Diversity Of Virome And Expression Of E3 Ubiquitin Ligase In Three Predominant Ticks Based On High-throughput Sequencing

Objective:Tick-borne arboviral diseases,such as SFTSV,CCHFV,TBEV,have increased their world-wide incidence in the past few years.The widespread distribution of the tick vectors,Ixodes persulcatus,Haemaphysalis concinna and Dermacentor silvarum are vital important vector factors contributed to the（re）-emergence of human arboviral diseases.Effective therapies and vaccines are still limited for most tick borne arboviruses.Accordingly,vector control is the primary way to prevent transmission of arboviruses to humans.A deep understanding of the interaction and co-evolution between viruses and tick vectors is expected to aid in the development of novel transmission control strategies.In the present dissertation,three predominant ticks were sampled for endemic areas to reveal their diversity of viruses to explore the potential tick borne arbovirus hazardous to human populations and pave a way to develop a reasonable clue to alert the health risk with prevent and control strategies.Methods:Three predominant tick species,Ixodes persulcatus,Haemaphysalis concinna and Dermacentor silvarum were sampled from Mudanjiang（Heilongjiang province）,Zhaluntun（Hulunbuir,Inner Monogolia）respectively.After morphological identifications,the samples were grouped and submitted to screen the potential virus infection by high-throughput sequencing.Total 8 groups were tested and about 90G sequences data were achieved.The sequenced data were trimmed by Trim Galore to remove adapters.After the quality of sequenced data was assessed with Fast QC-Illumina,the clean reads were analysis with the software CLC Genomics Workbench v3.6.1.The assembled contigs were then submitted to NCBI Genebank to search homologuous viral fragments through Blastx program in non-reductant libraries with an e-value of 1×10^-5 or better collated.As an additional screening test,all potential virus assemblies were screened against the Conserved Doman Database（www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi）with an expected value threshold of 1×10^-3 to identify viral gene segments.Finally,to assess mean sequence depth and relative frequency,the quality trimmed libraries were mapped back against all viral assemblies and the COX1 mitochondrial gene of target tick species using Bowtie.In the case of potentially multiple and/or overlapping open reading frames,FSFinder2（http://wilab.inha.ac.kr/fsfinder2/）was used to identify possible ribosomal frameshift.With similar procedures,the E3 ubiquitin ligase CDS of Dermacentor silvarum and Haemaphysalis concinna were also obtained from the transcripts of the tick samples.The amino acid sequence structure of E3 ubiquitin ligase was annotated accordingly and its functional site was inferred by using NCBI conservative domain database（CDD）.Results:More than 90G sequenced data were achieved by high throughput sequencing from 8 groups of tick samples.After annotated with bioinformatic tools,virus like fragments were found involved belonged to 6 orders,44 families and 54 genera.Among the virus,Bole Tick Virus 2（N,L）,Lesnoe mivirus（N,L,G）,Beiji nairovirus（N,L）,Norway mononegavirus（N,L）,Onega tick phlebovirus（N,L）,Xinjiang tick associated virus（orif1,orif2）and Ixodes scapularis bunyavirus（N,L）were identified along with other 21 species of virus,whose Rd Rp identities over 50 and the length of unique contigs beyond 250aa.In the group of I.persulcatus,Beiji nairovirus,Sara tick phlebovirus 1,Onega tick phlebovirus 1 and Lesnoe mivirus were shown predominant among 16 species virus found.And 7 virus discovered in the Hae.concinna group,with Manly virus as predominant one.As for Dermacentor silvarum group,9 virus were identified involving Bole tick virus 4,Xinjiang tick associated virus,Sara tick phlebovirus 1.The members of Rhabdovirus appeared in all the three tick species while Totivirus members were only discovered in Dermacentor silvarum and Hae.concinna.In addition,two E3 ubiquitin-protein ligase CDS were harvested from the transcripts of Dermacentor silvarum,which were identified as SH3RF3-like isoform X1with 99.30%-99.56%identity under the accession number MW354543 and MW354544.The rest one obtained from RNA transcipts of Haemaphysalis concinna were identified as SH3RF1-like isoform X2 with 88.83%identity under the accession number MW35455.The corresponding coding genes were also blasted and the deduced E3ubiquitin ligase CDS of Haemaphysalis longicornis,Ixodes persulcatus and Hyalomma asiaticum were obtained.The predicted E3 ubiquitin ligase of Hyalomma asiaticum was shown as a novel intermediated isoform other than the known ones in the available ticks species.Phylogenetic analysis of the above E3 ubiquitin ligase CDS indicated that an obvious root branch of family Argasidae formed represented with Ornithorodos turicata,while in the cluster of family Ixodidae,a distinctive branch formed by genus Ixodes and clades of Rhipicephalinae（Genus Dermacentor and Genus Rhipicephalus）,Hyalomminae（Genus Hyalomma）,Haemaphysalinae（Genus Haemaphysalis）were separated from each other and lined with systematic relationshipsConclusion:1.The diversity of viruses carried by the three ticks species includes 54 genera in44 families of 6 orders.The distribution of viruses varied with tick species,sex and their geographic locations.The specification of virus to tick species might be an important determines for its vector competence.2.Virus species in Rhabdovirus or Totivirus were found widely distributed across species,genera even families,suggested that the viruses might be less specific to tick species with high adaptions,while some species specific Ixodes scapularis phlebovirus and Beiji nairovirus are only found in the group of Ixodes persulcatus.3.The sequenced E3 ubiquitin-protein ligase CDS from the transcripts from the three tick species suggested that SH3RF3-like exist in Dermacentor silvarum,SH3RF2-like appear in Haemaphysalis concinna,while a novel intermediated isoform of E3 ubiquitin-protein ligase occur in Hyalomma asiaticum.4.The E3 ubiquitin-protein ligase CDS of ticks evolute following their systematic associations and shown specification in species,genera and families level.