家蚕二分浓核病毒感染家蚕后的释放动态研究

Release dynamics of Bombyx mori bidensovirus from virus-infected silkworm

摘要:

【目的】研究家蚕二分浓核病毒(BmBDV)感染家蚕后不同时间段内释放到环境中的病毒量，为通过蚕沙快速检测病毒提供方法和依据。【方法】选取家蚕二分浓核病毒基因组节段VD1上的ns1基因和VD2上的ns3基因中的一小部分片段，分别克隆到pMD18-T载体上，制备标准品质粒，并用实时荧光定量PCR的方法绘制ns1和ns3的标准曲线。通过检测BmBDV感染5龄家蚕后不同时间蚕沙中的病毒量来衡量释放到环境中的病毒的变化动态。【结果】在病毒感染家蚕第3天，大量的病毒随蚕沙释放到环境中，每微克蚕沙中约含1.38×106个VD1，6.54×105个VD2；从感染后第5天开始，释放到环境中的病毒量呈指数型增加；第7?8天病毒的释放量到达一个平台期，此时每微克蚕沙中约有2.12×107个VD1，1.34×107个VD2。普通PCR结果也反应出了类似的病毒释放动态。【结论】根据BmBDV感染家蚕后的释放动态，推测病毒的复制周期约60 h。利用实时荧光定量PCR检测蚕沙中的BmBDV，能简单、快速、准确地诊断病毒的感染。

Abstract:

[Objective] In order to investigate the amount of virus released from silkworm infected by Bombyx mori bidensovirus (BmBDV) at the different periods and to develop a method for rapid and accurate detection whether silkworm were infected by BmBDV or not. [Methods] A small fragment of ns1 gene of genomic segment VD1 and ns3 gene of VD2 were cloned to pMD18-T vector respectively, and then the standard curve of ns1 and ns3 was obtained. The release dynamics of BmBDV were investigated by detecting the amount of BmBDV in the faeces of 5th instar silkworm at different infection stage. [Results] The results shown that a lot of virus were detected in silkworm faeces at the 3 days post-infected about 1.38×106 copies/μg VD1, 6.54×105 copies/μg VD2. The amount of virus increased exponentially from 5 days post-infected. The amount of BmBDV reached a plateau at 7 to 8 days post-infected about 2.12×107 copies/μg VD1, 1.34×107 copies/μg VD2. The PCR results also indicated a similar dynamics. [Conclusion] The release dynamics of virus suggests that the replication cycle of BmBDV is about 60 h. The infection of BmBDV in silkworm can rapidly, simply and accurately be determined by detecting the virus in silkworm faeces using real-time quantitative PCR.