The Analysis Of Interactions Between Silkworm And Bombyx Mori Bidensovirus And Validation Of Viral Invasion In Resistant Silkworm

Bombyx mori,a model organism of Lepidoptera,is also a domesticated insect with important economic value.Bombyx mori bidensovirus(BmBDV)is one of the most serious pathogens of silkworm which cause losses in the sericulture industry.Bm BDV exclusively infect midgut of silkworm and mainly replicate in epithelial columnar cells,which results in the nuclear enlargement of target cells.Virus-infected silkworms showed typical flacherie symptoms.The virus particle is an icosahedral non-enveloping structure which contains two linear single-stranded DNA fragments VD1(Viral DNA 1,6.5 kb)and VD2(Viral DNA 2,6 kb).VD1 and VD2,and also their complementary strands,are encapsidated separately into four different types of particles.BmBDV was assigned to genus Bidensovirus within family Bidnaviridae,and is the only single-stranded DNA bipartite virus which encoded DNA plolymerase by itself as yet.However,up to now,the mechanisms of invasion of the virus and the mechanisms of host’s resistance to the virus have not been completely analyzed.In this study,susceptible Bombyx mori huaba35 and the anti-viral near-isogenic line huabaBC7 were used.Firstly,the high-throughput sequencing of RNA of the intestine tissues of BmBDV-infected silkworms was used to analyze the interaction between the virus and the host at the transcriptome level;we then studied whether the virus could invade the resistant silkworm;finally,we analyzed whether the virus’ s genome could integrate into the host’s genome.The results of this study are as follows:1.To accurately analyze the gene expression changes of Bm BDV-infected silkworm larvae,we first analyzed the expression of seven common candidate internal reference genes: ribosomal protein L3(RPL3),28 S ribosomal RNA(28S rRNA),actin(actinA3),TATA box binding protein(TBP),3-Glycerol phosphate dehydrogenase(GAPDH),α-Tubulin,protein(UBQ)in silkworm larvae infected or uninfected with Bm BDV,and evaluated their expression stability with three statistical methods(geNorm,NormFinder,and BestKeeper).The results revealed that the RPL3 was the most stable internal control,and UBQ was the least stable genes.Furthermore,the expression profiles of Bombyx mori serine protease(BmSP142)was assessed usingRPL3 as internal.The expression of BmSP142 was significantly different between Bm BDV-resistant and Bm BDV-susceptible silkworm after virus infection,suggesting that BmSP142 may be involved in the antiviral response.2.In order to understand the response of silkworm to BmBDV,the midguts of silkworm were extracted at 0 h,6 h,24 h and 48 h after BmBDV infection for RNA-seq,and comparative transcriptomic analysis was performed.We found that whole transcriptom of Bombyx mori genes had dramatically changed after BmBDV infection.Compared with healthy silkworm,we identified a total of 319 differentially expressed genes(DEGs),of which 138 were up-regulated and 181 were down-regulated.Via GO function analysis of DEGs,we found that 5 immune-related genes such as antimicrobial peptides and peptidoglycan recognition protein,and 9defense and stress response-related genes were up-regulated;4 genes related to chitin biosynthesis and six genes related to juvenile hormone were significantly downregulated.The KEGG analysis of DEGs revealed that these differential genes were involved in processes such as metabolism,N-glycan biosynthesis,and protein synthesis and processing,cytochrome c P450 metabolism,phagosome formation pathways and so on.Analysis of the virus transcripts indicated that the expression pattern of the virus genes exhibited a time sequence,the expression of genes(ns1,vp,etc.)in VD1 was earlier than that of in VD2(ns3,p133).Alternative splicing was absent during viral gene expression.3.Comparative analyses of viral genome replication,viral gene expression and histopathology between BmBDV-sensitive silkworms and-resistant silkworms were performed.The results revealed that BmBDV can invade resistant silkworms and its genome can be replicated at consistently low level in resistant strains;Viral genes could be expressed in resistant strains,but the expression of viral genes is abolished at the late stage of infection.Further studies by Southern blot and FIPN-PCR had found that the genome of the virus maybe not integrated into the genome of the host.