家蚕CVDAR品系对BmNPV的抗性特征分析

Characteristic analysis of resistance to BmNPV in CVDAR strain of silkworm

摘要:

[目的] 家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV）促使的血液型脓病是一种产业上非常严重的家蚕疾病，目前有效的防控方法较少。本研究以大造和CVDAR家蚕品系（对BmNPV有较强抗性的品系）为试验材料，通过分析CVDAR对BmNPV抗性特征，以期确定CVDAR对BmNPV的抗性机制。[方法] 本研究通过半致死剂量分析，发现CVDAR品系比大造品系对BmNPV感染的半致死剂量提高10倍以上；进一步HE染色分析大造与CVDAR品系病毒感染前后的中肠组织的变化，具体解析抗性品系CVDAR的抗BmNPV机制。[结果] 感染BmNPV 72 h后，大造中肠细胞细胞核明显膨大，着色变浅，到96 h后，细胞核持续增大有脱落趋势；而CVDAR抗性品系只在感染96 h后有中肠部分细胞核膨大，但排列整齐；同时通过荧光定量分析大造与CVDAR品系病毒感染后的增殖情况，结合各个时期代表病毒基因的转录水平分析比较发现，感染BmNPV后0-12 h也没有发现抗性品系CVDAR和大造之间的病毒拷贝数以及病毒基因转录水平的不同，但感染24 h后发现抗性品系CVDAR无论是病毒拷贝数还是病毒基因的转录表达水平都明显低于对照大造。[结论] 证明CVDAR口服添毒后中肠中病毒基因的转录在第一轮复制期间不受影响，之后转录水平降低。鉴定CVDAR品系抑制BmNPV增殖的关键时期是在感染BmNPV后的24 h，为解析抗性机制奠定基础。

Abstract:

[Objective] Bombyx mori nucleopolyhedrovirus (BmNPV) induced hematogenous sepsis is a common severe disease of silkworm, and there are few effective prevention and control methods. In this study, CVDAR andDaZao (lines with strong resistance to BmNPV) silkworm strains were used as experimental materials. By analyzing the resistance characteristics of CVDAR silkworm, the resistance mechanism of CVDAR to BmNPV was determined. [Methods] In this study, we found that the lethal dose of CVDAR strain to BmNPV infection was more than 10 times higher than that of DaZao through semi lethal dose analysis. Further HE staining was performed to analyze the changes of midgut tissues before and after the infection of CVDAR and DaZao strains, and analyzed the anti-BmNPV mechanism of the resistant CVDAR strain. [Results] The results showed that after 72 hours of infection with BmNPV, the nucleus of large intestinal cells was enlarged and the staining became lighter; after 96 hours, the nuclei continued to increase and tended to fall off. The CVDAR-resistant strains only had enlarged nuclei in part of midgut after 96 hours of infection, but they were well arranged. At the same time by fluorescence quantitative analysis of the CVDAR and DaZao strain proliferation after virus infection, combined with the analysis and comparison of the transcriptional levels of the representative virus genes in each period, no difference in virus copy number and viral genes transcription level between the resistant CVDAR strain and the DaZao strain were found between 0-12 h after BmNPV infection. However, 24 hours after infection, we found that the CVDAR of the resistant strain was significantly lower than that of the control strain, regardless of the number of viral copies or the transcriptional expression level of viral genes. [Conclusion] Our study demonstrated that during the first round of replication after oral addition of CVDAR, the transcription of midgut virus genes was not affected; and later the transcription level was reduced. The critical period for the inhibition of BmNPV proliferation by CVDAR strain was identified to be 24 h after BmNPV infection, laying a foundation for the analysis of the resistance mechanism.