Investigation Of Virus Spectrum Carried By Ticks In Hainan And Inner Mongolia And Establishment Of Multiple Detection Methods For Tick-borne Pathogens

Objective: Ticks are the second arthropod vectors of pathogens after to mosquitoes,and can carry and transmit a variety of infectious zoonotic pathogens such as bacteria,rickettsiae,parasites and viruses.With the continuous exploitation of natural resources,social and economic development,and increasingly frequent population movements,the contact between humans,wild animals,and vector insects has become increasingly close,and new tick-borne viruses(TBVs)have been discovered continuously.TBVs pose a serious threat to public health security.Hainan located in the tropical zone,with abundant rainfall and forest areas covering 95% of the islands.Due to the special climate and environment in Hainan,ticks here are active all year round,which is conducive to the spread of tick-borne viruses.The Hulunbuir region of Inner Mongolia is located in the most northern part of China.The rich forest resources and the half-year rainy season provide an excellent environment for the reproduction and transmission of ticks and tick-borne pathogens.This study investigates the species of ticks and the spectrum of tick-borne viruses in these two areas with special climate and ecological environment,comparing the differences and similarities between tick species and the important viruses carried by tickes in this the two areas;A multiple rapid diagnosis method for tick-borne pathogens is further established,which provides a highly specific and highly sensitive multiple detection method for the diagnosis of tick-borne pathogen infection.Methods:(1)We collected 855 ectoparasitic ticks from dogs and cattle from 4locations in Hainan and 500 free ticks from 10 locations in Hulunbeier,Inner Mongolia.The ticks were divided into different pools according to tick species,gender and geographic location,Viral RNA-sequence libraries were then subjected to transcriptome sequencing analysis using an Hiseq3000 platform(Illumina).Using the bioinformatics methods to analyze the metagenomics of tick-borne viruses,the important tick-borne viruses found in the samples were then verified by PCR method,and the molecular epidemiology and genetic evolution analysis of some important tick-borne viruses were also carried out.(2)Specific primers and probes were designed for the conserved regions of Orientia tsutsugamushi,Coxiella burnetii,spotted fever group Rickettsia,Rickettsia Mooseri,Erilichieae,Rickettsia Anaplasma,TBEV,and SFTSV.The specificity of the primers was verified by conventional PCR experiments.A liquid-phase gene chip detection method for multiple detection of tick-borne pathogens was obtained through probe synthesis,suspension liquid-phase chip preparation and detection condition optimization.Results:(1)According to tick species and geographical location,305 ticks were selected from 855 parasitic ticks of dogs and cattle in four places in Hainan,including20 Rhipicephalus microplus in Qiongzhong;60 Rhipicephalus microplus and 105 Rhipicephalus sanguineus in Tunchang;45 Rhipicephalus sanguineus in Lingao,75 Rhipicephalus sanguineus in Danzhou.These ticks were divided into 19 pools for high-throughput genome sequencing.The results of bioinformatics analysis showed that the viruses closely related to mammals carried by Hainan ticks belonged to 12 virus families.Among them,viruses potentially have human pathogenicity included:JMTV、Brown Dog Tick Phlebovirus1(BDTPV1)、BJNV and Quaranjavirus.The 855 ticks were divided into 57 pools according to tick species and geographic locations for JMTV prevalence screening.33 pools were JMTV positive,and the positive rate was 58%.The whole genome of JMTV was further amplified using positive sample RNA as temple,and the whole genome sequence of a JMTV strain was obtained,which was named JMTV-HMU.Phylogenetic analysis showed that the JMTV-HMU,Mogiana tick virus(MGTV)and Guangxi tick virus(GXTV)formed an independent branch that was separated from the ALSV in the deep root of the phylogenetic tree.In this study,we obtained a BDTPV1 partial L segment genome sequence(BDTPV1-HMU)of 3888 base pairs.Phylogenetic tree analysis showed that BDTPV1-HMU was closely related to BDTPV1 and formed a separate cluster with phlebovirus and Bole tick virus1(BLTV1);A total of 829 representative reads for Orthonairovirus of the Nairoviridae family were extracted.All reads were assembled into six contigs;the longest was 701 bp and the shortest was 314 bp.Among them,four contigs were matched to the RNA-dependent RNA polymerase of BJNV with98%～100% nucleotide identity.Two contigs were aligned to the RNA-dependent RNA polymerase of the Gakugsa tick virus with 99%～100% identity;A total of 219 representative reads for Quaranjavirus of the family Orthomyxoviridae were extracted.All reads were assembled into five contigs;the longest was 635 bp and the shortest was 246 bp.Among them,three contigs were matched to the polymerase basic protein1 of Zambezi tick virus 1 with 88.24%～91.46% amino acid identity.Two contigs were aligned to the hemagglutinin(HA)of the Granville quaranjavirus with60.71%～82.5% amino acid identity.(2)According to tick species and geographical location,200 ticks were selected from 500 ticks in ten places in Hulun Buir,Inner Mongolia,including 80 Ixodes persulcatus and 80 Haemaphysalis longicornis in Yiliekede,20 Dermacentor silvarum in Wunuer,20 Dermacentor silvarum in Meitian,These ticks were divided into 12 pools for high-throughput genome sequencing.The results of bioinformatics analysis showed that the virus-related sequences were annotated into 27 virus species which belonged to 9 families.Among them,four of which were proposed to be novel species of Flaviviridae(Wunuer tick virus and Meitian pestvirus),Phenuiviridae(Meitian tick virus)and Rhabdovirus(Yiliekedetick virus)as well as eleven new viral strains of six known viruses(Nuomin virus,Songling virus,Beiji nairovirus,Sara tick phlebovirus,Muwaka virus,and Onega tick phlebovirus).Divide 500 ticks into 32 pools according to tick species,gender and geographical location for screening of the prevalence of SGLV,BJNV,YLTV,Mt TV and NOMV,the positive rates were 9.38%,6.25%,12.5%,68.75% and 15.63%.(3)In this study,a method for multiple detection of 8 pathogens was established,the pathogens including Orientia tsutsugamushi,Coxiella burnetii,spotted fever group Rickettsia,Rickettsia Mooseri,Erilichieae,Rickettsia Anaplasma,TBEV,and SFTSV.The method designs primers and probes with good specificity and sensitivity for the conserved region of each pathogen,and can detect one of the pathogenic infections and multiple pathogenic co-infections,and the detection sensitivity can reach 10 copies.Conclusions: Based on the high-throughput sequencing analysis of ticks in Hainan and Hulunbeier,Inner Mongolia,we found that ticks in both areas carried a variety of viruses that are potentially pathogenic to humans,and a comparative research found that tick species in the two areas have significant differences.Ticks are mainly Rhipicephalus microplus and Rhipicephalus in Hainan,In the Hulunbuir,Inner Mongolia,ticks are mainly of Dermacentor.sp,Haemaphysalis.sp and Ixodes.sp.The dominant tick-borne viruses in Hainan are JMTV,BDPTV1 and Quaranjavirus,while the tick-borne viruses in Hulunbuir are mainly SGLV,NOMV,YLTV and Mt TV.Ticks in both regions were found to carry BJNV,suggesting that both regions are at risk of tick-borne viruses.This study established a high-throughput multiplex detection method for 8 tick-borne pathogens,which provides a faster method for the screening and diagnosis of tick-borne pathogens.The background screening of tick-borne viruses in Hainan and Hulunbeier,Inner Mongolia will deepen our understanding of tick-borne viruses and provide a theoretical basis for formulating effective prevention and control strategies for tick-borne diseases.