Characterization Of Genome And Expression Of Myzus Persicae Densovirus

In 2003,Myzus persicae densovirus(MpDV)was firstly isolated from infected Myzus persicae at Netherland.At that time,the partial genome sequence of this virus was determined.Presently,little is known about the phylogenetic evolution and infection mechanism of MpDV.Based on the genome sequence of MpDV that detected in Myzus persicae at Yangling,Shaanxi province,we amplified the partial genome sequence of MpDV in Myzus persicae that were collected from Kunming,Yunnan Province(the virus strain termed as MpDV-YN).We analyzed the genome charaters of MpDV-YN and the phylogenetic evolution of MpDV.Also,we detected the replication and transcription of MpDV-YN in different tissues of Myzus persicae and in the host plants of Myzus persicae(peppers and cabbages)by using quantitative real-time PCR.Our main results as following:1.Based on the MpDV-YL genome sequence,we designed the overlap PCR primers and amplified a fragment of 5483 bp length from M.persicae collected from Kunming,Yunnan province.Nucleotide sequence analysis revealed that different from the encoding strategy of MpDV-NL genome(which was predicted to contian 5 ORFs),the encoding strategy of MpDV-YN is similar with that of MpDV-YL,i.e.the genome of these two strains was predicted to contain 4 ORFs.The positive-sense strand contains two ORFs,which potentially encode the nonstructural proteins NS2 and NS1,whereas the antisense strand also contains two ORFs(ORF3 and ORF4)that was predicted to encode the structural proteins VP1 and VP2.2.Quantitative real-time PCR analyses showed that MpDV-YLis mainly replicated inthe intestinal tract of aphid larvae.3.To detect the transcript level of MpDV-YL NS1,NS2,VP1 and VP2 in the isolated tissues of head,gut,embryo and epidermis,quantitative real-time PCR analyses showed that the transcript level of VP1 is signifcantly higher than the other predicted viral genen in whole aphids.Meanwhile,the transcript level of all 4 predicted viral genes in intestinal tract of aphid larvae was significantly higher than in other tissues(head,embryo and epidermis).4.To detect whether MpDV-YL can be transfered in the host plants of Myzus persicae,the genome copy number of MpDV-YL in leaves,stems and roots of the plants(pepper and cabbage)was detected after feeding with infected peach aphids.Quantitative real-time PCR showed that a significant high amout of MpDV-YL was detected in leaves of two host plants.However,after treatment of the leveaves with DNase I,the genome copy number of MpDV-YL in these leaves was dramatically decreased.We proposed that the MpDV-YL in the leaves of host plants might be contaminatedfrom the aphid honeydew.5.We constructed the transient expression plasmids of MpDV-YL VP1 and VP2 genes.In transfected Sf9 cells,the bimolecular fluorescence complementation(Bi FC)assasy showed that there might be no interaction between VP1 and VP2.Further analysis indicated that,in transfected Sf9 cells,6-His tagged MpDV-YL VP1 and VP2 were transiently expressed as the predicted molecular weight size.We proposed that these two viral proteins were not cleaved in Sf9 cells.In summary,our results suggest that the encoding strategy of MpDV in Myzus persicae collected from China is different from that of MpDV strain of Netherland.MpDV might be involved as a separate line in parovirus.MpDV-YL is mainly replicated in the internetal tract of peach aphid larvae and this virus might not be transferred by aphid to the host plants of Myzus persicae.Overall,these resultsshould be shed light on further studies on the infection mechanism of MpDV.