| Sacbrood disease is a viral disease caused by sacbrood virus(SBV).It is especially harmful to Apis cerana cerana and seriously affects the sustainable development of Chinese beekeeping.However,the mechanism of SBV infection and the immune response of larvae for the virus are still unclear.Therefore,this study was conducted using in vitro culture of the larvae and SBV artificial inoculation to investigate the infective characteristics of SBV to larvae.RNAscope-ISH technology was used to locate and track SBV in host infection.RNA sequencing was used to analyze the differential gene expression characteristics of SBV infected larvae with emphasis on the immune response and pathogenesis-related gene expression of the Apis cerana cerana larvae.The major results of this research were listed below:1.The experimental conditions for the larvae in vitro culture and artificial inoculation with SBV were optimized,and the infection characteristics of SBV was analyzed.The results showed that the survival rate of larvae dropped sharply on the third day(3 dpi)after SBV infection,and showed a typical"sac" shape consistent with bee colony infection under the experimental conditions.Using absolute quantitative qPCR method,the quantitative analysis of SBV nucleic acid found that SBV remained at a low level on the first day post infection(1 dpi,1 day post infection),and showed exponential growth on the second and third days with the number of virus copies reached up to 105,and SBV proliferated slowly on the 4th day(4 dpi)after infection.2.RNAscope-ISH analysis showed that the hybridization signal of SBV positive-strand and SBV negative-strand increased continuously from 1 dpi to 3 days post infection,indicating that the amount of SBV proliferation and virus replication continued to increase at these stages.The results of this study found that SBV first enters to the peritrophic membrane of the midgut,then enters the fat body trophocyte,and replicates and enriches in large number of the trophocyte,thereby destroyed the stability of the insect tissue structure.Moreover,cytological methods were used to obtain the replication process of the negative-strand of SBV,which laid the foundation for effective prevention and control of virus infection in the future.3.Through RNA sequencing,the gene expression profile of larvae from 1 dpi to 4 dpi showed significant differences after SBV inoculation,with the most differential gene expression at 3 dpi.From ldpi to 4 dpi,there were 20 immune-related genes that were differentially expressed in common.Among them,the myosin regulatory light chain 2-like(LOC107996620)was continuously up-regulated and has the function of calmodulin(CaM),this gene was enriched in the TRP pathway by inflammatory mediators’regulation and cAMP signaling pathway.The expression of APN gene(LOC107999228)was continuously down-regulated,enrichment analysis showed that APN protein is an inherent component of the membrane and has catalytic activity.Analysis of the unique genes of 3 dpi,4 dpi live larvae and dead larvae found that the larvae that survived SBV infection mainly depend on the regulation of genes in the spliceosome pathway and the regulation of genes in various metabolic pathways.The larvae that die post infection with SBV were closely related to the differential expression of genes in the tight junction pathway,Wnt signaling pathway,cGMP-PKG signaling pathway,and Hippo signaling pathwayThis study primarily revealed the infection characteristics of SBV and the immune response of larvae to SBV infection,and the research results laid the foundation for the effective prevention and control of Chinese sacbrood disease in the future. |
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