| Research objective:Mosquito-borne infectious diseases,is the natural focal disease of mosquito-borne diseases,including japanese encephalitis,malaria,dengue fever and other harmful infectious diseases.(1)To study the composition of mosquito species and mosquito-borne virus in Haikou C ity,Hainan Province,and to provide important information resources for the prevention and control of mosquito-borne viral infectious diseases.(2)Established a rapid detection method for zika virus,to provide technical support for the prevention and monitoring of zika virus in the Hainan province.Research methods:(1)Five representative geographical environments in the eastern,western,southern,northern and central areas were selected according to the characteristics of the ecological environment in Haikou C ity as sample collection sites.Adult mosquitoes were caught by light trap and human lure,and then classified and preserved at-80℃.The nucleotide was extracted from the mosquitoes and after reverse transcription,specific primers were used for scanning of the target viruses in the c DNA.The primers used were designed according to the NS3 gene of dengue virus and the yellow fever virus;the NS5 gene of zika virus,the E gene and Pr M gene of japanese encephalitis virus of Flavivirus;the E gene of sindbis virus and chikungunya virus,the NS2 gene of M1 virus of Alphavirus.At the same time,the nucleic acid of the mosquitoes collected at the same sites were pooled together to construct the mosquito library,and five pooled were constructed in this study.The Hiseq2000 sequencer were then used for sequencing.The data generated in the metagenomic sequencing were then subjected to bio-informatic analyses.(2)Establish of zika virus rapid detection method,specific primers were designed based on the conserved regions of zika virus NS5 gene sequence from representative Asian and African genetype and designated ZK-1F/R and ZK-2F/R.The specificity,sensitivity and repeatability of the primers were evaluated and optimized using PC R and real-time PCR.The method was validated using ZIKV infected cells.Research result:(1)A total of 6283 mosquitoes were collected from 4 genera,1551 were Aedes mosquitos;4595 were Culex mosquitos;92 were Anopheles mosquitoes and 45 were Armigere mosquitoes.Mosquito samples were analyzed by PCR for Japanese encephalitis virus,dengue virus,yellow fever virus which member of Flavivirus and sindbis virus,chikungunya virus,M1 virus which member of Alphavirus,no positive results were found.Metagenomic sequences of five mosquito libraries were sequenced by Hiseq2000 sequencer,the results showed that 65110 virus sequences were found and the average length was 102.9 nt.The virus sequences matched to viruses,unclassified viruses and bacteriophages in NCBI,the viruses found belonged to 26 families and genera,including Parvoviridae,Flaviviridae,Rudiviridae,Adenoviridae,Ascoviridae,Ifaviridae,Baculoviridae,Nanoviridae,Rhabdoviridae,Iridoviridae,Mimiviridae,Nimaviridae,Papillomaviridae,Phycodnaviridae,Poxviridae,Polyomaviridae,Chrysoviridae,Dicistroviridae,Partitiviridae,Reoviridae,Retroviridae,Anelloviridae,Circoviridae,Geminiviridae,Caulimoviridae,Orthomyxoviridae,etc.,among which insect virus accounted for 63.0%,plant virus accounted for 13.2%,vertebrate virus accounted for 16.3%,phage accounted for 5.7%,and other viruses accounted for 1.8%.32 kinds of densovirus were found in invertebrate viruses;porcine parvovirus was found and human pathogenic viruses like cowpox virus,monkeypox virus were found in vertebrate viruses as well.(2)The results of PCR showed that the specificity of two pairs of primers of the zika virus was both good,the Real-time PCR detection sensitivity was 1.0×10~3copies/μl and 1.0×10~1 copies/μl.The amplification efficiency was 0.68 and 0.90,indicating that the combined effect of primer ZK-2F/R was better than that of ZK-1F/R.A Real-time fluorescence quantitative PCR method based on primers ZK-2F/R was established.The PRVABC59 strain and MR766 strain of zika virus were detected with the results of 9.0×10~4copies/μl and 8.5×10~4copies/μl,respectively.Conclusion:(1)Mosquito samples were screened by PCR and metagenomic sequencing for japanese encephalitis virus,dengue virus,yellow fever virus which are members of Flavivirus.Sindbis virus,chikungunya virus,M1 virus which are members of Alphavirus,no positive results were found,indicating that the collected mosquitoes did not carry the above-mentioned known important virus,inferred that a low possibility of the outbreak of the viruses in Haikou city,but still should be continuously monitor to prevent its outbreak.In this study,metagenomic sequencing results found a wide range of viruses,most of the m are insects and plants viruses.Human pathogenic viruses like cowpox virus,monkeypox virus were found in vertebrate viruses,which can cause rash,blisters and other clinical symptoms,but whether it can be transmitted to humans through mosquitoes remains to be studied.This metagenomic sequencing also found 180 kinds of viruses belong to other families and genera in the mosquitoes,it provided the basis for the initial understanding of Haikou mosquitoes-borne virus information and built Haikou mosquitoes virus spectrum,but also provide the important basic data of Haikou mosquito-borne viral infectious diseases for the future.This study reflects the metagenomic sequencing technology has a good practical value in mosquito-borne virus detection,it has a broad prospect of application.In addition,the detailed distribution and composition of the virus groups and the situation of evolution of the virus in mosquitoes which detected in this study have yet to be studied further.(2)A rapid detection of zika virus method was established in this study,It can be used for qualitative and quantitative analysis of zika virus in various samples,to provide reliable technical support for the rapid diagnosis of zika virus cases in Hainan province. |
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