A Preliminary Study On The Molecular Mechanism Of Infectious Difference Between Two BmNPV Strains On Bombyx Mori

Bombyx mori,is a silk-secreting insect with high economic value.It is one of the main economic resources of more than 30 million families in China,India,Vietnam and Thailand.However,sericulture production is affected by bacteria,fungi and viruses every year.Bombyx mori nuclear polyhedrosis virus（BmNPV）belongs to the alphabaculovirus.As a pathogenic microorganism that specifically infects B.mori,BmNPV caused 86.92%of the loss of silkworm production due to hemolynamic septic disease caused by BmNPV infection every year.It is generally believed that cultivating silkworm varieties with high resistance to BmNPV is the most economical and effective means to control the harm of the disease to silkworm production.In the past 10 years,several BmNPV-resistant varieties have been cultivated by some research institutes in China,and the results of production application also proved that the disease resistance effect is remarkable.However,in recent years,some researchers have found that BmNPV from different regions has different infection to the same variety of silkworm,and even the pathogenicity of BmNPV from the same province is also different,which puts forward new challenges for silkworm disease resistance breeding.It is also of great significance to study the causes and mechanisms of differences in the pathogenicity of BmNPV in different geographical strains to silkworms.Therefore,two BmNPV strains were selected in this study:Zhenjiang strain（BmNPV ZJ）and Yunnan strain（BmNPV YN）;the parent of the control variety of silkworm varieties approved by the state for summer and autumn:Baiyu were used as the host infected by BmNPV.The molecular mechanism of pathogenicity difference of BmNPV strains was explored from the perspective of viral genome and host response.The main results are as follows:（1）Determination and comparison of pathogenicity of two BmNPV strainsBaiyu as experimental material.When the B.mori reached the 2^ndinstar,BmNPV ZJ and BmNPV YN with different gradient concentrations were inoculated orally to determine and compare the pathogenicity of the two BmNPV strains.The results showed that the LC₅₀of BmNPV ZJ to B.mori of Baiyu variety was 6.45×10⁶PIB/m L,and that of BmNPV YN was 3.62×10⁵PIB/m L,indicating that BmNPV YN had stronger pathogenicity.Four genes ie-1,p143,vp39 and polh of BmNPV at very early,early,late and very late stages were selected for q PCR to detect the proliferation of the two BmNPV strains.The results showed that the relative expression levels of the four genes above BmNPV ZJ were higher than those of BmNPV YN in the midgut of Baiyu within 12～96 h,indicating that the lethal concentration of BmNPV ZJ on Baiyu was higher than that of BmNPV YN,and BmNPV YN strain might belong to the virus type that could kill the host with only a small amount of proliferation.（2）Molecular mechanism of infectious difference between the two BmNPV strains based on genome analysis of BmNPVThe whole genome sequencing of the two viruses was performed using the combination of Illumina second-generation sequencing technology and Pacbio third-generation sequencing technology.The results showed that the genome sizes of BmNPV ZJ and BmNPV YN were 135 895 bp and 143 180 bp,with 141 and 145 CDS sequences,and the GC contents were 40.93%and 39.71%,respectively.By comparing the genome sequences of the two BmNPV strains,it was found that there were a large number of SNP mutations and In Del mutations between their genomes,which was also the reason for the change of gene length,number and coding gene number of the two BmNPV strains.According to statistics,there were 76 genes with amino acid sequence differences,including22 core genes,36 non-core genes and other genes with unclear classification,which involved many genes such as per os infectivity factor,viral replication,and hindering host molting and pupation.Through bioinformatics analysis,it was found that the differences in nucleotide sequences of some genes of the two viruses caused in changes in their secondary structure.Therefore,we speculated that the mutation and rearrangement of some BmNPV genes may be changed the function of the encoded protein due to the difference of geographical environment,and eventually lead to the difference of pathogenicity between the two BmNPV strains.（3）The post-transcriptional level of B.mori infectied with BmNPV was used to explore the reasons for the difference in infectivity between the two BmNPV strainsThe two BmNPV strains were orally fed to the 5^thinstar larvae,and the midgut tissues at 12 h,24 h and 48 h were taken for transcriptome sequencing.A total of 2592differentially expressed genes were found.Among them,there were 1702,847 and 443differentially expressed genes at 12 h,24 h and 48 h,respectively.It can be seen that with the passage of time,the effect of different strains of BmNPV on silkworm became smaller.GO annotation was performed on the differentially expressed genes among samples,and a total of 1690 differentially expressed genes were obtained for GO annotation,involving 52secondary functional items.KEGG enrichment analysis showed that 1037 differentially expressed genes were enriched in 117 metabolic pathways after feeding BmNPV ZJ and BmNPV YN.Further analysis showed that differentially expressed genes were enriched in Lysosome pathway at 12 h,24 h and 48 h.Among them,the gene expression levels of CD63antigen（KWMTBOMO14236）,Cathepsin B（KWMTBOMO10376）and Lysosomal acid lipase（KWMTBOMO06653）were significantly different.The above results suggested that after BmNPV infection of different strains,the BmNPV resistance levels initiated by B.mori were different.Lysosome pathway may be directly or indirectly involved in the response of BmNPV infection in B.mori.Among them,compared with BmNPV ZJ,BmNPV YN strain infection,the expression of lysosomal main membrane protein CD63-related genes was down-regulated,and the expression of lysosomal cathepsin B-related genes was up-regulated,which affected lysosomal-mediated autophagy/heterophagy and apoptosis,and made B.mori show inconsistent resistance to different strains of BmNPV infection.The results can provide new ideas for further exploring the molecular mechanism that causes the difference in pathogenicity of BmNPV in different strains.In summary,we speculated that due to the differences in geographical environment,some genes of the two BmNPVs were mutated and rearranged.Among them,the difference in the gene sequence of per os infectivity factor（PIFs）may affect the process of BmNPV infection of silkworm midgut epithelial cells.With the extension of time,BmNPV began to invade the silkworm body,the natural immune response began to start.Lysosome pathway may be directly or indirectly involved in the response of BmNPV infection in B.mori.However,after BmNPV infection of different strains,the BmNPV resistance levels initiated by B.mori were different.Among them,after the infection of the two BmNPV strains,the expression levels of related genes in lysosomes were different,which affected a series of immune responses mediated by lysosomes,and ultimately led to inconsistent resistance of B.mori to the infection of the two BmNPV strains.