Virome Analysis In Ticks From Zhoushan And Detection Of Dabieshan Virus By Indirect ELISA

ObjectiveTciks are the second largest group of arthropods after mosquitoes,which have long life cycle,wide distribution and multiple selection of hosts.Many kinds of microorganisms,such as bacteria,viruses,rickettsia,spirochete,protozoa and parasites,can be spread by ticks.Many pathogens of them showed highly pathogenic to humans and animals.With the rapid development of Next-generation sequencing technology and combination with Viral Metagenomics,more and more novel viruses have been discovered.In this way,the door to a brand new‘virus world'has been opened.In order to full understand the virus spectrum of ticks from Zhoushan Islands Zhejiang Province,enouth ticks were collected in Zhoushan Islands including Qushan,Zhoushan and Daishan three locations in September 2016.Method&Result1.A total of 421 ticks were collected from the three locations.The tick species were calssified with morphological method for preliminary analysis and identified with PCR(Polymerase Chain Reaction)methods by targeting at 16 s r RNA.The PCR results showed that there were only two species being identified as Haemaphysalis longicornis and Rhipicephalus haemaphysaloides.We used totally 60 ticks(20 ticks for each location)for the into high-throughput sequencing.We obtained a total of 23,915,810 reads as raw data.After data processing,10,106,532 paired reads were spliced as overlapping sequence,42.26% of the data were labeled as valid tags.Among them,101,282 reads were predicted to be ORF(Open Reading Frame which codes for protein),and virus ORF accounted for 0.015%(15/101,282).These reads were aligned by NCBI and identified to be four viruse species belonging to genus Buenavirus.Three novel viruses shared the highest homology with Bronnoya virus,MG22 virus and Odaw virus respectively,the fourth virus involved the highly homologous with the known virus named Dabieshan virus.This is the first time to applied high-throughput sequencing technology into explore the virus spectrum carried by ticks in the Zhoushan Islands.It is of guiding significance for understanding and mastering the diversity of tick–born virus in the region,monitoring and preventing the spread of tick-borned disease.2.PCR verification of the discovered virus according to the results of Next-generation sequencing.Based on the spliced sequence,specific primers targeted at the Rd Rp(RNA-dependent RNA polymerase)region.These primers were designed by primer 5.0 to verify the accuracy of high-throughput sequencing results and to detect the positive rates of each virus at the three sampling sites.The PCR results showed that the presece of the above virus was not detected in Qushan.Three strains were detected in the Daishan island,and the bronnoya-like virus was not detected.Only Dabieshan virus was detected in Zhoushan island.3.Using the genomic sequence of Dabieshan virus published by NCBI virus database as the standard sequence,primers were designed to amplify the genomic sequence of Dabieshan virus we detected through positive samples.AS the result of amplification,the coding region sequences of l segment and S segment were successfully obtained.The partial L-segment genenomic sequences of DSP4 and ZSP7 viruses were also obtained by genomic amplification.The Phylogenetic analysis tree was constructed based on the l segement of the above viruses.It was found that all four viruses belonged to the fam Liy phlebovirus,the discovered viruses located far away from the known viruses and formed a brand new branch.4.After obtaining the genomic sequence of the S fragment,the nucleocapsid protein(NP)of Dabieshan virus was expressed recombinantly.The polyclonal antibody was prepared suceessfully with high titer by immunizing Balb/c mice and New Zealand white rabbits.The method of indirect ELISA was established using recombinant NP protein to analyze the distribution of corresponding viral antibody titers in serum of the population in this area.However,the result shown that the serum antibody of the population detected by ELISA was at a low level.The next step we need to do is to improve the ELISA method and expand the range of detection.ConclusionIn this study,four novel viruses were discovered by high-throughput sequencing technology,and specific primers were designed to identify and calculate positive rate for each virus— Dabieshan virus,ZSP7 virus and DSP4 virus and the positive rates were 33.2%(81/244),0.41%(1/244)and 1.2%(3/244)respectively.Through the recombinant expression of Dabieshan virus nucleocapsid protein(NP),an indirect ELISA method using NP as a solid phase antigen was established.The result of detection has found thatthere is no significant difference of anti-NP antibody titer in the serum between Zhoushan and Yancheng populations.The Viral Metagenomics research based on high-throughput sequencing technology has greatly improved the efficiency and accuracy of known virus detection and unknown virus investigation.Combined with specific PCR methods,the research of viral spectrum of ticks has become targeted,reducing the blindness of the experiment and greatly improving the efficiency of scientific research experiments.The application of the NP recombination protein of Dabieshan virus for population serological analysis is helpful to understand whether there has the case that Dabieshan virus is transmitted from ticks to humans in this region.Besides,serological analysis can reveal the pathogenicity of this virus and identify whether this virus can infect human and host animals.