Isolation And Identification Of Tick - Borne Encephalitis Virus Xinjiang Strain And Evaluation Of Two Encephalitis Diagnostic Antigens

Arthropod-borne viruses (arboviruses) which can cause serious zoonotic diseases arespread by susceptible vertebrates bited by blood-sucking arthropod. Many novel arbovirusesemerged in recent years accompanied with the global warming and changes in natural foci.Spreading and prevalence of arboviruses present more and more effects on human health andeconomy development. To rapidly and accurately detect arboviruses has importantimplications on clinical diagnosis and monitoring of epidemic status. There are more than ahundred known arboviruses species currently, including Flaviviridae, Bunyaviridae andTogaviridae. High diversity of arboviruses is a challenge for of pathogen identification. Theclassical virus isolation is not only time-consuming and labor-consuming, but also requiringhigh-skilled operators. Serological analysis needs specific virus antigen or antibody. Viralnucleic acid detection is frequently used in recent years, such as PCR, real-time PCR, nucleicacid hybridization and microarray. However, these methods are genome sequence dependentand have great limitations on detecting of novel or unknown pathogens. High-throughputsequencing technology developed in recent years can simultaneously sequence numerousnucleic acid molecules from samples without pre-treatment or virus isolation. Hence,high-throughput sequencing technology become the most effective means of identification ofunknown pathogens.In ticks and other arthropods vectors, arboviruses can be identified by host cells and alarge number of vsRNAs (virus derived small interfering RNAs) were produced.It is animportant immune defense mechanism against viral infection in hosts. In essentially, vsRNAsare small fragments of viral genome processed by host cells. Viral siRNAs sequenced frommosquito and other arthropods vectors are all overlapping in sequence, and can be used forviral genome assembly and virus discovery. There are many natural foci and vectorarthropods in Xinjiang province due to its diversity ecological landscapes. In this study, tickswhich collected in XiErXiLi Nature Reserve of xinjiang province were sequenced byhigh-throughput methods. Through analysis of high-throughput sequencing data, the potentialexisted arboviruses in natural foci could be rapidly and accurately screened. Thehigh-throughput results could be further validated by using conventional methods.High-throughput sequencing technology based on vsRNA provides a new way for detectionof arboviruses and surveillance of natural foci.Total RNA and small RNA were extracted from ticks. After high-throughput sequencing,the siRNA-seq was constructed containing20-30nt fragments. The high-throughputsequencing data were analyzed. sRNA-seq of ticks includes17558502clean reads.The redundant reads were excluded and the remaining reads were compared to the viral genomein Genbank and used for viral genome assembling. After comparison,32631reads weremapped to viral genome and ranked firstly to tick-borne encephalitis.Cytopathic effect was observed when grinded supernatants of ticks were inoculated intoBHK-21cells. The virus nucleic acid was positive by RT-PCR amplification with universalflavivirus primers and specific tick-borne encephalitis primers. The new isolated virus wasconfirmed to be a tick-borne encephalitis virus. The whole genome was sequenced and theisolate was named as TBEV-XinJiang01and the sequence information was uploaded inGenbank with the Genbank Number GI: JX534167. Total length of TBEV-XinJiang01is11103nt. The length of5’UTR is131nt and the length of3’UTR is727nt. Phylogenetic treewas built by TBEV-XinJiang01’s genomic coding region and it belongs to the far-easternsubtype. It is more similar to the two strains in Russia and there are some differences betweenTBEV-XinJiang01and strains of Northeast China Some amino acid mutation sites may havebiological significance. Afterwards, phylogenetic tree was built by3’-untranslated region. It ismore similar to European subtype and Siberian subtype. The differences between the strainsin northeast China and TBEV-XinJiang01are significant while TBEV-XinJiang01has alonger3’UTR sequence. The difference of replication between TBEV-XinJiang-01and TBEVMDJ-01was compared. The proliferation of TBEV-XinJiang01is weaker than TBEVMDJ-01under the same conditions The TBEV-XinJiang-01isolate is the first report of a newtype of tick-borne encephalitis virus in xinjiang province.This study also explores the establishment of a simple and sensitive method forserological detection of tick-borne encephalitis virus and Japanese encephalitis virus. ED IIIdomain of TBEV and JEV were selected as potential diagnostic antigens for detection ofantibodies from clinical infected patients. RNA was extracted from cultured TBEV and JEV,and specific PCR was performed to obtain the relative nucleic acids.The recombinantplasmids were constructed and then successfully expressed in E.coli BL21. The purifiedproteins were printed on the microwell plate after dilution and Array-ELISA technique wasemployed for simultaneous detection of TBEV or JEV infected serum.36of the55clinicalTBE serum samples were detected positive and the detection rate is65.5%.5of the8clinicalJE serum samples were detected positive and the detection rate is65.5%. All of the normalserum were detected negative.The seurm infected with TBEV or JEV keeped in ourlaboratory were detected positive by IFA before. The results showed that both antigens have abetter diagnosis specificity for the detection of clinical patients and have a high detection rate.But there will be some omissions.High-throughput sequencing technology was used to detect the possible viruses carried by ticks in our study. The RNA-seq and sRNA-seq libraries were constructed throughhigh-throughput sequencing. The resulting sequence reads were compared to the host andviral genome and used for assembling virus genome. The results were then validated byconventional methods. Combined with these strategies, a novel TBEV strain was confirmedand isolated in this study. In addition, ED III domain of TBEV and JEV were expressed inE.coli and were purified. We evaluate the diagnostic value of the ED III domain of TBEV andJEV which provides theoretical guidance for the development of the clinical serologicaldiagnostic techniques and serological detection reagent.