| BackgroundArboviruses are a group of viruses that transmit disease by biting sensitive vertebrates from blood-sucking arthropods such as sandflies,mosquitoes,midges,ticks,and other species.Therefore,arboviruses and arboviruses have become a hot public health issue in the international community.Shanxi Province is surrounded by mountains and water in four circles,with complex terrain,abundant sunshine and suitable temperature,which contribute to the diversity of local insect vectors.Sandflies and mosquitoes are the important vectors of blood-sucking insects in Shanxi Province.The research team reported in 2018 that a new type of Phlebovirus,Wuxiang virus(WUXV),was isolated from sandfly specimens collected in Wuxiang County,Shanxi Province,China.This is the first time that the Phlebovirus was isolated from sandfly samples collected in nature in China,suggesting that the virus transmitted by sandflies not only exists in the Mediterranean basin region,but also has appeared in the Chinese mainland located in East Asia.Although the Phlebovirus has been isolated from sandfly samples collected in nature in China,however,the seasonal fluctuation of local sandflies and the persistent existence of virus carried by sandflies are still unknown.In addition,mosquitoes,as important vectors of blood-feeding insects in Shanxi Province,may carry a variety of arboviruses.The investigation of local mosquitoes and arboviruses carried by mosquitoes in Wuxiang County is beneficial to the prevention and control of arboviruses and related diseases in China.ObjectiveThe research group continuously collected sandfly specimens and mosquito specimens in Wuxiang County,Changzhi City,Shanxi Province from June to August2019,and carried out virus isolation and identification work to clarify the seasonal fluctuation of sandflies in Wuxiang County and the persistence of virus carried by sandflies,while exploring and researching Arboviruses carried by local mosquitoes in Wuxiang County provide basic data for the local control of blood-sucking insects and the public health hazards of the viruses they carry.MethodsFrom June to August 2019,a total of four fixed collection points of blood-sucking insect specimens were set up in two villages(Moyu Village and Qianshenlianggou Village)in Wuxiang County,Shanxi Province.In other words,four consecutive collection works from June to August were all carried out at the fixed collection points.One blood-sucking insect collection tool(Mrs.Mosquito lamp)was set at each collection point.The collection time of hanging lights is 18:00 pm to 6:00 am the next day.All the collected blood-sucking insect specimens were placed in a low-temperature refrigerator for 30 minutes and then taken out under ice bath conditions for specimen processing.According to the specimen morphology,collection time,collection environment and other information of the blood-sucking insects,they were packaged and numbered and registered.The collected samples were stored in liquid nitrogen and transported through dry ice to the Beijing laboratory for examination.The samples were ground and centrifuged,and the supernatant was inoculated parallel with BHK-21 cells(golden hamster kidney cells)and C6/36 cells(Aedes albopictus egg cells)for virus isolation.The positive isolates were identified by molecular biological methods.The homology analysis and molecular genetic evolution analysis of the nucleotide sequence of the virus genome were carried out by biological software.Results1.Wuxiang virus is a persistent virus population in the vector of sandflies in Wuxiang County,Shanxi ProvinceA total of 7466 sandflies were collected from June to August 2019 in Wuxiang County,Shanxi Province.The numbers of sandflies collected on June 14,June 26,July 19 and August 16 were 1835(24.58%),4465(59.80%),1136(15.22%)and 30(0.40%),respectively.In this study,91 pools of sandflies were isolated and identified,and 33 strains of Wuxiang virus isolates which could cause mammalian cell cytopathic effects(BHK-21cells)were obtained.The isolation rates of Wuxiang virus in sandflies collected on June14,June 26,July 19 and August 16 were 24%(6/25),39.22%(20/51),50%(7/14)and0%(0/1),respectively.2.Two rhabdoviruses were isolated from Armigeres subalbatus in Wuxiang County,Shanxi ProvinceA total of 782 mosquito specimens of 4 genera and 4 species were collected from June to August 2019 in Wuxiang County,Shanxi Province.Among them,Anopheles sinensis and Armigeres subalbatus were the dominant species in Wuxiang County.In this study,we reported that a virus isolate(SX1916),which can cause mosquito cell cytopathic effects(C6/36),was isolated from a sample of Armigeres subalbatus collected in Shanxi Province,China in 2019.Sequencing(NGS)analysis revealed the presence of two rhabaviruses in SX1916 isolate.One of the viruses(Shanxi Arboretum virus(SXABTV))was 11480 nt,which is similar to Arboretum virus of the Rhabdoviridae Famliy.The genome sequence of the other virus(Shanxi Armigeres subalbatus rhabdovirus(SXARV))was 9590 nt.The SXARV genome encoded five proteins(N,P,M,G and L)plus a small hydrophobic protein(U1).Molecular genetic analysis showed that SXARV belonged to the Almendravirus Genus.Further analysis showed that the full length of the six known Almendravirus genomes ranged from 10735nt(Rio Chico virus)to 11876nt(Puerto Almendras virus),while the full length of the SXARV genomes was only 9590 nt,which was the shortest genomes of the viruses in this Genus(N and P genes were significantly shorter).The L gene of SXARV virus(RDRP)has the highest amino acid homology with the Rio Chico virus isolated from Panama mosquitoes in Almendravirus Genus,with only 63.3%(the amino acid homology of the coding region of L protein is less than 90%,which can be considered as a new species of rhabavirus).In conclusion,the SXARV virus isolated from Armigeres subalbatus collected in Shanxi,China is a new virus species in Almendravirus Genus.ConclusionIn this study,several strains of Wuxiang virus(Phlebotomus chinensis)and rhamnavirus(Armigeres subalbatus)were isolated from natural samples of blood-sucking insects in Shanxi Province,China from June to August 2019.(1)May may be the onset time of sandflies in nature in Wuxiang County,the density peak of sandflies in June and July,and the end of September,suggesting that the local sandflies had one-generation breeding throughout the year in Wuxiang County;(2)Wuxiang virus can be isolated from local sandflies at different collection time(on the premise that the number of sandflies collected must meet the grinding standard),which indicated that Wuxiang virus was a persistent virus population in the natural sandfly vector in Wuxiang County;(3)SXABTV and SXARV,two kinds of rhabdoviruses,were detected in the pool SX1916 collected from Shanxi,China.SXABTV and Arboretum virus of Almendravirus Genus belong to the same species.SXARV is a new virus species in Almendravirus Genus;(4)Only SXARV virus was isolated in the two plaque purification experiments.Considering the presence of SXARV virus and SXABTV virus in the infection fluid of SX1916 cells,further research is needed to explore whether the presence of SXARV interferes with the plaque formation ability of SXABTV virus or whether SXABTV does not have the plaque formation ability at all;(5)The discovery of SXABTV and SXARV rhabdoviruses has further enriched our understanding of viruses in the Almendravirus Genus of Rhabdoviridae Family. |
