Molecular Characteristics Of Jingmen Virus Among Rodents In Xinjiang And Ticks In Northern Region,China

Background and Aims:Jingmen viruses(JMV)are a group of newly discovered tick-borne single-stranded positive-sense segmented RNA viruses.Due to their homology in NS3 and NS5 fragments of flaviviridae,they are currently classified into the Jingmenvirus group of flaviviridae.JMV is the first+RNA viruses reported in the world that connected the gaps between segmented and unsegmented flaviviruses.JMV is a group of arboviruses with arthropods as vectors.Jingmen tick virus(JMTV),the reference strain of JMV,was first identified from Rhipicephalus microplus in Jingmen City,Hubei Province in 2010.Subsequently,this virus was reported worldwide,including in Asia,South America,Africa,Caribbean and Europe.The virus exhibited significant genetic diversity and intercontinental geographic clustering.As the vectors of JMTV,ticks play an important role in transmission JMTV from arthropods to mammals.At present,it has been reported that JMTV infected cattles,monkey and other mammals.More importantly,the virus was detected in sera of patients with Crimean Congo haemorrhagic by Germany and Kosovo's scientists.Subsequently,human infections of JMTV and JMTV-like(Alongshanvirus,ALSV)were successively reported by two research teams from China.Rodents,as the largest group of mammals in close contact with humans,carry a variety of pathogens and also are hosts of ticks.Therefore,characteristic JMTV in rodents and monitoring the prevalence of the virus in different tick species from different areas will help to understand the origin,spread and evolution of this new human pathogen,and will have important public health significance for prevention and control of the virus.Methods:1)Ticks(n=277)were collected from Bole City of Xinjiang,Shenyang City of Liaoning Province and Changping District of Beijing,including 83 Hyalomma trititum from Bole City,98 Haemaphysalis ionicornis from Shenyang and 96 Dermacentor sinicus from Changping District in 2011-2012;2)Rodents(n=164)were captured by cages trapping method in Qapqal Xibe Autonomous County,Xinjiang Uygur Autonomous Region in 2016.Total RNAs of homogenized heart,liver,spleen,lung,kidney of rodents and individual tick were extracted.RT-PCR was used to detect JMTV and genome was amplified from JMTV positive samples then sequenced by standard Sanger sequencing.Sequence comparison were carried out with published genome of JMTV.Phylogenetic trees were generated by the Maximum Likelihood method with PhyML version 3.0,Bootstrap analysis was performed with 1,000 replicates with bootstrap values>70%considered significant.Results:1)Total of 164 rodents representing 8 of species were collected in Qapqal Xibe county of Xinjiang Uygur Autonomous Region of China and screened for JMTV by RT-PCR.JMTV was identified in 42 rodents in liver(25.6%),including 23 in Microtus arvalis voles(24.5%),9 Apodemus uralensis mice(29.0%),5 Mus musculus mice,1 Rhombomys opimus gerbil,1Meriones tamariscinus gerbil,1 Meriones libycus gerbil,1 Cricetulus migratorius hamster and 1 Microtus gregalis vole.The 42 JMTV positive samples were further amplified for full length of genome.As a result,7 of nearly full length and 5 of partial genomes were recovered.The newly discovered viruses have the highest homology with known JMTV in China.The nucleotide difference of the 7 voles JMTV each other in four segments was 0.1%?1.5%,the difference between vole JMTV and the known JMTV in China was 8.9%,the nucleotide similarity between vole JMTV and the JMTV from Africa,South America and Europe was 77.8%-92.1%,and the nucleotide identity to Alongshan virus(JMTV-like virus)was relatively low(57.5%?72%).Phylogenetic analysis of S1,S2 and S3 segments revealed that the newly identified rodent viruses clustered with JMTV reported from China and branched into two genetic groups.One group comprised of viruses only found in M.arvalis,while the other group included viruses in A.uralensis,C.migratorius and M.gregalis.However,all rodent JMTV were clustered into one branch on the S4 tree,suggesting the diversity of JMTV.2)JMTV were have been detected from organs of rodents,include heart,liver,spleen,lung and kidney indicating that JMTV was widely distributed in host organs.But the positive rate of JMTV in all organs was various.Liver exhibited the detected rate of 25.6%,lung and spleen were 10.8%and 2.6%,respectively.Data from kidney and heart were absent due to lack of samples.Sample size of common vole was the largest among the 8 rodents species.The positive rate of JMTV in liver,lung and spleen of voles was 24.5%,19.1%and 15.7%respectively.Systematic analysis of organs infection of JMTV in 8 rodents species revealed that one organ infection(liver)was 29 out of 42,two or more organs 13/42.Only 2 of them were multiple organs infection in addition to liver.3)Among the 277 ticks collected from 3 cities of Northern region of China(Bole City,Xinjiang Uygur Autonomous Region;Shenyang City,Liaoning Province and Changping District,Beijing City),67 were JMTV positive(24.2%).Carrying rates vary in different ticks,as 41(42.7%)were in Dermacentor sinicus,12(14.5%)in Hyalomma detritum,14(14.3%)in Haemaphysalis longicornis.Four near full-length JMTV genome sequences were obtained by genome-wide amplification sharing identity of 91.4%?99.8%.The newly recovered JMTV processed the highest homology with tick JMTV discovered in China.Phylogenetic tree demonstrated that the 4 new stains were clustered into the same lineage of rodent borne JMTV from Xinjiang based on S1,S2,S4.However only two of the newfound viruses were closed to JMTV from Xinjiang rodent on S3.The other two were fallen in the tick borne JMTV lineage from other regions of China,suggesting a possible occurrence of gene reassortment in S3.Conclusion:1)This is the first report that JMTV was detected among rodent species in Xinjiang.Genomic analysis demonstrated that the newly discovered rodent JMTV showed a high genetic diversity,especially up to nearly 10%nucleotide difference in both S2 and S4 segments among 12 rodent JMTV strains.Phylogenetic analysis revealed that rodent JMTV reported here clustered with known tick JMTV from China.These viruses branched into two genetic groups in S1,S2 and S3 segments.However,all rodent JMTV were clustered into one branch on the S4 segment,suggesting the diversity of JMTV.2)Data of JMTV identified in different organs of rodents showed the different detected rate.This suggest that there may be organ preference of JMTV infected in rodent although this is needed to be further investigated.Anyways,multiple organs infected by JMTV in the same rodent indicated systematic infection of JMTV in rodents.3)The newfound tick JMTV strains identified in this study exhibited genetic diversity(90.1%?99.8%).Phylogenetic analysis showed that all the newly discovered tick JMTV strains were clustered with JMTV in China.However,two of them probably have a gene reassortment in S3 segment.As the vectors of JMTV,ticks play an important role in transmission JMTV from arthropods to mammals.