published in 2014 by Firth,who claimed detecting pegivirus in Norway rats from New York City.The epidemiological information of RPgV in China is blank.Numerous valuable research methods were used in epidemiology,especially in seroprevalance research.Burbelo has developed an exclusive testing method which is relied on a luciferase expression vector producing antigen.This method has been successfully conducted by testing antibodies to Hepacivirus helicase in equine sera samples.Notably,this methods is convenient and higly sensitive without acquiring particular second antibodies.Serological studies of hepaciviruses have been described conclusively.Recent advances in serological assay using a sensitive luciferase reporter have been established for screening different animal species of virus infection.On the contrary,few evidence on the animal-borne seroprevalence was demonstrated,despite one assay detected antibodies to EPgV by E.coli expressed antigen.Recognition of novel viruses or new rodent-borne and insectivore-borne viruses has advanced our understanding of host range and epizootiology.HPgV type 2 has been derived using metagenomic method.Characteristic analysis of HPgV-2 reveals it shares partially hepacivirus genome and pegivirus genome.Viral sequences detected in bats display a diversified phylogenetic result.ObjectiveIn the present study,commensual rats were captured from Guangzhou and Xia'men cities in Southern China.We attempt to conduct molecular epidemiological research for detecting pegivirus in rat sera samples and establish a in-house luciferase immunopreciptation assay for antibodies to rat pegivirus.Thirdly,to generate full length genome sequences might provide insights into viral mutation and transmission route.Methods1 Rat sample collectionBetween August 2015 and March 2016,a total number of 226 rats were captured using cage trapsfrom Guangzhou(Baiyun,Yuexiu districts,n=202)and Xia-men(Tong-an,Huli districts,n=24)cities,which belong to the subtropical southern provinces of Guangdong and Fujian respectively.2 Targeted molecular analysis200?L of each blood sample were extracted and transcripted.Nested PCR was conducted to amplify the partial helicase genes of pegivirus.To confirm the presence of pegivirus sequences in the positive rats,RNA-dependent RNA polymerase genes were amplified and compared to the previously identified homologous regions.Sequences were analyzed by multiple bioinformatics methods.3 Seroprevalence research for rat pegivirusThe NS3 and E2 antigen expression plasmid was constructed,designated as RPgV-NS3-pNLF and RPgV-E2-pNLF.To evaluate antibody levels against pegivirus in rodent serum samples,luciferase immunoprecipitation system assay was developed with modification to the previously reported assay for NPHV.After trasfeccted into 293T cells,1×107 light units(LU)of crude cell extracts containing either LU-RPgV-NS3 or LU-RPgV-Env antigen were diluted 1000 fold in 50?L of assay buffer.After twice washing with PBST,each well of the plate was added with diluted antigen at 37? for 30min then rinsed five times with PBST.Finally,the LU were measured using furimazine substrate mix in the Nano-Glo(?)Luciferase assay kit.All LU data were obtained from the average of triplicated experimental results.The statistical analysis was conducted using SPSS 22.0 software package.4 Phylogenetic analysis of full length pegivirus genomeThree near full length genome sequences of rat pegivirus isolated in this study were generated by multiple nested PCRs.Bioinformatics methods such as similarity and phylogenetic tree,evolution rate were performed by MEGA,clustalX,and The Bayesian MCMC method.Results1 Detection of pegivirus in rodents and shrewsTo investigate the frequency of pegivirus infection,each of 315 samples was individually processed and screened by nested PCR programs using primers from NS3 region.21.6%(68/315)of the serum samples from both Guangzhou and Xiamen cities yielded a positive result for pegiviruses.Noting that the majority specimens consisted of Norway rats(219/315),62 positive samples were detected within specific species,which may have induced sampling bias.Interestingly,one positive sample came from Rattus Tanezumi(1/13)while the Rattus losea and insectivore Suncus Murinus,by contrast,were detected as 5.88%and 6.25%for pegivirus,respectively This is the first report of pegivirus infection in R.Tanezumi,implicating this species as additional reservoirs of pegivirus.All the positive data set of rodent pegiviruses implied close relationship with two previously reported rodent pegiviruses(Accession number KJ950934;KC815311).Obviously,all the variants in NS3 genome region were congruent with branching orders in NS5B region,denoting lack of recombination in these sequences.In general,pegiviruses formed three distinct phylogenetic lineages.2 Seroprevalence of rat pegivirusA subset of 212 rat and shrew with applicable blood samples were tested by modified luciferase immunoprecipitation seroscreening approach.All of 212 serum samples were screened for the nucleic acids of pegivirus using targeted PCR as described above.The results revealed that antibodies against the R.Norvegicus pegivirus NS3 antigen were found in 109 from 212 serum samples tested positive(57.4%),E2 antibodies were found in 97 out of 207 serum samples.Significant differerences was found between two tests.3 Phylogenetic analysis of full genome sequenceFull length amino acids alignment was conducted and generated as three clusters.The above lineage comprised variants from human,primates and bats,while the novel porcine pegivirus,on the other hand,together with variants from bat and primates,constituted the second lineage.The human pegivirus-2 and variants from rodents and bats fell into the bottom lineage.Specifically,all sequences revealed strong nucleotide identities(bootstrap>70)to each other over the NS3 and NS5B regions using multiple sequences alignment.Together with other two distinct rodent pegivirus which was identified from blood samples of wood rats and Norway rats,these sequences formed a separate clade within the genus Pegivirus.Beysian MCMC methods reveals that the substitution rate of human pegivirus was 6.061 X 10 4/site/year.Conclusions1 We identified the first rat pegivirus in China,the prevalence of rat pegivirus was about 21.6%,which is a little bit higher than rat pegivirus detected in New York Cities.Secondly,Rattus norvegicus,Rattus tanezumi/R.brunneusculus,Rattus losea/R.rattus from Rodentia were tested positive with rat pegivirus,expanding the taxonomy of species carrying rat pegivirus.In addition,Suncus murinus from Insectivora was test positive in sera samples.All pegivirus derived from our study were sharing high identity in NS3 and NS5B gene.2 We have successfully established in-house seroprevalence testing methods using luciferase Eukaryotic expression vector,and provide rat pegivirus antibodies epidemiological data for the first time.Antibodies to rat PgV NS3 helicase were tested about 57.4%,Antibodies to rat PgV E2 glycoprotein were 46.9%.3 Three near full length genome sequences were derived in this study.Overall,the three rat pegiviruses generated in our study branch very deeply next to Ruttus Norvegicus,Neotoma Lepida,and divergent highly among human,primate and other original hosts pegiviruses.All of the pegiviruses variants found in Ruttus Norvegicus clustered together.However,diversified amino acids sequences were observed in structural protein.Bayesian MCMC analysis reveal a slow evolution rate within human pegivirus.