Investigation On Mosquitoes And Mosquito-borne Viruses At Lancang River Watershed In Yunnan Province

Yunnan province is core areas of the mosquito fauna species and distribution.There are many mosquito-borne diseases in Yunnan province. To date,11species virusin5families including Sindbis virus (SINV), Chikungunya virus, Getah virus(GETV),Japanese encephalitis virus (JEV), Dengue virus(DENV), Yunnan orbivirus(YUOV),Colti virus, Kadipiro virus, Banna virus(BAV) and Densonucleosis virus were found.JEV had been isolated from15kinds of mosquitoes in Yunnan province. Length ofchannel of Lancang River in Yunnan province is1247km, running through north andSouth Yunnan. Basin area is about90000km2which is one of the core areas and keyareas of the mosquito fauna and species distribution in China. Mosquito andmosquito-borne virus investigation were concentrated in the Southwestern Yunnanborder area to date. Investigation on mosquito and mosquito-borne viruses were lessperformed in Northwestern Yunnan. With global warming, increased populationmobility, people’s production and life style change, and the Lancang Mekong Riverinternational shipping, it is necessary to make an investigation on mosquito andmosquito-borne virus distribution in the Yunnan Lancang River watershed.Mosquitoes samples were collected at10settlements in the upper reaches, middlereaches, and downstream of Lancang River watershed with UV lamps in the seasonalpeak of mosquito activity in livestock shelters and human houses during2007-2009.Viruses were isolated from the mosquitoes and serum and cerebrospinalfluid specimens collected from suspected patients by inoculating on C6/36cells.Virus isolates were identified by RT-PCR and morphological observation. Homologyand phylogenetic analysis were performed on the nucleotide sequences of isolatesusing Mega5.0software.A total of112344mosquitoes, belonging to40species in8genera, werecollected in livestock shelters in the Yunnan Lancang River watershed. Among thecollected mosquitoes, Cx.tritaeniorhynchus was the most abundant species,accounting for80.26%, followed by An.sinensis(8.35%), Cx.annulus（5.41%）and Cx.theiler（i2.42%）. A total of4427mosquitoes, belonging to32species in7genera,were collected in the house. Among the collected mosquitoes, Cx.tritaeniorhynchuswas the most abundant species,accounting for35.71%, followed by An.sinensis（27.51%）, Cx.theileri（10.68%）andAr. subalbatus（3.55%）; In the upper reaches,a total of20species in6genera, were collected in livestock shelters. Among thecollected mosquitoes, Cx. theileri was the most abundant species, accounting for36.47%(1680/4607), followed by Cx.tritaeniorhynchus （30.63%）, An.sinensis（14.85%）and An.kummingensis（11.63%）. A total of6species in4genera, werecollected in the house. Among the collected mosquitoes, Ar. subalbatus was the mostabundant species, accounting for29.41%, followed by Cx. pipiens quinguefasciatus（23.53%）, An.sinensis（17.65%）, An.maculata Cx.pallidothorax s（11.76%）; Inthe middle reaches, a total of28species in6genera, were collected in livestockshelters. Among the collected mosquitoes, Cx.tritaeniorhynchus was the mostabundant species, accounting for84.03%, followed by An.sinensis （6.67%）,Cx.annulu（s5.49%，）and Cx.theiler（i1.26%，）. A total of20species in5genera, werecollected in the house. Among the collected mosquitoes, Cx.tritaeniorhynchus was themost abundant species, accounting for36.15%, followed by An.sinensis（29.09%）,Cx.theiler（i12.15%）and Cx. pipiens quinguefasciatus（11.82%）;In downstream area,a total of35species in7genera, were collected in livestock shelters. Among thecollected mosquitoes, Cx.tritaeniorhynchus was the most abundant species,accounting for77.15%, followed by An.sinensis（12.53%）, Cx.annulus（5.76%）andCx.theileri（1.26%）. A total of25species in6genera, were collected in the house.Among the collected mosquitoes, Cx.tritaeniorhynchus was the most abundant species,accounting for33.59%, followed by Cx. pipiens quinguefasciatus （19.69%）,An.sinensis （16.60%）and Cx.annulus（8.49%）.A total of55positive isolates with CPE were obtained from193pools of14706mosquitoes, collected in in10counties in the Yunnan Lancang River watershed.Foutyisolates belong to5families (families Flaviviridae, Togaviridae, Reoviridae,Totiviridae, Mesoniviridae）were indentified. Nine were negative to all RT-PCR usingall primers used in this study. Of the40strains,11were identified as JEV,6wereidentified as Culex flavivirus(CxFV),6were identified as Aedes flavivirus,5were identified as GETV,3were identified as BAV,5were identified as YNOV,6wereidentified as Toti virus,1were identified as Alphamesonivirus1-like virus. Twoisolates were detected BAV and Toti virus simultaneously and one strain was detectedCxFV and Toti virus simultaneously. Seven species viruses were isolated fromCx.tritaeniorhynchus,3species from An. sinensis,1species from Ae. albopictus andunclassified Aedes mosquitoes. A strain of DENV type2was isolated from animported patient traveling to Laos.Eleven strains of JEV were isolated from mosquitoes sampling in the upperreaches, middle reaches, and downstream of Lancang River watershed. The minimuminfection rate (MIR) of JEV in Cx. tritaeniorhynchus in the upper reaches, middlereaches, and downstream areas were2.46‰、0.72‰and0.90‰, respectively. Tenstrains of JEV had98.0%-100%similarity of envelope (E) gene at the nucleotide leveland99.8%-100%identity at the deduced amino acid level. The homology ofnucleotide and the amino acid sequences of the E gene of the new JEVs with the liveattenuated vaccine strain SA-14-14-2in China were86.1%-86.9%and96.8%-97.2%,respectively. Phylogenetic analysis showed that the new JEV isolates all belong togenotype1.Twelve insect-specific flaviviruses were isolated from mosquitoes collected in theupper reaches, middle reaches, and downstream of Lancang River watershed. Theviruses were isolated from Cx. tritaeniorhynchus, An. Sinensis, Ae. albopictus andunclassified Aedes mosquitoes. The phylogenetic tree based on the partial nonstructural protein5(NS5) gene showed that the viruses isolated from Culex and Aedeswere clustered into different clades. Newly isolated flaviviruses from Cx.tritaeniorhynchus and An. Sinensis were of closed evolutionary relationship withCxFV. While isolates from Aedes were different from Cell fusing agent virus andKamiti River virus both isolated from Aedes and were grouped into different cladeswith the two viruses.Six strains of Totivirus were first isolated from Cx. tritaeniorhynchus collected inthe middle reaches of Lancang River watershed. The phylogenetic analysis based on169aa of partial RNA-dependent RNA polymerase (RdRp) gene showed that thenewly Totiviruses formed a cluster with the Omono River virus（OMRV）. The newlyisolates belongs to the family Totiviridae. This is the first isolation of Totivirus from Cx. tritaeniorhynchus in China.A strain of Alphamesonivirus1-like virus named as NDiV-NJ8-09was isolatedfrom Cx. tritaeniorhynchus collected in the middle reaches of Lancang Riverwatershed. Phylogenetic analysis based on the complete nucleotide sequence of thenew isolate was performed with the fifteen taxonomically relevant virusesrepresenting the four virus families of the order Nidovirales as proposed by theInternational Committee on Taxonomy of Viruses. This analysis showed that the newisolate was most closely related to Nam Dinh virus (NDiV)02VN178and Cavallyvirus (CAVV) strains. These three viruses formed a clade, which means new isolatesNDiV-NJ8-09belonging to family Mesoniviridae of order Nidovirales.Three strains of BAV were isolated from Cx. tritaeniorhynchus collected in themiddle reaches of Lancang River watershed. Five strains of YUOV were isolated fromCx. tritaeniorhynchus collected in the downstream of Lancang River watershed. Fivestrains of YUOV were isolated from Cx. tritaeniorhynchus collected in the upperreaches, downstream of Lancang River watershed.A strain of DENV2was isolated from imported patient who was negative toDENV IgM detection. The phylogenetic tree based on the full E gene showed that theisolate was grouped into the Asia1clade, and had the greatest nucleotide sequencesimilarity (99.6%) with the D2/Thailand/0606aTw strain isolated from Thailand in2006.These results suggested that not only mosquito species diversity, but alsomosquito-borne virus diversity exist at the Lancang River Watershed. In conclusions:1. Cx. tritaeniorhynchus and An. sinensis were the local dominant species inlivestock shelters houses in Lancang River watershed. Culex tritaeniorhynchus andAnopheles sinensis were also the local dominant species in houses in the middlereaches and downstream of Lancang River watershed. Types of mosquito to the regionfrom the high latitude to low latitude area gradually increased.2. Cx. tritaeniorhynchus can be infected more moaquito-borne virus, suggestingthat the vector of arbovirus was widely distributed in Lancang River watershed.3. Mosquito flavivirus, Totivirus and Alphamesonivirus1-like virus were firstisolated in Yunnan. Further research is required on their biological characters andrelationship with human disease. 4. BAV was isolated from mosquitoes collected in middle reaches of LancangRiver watershed, BAV distribution in the entire basin can be speculated combinedwith the previous survey data. JEV was widely distributed in the Yunnan LancangRiver watershed, suggesting that vector control measures should be enhanced toensure the health of residents.5. GETV was widely distributed in Lancang River watershed. The animalhusbandry department should pay more attention to the prevention and control of thedisease in the corresponding area.6. A strain of DENV2was first isolated from imported patient traveled to Laos.Dengue fever surveillance and case management should be strengthen amongentry-exit persons in border areas of Yunnan to avoid local dengue fever outbreakscaused by imported dengue patients.