Mosquitoes And Mosquito-borne Arboviruses In Shandong Province

BackgroundArbovirus refers to a class of viruses that can spread diseases by blood-sucking arthropod bites vertebrates,including mosquitoes,ticks,sand flies,midges and mites.Shandong province is located on the east coast of China,and in the lower of Yellow River.The river system is developed in the province,and the lakes are concentrated between the hilly area of the south and the southwest plain of Shandong Province.The climate of Shandong province is warm temperate humid and semi-humid monsoon,the geographical condition is complexity,and the climate is warm and humid.These situations are suitable for insect vectors.Shandong was a area with high incidence of JE in the history.In addition,in recent years,there has been an increasing trend in adult cases of JE.The research on arbovirus about Shandong is limited and unexplained fever and encephalitis cases are exist.Thus we collected mosquitoes in Dongying,Yantai,Jinan,Zibo,Linyi and Jining city of Shandong Province during summer in 2016-2017,isolated and identified the viruses as well.ObjectiveThrough a systematic survey of arbovirus in Shandong province to provide biasis for prevention and control for local arbovirus disease and it can also provide diagnoses information for unknown fever cases in summer and autumn in Shandong.MethodsMosquito-grinding supernatant was inoculated into C6/36 cells and BHK-21 cells.The positive isolates were identified using molecular biology techniques;New virus genome were obtained by using the next generation sequencing;The specific gene segment was amplificated and sequenced directly,amino acid sequence analysis and phylogenetic analysis were carried out by using ClustalX2.0,MegAlign,Mega5 and BioEdit software.Electron microscopy was applied for morphological study on virus.Results1.We collected mosquitoes in Ji'nan,Yantai,Dongying,Zibo,Linyi and lining City in Shandong during 2016-2017.Totally 48139 mosquitoes include 4 genera and 5 species.Culex tritaeniorhynchus was the dominant specie in Shandong.The number of mosquitoes collected in Jining is the largest,accounting for 25.77%of the total,Dongying,Linyi,Ji'nan,Yantai and Zibo accounts for 24.49%,17.05%,5.77%,3.58%and 0.49%,respectly.The collection of habitats is concentrated in the piggery,and most of the mosquitoes that sucked blood.2.23 strains of viruses were isolated from the mosquitoes collected in Shandong province during 2016?2017,including 21 strains of JEV,1 CppDNV and 1 KDV.21 new isolates of JEV were all genotype I.The results of phylogenetic analysis showed that the 21 newly isolates are closed to two JEV strains isolated from Shandong in 2013.Real time fluorescent RT-PCR method was used to detect JEV nucleic acid in 274 pools of mosquito specimens.53 pools of samples were positive,with a positi've rate of 19.34%(53/274).All positive results were detected in Culex tritaeniorhynchus.The nucleotide sequence analysis of newly isolated CppDNV non structural protein NS1 and NS2 showed that the nucleotide homology between the SDKL1604 and other isolates in China was 98.9%-99%.Phylogenetic analysis showed that all Chinese isolates were in the same evolutionary branch.The newly isolated KDV is a segmental virus.The mucleotide and amino acids homology with isolate from dragonflies in China are 86%-99%and 88%-99%,respectively.ConclusionIn this study,4 genera and 5 species of mosquitoes were collected in Shandong province.It was proved that Culex tritaeniorhynchus was the dominant mosquito in the local area.JEV,KDV and CppDNV were isolated from 2 species mosquito samples.JEV infection was detected in multiple mosquitoes in many regions of Shandong Province,suggesting a high risk of JEV infection in Shandong province.KDV was isolated in Shandong province for the first time.The next generation sequencing technology played an important role in the analysis and identification of rare viruses and improved the efficiency and accuracy of molecular biological analysis.