Study On Mosquito Metavirus Genomics From Yunnan Province And Its Characteristics Of Newly Isolated Japanese Encephalitis Virus

Aims:By studying the characteristics of mosquito metavirus genomics and newly isolated Japanese encephalitis virus in Yunnan region of China,the species and distribution of latent viruses in Yunnan Province and the variation and epidemiology of newly isolated Japanese encephalitis virus were analyzed for future prevention and control of virus disease in this area and to provide reference for the control of viral spread to other areas.Materials and methods:(1)Metagenomics:Mosquito samples were collected on sites.Mosquito species were laboratory identified and grouped.Mosquito samples were ground.Supernatant of ground liquid was filtrated for sterilization.Host nucleic acid was degraded using nuclease.Mosquito sample RNA extraction.Metavirus nucleic acid was reverse transcribed.The sscDNA metavirus was synthesized using SISPA amplification of the nucleic acid sequence.The dscDNA metavirus nucleic acid sequence was synthesized using klenow fragment.Oligonucleotide primers and free nucleotides were removed using Exon I and SAP.The dscDNA metavirus nucleic acid sequence SISPA amplification.The dscDNA metavirus nucleic acid sequence was purified for removing the oligonucleotide.Agarose gel electrophoresis was performed to confirm the length range of SISPA amplification products.The concentration of dscDNA metavirus was detected by nanodrop.The second generation high-throughput sequencing technique was used to detect metavirus.Viral sequence information was annotated.Specific virus PCR/RT-PCR assays.The amplified sequences were analyzed using Neighbor-joining evolutionary.(2)Virus characterization:Mosquito samples were collected on-site.Mosquito species were laboratory identified and grouped.Mosquito samples were ground.Virus species were identified using nested-PCR.The supernatant filtration of specific virus mosquito samples for sterilization.BHK-21 cells were infected using sterile supernatant.Specific virus proliferation was identified by CPE,nested-PCR,IFA and negative-staining electron microscopy.The newly isolated Japanese encephalitis virus and its epidemiological analysis were explored using Neighbor-joining Phylogenetic analysis.Results:Illumina sequencing revealed the composition of mosquito virus from Yunnan province in China.More than 50 viral families were found in mosquitoes through gene annotation,including Circovirus,Anelloviridae,Parvovirus and Flaviviridae and so on.The Illumina sequencing results showed that 22,964,305 reads were obtained,with an average length of 186 nucleotides.The number of contigs was 10,538,636 and the overlapping rate was 91.78%.Through sequencing and analysis of Illumina sequencing results,the virus sequences of three samples accounted for 3.84%,17.84%and 0.17%,respectively.According to the classification of viral hosts,87.4%of which were vertebrate viruses,followed by 5.4%of plant viruses and 1%of fungi viruses being found,and Insect virus and phage accounted for 0.8%and 0.6%respectively.On this background,three segments of DENV sequences,two ZIKV virus sequences and one JEV virus sequence were identified by BLAST alignment and nested-PCR.The DENV virus sequences,one 1231-nucleotides-long segment(DENY-China/YN2016-1),one 491-nucleotides-long segment(DENV-China/YN2016-2),and one 939-nucleotides-long segment(DENV-China/YN2016-3),which encode partial non-structural proteins NS4A to NS5,partial envelope protein E and partial non-structural protein NS5 respectively.Phylogenetic analysis showed that DENV-China/YN2016-2 belonged to genotype IV DENV.The sequences of ZIKV,one 694-nucleotides-long segment(ZIKV-China/YN2016-1)and one 470-nucleotides-long segment(ZIKV-China/YN2016-2),which all encoded partial non-structural protein NS3.Phylogenetic analysis showed that ZIKV-China/YN2016-1 belonged to ZIKV of genotype Asian.Nucleotide identity analysis showed that DENV-China/YN2016-2 has the closest relationship with DENV from Thailand isolated in 1991 with the highest nucleotide identity of 95.3%.ZIKV-China/YN2016-1 had the closest relationship with ZIKV from Thailand isolated in 2004 with the highest nucleotide identity of 97.1%.JEV-China/YN2016-1 strain was identified by IFA and negative-staining electron microscopy.The viral titer of JEV-China/YN2016-1 was 6.30×105 TCID50/0.1mL and the Median Lethal Dose was 3.16×103 LD50/0.03mL.After infection with JEV,liquefaction necrosis,nervous necrosis,ring bleeding,monocytosis and glial cell nodule hyperplasia appeared in cerebrum of BALB/c suckling mice.Phylogenetic analysis of JEV-China/YN2016-1 manifested that it belonged to genotype IJEV.Nucleotide identity analysis showed that JEV-China/YN2016-1 had the closest relationship with JEV from Laos isolated in 2009 with the highest nucleotide identity of 98.8%Amino acid mutation analysis found that valine or serine had mutated to Alanine at the position of amino acid 340 of the E protein.Conclusions:(1)Illumina sequencing revealed the abundant viral species in mosquito from Yunnan province in China.(2)Genotype IV DENV and genotype I JEV viruses are still prevalent in Yunnan province.The trace of transmission of ZIKV had already appeared in this area.(3)The isolation and variation of JEV in Yunnan Province indicates that it may form a new epidemic situation.