Study On The Latent Infection Mechanism Of BmMLV

Virus can establish different infectious status on the host,depending on the virus types,pathogenicity,the host immunity and other factors.These status mainly include pathogens being cleared,recessive infection（subclinical infection）,apparent infection（clinical infection）,pathogen carrying state,latent infection.Recently,a lot of studies about viral apparent infection have been reported,but reports about virus recessive infection is rare.Recessive infection means that pathogens cause the host generating specific immune response,not or only cause minor tissue damage.Recessive infection does not show any clinical symptoms,signs,biochemical changes,even can only be found by immunological tests.At present,Bombyx mori macula-like virus is a new kind of virus that can establish a latent infection on silkworm and its cell lines,withouting causing the pathological changes in host cell.However,it is still unclear about the latent mechanism of BmMLV infection.This study proposed to detect the response to BmMLV virus particles,genomic RNA and genome cDNA infection of silkworm and its cells based on our completed work for BmMLV RNA genome sequencing.Also,we expected to explore the molecular mechanism of BmMLV recessive infection and the ability of gene transferring genes to silkworms based on BmMLV carrier system.In this way,it will not only provide new clues for the deep understanding of the interaction between virus and the silkworm,but also can provide the new technology for the gene transferring to silkworm.1.The sequencing and bioinformatics analysis of BmMLV-SZ full genome.We applyied RT-PCR to amplify genomic cDNA of Bm MLV,the fragments were cloned into sequencing vector,sequencing respectively,finally joining together for whole genome RNA sequences of BmMLV namely BmMLV-SZ virus.Its genome contais 6535 nt,5’end 57 nt for non-coding regions,3’end 67 nt for the non-coding regions（containing polyA）₂₂),has the similar characteristics to the eukaryotic mRNA.It was speculated that there are three open reading frames:ORF1 58-5304（nt）,composed of 5247 nt,encoding 1748 amino acid,ORF2（5315-6028 nt）,composed of714 nt,encoding 238 aa,ORF3（6061-6468 nt）,composed of 408 nt,encoding a p15polypeptide（136 aa）.The homology of BmMLV genomic sequence alignment was 99%,when compared to the wild type BmMLV.There are a lot of base mutations and deletions in RdRp coding region,indicating it was in relation to the recessive exists of BmMLV in culture Bombyx mori cells.Based on the analysis of the evolution of the RdRP and CP amino acid sequence,Bm MLV has a high similarity to the members of the Tymoviridae family speculating BmMLV probably originated from plant virus.2 The preparation of BmMLV p15 and Bm MLV-cp polyclonal antibody and p15and subcellular localizationBmMLV p15 and cp genes were cloned into pET28a（+）to build a prokaryotic expression vector and express recombinant p15 and cp protein in Escherichia coli.Then we prepare polyclonal antibody by immuning mice with the recombinant protein.Westernblottingconfirmedthatthepolyclonalantibodyarespecific.Immunofluorescence showed that BmMLV-p15 in silkworm ovarian sources of Bm N cells can express,locate in cytoplasm and nucleus with a few distribution;CP located in the cytoplasm.3 Bm MLV infection silkworm and the effect of temperature and nutrition conditions on BmMLV proliferationReal-time PCR was appied to detect the RNA level of BmMLV in each organization of silkworm after inoculated BmMLV.The results indicated Bm MLV RNA can be detected in the midgut,silk gland,and gonads and the highest level of viral RNA was in the intestinal tissue.Under the condition of different pressure,temperature,medium),the results showed high temperature of 37°C and mammalian cell culture incorporating in insect TC-100 medium can could promote the proliferation of the virus,meaning that high temperature and the change of nutrition conditions can affect proliferation of BmMLV in Bm N cells and Sf9 cells.4 Sf-9 cells infected silkworm to macular virus（BmMLV）after the transcriptome analysisIn order to further explore the latent infection mechanism of sf-9 cells,we made a transcriptome analysis between normal sf-9 cells and sf-9 cells infected with BmMLV.There are a total of 5670 differentially expressed genes in normal sf-9 cells and sf-9 infected with Bm MLV,among them 3180 genes were upregulated and 2490genes.743 differentially expressed genes were in fit with strict common expression of t test,among them 300 genes were upregulated,443genes were downregulated.The GO enrichment analysis of all the differentially expressed genes（DEGs）showed that the upregulated differentially expressed genes were mainly related to such functions,transport,transmembrane transport,cell autophagy and downregulated genes were related to protein hydrolysis,activity of hydrolytic enzyme,metabolism of small molecules,the negative regulation of apoptosis process,etc.The KEGG enrichment analysis of all the differentially expressed genes（DEGs）showed the upregulated differentially expressed genes enriched in RNA degradation,the ribosome biogenic pathways and downregulated genes enriched in proteasome,metabolic pathways of exogenous substancescaused by the cytochrome P450.5 BmMLV infection to mammalian cellsBmMLV originates from plant virus,whether it could establish a persistent infection on mammalian cell line is still unclear.This study focused on exploring whether BmMLV across"border"between the plant and animal virus to infect vertebrate cells.We inoculated lung adenocarcinoma cell line（H1299）with Bm MLV.The detection by real-time PCR revealed Bm MLV level reduce gradually,and remained in a low level5 days later,We couldn’t detect Bm MLV up to 21days.The result indicated it is difficult for Bm MLV to exist in H1299 cells.6The construction of gene transfer vector based on BmMLV genomeThe full-length single cDNA and genomic RNA of BmMLV were transfected into Sf9 cells.We can detect a low level of proliferation in Sf9 cells,proving the RNA and the full-length of single cDNA of BmMLV has the ability to infect.Given the BmMLV viral genome was relatively small,easy for genetic operation;Genomic cDNA with infectivity and without integrating into the host chromosome can avoid the potential gene mutation and random effects.The full-length cDNA plasmid was cloned in pBlueScript SK vector,constructing recombinant virus vectors and gene transfer vectors with fluorescent reporter gene,different promoters control over the full-length cDNA of BmMLV,expecting to lay the foundation for development of subsequent gene transfer viral vector.