Identification Of A Novel Tick-borne Virus MIV-2 And Investigation On The Virus Prevalence In Different Tick Species

Ticks are arthropods that can transmit a variety of pathogens.Tick-borne viruses(TBVs)are insect-borne viruses that persist in nature by circulating between the vector of Tick and the reservoirs.With the development of molecular biology,many new species,strains or genotypes of tick-borne viruses have been discovered.Negative-sense RNA viruses have a wide range of hosts,including plants,arthropods,mammals,etc.,so they can cause diseases to humans and losses to animal husbandry and agriculture.In recent years,many new negative stranded RNA viruses have been discovered by scientists,but no further studies have been carried out since they were not isolated.In this study,77 isolated ticks from five collection sites in Heilongjiang province of China in 2018 were selected.After nucleic acid extraction,based on the second-generation sequencing technology,Jingchuvirales and Mononegavirales viruses were explored and their evolutionary status was analyzed.Ixodes persulcatus mononegavirales-like virus HLJ,Ixodes persulcatus chuvirus HLJ and Ixodes persulcatus dimarhabdovirus HLJ were detected.Ixodes persulcatus mononegavirales-like virus HLJ was found to belong to the unclassified mononegavirales,and was most recently related to the Deer tick mononegavirales virus reported from Heilongjiang province,China.Ixodes persulcatus dimarhabdovirus HLJ belongs to the family Rhabdoviridae of Mononegavirales.Ixodes persulcatus chuvirus HLJ belongs to Jingchuvirales,and has the closest relationship with a chuvirus-like virus 3 and Wuhan Mosquito 8.We tried to isolate the three novel viruses,and isolated a virus close to Ixodes persulcatus chuvirus HLJ,tentatively named MIV-2.Cell lines VERO,HUVEC,BME/CTVM23 and IDE8 were selected for in vitro cell isolation and culture of MIV-2,and it was found that BME/CTVM2 may be its sensitive cell line.On this basis,sequencing,detection method,evolutionary analysis and preliminary structural study were carried out on MIV-2.We have separated to four strains of MIV-2,respectively is Ixodes persulcatus,Dermacentor silvarum,Haemaphysalis longicornis and Rhipicephalus microplus ticks,MIV-2 present a genome of 11.36 kb.On the basis of obtaining the whole genome sequence,specific realtime fluorescence quantitative PCR primers were designed,detection methods were established and evolutionary analysis was carried out.The MIV-2 found in France,Brazil,Trinidad and Tobago,China,Thailand and this study came from different countries and regions,while the gene sequence of MIV-2 remained highly consistent.The homology of MIV-2 was also high in different tick species.We have preliminarily verified the circular structure of MIV-2,so we believe that the conservatism of MIV-2 may be related to its circular structure.Structures of all complete chuvirus genomes were analyzed and it was found that they have similar structural characteristics.To investigate the prevalence of MIV-2 carried by 15 species in seven provinces and cities including Yunnan and Guizhou provinces.MIV-2 was found in eight tick species,Ixodes granulatus,Rhipicephalus microplus,Ixodes ovatus,Amblyomma testudinarium,Haemaphysalis montgomeryi,Haemaphysalis longicornis,Haemaphysalis concinna,Dermacentor silvarum.Through analysis,it was found that MIV-2 existed in many tick species in many places in China,among which Jilin province and Yunnan province had more advantages.Through statistical analysis,the volume of wuhan chu virus carried by different tick species was different.However,due to the limited sample size,it could not be excluded that other detected tick species did not carry MIV-2.In summary,this research through high-throughput sequencing,comparing the sequence and gene annotation methods to find new viruses.We found three new tick-borne viruses in Ixodes persulcatus in Northeast China,and obtained MIV-2 isolates.Through the evolutionary analysis and the epidemic rate of the virus and the investigation of carrying ticks in other areas of China,the basic data were provided for the understanding of the evolution rule,the tick species and the infection status.