Molecular Epidemiological Investigation And Transmission In Vector Ticks Of Jingmen Tick Virus

BackgroundJingmen tick virus（JMTV）is a newly discovered human pathogen in recent years,and ticks are its main vector of transmission.First identified in 2010 in Rhipicephalus microplus from Hubei province,China.So far,there are few clinical reports about JMTV,its variants in non-human primate host and deadly Crimean-Congo hemorrhagic fever（CCHF）patients were found for the first time,and emphasized the JMTV relevance to animal and human health.Subsequently,JMTV was detected in ticks taken from the patients and the patients bitten by ticks that provided direct evidence that JMTV could infect humans via vector ticks in Mudanjiang Forest Central Hospital,Heilongjiang province,China.JMTV is a tick-borne virus distributed in the world,mainly in Brazil,Southeast Finland,Uganda,France,Kosovo,and Laos,etc.And recombination occurred during evolution.At present,there are few epidemiological data about JMTV in China,mainly found in Hubei,Zhejiang,Heilongjiang,and Guangxi.It can cross species barriers to infect multiple animal hosts and cause symptomatic infections in humans,such that fever,headache,asthenia,and myalgia.At present,the growth of JMTV in cells and the colonization of Amblyomma javanense tick organs have been studied only in China.How the virus is transmitted by Dermacentor silvarum ticks and its colonization in viscera has not been studied.Therefore,in addition to the study on the distribution range,geographical characteristics,genetic characteristics,this study also used the isolated JMTV to study its transmission in vector ticks,these will be helpful to the prevention of tick-borne viruses and the assessment of risk.Genome recombination is also a key issue in the study of pathogenicity and infectivity of viruses.Research purposeTo understand the prevalence of JMTV in sampling sites and vector ticks in multiple areas,to try to find out whether new tick species can infect JMTV,and to increase the epidemiological data of JMTV in China.Phylogenetic analysis was used to understand the geographical distribution characteristics,genetic diversity,similarity,and potential genome recombination of JMTV in the world.To determine the propagation and transmission dynamics of JMTV in unfed Dermacentor silvarum under laboratory conditions.Methods1.Epidemiological investigation methods:In this study,ticks were collected from multiple sampling sites,viral nucleic acids were extracted from ticks and detected using specific primers by the combination of Quantitative Real-time PCR（q PCR）,and JMTV was screened and confirmed by means of sequencing technology and database comparison.2.Phylogenetic and recombination analysis method:All JMTV sequences and some related sequences were collected from database,and sequence comparison was conducted with the biological information analysis software MAFFT 7.450 and MEGA 7.0.Phy ML 3.0 software was used for phylogenetic tree reconstruction.Virus recombination analysis was performed using RDP 4.0and Simplot 3.5.1.3.Transmission research methods:（1）Dermacentor silvarum ticks were fed with six-week-old male Balb/c mice and goats;（2）the vector ticks were soaked by the virus;（3）q PCR was used to detect the virus copy numbers at different time points in different stages of soaked ticks and the virus infection in tissues and viscera of mice after being bitten by soaked ticks;（4）Indirect immunofluorescence assay（IFA）was used to detect the specific antibody levels in the serum of mice after being bitten by soaked ticks;（5）RNA in situ fluorescence hybridization（FISH）confirmed the localization of virus in vector tick organs.Research results1.Epidemiological investigation results:A total of 1040 ticks were collected,623 unfed ticks,417 fed ticks from sheep and cattle,including 14species of 6 genera,covering 28 sampling sites in 12 provinces.JMTV was detected in Haemaphysalis longhornides,Rhipicephalus microplus and Rhipicephalus sanguineus in three regions,with a total positive rate of 2.69%（28/1040）,among which the positive rate of JMTV was 3.85%（7/182）in,Shandong province’s sampling points.The positive rate of JMTV was 6.90%（2/29）in Deyang city sampling point,Sichuan province,and 79.17%（19/24）in Nanchang city sampling point,Jiangxi province.2.Phylogenetic and recombination analysis results:JMTV has been found in many regions of 9 countries in the world,and JMTV found in different countries formed a good geographical cluster,its host including ticks,microtus obscurus,mosquitoes,non-human primates,cattle,pteropus lylei（a kind of bat）and humans.There was no obvious clustering among the species of vector ticks,and a variety of ticks,such as Haemaphysalis longhornithus,Rhipicephalus microplus,Amblyomma javanense,Ixodes ricinus,Rhipicephalus sanguineus,etc.,could infect JMTV and JMTV could spread frequently among different tick species.Phylogenetic trees suggest that in ticks and their hosts or different tick species could have more close evolutionary relationships of JMTV.The inconsistencies in the topological structure of the genome fragments and sequence recombination analysis of JMTV＿85 genome indicate that genome recombination events may have occurred during the evolution of the virus.In segment 1,2,and 4,there was a high similarity with the sequence JMTV＿YJ3-3,and there was an obvious break point near segment3,which had a high sequence similarity with the sequence of JMTV＿X84-3,with breakpoints at 2336 and 3431,respectively.3.Transmission research results:Under laboratory conditions,we used in vitro immersion method to study the transmission via vector ticks of the virus.Virus copy numbers in the 3 groups of nymphs ranged between 10^3.5～10^4.5copies/m L,we observed that trend of increasing and then decreasing virus copy numbers of nymphs in 15-25 days.The copy number of virus in 3 groups of adults was between 10⁴～10⁵copies/m L.However,from the overall point of view,it was found that the virus copy numbers of nymphs and adult ticks were lower and did not change regularly with time.JMTV was not detected in second-generation adult ticks after nymphs molting with immersed,the virus could not complete the transmission from nymphs to adult ticks.Molecular biology and serological tests showed that no virus infection occurred in mice bitten by immersed ticks,indicating that ticks did not transmit the virus to mice during feeding,and no virus colonization was observed in the midgut and salivary glands of Dermacentor silvarum ticks.Research conclusions1.Conclusions of epidemiology research:We did not find JMTV in the new tick species,and the discovery of JMTV in Shandong,Sichuan and Jiangxi province has increased the understanding of the scope of JMTV in China.The epidemiological data show JMTV lower infection rate of sample collecting regions in the 12 provinces.JMTV found in Rhipicephalus microplus tick’s eggs,indicating that the virus can transovarial transmission in Rhipicephalus microplus ticks.To some extent,it increased the risk of tick-borne virus in areas where Rhipicephalus microplus ticks are dominant.We should continue to expand the research scope,strengthen the monitoring efforts.2.Conclusions of phylogenetic and recombination analysis:JMTV is widely distributed in the world,with increasing host range and diversity,and can spread across species and frequently between ticks.JMTV has significant genetic similarity and diversity,and genome recombination may occur during evolution.3.Conclusions of transmission research:Under laboratory conditions,Dermacentor silvarum ticks from nymphs to adults have poor ability to maintain JMTV,they might not be easy to be infected JMTV,and be less likely to carry and transmit JMTV.