The Infectious Mechanism And Transcription Analysis Of Junonia Coenia Densovirus

The Junonia coenia densovirus (JcDNV) is a member of the densovirus genus of the Parvoviridae family and infects the larvae of Junonia coenia (the Common Buckeye butterfly). JcDNV was first isolated from the tissue of nymphalid in1972. The JcDNV is a small icosahedral nonenveloped insect viruses containing a linear single-stranded DNA genome of6kb in length. The pathogenesis associated with the disease was described as a "cellular dense nucleosis". Because of their high virulence and efficient transmission among insect pests as well as disease-vectors, the JcDNV was considered as biological control agents. In the present study, we investigated the mechanism infection and transcritption stratagies of the JcDNV. The main results are summarized as following:1. Junonia coenia densovirus crosses the insect mid-gut barrier by transcytosisDensoviruses are lethal to insects of different orders in larval stages. Although the horizontal transmission mechanisms are poorly understood, pathogenesis caused by densoviruses usually starts with the ingestion of contaminated food by the host and leading, depending on the virus, to a replication restricted to the mid-gut or to exclude it. Using the JcDNV and its lepidopteran host Spodoptera frugiperda as an interaction model, we focused on the mechanisms of infection during the early stage in which the JcDNV has to overcome the intestinal epithelium barrier to reach target tissues(in vivo and in vitro) and ultimately replicate within them. We studied4major aspects of interaction between the virus JcDNV and the mid-gut of insect:ⅰ) the kinetic analysis of mid-gut cells infection ⅱ) the mid-gut cell type targeted by the viral particles; ⅲ) the JcDNV transport mechanism and ⅳ) the physiological consequence of infection on the integrity of the epithelium cells. Using several approaches including in vivo and in vitro assay, as well as molecular and physiological tools, we showed that JcDNV entry occured specifically in columnar cells by endocytosis and further crossed the epithelium by transcytosis. As a consequence, viral entry triggered an increased permeability of the epithelium although we excluded a direct passage of the virus through the paracellular pathway. Finally we demonstrated that four mutations on the capsid gene of JcDNV significantly affected its transport across the mid-gut but not its internalisation.2. The use of silkworm (Bombyx mori) as a new model to study the JcDNV virulenceTo evaluate the suitability and capacity of using DNVs in lepidopteran pest control and the risks of invading new hosts, mechanisms underlying specificity and pathogenesis need to be deciphered carefully. In this study,we described Bombyx mori as a new model to evaluate the JcDNV pathogenesis and tissue tropism. The similar tissue tropism was detected in Bombyx mori and in Spodoptera frugiperda. However, some significant disparities were observed:i), the JcDNV highly replicated in Malpighian tubes and fat body in B. mori that did not occur in S. frugiperda. ii), midgut cells of B. mori were more severely impaired than those of S. frugiperda. iii), in Ussing Chamber system, it was shown that JcDNV could cross the midgut of both species in10min, but with nearly30-fold more virus remained in midgut of B.mori than that of S. frugiperda by quantitative PCR. This result suggests that JcDNV is more pathogenic to Bombyx mori than Spodoptera frugiperda.3. Analysis of the transcription strategy of the Junonia coenia densovirus (JcDNV) genomeThe JcDNV has an ambisense genome with the structural (VP) and nonstruc-tural (NS) genes located in the5’half on opposite strands. Northern blot analysis of Ld652cells and Spodoptera littoralis larvae transfected with plasmid pBRJ encompassing an infectious sequence of the JcDNV genome revealed three transcripts, an unspliced2.5kb VP mRNA encoding capsid proteins and two NS mRNAs, one unspliced2.5kb mRNA encoding NS3, the other of1.7kb resulting from the splic-ing out of the NS3coding sequence and expressing NS1and NS2. Analysis of translation products of VP mRNA clearly showed that the4capsid proteins are generated by a leaky scanning mechanism. RT-qPCR was performed to study the time course of ns1, ns3and vp gene expression. We found that as early as2h post-transfection, nsl gene starts to be transcribed and keeps increasing until48h post-transfection. For the ns3gene, transcription also starts at2h post-transfection, but it increases slowly compared to the nsl and the amount of transcripts is always less than that of nsl. The transcription of vp gene starts at3h post-transfection and is a little bit late than that of nsl and ns3genes. However, the amount of vp gene transcripts increases rapidely from12h post-transcfection.