Screening And Identification Of Arboviruses Carried By Ticks And Mosquitoes In Some Parts Of Yunnan

Arboviruses are transmitted through the bite of vertebrates by hemophilic arthropods,causing zoonotic diseases that not only have serious impact on animal husbandry but also pose a threat to human life and health.Yunnan is located in southwestern China and is tropical-subtropical,with high average annual temperatures and abundant precipitation providing favorable conditions for arthropod breeding.Southeast Asia bordering Yunnan Province is a high prevalence of many arboviruses such as dengue virus,chikungunya virus and Zika virus,and the convenient transportation location further facilitates the cross-border introduction of arboviruses into Yunnan and increases the risk of arboviral disease epidemics in the province.In this study,extensive sample collection of vector insects was conducted in the southern prefecture of Yunnan Province,and detection of arboviruses was screened by established Pan-PCR methods and Metaviromics.It was found that the carriage rate of Jingmen tick virus was 56.67%(68/120)in live cattle ticks near the Mohan port in Yunnan Province.After homologous evolutionary relationship analysis of the nucleic acid sequences of its four full-length genes indicated that this strain showed closer affinity with the Jingmen tick virus strain from the neighboring country Laos suggesting a higher possibility of Jingmen tick virus transmission.In this study,the results of generation sequencing screening of 3510 mosquito samples collected between July 2020 and July 2022 from seven states/cities in Yunnan Province showed that seven viruses were screened,two of which were zoonotic viruses,Japanese encephalitis virus and Mangshi virus,and four insect specific viruses,Culex flavivirus,Quang Binh virus,Armigeres subalbatus totivirus and Yunnan Picorna-like virus.The infection rates of positive specimens for each virus were 5.56%(2/36)for Japanese encephalitis virus,2.27%(1/36)for Mangshi virus,2.27%(1/36)for Armigeres subalbatus totivirus,Culex flavivirus for 2.27%(1/36),Quang Binh virus for 2.27%(1/36)and for Yunnan Picorna-like virus 5.56%(2/36).Among them,the homology evolutionary tree constructed for the E gene of Culex flavivirus found in this study screened for Culex flavivirus showed closer affinity with the Culex flavivirus found in the three-banded beaked Culex flavivirus of Guangdong,and the Mangshi virus exhibited the highest similarity to the most primitive Dehong isolate,DH13M041.Analysis of the E gene of Culex flavivirus revealed a possible new subtype of Culex flavivirus.The NS5 gene evolutionary tree constructed by Quang Binh virus revealed that the JM17156 virus strain from Guangzhou was more closely related to the strain detected in this study,and the fulllength similarity between the harassed Armigeres subalbatus totivirus and Sa X06-AK20 strain was 91.89%,and there was only one similar strain in the database.The virus was isolated from 36 groups of mosquito samples using C6/36 cells,and two strains of virus were isolated that could be stably transmitted,one strain of Armigeres subalbatus and one strain of Culex flavivirus,which is the first isolation of Aedes Armigeres subalbatus totivirus in Yunnan Province.We selected 10 mosquito sample pools for Metaviromics analysis,in which the most abundant virus families were Partitiviridae,Totiviridae and Chrysoviridae,and the viruses screened by generation sequencing were detected in the Metaviromics sequencing results.In this study,Picorna-like virus was suspected to be a novel virus.To further elucidate the genomic and pathogenic characteristics of this virus strain,this study focused on the genetic annotation and isolation culture of this virus.The results revealed that the genome of the virus was mapped by open reading frame search and identification of conserved structural domains.The viral genome was 9035 bp in length,including three open reading frames and three structural domains,namely,RNA-helicase,RNA-dependent RNA polymerase(Rd Rp),Caliciviridae capsid protein(Cali-coat),and.An evolutionary tree was constructed based on these three structural domains to analyze their viral types and homology,and the results showed that the virus belongs to the small RNA virus-like superfamily,independent of the currently classified families in the small RNA order.In this study,we established a q RCR assay for this virus to assess its proliferation in C6/36 cells and found that the virus did not proliferate in C6/36 cells,but could infect C6/36 cells and did not infect Vero E6 cells,which together with previous studies suggested that the virus belongs to an insect-specific virus.In conclusion,Jingmen tick virus was detected in ticks,and Japanese encephalitis virus,Mangshi virus,Culex flavivirus,Quang Binh virus,Armigeres subalbatus totivirus and Yunnan Picorna-like virus were detected in mosquitoes,where Jingmen tick virus was detected to have a closer evolutionary relationship with virus strains from the Laos region of Southeast Asia.There is a high diversity of arboviruses carried in mosquitoes and ticks in Yunnan Province,and detection of inbound arboviruses should be strengthened,as well as continuous monitoring of arboviruses in the Yunnan region to prevent new and re-emerging arbovirus outbreaks in Yunnan Province and to ensure the health of people as well as the safety of livestock in the Yunnan region.