Discovery,Isolation,Identification,Establishment Of Detection Methods And Preliminary Prevalent Investigation Of A New Tick-borne Virus

Ticks are obligate blood?sucking ectoparasites,which are considered as the second only to mosquitoes as vectors of human diseases,and also the most important vector of pathogens infecting domestic and wild animals.Ticks can transmit a variety of pathogens including:viruses,bacteria,rickettsia and parasites,most of which can cause serious diseases to humans and domestic animals.Chuvirus is a new group of viruses discovered in arthropods and named in 2015.These viruses belong to Mivirus,family Chuviridae,order Jingchuvirales.The present study isolated a new virus from ticks by high-throughput sequencing and reported its genome and biological characteristics,the establishment of etiological and serological detection methods,its distribution in ticks,human,cattle and sheep from the northeast in China.Findings from this study provide valuable information for the pathogenic mechanism and prevention and control of the diseases caused by the virus.In the present study,290 ticks from the northeast in Inner Mongolia were used for metagenomic sequencing,and four contigs with a total of 2109 bp were obtained by sequence assembly.The obtained sequences had the highest identities with Suffolk virus isolate F13 from Chuviridae through NCBI blast,the identities were 65.53-75.40%indicating that it was a new virus in Chuviridae.The virus was named Nouming virus(NOMV)according to its discovery site.Then,the primers were designed based on metagenomic sequences to screen positive tick samples from Inner Mongolia.The complete genome of NOMV was obtained by nested-PCR,genome walking and RACE,which was circular with 10900 bp in length,encoding one 6516 bp RNA-dependent RNA polymerase(Rd Rp),one 2001 bp glycoproteins(GP),one 1278 bp nucleoprotein(NP)and one viral protein 4(VP4).Comparative analysis of NOMV complete genome nucleotide sequences and Rd Rp,GP and NP nucleotide and amino acid sequences showed that the highest identity was all between NOMV and Suffolk virus isolate F13.The nucleotide sequence identity of complete genomes was 67.2%between the two viruses,and the identities of Rd Rp,GP and NP nucleotide and amino acid sequences were 68.2%,68.8%,63.5%,and 75.9%,74.8%,61.9%,respectively.Phylogenetic analysis based on nucleotide sequences of complete genome and amino acid sequences of Rd Rp,GP and NP by ML method showed that NOMV all clustered together with Suffolk virus isolate F13,indicating that it was closely related to Suffolk virus isolate F13.The analysis of complete NOMV genome sequence showed that there were only three conserved domains located in Rd Rp,namely Mononeg RNA pol super family in 477-3038 bp,Mononeg?m RNAcap super family in 3198-3938 bp and G-7-MTase super family in 5346-5711 bp.The bioinformatics analysis of three ORFs showed that there were four potential N-glycosylated sites,no signal peptide and transmembrane domain in Rd Rp;two potential N-glycosylated sites,one signal peptide located between 17-18 bp and two transmembrane domains in GP;two potential N-glycosylated sites,no signal peptide and transmembrane domain in NP.Cell culture of NOMV-positive blood showed that the virus could be detected in BHK,Vero?SMMC and Wish cells after passaged three times,which indicated that NOMV could be cultured in BHK,Vero?SMMC and Wish cells.Moreover,cytopathic effect could not be detected after inoculation.Purified virions in BHK cell showed NOMV is an enveloped spherical particle,with a diameter of approximately 120-150 nm under electronic microscopy.Viral particles in ultrathin sections could be observed in the cytoplasmic vacuoles.In order to test the nucleic acid of the virus,two PCR-based methods for detecting NOMV were established,including nested PCR and q RT-PCR.Two pairs of specific primers for nested PCR were designed based on Rd Rp sequence.PCR conditions were optimized based on annealing temperature,specificity,sensitivity and repeatability tests.The results showed that the best annealing temperature of 1^stround was 55?and of 2^ndround was 52?,and the method had high specificity with no target bands of tick-borne encephalitis virus,Alongshan virus,severe fever with thrombocytopenia syndrome virus,Rickettsia typhi,Borrelia burgdorferi,Borrelia miyamotoi,Anaplasma bovis and Babesia ovis.Sensitivity test showed that the lowest detection limit of this method is 2.4×10²copies/?L,and the method had a good repeatability.The specific primers for q RT-PCR were designed based on NP sequence.Based on amplification system and condition optimization,standard curve construction,specificity,sensitivity and repeatability tests showed that the lowest detection limit of this method is 2.8×10¹copies/?L,the method had good sensitivity,specificity and readability.In order to know the epidemiology of NOMV,the prevalences of NOMV in ticks,human,cattle and sheep were investigated based on the established PCR methods.Nested PCR was used to detected the virus in ticks from Heilongjiang province,Inner Mongolia and Jilin province,the total positive rate was 3.9%(3.1-5.0,95%CI),including the highest prevalence rate with 9.4%(6.6-13.3,95%CI)in Heilongjiang province,followed by 4.0%(2.0-7.4,95%CI)in Inner Mongolia and2.0%(1.3-3.0,95%CI)in Jilin province.For prevalence in tick species,the highest prevalence rate was detected in Ixodes persulcatus with 8.2%(5.3-12.6,95%CI),followed by Dermacentor crenulatus with 7.8%(4.9-12.2,95%CI),Haemaphysalis concinna with 2.8%(1.0-7.0,95%CI),and Haemaphysalis longicornis with 1.9%(1.2-3.0,95%CI).q RT-PCR was used to detect NOMV in 830 patients,252 cattle and 184 sheep in Inner Mongolia.The positive rates were 18.9%,31.3%and 21.7%,respectively.The positive samples from different hosts were selected to amplify the partial sequences of Rd Rp,the identity and phylogenetic analysis showed that the identities of NOMV from different hosts were 96.7-99.7%,and phylogenetic analysis showed that all NOMV Rd Rp sequences clustered together but distinct from that of Suffolk virus isolate F13.The copies of NOMV in patients,cattle and sheep were all in 10³-10⁵copies/m L,which indicated that the virus exiting in these hosts but with low quantities.The geographical distribution,clinical symptoms and biochemical results were analyzed in 54 NOMV-positive patients,revealing that most of them were from Heilongjiang province and Inner Mongolia.Most of them were male with40-60 years old.The disease usually occurred from May to July.Of 54 patients,all patients had been bitten by ticks and 48 of them were field workers.The clinical symptoms of 54 patients including 79.6%presented with headache and fever,57.4%with low spirits,48.1%with dizzy,38.8%with fatigue,muscle or joint pain,33.3%with loss of appetite,and 20.3%with cough,chest tightness or poor sleep.Laboratory examination showed that infection with this virus included an increase in monocytes and neutrophils,and a decrease of lymphocyte and hemoglobin.Some patients showed the changed of coagulation function,such as the rise of fibrinogen,activated partial thromboplastin time and prothrombin time.Changes in liver function were also presented with the rise of alanine aminotransferase,aspartate aminotransferase,L-?-glutamine,and direct bilirubin.Thus,the results of epidemiological investigation showed that the two established methods in the present study could be used for the detection of NOMV.This is the first report on the dectection of a Chuviridae virus in human,cattle and sheep and it also suggested that the virus might be tick-borne,causing a tick-borne disease.In order to detect antibody of NOMV-positive human and animals,the specific primers of N protein of NOMV were designed,and the recombinant N protein was expressed successfully in E.coli.The purified protein was used for the establishment of indirect ELISA method with a concentration of 0.530 mg/m L of recombinant N protein.The best conditions for the method included:coating buffer as coating fluid,5?g/m L protein coating concentration,5%BSA was used as blocking fluid and incubated at 37?for 1 h,the dilution ratio of primary antibody was 1:10 and the dilution ratio of secondary antibody was 1:10000.Under the conditions,the indirect ELISA method had good specificity,sensitivity and repeatability,and the cut-off value is 0.4585.The method was used to detect antibody in 192 cattle sera and 191 sheep sera from Inner Mongolia.The total positive rates of cattle and sheep were 8.4%and 20.4%,respectively.At the same time,30 sera of NOMV-positive patients were tested for NOMV,the positive rate was 70%.The positive rates of 43 sera of NOMV–positive cattle and 22 sera of NOMV-positive sheep were 11.6%and31.8%,respecteively.The results suggested that the method could be used to detect NOMV and it could be used as an assistant method for NOMV detection.To sum up,this study identified a new virus from family Chuviridae for the first time and named it Noumin virus.The analysis of genomic characteristic showed that the complete genome of NOMV is circular,which is closer related to Suffolk virus in the same family.The analysis of biological characteristics showed that the virus can grow in BHK,Vero,SMMC and Wish cells,and it has an enveloped spherical particle with a diameter of approximately 120–150 nm.Meanwhile,etiological and serological detection methods were established to know the epidemiological investigation,the results showed that NOMV is prevalence in ticks,patients,cattle and sheep,and Heilongjiang province is the main endemic area,I.persulcatus and D.crenulatus are the main source of NOMV.This study is the first time to report Chuvirus from humans,cattle and sheep,which provided a new idea for the exploration of tick viruses and provide a basis for its diagnosis and prevention.