Investigation Of Mosquitoes And Mosquito-borne Viruses In Shanghai

Mosquitoes belong to arthropoda,insecta,diptera,long Angle.Mosquitoes are divided into 3 subfamily,namely Toxorhynchitinae subfamily,Anophelinae subfamily and Culicinae subfamily.The type of mosquitoes are various,there are 40 genera,200 kinds have been known in the world.Mosquito-borne virus is transmitted and propagation by blood-sucking insects as a medium,mainly through insect bites sensitive vertebrate.Mosquito-borne virus firstly in vivo,insect,then the virus transmit to vertebrate,eventually causing zoonotic diseases,which is a very serious public health problem.Mosquito-borne viruses mostly belong to natural focal disease,capturing mosquito samples from nature is the popular method to know the local mosquito-borne viruses.Shanghai has one of the world’s largest port,with big population density,strong liquidity,people from all over the world and their supplies could carry a variety of mosquito-borne viruses to Shanghai.Although there are only a small amount of imported mosquito-borne disease cases reported each year,in the current environment,shanghai still have the potential risk of mosquito-borne diseases.To provide evidence for prevention and control of mosquito-borne diseases,and to help the government to carry out the public health safety security work,this study was carried out to investigate the distribution of mosquitoes and mosquito-borne viruses in Shanghai pigfarms.The experiments are described as followings:Experiment Ⅰ Investigation of mosquitoes in ShanghaiIn 2014,mosquito samples were collected from 11 pigfarms belonging to four urban sites of shanghai districts.Light traps were operated from 6 pm until 6 am on each study day at each collection site.Light traps were hung away from interference by light sources,3.0 m above the floor.The mosquito samples should carry into a small refrigerator,then back to the laboratory immediately.Mosquitoes were first classified,stored in laboratory liquid nitrogen tank.The results showed that:A total of 30396 mosquitoes of 3 genera and 7 species were collected.Cx.tritaeniorhynchus was the major species,comprising 95%of the total population,28856.The number of Culex bitaeniorhynchus is 688 and comprise 2.26%of the total population.An.Sinensis comprising 2.6%of the total population is 789.Other species comprising 0.18%of the total population is 63.Experiment Ⅱ Isolation and identification of mosquito-borne viruses in shangai Mosquito samples which have been classified according to species must be fully grinded.Inoculated suckling mice and culture supernatant fluids were harvested(BHK-21/C6/36).Using primer 5.0 design primes according to sequences in Genbank,targeting JEV,flavivirus,alphavirus,SINV,and the 12th segment of Banna virus(BAV).Using RT-PCR to amplify the gene,and recycling products,after connecting carrier to sequencing.Then identified by IFA.The results showed that:Sixteen strains of viruses were isolated from the mosquito pools,revealed that eight viral strains identified as Japanese encephalitis virus(JEV,three belonging to Genotype Ⅰ;three belonging to Genotype Ⅲ).Eight strains isolated were identified as Banna virus(BAV,all from mosquito samples).Experiment Ⅲ Molecular biological characteristic analyses of Japanese encephalitis viruses isolated from mosquitoesAccording to sequences in Genebank,Using primer 5.0 design two specific primers.Extraction of viral RNA use the QIAamp Viral RNA Mini Kit(Qiagen).The RT-PCR reaction was carried out using the Superscript Ⅲ One-Step RT-PCR system with Platinum Taq High Fidelity(Invitrogen).The cDNA was then amplified.Sequences were compared with the GenBank database using the BLAST program and were aligned,edited,and analyzed using the DNA Star/MEGA 4.0 software package.Conduct Phylogenetic tree of JEV strains and compare nucleotide sequences and amino acid sequences.Internal Brain toxicity test in mice and the TCID50 test results show this eight isolates all have Strong pathogenicity.Conduct the phylogenetic tree us the method of Chen,The results showed that:there are 8 isolates.Four isolated belong to GⅠ(the source of three isolates are from mosquitoes while the other one is from swine serum).Four isolated belong to GⅢ(the source of three isolates are from mosquitoes while the other one is from swine serum).Sequence analysis based on the amplified E gene indicated that the homology of the nucleotide and amino acid sequences between the JEV isolates and stain SA-14-14-2 were 88%-98.2%and 86.5%-97.9%respectively.The homology of the nucleotide and amino acid sequences between the JEV isolates were 88.0%-100%and 86.3%-100%.Experiment Ⅳ Molecular biological characteristic analyses of Banna viruses isolated from mosquitoesAccording to sequence in Genebank,Using primer 5.0 design the 12th segment of Banna virus(BAV)primers.The cDNA was then amplified.Sequences were compared with the GenBank database using the BLAST program and were aligned,edited,and analyzed using the DNAStar/MEGA 4.0 software package.Conduct Phylogenetic tree of BAV strains and compare nucleotide sequence and amino acid sequence.The results showed that:Banna virus was isolated for the first time from mosquitoes in Shanghai.The homology of the nucleotide acid sequences between the BAV isolate were 99.5%-100%,and the homology of the nucleotide acid sequences between the Yunnan isolate were 94.2%-98.7%,between the Beijing isolate is 98.3%-98.5%,between the Inner Mongolia isolate were 98.5%-98.8%,between the Indonesia isolate were 89.7%-90.7%,this 8 Banna viruses have farthest homology of the nucleotide acid sequences between Hungary were 44.5%-44.6%.This 8 isolates were in a relative independent branch A2,very close to Beijing Yunnan Inner Mongolia,but has distinguish difference among other isolates.