Isolation,identification And Epidemiological Investigation Of Tick-borne Nairovirus Yezo Virus In China

Background Ticks are obligate blood-sucking arthropods of Acarridea and important zoonotic vectors.They can transmit a wide range of pathogens such as viruses,bacteria,fungi and protozoan to animals and humans through biting.Factors such as increased population mobility,climate change and human encroachment on protected areas for wildlife have increased the chances of people being bitten by ticks.Tick-borne diseases have caused great harm to human health,China’s large livestock industry and wild animals.Nairoviridae belong to Bunyavirales,including 47 species in 7 genera,including 41 species in Orthonairovirus,which can cause serious diseases in humans and animals,such as Crimean–Congo Hemorrhagic Fever Virus(CCHFV),which has a fatality rate of more than 30 percent in humans,and Nairobi sheep disease virus(NSDV),which has a huge impact on animal husbandry.With the rapid development of metagenomic and macro-transcriptomics high-throughput sequencing technologies,more and more microorganisms have been discovered from ticks.However,most of the new tick-borne pathogens have not been isolated and cultured to obtain strains,so the studies on pathogen growth,replication and pathogenic mechanisms are extremely limited.In recent years,a variety of tick-borne Nairovirus has emerged in China,such as Tacheng tick virus 1(Tc TV-1),Songling virus(SGLV)and Beiji nairovirus(BJNV),etc.The natural foci of tick-borne infectious diseases in China is complex,tick-rich and precarious,so the emergence of these viruses in Asian countries may cause a huge burden on Chinese public health far beyond the current understanding,and a comprehensive understanding of the characteristics and pathogenic mechanisms of these new Nairovirus is an urgent need to develop prevention and treatment methods.Objectives Firstly,obtain Nairovirus isolates.Secondly,understand the genomic and biological characteristics of Nairovirus isolates.Thirdly,learn about the prevalence of Nairovirus in tick bite patients,ticks,and animal reservoirs.Finally,to investigate the mechanism of Nairovirus escape Ubiquitin(Ub)and Interferon Stimulated Gene 15(ISG15)dependent innate immune responses.Contents Firstly,collection of tick samples from representative areas and isolation and identification of Nairovirus.Secondly,the genetic characteristics of Yezo virus(YEZV)and its biological characteristics were studied.Thirdly,a retrospective survey of tick bite patients at sentinel hospitals and a natural focus investigation study of Yezo virus.Finally,study the mechanism of Nairovirus escape Ub and ISG15-dependent innate immune responses.Methods Firstly,the collected ticks were morphologically identified,and then the live ticks were ground and seeded into various cell lines,isolated and cultured for 4 weeks and tested by RT-q PCR to determine the virus isolates.Secondly,the whole genome sequencing of Nairovirus isolates was performed to understand their genomic characteristics.Electron microscopy was used to observe the morphological features of Nairovirus.Indirect immunofluorescence assays and fluorescence in situ hybridization experiments were performed on cultured Nairovirus to identify its growth on sensitive cells.The growth curves of Nairovirus isolates on different cell lines were plotted to understand their growth patterns.Thirdly,through RT-q PCR detection method,a variety of tick specimens in many regions of China were screened,and retrospective screening of serum from tick bite patients in Northeast Sentinel Hospital,as well as serum from Yunnan cattle and sheep to understand the prevalence of Nairovirus in patients,ticks and host animals.Finally,the mechanism of viral escape from Ub and ISG15-dependent innate immune responses in host cells was studied by virus isolates infecting sensitive cells,transfecting target gene plasmids overexpressing relevant proteins and western blotting.Results Firstly,a strain of Nairovirus YEZV was isolated.The collected live ticks were morphologically identified.It was trivially seeded into different cell lines by site and tick species,and finally a strain of Nairovirus was isolated from the Ixodes persulcatus in the Greater Khing’an Mountains,Heilongjiang Province and the whole genome sequence was obtained.Through the phylogenetic analysis of the gene fragments of the isolated virus strains,it was found that they were most closely related to the Yezo virus HH003-2020 virus strain reported in Japan in 2021.The sequence homology comparison results: L segment was 98.41%,M segment was 98.13%,S segment was 99.09%,which proved that the isolated Nairovirus was Yezo virus(YEZV).It was identified by transmission section electron microscopy,indirect immunofluorescence experiment,and fluorescence in situ hybridization experiment,and the cultured higher concentrations of virus were reseeded into various cell lines,and the growth of YEZV in different cells was mapped by real-time RT-PCR detection.Secondly,genomic and biological characterization studies of the YEZV.Next-generation sequencing of YEZV isolates yielded a complete genome.The genome is divided into three segments,L(12089 nt),M(4228 nt),and S(1650 nt)in length order.The total length consists of 17,967 nucleotides,of which the guanine and cytosine(G+C)content is 44.98 %.The analysis of the three genome-wide segments of the obtained YEZV isolate with the genome-wide segments of the existing Nairovirus confirmed that the isolate was YEZV,and with the exception of YEZV,which was found in Japan,it was found to be most closely related to the evolutionary relationship of the YEZV strain reported in 2021 from fever patients in Hokkaido,Japan.The negative stained samples and ultrathin sections of YEZV were observed by transmission electron microscopy,and the virus was spherical with a diameter of 60~180 nm,mainly distributed in the cytoplasm.Infection with YEZV in BHK-21 cells was identified using indirect immunofluorescence assays(IFA)and fluorescence in situ hybridization assays(FISH).According to the growth curves of YEZV on different cell lines,it was found that it can grow in Huh7,BHK-21,HUVEC,Vero81,IDE8 cells,suggesting that it may infect human and host animals.Growth is most rapid in BHK-21 cells,and the highest load after reaching the plateau phase,but almost no growth in Thp-1 and CTVM23 cells.By comparing the cytogenic effects of YEZV in the above seven different cells,it was found that no obvious cytopathic effect was observed after 7 days of culture,indicating that the cytotoxicity of YEZV was small.Thirdly,an epidemiological study of the Yezo virus.Indirect immunofluorescence experiment(IFA)screening was carried OTU on 617 tick bite patient serum from Mudanjiang Forestry Hospital in northeast China in 2012~2016,and a total of 4 tick bite patients were detected as convalescent seropositive,proving that YEZV is likely to have caused Nairovirus infection in northeast China.Indirect immunofluorescence test was carried out on 164 serum samples of cattle and sheep from Tengchong,Yunnan Province in southern China in 2017~2019,and the results were negative.A total of 764 ticks were screened for single nucleic acid by RT-q PCR,and the samples were divided into 6 tick species involving 8 provinces.Three kinds of Nairovirus were screened for infection with Yezo virus(YEZV),Yunnan tick virus(YNTV),and Beiji nairovirus(BJNV).The results showed that the total infection rate of YEZV was 9.55%(73/764),the total infection rate of YNTV was 10.73%(82/764),and the total infection rate of BJNV was 9.55%(73/764),and the infection rate of Nairovirus was different in different tick species in different regions.According to the tick species and location information of nucleic acid samples,YEZV had the highest positive rate among Ixodes persulcatus in Heilongjiang,China,with a positive rate of 31.41%(60/191).Secondly,the positive rate in Detmacentor nuttalli Olenev in Inner Mongolia was 10.53%(10/95).The positive rate in Rhipicephalus microplus in Jianchuan,Yunnan Province was 2.70%(2/74).The positive rate in Rhipicephalus microplus in Nanjing,Fujian Province was 1.09%(1/92).Finally,a study of the YEZV escaping Ub and ISG15-dependent innate immune response.Huh7 cells were infected with live virus strain YEZV,or Huh7 cells were co-transfected with synthetic YEZV and CCHFV Ovarian tumor(OTU)structural plasmids and ubiquitin-associated gene plasmids,followed by western blotting experiments to detect changes in intracellular amounts of Ub and Interferon Stimulated Gene 15(ISG15).In the live strain infection experiment of Nairovirus YEZV,it was found that it could not significantly reduce the expression of Ub and ISG15 in host cells Huh7,but in the experiment of transfecting YEZV OTU structural plasmids,it was observed that overexpression of Nairovirus OTU gene could significantly reduce the expression of Ub and free ISG15 in Huh7 cells,which proves that YEZV can achieve immune escape by inhibiting the innate immune response of host cells Ub and ISG15.Conclusions Firstly,a strain of YEZV was isolated from the Ixodes persulcatus in the Greater Khing’an Mountains,Heilongjiang Province.Secondly,YEZV grows in Huh7 cells,HUVEC,and other murine and monkey-derived cell lines,suggests its potential to infect humans and animals.Thirdly,from the retrospective screening of tick bite patients in sentinel hospitals in northeast China,it was found that 4 convalescent serum had positive conversion of YEZV antibody,suggesting that YEZV infection may have occurred in the population in this area.Fourthly,the main distribution tick species of Yezo virus are Ixodes persulcatus and Detmacentor nuttalli Olenev in northeast China,and Rhipicephalus microplus in southern China.Finally,the OTU structure of YEZV can significantly reduce the expression of Ub and ISG15 in Huh7 cells,and it is preliminarily proved that YEZV can achieve immune escape by inhibiting the Ub and ISG15-dependent natural immune response in host cells.Significance and innovation This study is the first time that the Nairovirus Yezo virus has been isolated and patients have been found in China.Then,the genomic characteristics and biological characteristics of YEZV isolates in China were studied,and their prevalence in tick bite patients,ticks and host animals was investigated,and the effect of YEZV on the expression of Ub and ISG15 proteins in host cells was studied,which laid a foundation for a more in-depth study of the immune escape mechanism of Nairovirus.