Investigation Of Arboviruses From Ticks And Rodents In The Surrounding Region Of Xinjiang Junggar Basin

Xinjiang Uygur Autonomous Region is located in China’s northwest border, the hinterland of the Eurasian continent, and adjacent to eight countries, such as Russia, Mongolia, Pakistan and so on. It has a lot of boundary port and the economic exchanges with neighboring countries frequently. Xinjiang’s ecological environment geographic is diversity, and species widely distributed in this area are significantly different with the other provinces in China. So, It is suitable for growth and reproduction of ticks, which are widely distributed and have many categories and spread many kind of arboviruses. Sometimes, people had unexplained fever and encephalitis sporadic or epidemic in Xinjiang, caused by suspected arboviruses. However, due to various reasons, it has not yet been systematically and in-depth investigation. So we should pay more attention to it. There are four kinds of arbovirus disease epidemic in China. Xinjiang hemorrhagic fever and tick borne encephalitis have been found in Xinjiang. Arbovirus investigation in the past in Xinjiang indicated that the tick borne encephalitis virus, eastern equine encephalitis virus, Xinjiang hemorrhagic fever virus and new orbivirus had been already isolated from Ixodes persulcatus, Dermacentor silvarum, Hyalomma asiaticum asiaticum, Dermacentor marginatus and Dermacentor nuttalli, respectively. The Sindbis virus, western equine encephalitis virus, Liaoning virus and Tahyna virus were isolated from Anopheles, Culex and Aedes, respectively. But there is still a lack of in-depth investigation of arbovirus in surrounding region of Xinjiang Junggar basin. In order to understand arboviruses carried by ticks and rodents in the surrounding area of Junggar basin, in this paper, we used RT-PCR, cell culture and biological information science methods to investigate arboviruses from the ticks and rodents(rat) in this area. The results of this study could enrich arbovirus resources information for Xinjiang area and China, and it has important significance for the prevention and control arbovirus disease in Xinjiang area. The results in this study are mainly as follows:1. Xinjiang’ rodent sample collection and preliminary testing carried arboviruses statusIn 2014 and 2015, we collected about 1054 rodents in the surrounding areas of Junggar Basin, including Wusu, Fukang City, Qitai, Jimsar and Shihezi. 684 and 370 rodents were collected in 2014 and 2015, respectively.. The main rodent species have Spermophilus undulatus, Meriones meridianus Pallas, Rhombomys opimus and Dipus sagitta. The liver, spleen, lung and intestinal of samples were taken out for testing. The primers were designed and RT-PCR assay were used to detect the Severe Fever with Thrombocytopenia Syndrome Virus(SFTSV), Xinjiang Hemorrhagic Fever Virus(XHFV), Xinjiang Tick borne Virus(XJTV) and Guertu virus of samples. The parts of the samples were detected for Hantavirus(HV), Tick Borne Encephalitis Virus(TBEV) and flavivirus. Initial detection results of RT-PCR from animal tissue specimens showed that positive rate of Guertu virus and sftsv were 9.3%(18/193) and 6.2%(12/193), respectively. Guertu virus and sftsv mainly concentrated in Wusu region which positive rate were 7.3%(14/193) and 3.5%(7/193), respectively. In 2015, A total of 15 XHFV positive samples, 10 of SFTSV positive samples, 29 of Guertu virus positive samples were detected. The positive samples detected mainly concentrated in Wusu area. The positive rate of XHFV, SFTSV and Guertu virus were 6.6%(9/136), 4.4%(6/136) and 16.9%(23/136), respectively. It indicated that wild animal rodents might have been infected with XHFV, SFTSV and Guertu virus in the dictrict under survey..2. Identification and isolation of a new virus from Xinjiang tick and its epidemiological investigation.In 2014, we collected 14726 tick(Dermacentor nuttalli) in Xinjiang Wusu region. According to the different area and tick species group, ticks were divided into 68 groups, then we selected 8 groups of samples for 454 high-throughput sequencing, of which there are 7 groups detected flavivirus gene sequence. After the comparison of Contig sequences obtained in NCBI, it was found to have the highest similarity with Jingmen tick borne virus(JMTV) in 65%-84%. The new virus is temporarily named as Xinjiang tick virus(XJTV). 3 goups of positive samples with 454 high-throughput sequencing were selected for using Vero cells to culture XJTV, two generations of the Cell supernatant sample was positive by RT-PCR detection, but XJTV were not detected in cells supernatant after 3-6 generations. XJTV was observed in the first generation of cell culture by electron microscope, and some of the suspected virus particles were found in the cell. The epidemic situation of XJTV was investigated by RT-PCR. 54 groups XJTV positive samples of 68 groups of ticks(Dermacentor nuttalli) were detected in Wusu region in 2014, the positive rate was 79.4%. The results indicated that Dermacentor nuttalli was infected with XJTV, which may be distributed in Wusu region.3. Obtaining XJTV complete gene sequence informationSeven contig sequences of XJTV were obtained through the Wuhan virus 454 High-throughput sequencing. After the sequence alignment, JMTV S1 and S3 part of genetic information were only found. The amplified primers were designed according to the sequence obtained, and then complete sequence of the virus S1 and S3 by RT-PCR were gotten. One of the positive samples collected at Yanggou district in Xinjiang was send to Shanghai Bohao company to sequence. Based on the information of the sequence of S2 and S4 obtained by RNA-Seq technology, the specific primers were designed, and the the virus S2 and S4 sequence information were obtained by RT-PCR. S1, S2, S3 and S4 were cloned into p GEM-Teasy vector using RT-PCR and overlapping RT-PCR methods. This provides a basis for better preservation of viral genome information, as well as for the study of the molecular characteristics of the virus and related gene function.4. Bioinformatics analysis of XJTV non structural proteins and structural proteins sequencesThe non structural proteins sequences(NSP1 and NSP2) and structural proteins sequences(VP1, VP2 and VP3) of JMTV were analyzed by the biological software Signa IP4.1, DNAStar, Phrye,TMHMM, Serve, MEGA5, Net NGlyc1.0, and so on. XJTV and the representative of Flaviviridae strains have many conserved motif in the functional areas of higher non-structural proteins. Dengue virus(DENV-4) and Japanese encephalitis virus(JEV) were used as a template to simulate the three-dimensional structure of S1 and S3 encoded proteins of XJTV. The results showed that XJTV based on the two level structure of the motif in the space position is also corresponding with the template protein motif, suggesting that these conservative structures in the virus’ s life cycle may perform the same biological function. After using biological software to analyze the transmembrane regions, signal peptide and glycosylation sites of structural protein VP1, VP2 and VP3, we preliminary predicted that the structural protein VP1, VP2 and VP3 of the virus have the function of envelope protein, nucleocapsid protein and membrane protein function respectively. Based on the development phylogenetic tree about non structural proteins S1 and S3 of XJTV, the results showed that XJTV does not belong to the Flaviviridae existing three genera after compared with the choice of the family Flaviviridae part of representative strains, which stay with JMTV and MGTV on separate branch of the phylogenetic tree. It suggested that XJTV may be a new genus of the Flaviviridae family.5.Construction of recombinant expression carrier of XJTV nucleocapsid protein and preparation of rabbit polyclonal antibody angainst nucleocapsid proteinXJTV and JMTV structural protein coding gene sequences were compared, relatively conservative nucleocapsid protein(capsid protein ORF sequence was selected and constructed into prokaryotic expression vector p ET28 a and pet32 a, also constructed into the eukaryotic expression vector pc DNA3.1. By sequence analysis, the results showed that three kinds of expression plasmids p ET28a-CP, p ET32a-CP and pc DNA3.1-CP were successfully obtained. p ET28a-CP plasmid was transfected into E.coli to express the recombinant protein. After the prokaryotic expression, the protein was purified to prepare for the antibody by immunization of New Zealand white rabbits in the subsequent experiment. The obtained antibody titer reached 1:30000 above. By Western blot test, it was proved that the rabbit antibody had a good binding affinity to antigen. The construction of prokaryotic expression plasmid and the preparation of the antibody in this study can provide the conditions for the further serological detection of virus and the immunological characteristics study of XJTV. The eukaryotic expression vector p CDNA3.1-CP was transfected into 293 T cells, tested by IFA. The results indicated that the VP2 gene encoding the nucleocapsid protein was expressed successfully in the cells, which provides the basis for the subsequent virus packaging and on the mammalian infection mechanism research of virus.