Identification And Biological Function Analysis Of A Novel Picornavirus RoWV-1 From Pachycrepoideus Vindemmiae(Rondani)

With the development of sequencing technology and virome,more and more novel viruses have been discovered.Parasitoid wasps are one of the most abundant species of insects,and there must be a lot of viruses on them.As a kind of ectoparasitoid wasp in the pupal stage of Drosophila melanogaster(Meigen,1830),the Pachycrepoideus vindemmiae(Rondani,1875)hosts a wide range.Besides fruit fly,it also hosts agricultural pests and health pests such as citrus fruit fly,housefly,etc.It is a biological control resource with great potential for development.In this paper,we conducted a series of studies on a novel RNA virus,Rondani's wasp virus 1(RoWV-1),which was found in the P.vindemmiae,mainly including the basic molecular biological characteristics of the virus and the biological relationship between the virus and its host,P.vindemmiae and D.melanogaster.The main research results are as follows:1.Discovery and identification of a novel picornavirus RoWV-1 in the wasp P.vindemmiaeWe identified a 9,332-nucleotide-long novel picornaviral genome sequence in the transcriptome of an agriculturally important parasitoid wasp(P.vindemmiae).The genome of the novel virus,Rondani's wasp virus 1(RoWV-1),contains two open reading frames encoding a non-structural and a structural protein,respectively,and is 3'-polyadenylated.Phylogenetic analyses firmly place RoWV-1 into the dicistrovirid genus Cripavirus.We detected RoWV-1 in various tissues and life stages of the parasitoid wasp,with the highest virus load measured in the larval digestive tract.We demonstrate that RoWV-1 is transmitted horizontally from infected to uninfected wasps but not vertically to wasp offspring.Comparison of several important biological parameters between the infected and uninfected wasps indicated that RoWV-1 had no obvious effect on wasp.2.Effects of RoWV-1 on the biological parameters of D.melanogaster,the host of P.vindemmiaeRoWV-1 can also infect wasps' host D.melanogaster and can replicate and proliferate in large quantities in D.melanogaster.Tissue distribution and developmental dynamics of RoWV-1 in D.melanogaster showed that RoWV-1 was widely distributed in various tissues of D.melanogaster,and the virus content was the highest in the intestinal tract of females.Transmission analysis indicated that RoWV-1 could not be transmitted vertically,but could be transmitted horizontally through feces and cadavers of infected D.melanogaster.Comparison of several important biological parameters between the infected and uninfected flies indicated that RoWV-1 could increase the fecundity and prolong the developmental duration of D.melanogaster,but reduce the eclosion rate of D.melanogaster.3.Explore the transmission route of RoWV-1 between P.vindemmiae and D.melanogasterRoWV-1 can be transferred from the parasitoid wasps to D.melanogaster pupae,and RoWV-1 can also be detected in the venom of parasitoid wasps.However,the viral titer kept a low level in D.melanogaster pupae and did not replicate significantly in a short period of time.On the other hand,parasitoid wasps can be infected via feeding on infected D.melanogaster pupae.Although RoWV-1 cannot be transmitted from parent to offspring by vertical transmission in P.vindemmiae,the wasp offspring could be infected with the help of infected D.melanogaster pupae.What's more,when the parasitoid wasps parasitized the infected D.melanogaster pupae,the parasitoid wasps were also infected with RoWV-1,and the amount of the RoWV-1 in the parasitoid wasps' abdomen increased with the extension of parasitism time.By comparing some biological parameters when parasitoid wasps to parasitic infected D.melanogaster pupae and uninfected D.melanogaster pupae,the results showed that parasitoid wasps parasitized infected D.melanogaster pupae could significantly improve the success rate of parasitoid wasps.4.Preliminary study on suppressor of RNAi encoded by RoWV-1Viruses are targets of the insect host RNA interference(RNAi)pathway.As a counter defense,viruses encode suppressor to counteract this antiviral response.Here we show that one of core component of the RNAi antiviral response Dcr-2 is essential for antiviral defense in adult D.melanogaster.Dcr-2 mutant flies are hypersensitive to infection with RoWV-1.The mortality in Dcr-2 mutant flies as well as the viral RNA increased siginificantly.Moreover,RoWV-1 encode a conserved ds RBD domain in ORF1.The RoWV-1-1A protein which contains ds RBD domain could suppress RNAi efficiency induced by long double-stranded RNA(ds RNA)in S2 cell.This indicated this RoWV-1-1A protein is a potent suppressor of RNA interference(RNAi) encoded by RoWV-1.In summary,a novel picornavirus RoWV-1,which belongs to the family Dicistroviridae,was found in the P.vindemmiae,and is a new member of the genus Cripavirus.The infection of RoWV-1 had no obvious biological effect on the P.vindemmiae.However,when RoWV-1 infected the parasitoid host D.melanogaster,its oviposition quantity was increased,the developmental duration was prolonged,and the eclosion rate decreased.In order to further clarify the relationship between RoWV-1 and parasitoid wasps and host D.melanogaster,this study also identified the complex transmission mode of RoWV-1 between parasitoid wasps and D.melanogaster,which provided some reference value for the study of cross-species transmission of virus using parasitoid wasps and host system.In addition,this study also identified a viral suppressor of RNA interference encoded by the virus RoWV-1,revealing the game process between the virus and the host.In conclusion,this study systematically elaborated the biological relationship between ROWV-1 virus and host fly parasitoid wasps and D.melanogaster,providing an important reference for indepth understanding of the internal relationship between parasitoid wasps,viruses and pests.