Sequenceing, Analysis Of Antheraea Pernyi Iflavirus Genome, And Studies On Its Transmission

Antheraea pernyi(A.pernyi) is an economically important insect and organisms for basic research. In the wild filed,larvae of the Antheraea pernyi are often affected by A.pernyi vomiting disease(Antheraea pernyi vomiting disease,AVD), which cause a serious economic loss In north China. In this paper,we found a novel picorna-like virus and demonstrated that it is the pathogeny of AVD. This finding will greatly exert an influence on AVD controlling. Contents and results are as follows:(1) A novel positive sense single stranded RNA virus was purified from Chinese oak silkmoth larvae and pupae. Healthy A.pernyi larvae injected with this virus reproduced typical A.pernyi vomiting disease symptoms within3days after infection. This virus is inferred as the pathogeny of AVD.(2) The genome of this virus is10,163nucleotides long, has a natural poly-A tail, and contains a single, large open reading frame flanked at the5’and3’ends by untranslated regions containing putative structural elements for replication and translation of the virus genome. The open reading frame is predicted to encode a3036Amino acid polyprotein with four viral structural proteins (Viral protein1-4) located in the N-terminal end and the non-structural proteins, including a helicase, RNA-dependent RNA polymerase and3C-protease, located in the C-terminal end of the polyprotein. Putative3C-protease and autolytic cleavage sites were identified for processing the polyprotein into functional units. The genome organization, Amino acid sequence and phylogenetic analyses suggest that the virus is a novel species of the genus Iflavirus, with the proposed name of Antheraea pernyi Iflavirus (ApIV).(3) ApIV2C protein were expressed by using Bac-to-Bac system in Sf9cells. The ApIV2C protein was purified on Ni-NAT column. Furthermore, we detected the NTPase activity and helicse activity of ApIV2C protein. ApIV2C protein NTPase activity was found to depend on the presence of Mg2+. The ATPase activity of ApIV2C protein could reach the maximum at50mM Tris-HCl(pH8.0),2mM MgCl2,50mM NaCl,1mM ATP, the optimal temperature is37℃.(4) Laboratory infection assays were performed with larvae, pupae and adult moth. ApIV replication were investigated by reverse transcription-PCR methods. We could demonstrate that Antheraea pernyi injected with ApIV or fed with ApIV developed overt infections suggesting that ApIV can be transmitted by horizontal transmission route. Further more,ApIV replication was demonstrated Not only in the head, epidermis, hemocyte, gut, fat body, but also in the ovary and testis. When moths were identified as positive for ApIV, the same virus were detected in their offspring eggs also. The presence of viruses in the tissue of reproductive organs and the detection of the same virus in their eggs suggest the vertical transmission of viruses from ApIV infected moth to progeny.