A Novel Densovirus Isolated From The Asian Tiger Mosquito Displays Varied Pathogenicity Depending On Its Host Species

Background:Mosquito-borne viral diseases(MBVDs)continue to pose a significant global public health burden.Mosquito control remains a core intervention strategy in integrated mosquito management(IMM)programs to reduce the transmission of MBVDs.However,the commonly and widely used of chemical pesticides has generated complex problems because of the high level of toxicity caused to the environment,consequent safety risks for humans,and insecticide resistance(IR)in mosquitoes which means that the development of new mosquito control method is of great importance.The research and application of biocides will provide new strategies for mosquito control.Mosquito densoviruses(MDVs)are mosquito-specific entomopathogenic viruses and can spread vertically and horizontally in nature.They infect and kill larvae in a dose dependent manner in the aqueous larval habitat.Infected larvae that survive to become adult mosquitoes exhibit a shortening of lifespan and most of them do not survive longer than the extrinsic incubation period for arboviruses.Thus they may have a significant impact on transmission of pathogens.And the availabity of infectious clones of MDV genomes allows the development of densovirus vectors for expressing genes of interest in mosquitoes and mosquito cell lines.Therefore,isolation and identification of MDVs and analysis of the molecular biology and pathogenic characteristics will provide a theoretical basis for the development of MDVs as biocides and gene expression vectors.Objective:we report the isolation and characterization of a novel Aedes albopictus densovirus-7(AalDV-7)isolated from wild-caught adult Ae.albopictus in the city of Guangzhou in Guangdong Province,China.We analyzed the pathogenicity of Aedes albopictus densovirus-7 to larvae of the major dengue and Zika virus vectors Ae.albopictus and Ae.aegypti and the potential Zika virus vector Culex quinquefasciatus.The infection rate for the host and the viral concentrations of AalDV-7 in vivo were also evaluated to explore its potential for vector control.Methods:During a recent outbreak of dengue virus in Guangdong Province,field-collected Ae.albopictus were received by our laboratory from the cities of Guangzhou and Shenzhen in Guangdong Province for the detection of arboviruses.A novel MDV isolated from wild-caught adult Ae.albopictus by ultra-high speed density gradient centrifugation was designed Aedes albopictus densovirus 7(AalDV-7)after the vector from which it was isolated.The feature of the genome,the transcripts and the proteins of AalDV-7 were analyzed.What's more,newly hatched 1st instar larvae were exposed to AalDV-7(n=100 per group)at concentrations ranging from 1×108to1×1011 geq/ml to analyze the pathogenicity of AalDV-7.Larval mortality was scored every 24 h until the emergence of the adults.And the infection rate and viral titer were evaluated using the method described above.Results:The complete genome sequence of the virus has been submitted to GenBank(GenBank:MK1823 84)and was termed Aedes albopictus densovirus-7(AalDV-7).It is 4,048 nt in length and contains three overlapping ORFs encoding the nonstructural 1(NS1)protein,the NS2 protein,and the viral capsid protein(VP).It was proved that the transcription initiation site(TIS)of VP protein locates 158 nt or 1 nt upstream the ATG and NS1 and NS2 gene share a common TIS located 6 nt upstream of the ATG of the NS1/NS2 gene and the NS protein shares the same transcription termination sites(TTS)with VP protein just like other MDVs.By molecular phylogenetic analysis,the AalDV-7 was clustered into one clade with Aa1DV-2,AalDV-5,AaeDV,CppDV and AgDV,most of which were isolated from indigenous mosquitoes.The anal papillae and bristle cells were major portals of entry in larvae.Red fluorescence was observed spread to other tissues from its portal of entry,including muscle fibers,foregut,midgut,hindgut,malpighian tubule,etc.The pathogenicity of AalDV-7 to Ae.albopictus,Aedes aegypti and Culex quinquefasciatas larvae was completely different,and the LD50 of AalDV-7 in Ae.albopictus which was 109.48 genome equivalents per ml(geq/ml)was 12 and 46 times more toxic than those in Ae.aegyvptr(1010.56 geq/ml)and Cx.quinquefasciatus(1011.15 geq/ml).Furthermore,the LT50 value in Ae.albopictis(7.72 days)was 25%and 26%shorter than those in Ae.aegypti(10.24 days)and Cx.quinquefasciatus(10.42 days)at a titer of 1011 geq/ml.Furthermore,the mortality of AalDV-7-infected mosquitoes increased in a dose-dependent manner,and the highest mortality was found in Ae.albopictus larvae infected with 1011 geq/ml AalDV-7(82.00%).Sublethal effects analysis also showed that AalDV-7 infection significantly decreased pupation and emergence rates.The 1st-2nd instar larvae of all three mosquito species showed a near 100%infection rate,and the highest relative vial titer(305.97 ± 67.57 geq/ng)was observed in the 1St-2nd instar larvae of Cx.quinquefasciatusConclusion:AalDV-7 displayed varied pathogenicity depending on its host species.These pathogenic characteristics make AalDV-7 a potential flavivirus control agent in China,whereas its negligible pathogenicity and high infection rate and viral titer im vivo make it a good candidate for gene delivery vectors in Cx.quinqzuefasciatus gene function analysis.In conclusion,the continuous discovery and isolation of new MDVs enrich the pool of mosquito entomopathogenic viruses and provide a variety of choices for optimal MDVs or combinations of MDVs to target certain mosquitoes.