Isolation,identification And Pathogenicity Analysis Of A Mosquito-derived Tembusu Virus In Mice

Arbovirus can be transmitted to susceptible vertebrate hosts through the bite of bloodsucking arthropods such as mosquitoes.Yunnan Province locates in the southwest area of China with a unique geographical location,warm and humid climate,many rainforests,concentrated precipitation,large temperature differences between day and night,distinct wet and dry seasons,and significant vertical variations in temperature with topography,which makes Yunnan region have large differences in climate,ecology,species with the interior.The special environmental conditions in Yunnan province provide advantages for mosquitoes breeding,and it is also a region where arbovirus transmission and arboviral diseases are more widespread in China.The background investigation of arboviruses in this region needs to be improved,and long-term continuous monitoring is required.In this study,we collected mosquito samples from July to August 2020 in Nanshia Village,Yuanjiang County,Yuxi City,Yunnan Province,and carried out virus isolation and identification subsequently.5 strains of Denso virus(DNV),1 strain of Nam dinh virus(NDiV)and 1 strain of Tembusu virus(TMUV)were successfully isolated,providing basic data for identifying the species and prevalence of local arboviruses.The whole genome sequence of TMUV was obtained by Next-generation sequencing,and the cell biology,molecular biology and pathogenicity of the TMUV were analyzed to provide the basis for the prevention of TMUV in this region.The specific contents of this study are as follows:This work provides basic data to identify the types and prevalence of arboviruses,and to provide more reference for preventing and controlling arboviruses in Yunnan province.1.Epidemiological survey of mosquito-borne virus in Yuanjiang County,Yuxi City,Yunnan ProvinceA total of 4996 mosquito samples were collected from July to August 2020 in Nansha village,Yuanjiang County,Yuxi City,Yunnan Province,and divided into 94 pools according to 50-100 mosquitoes/pool.The virus was isolated by cell inoculation and identified by RTPCR and RT-qPCR.RT-qPCR results showed that the collected samples carried Getah virus and Japanese encephalitis virus.The results showed that a total of 7 positive isolates were obtained(YN2020-14,YN2020-19,YN2020-20,YN2020-49,YN2020-60,YN2020-70,YN2020-76).We identified that YN2020-20 was Tembusu virus(TMUV),YN2020-49 was Nam dinh virus(NDiV),YN2020-14,YN2020-19,YN2020-60,YN2020-70,and YN202076 belonged to Denso virus(DNV).2.Isolation,identification,and phylogenetic analysis of a mosquito-derived TMUV strain.Cell biological characterization of isolated TMUV-YN2020-20 showed that TMUVYN2020-20 was able to proliferate in BHK-21,VERO,DF-1,and C6/36 cells.We then identified the genome sequence of TMUV-YN2020-20 using amino acid homology analysis,amino acid variation analysis,and phylogenetic analysis.The results showed that TMUVYN2020-20 had 89.7%(97.6%)and 88.3%(97.3%)nucleotide(amino acid)homology with MM1775 and Sitiawan strains,respectively.We constructed phylogenetic trees based on both ORF region and E gene of 148 TMUVs.The genotyping phylogenetic tree showed that TMUV-YN2020-20 strain was closely related to the two Yunnan mosquito-derived isolates obtained in 2012.In addition,there were five novel mutations in TMUV-YN2020-20(EV358I,NS1-Y/F/I113L,NS4A-T/A89V,NS4B-D/E/N/C22S and NS5-E638G),which were relatively conserved in previous studies.3.Pathogenicity of TMUV isolate in mice6-week-old BALB/c mice were infected with TMUV-YN2020-20 with a viral titer of 1×105.8 PFU/ml via intracranial route(50 μl).The mice in the infected group showed weight loss,difficulty in movement and paralysis.The survival rate was 43%in virusinfected mice.Histopathological examination showed that mice infected with TMUVYN2020-20 had more obvious histopathological changes in the heart,spleen,lung,brain,and other tissues,mainly manifesting organomegaly,hyperemia,congestion and infiltration of neutrophils in the tissues and organs.Moreover,the viral load in the brain was much higher than that in other tissues.These results suggested that mosquito-derived TMUV pathogenic to mice.