| [Background]Ticks are the first recognized arthropods that can transmit pathogens to humans,and the second largest vector of infectious diseases after mosquitoes in the world.They can carry and transmit various pathogens,such as viruses,bacteria and protozoa,and usually harbor diverse pathogens at the same time.Viruses are important tick-borne pathogens.Currently,at least 160 viruses mainly transmitted by various hard ticks have been identified,of which about 25%are related to viral infectious diseases of humans and animals.Tick-borne orthonairoviruses belong to the family Nairoviridae of the order Bunyavirales,whose genomes consist of three single-stranded negative-sense RNA segments(L,M and S),and some of them can cause serious diseases in humans and animals.Tamdy orthonairovirus includes at least five different genotypes,which are widely distributed in a variety of tick species and have been reported to be related to human diseases and vertebrate infections.In addition,there are two tick-borne segmented RNA viruses,Jingmen tick virus(JMTV)and Alongshan virus(ALSV),which belong to the Jingmenvirus group in the family Flaviviridae and have been found to be associated with human febrile illness in recent years.There are also some emerging tick-borne viruses whose pathogenicity are not clear,such as South Bay Virus(SBV)and Black-legged tick phlebovirus(BTPV).At present,the research on these novel tick-borne viruses is relatively less,so it is necessary to explore and study them.Since 1982,33 emerging tick-borne pathogens,transmitted by nearly 30 different tick species,have been reported in China's mainland.The majority of them were bacteria and protozoa,for example,Rickettsia spp.,Anaplasma spp.,Ehrlichia spp.,Borrelia spp.,Babesia spp.,and Theileria spp.,which can cause a variety of human and animal diseases,including rickettsiosis,anaplasmosis,ehrlichiosis,Lyme disease,tick-borne relapsing fever,babesiosis,and theileriosis.Moreover,one tick species can harbor and transmit diverse pathogens,and several tick-borne diseases often co-exist in the same natural foci.Humans and animals bitten by co-infected ticks would have more complicated conditions.Thus,emerging tick-borne pathogens as well as their co-infections will pose a great threat to human and animal health.Metaviromics has the advantages of high accuracy,high throughput,high sensitivity and low operating cost.It can effectively capture the whole genome information of the virus when combined with bioinformatics tools.Additionally,it can also quantify virus abundance without bias in the context of conserved host genes,which provides important technical support for revealing the genetic diversity of viruses,finding novel viruses,and preventing and controlling emerging infectious diseases.At present,a comprehensive and systematic investigation of natural foci combining etiology(the characteristics and pathogenicity of agents)with ecology(ecological environment,vector and animal hosts)has become a trend in the field of tick-borne disease research.China is rich in tick species and has complex natural foci of tick-borne infectious diseases.Thus,active surveillance on ticks from the natural foci is very necessary for the public health authority.[Objectives]1.To obtain the virus spectrum in ticks in southern China and identify novel viruses.2.To obtain the newly identified tick virus isolates,further study their genetic and biological characteristics,and evaluate the infectivity and pathogenicity to humans.3.To achieve a more comprehensive understanding of the prevalence of Yunnan tick virus and other emerging tick-borne pathogens as well as their co-infections in different regions and different tick species.[Methods]1.Tick collection and identification.Representative landscapes in different regions were selected as sampling sites,and ticks were collected during their peak seasons from 2017 to 2019.Questing ticks were collected by flagging vegetation,and infesting ticks were collected from body surfaces of livestock or wildlife.All ticks were identified morphologically to species level and different developmental stages by entomologists.2.Metatranscriptome sequencing and analyses.RNA extracted from ticks was firstly tested for its concentration,purity and integrity,and then used to construct the sequencing library and sequenced.Performing viral genomes discovery and assembly,and relative transcript abundances quantification on the sequencing data via bioinformatics software.3.Virus isolation and identification.Ticks were ground and inoculated onto HUVEC,Vero 81 and IDE8 cells for virus isolation.After three consecutive passages,RNA from cell culture was extracted for high-throughput sequencing.When the virus isolate was obtained,its whole genome was sequenced subsequently,and performed structure and function predictions,homologous,phylogenetic and recombination analyses using bioinformatics software.Establishing detection methods and growth curves of the virus.Observing the morphological characteristics of the virus by ultra-thin section electron microscope.Observing virus infections in different cells by indirect immunoinfluscent assay(IFA)and RNA fluorescence in situ hybridization(FISH)assay.And the double serum samples of tick-bitten patients collected from the sentinel hospital were used for virus screening via IFA.4.Molecular examination of tick-borne pathogens.Total nucleic acid was extracted from each tick homogenate.YNTV was examined by one-step fluorescence quantitative RT-PCR with specific primers,and its longer large,medium and small segments were amplified by one-step RT-PCR.Tacheng tick virus 1(TTV1),Tamdy virus(TDYV),JMTV,ALSV,SBV and BTPV 1-3 were examined by SYBR Green one-step RT-PCR.Rickettsia spp.,Anaplasma spp.,Ehrlichia spp.,Borrelia spp.,Babesia spp.,and Theileria spp.were examined by conventional/semi-nested/nested PCR.Phylogenetic analyses were conducted through Mega 7.0.Differences in the prevalence of pathogens in different regions and different tick species were evaluated by Chi-square test or Fisher's exact test.[Results]1.Metatranscriptome sequencing results.In this study,a total of 93 sequencing libraries were constructed,and 3473838929 raw reads,3362776236 clean reads and 28413469 contigs with an average length of 592 nt were obtained.Of them,22930535 reads could be annotated to virus-related sequences.The community composition of tick-borne viruses was dominated by unclassified viruses,and the remaining viruses could be divided into 65 families.2.Isolation and identification of Yunnan tick virus.A novel orthonairovirus was isolated from HUVEC cell culture of larval Haemaphysalis montgomeryi from Jianchuan,Yunnan,China,and it was provisionally designated as Yunnan tick virus(YNTV).(1)YNTV was a negative-sense RNA virus.Its genome consisted of large,medium and small segments with the length of 12077 nt,4489 nt,.and 2057 nt,respectively,encoding large protein,glycoprotein precursor and nucleocapsid containing 3255,1357,and 492 amino acids,respectively;(2)Only the medium segment had signal peptide and transmembrane regions.There were 6,9 and 1 potential glycosylation sites in the large,medium and small segments,respectively.In addition,there were 6 conserved motifs(Pre-Motif A and Motif A-E)in the large protein;(3)Homologous comparison demonstrated that YNTV was more closely related to the Tamdy orthonairoviruses(41.8%-68.0%nt and 44.2%-63.2%aa similarities)than to other genogroups of orthonairoviruses,with 29.3%-44.2%nt and 18.4%-32.2%aa similarities;(4)Phylogenetic analyses showed that YNTV was grouped into the Tamdy genogroup.Amino acid sequences of its large protein and glycoprotein precursor were phylogenetically clustered with Wenzhou tick virus(WTV)from Haemaphysalis hystricis in China,while the sequence of its nucleocapsid exhibited a closer relationship with TTV1 isolated from a tick-bitten patient with febrile illness in northwestern China;(5)Recombination analyses showed that the complete large,medium and small segments of YNTV had the highest homology with WTV as a whole.However,its large segment had higher homology with TDYV and Songling virus(SGLV)at the positions of 6750-7000 bp and 8000-8250 bp,while its medium and small segments had higher homology with TTV1 and SGLV at the positions of 2700-3200 bp and 625-850 bp,respectivly,showing that there were recombination signals in all three segments of YNTV;(6)The standard curves based on the large and small segments of YNTV were x(L)=(39.99-y(L))/3.390 and x(s)=(38.48-y(s))/3.152,where y represented cycle threshold and the viral load was 10x;(7)The growth curves of YNTV showed that it grew hardly in IDE8 and C6/36 cells,and grew slowly in Vero 81 cells and reached the peak of growth at the 144th hour after inoculation.However,it could replicate rapidly in both BHK-21 and HUVEC cells and reached the peak of growth at the 168th hour after inoculation.The trend of YNTV viral load in the freeze-thaw lysate of five infected cells was basically consistent with that in the supernatant of cell culture;(8)Virus-induced cytopathogenic effects including cell shrinkage,rounding and higher cell refractive index,were observed in infected HUVEC cells but not in Vero 81 and BHK-21 cells;(9)YNTV virions were enveloped spheres,with a diameter of approximately 80-100 nm;(10)YNTV could be detected in HUVEC,BHK-21 and Vero 81 cell lines using IFA and FISH;(11)YNTV was negative in the acute and convalescent serum samples of 93 tick-bitten patients from Mudanjiang Forestry Central Hospital,indicating that none of these tick-bitten patients were infected with YNTV3.Detection of tick-borne viruses.The overall prevalence of YNTV was 1.34%,which was only detected in Ixodes ovatus,Amblyomma javanensis and Haemaphysalis montgomeryi ticks from Guangxi and Yunnan provinces,and YNTV fragments detected in ticks were phylogenetically clustered with the YNTV isolate,forming a unique clade distinct from other Tamdy orthonairoviruses.The overall prevalence of JMTV was 3.23%,which was detected in Amblyomma testudinarium,Haemaphysalis hystricis,Amblyomma javanensis,Ixodes persulcatus and Rhipicephalus microplus from Guangxi,Chongqing,Guizhou and Heilongjiang provinces,BTPV 1 was only present in one Haemaphysalis concinna tick from Jilin Province.The presence of TTV1,TDYV,ALSV,SBV,BTPV 2 and BTPV 3 in ticks were also investigated and the results were all negative.4.Detection of tick-borne bacteria and protozoa.In general,24 known pathogens(6 Rickettsia spp.,4 Anaplasma spp.,4 Ehrlichia spp.,3 Borrelia spp.,3 Babesia spp.and 4 Theileria spp.)and 17 sequence variants(4 Rickettsia spp.,4 Anaplasma spp.,3 Ehrlichia spp.,2 Borrelia spp.and 4 Babesia spp.)were detected from 356 ticks(47.85%)of 14 species(except Rhipicephalus haemaphysaloides and Haemaphysalis hystricis)in 8 provinces.The overall prevalemce of Rickettsia spp.,Anaplasma spp.,Ehrlichia spp.,Borrelia spp.,Babesia spp.,and Theileria spp.was 33.87%,10.89%,2.15%,4.44%,1.75%and 3.23%,respectively,and the differences in infection rates among sampling sites and tick species were statistically significant.5.Co-infections.Of the total 744 ticks,76(10.22%)of nine species showed the presence of co-infections.Frequent co-infections were observed in Amblyomma testudinarium,Amblyomma geoemydae and Haemaphysalis montgomeryi.Simultaneous infection with two pathogens were observed in 69 ticks,while the other 7 ticks co-infected with three pathogens.And co-infection of Rickettsia spp.and Anaplasma spp.was the most common.In addition,co-infections of YNTV and Rickettsia spp./Anaplasma spp.in Haemaphysalis montgomeryi,JMTV and multiple bacteria/protozoa in Amblyomma testudinarium,Ixodes persulcatus and Rhipicephalus microplus were also identified.[Conclusions]1.The viruses harbored by ticks in southern China were abundant and dominated by unclassified viruses.2.A novel Tamdy orthonairovirus was isolated from Haemaphysalis montgomeryi in Yunnan Province,and provisionally designated as YNTV.Its genomic structure and function were similar to those of typical orthonairoviruses,and the recombination signals were found in all three segments of YNTV.3.The morphology of YNTV was consistent with that of the Nairoviridae family.It could infect a variety of mammalian cell lines and cause HUVEC cells to produce CPE,suggesting its potential pathogenicity.4.YNTV was mainly distributed in ticks in Yunnan and Guangxi provinces.Ixodes ovatus,Amblyomma javanensis and Haemaphysalis montgomeryi were possible vectors of YNTV,and it could co-exist with Rickettsia and Anaplasma in the same tick.5.Diverse pathogens and some potential new variants,as well as multiple co-infections were identified,reflecting high diversity and complexity of pathogens in ticks in our country.[Innovations]1.In this study,a novel orthonairovirus was isolated from Haemaphysalis montgomeryi ticks in Yunnan Province,and its related characteristics were studied before it was found to be capable of infecting humans,which laid a foundation for the subsequent evaluation of its pathogenicity.2.In this study,the virus community carried by nearly 10 species of ticks from about half of the provinces in southern China were investigated,which could provide theoretical basis for the further research on tick-borne viruses and the prevention and control of tick-borne viral diseases.3.In this study,an extensive investigation was conducted on the infection and co-infection of 15(re)emerging tick-borne pathogens(including 9 viruses,6 bacteria and parasites)carried by nearly 20 species of ticks from three different animal geographical regions in China,providing a scientific basis for understanding the risk areas of tick-borne infectious diseases and designing corresponding precise prevention and control strategies and measures in our country. |
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