家蚕浓核病毒 (中国镇江株)在不同感受性宿主体内的复制

Replication of Bombyx mori Densonucleosis Virus (Zhenjiang isolate) in Different Silkworm Strains

摘要:

家蚕浓核病毒中国镇江株是一株双生浓核病毒(bidensovirus)。其宿主感染后的病症与典型的家蚕浓核病毒（BmDNV-1 伊那株）表现相似，病蚕软化，中肠的圆筒型细胞呈浓核症。该病毒的最大特点是基因组中含有二套DNA分子( VD1, VD2)，这两种核酸分子以单链（+VD1, -VD1,+ VD2, -VD2）线型方式被分开包装在各自的衣壳蛋白中，成为四种病毒体，而且它自身编码DNA聚合酶。有部分蚕品种对该病毒表现完全抗性，即不发病。分别对敏感性家蚕品种(华八35)和抗性家蚕品种(秋丰d)的幼虫进行经口接种病毒。在接种后，从2h到96h分9个时间点，对中肠组织进行取样。以家蚕细胞质肌动蛋白A3(actin A3)基因作为参比基因，用来标定取样组织细胞数。针对VD1和VD2分别设计特异引物，用荧光定量PCR的方法分别检测各个时间点的样品中的病毒基因组VD1和VD2拷贝数。结果表明：无论是在感性还是在抗性宿主体内，家蚕浓核病毒中国株的基因组 VD1和 VD2在各时间点拷贝数相近，表现出VD1和 VD2是同步复制的；病毒侵入两种宿主中肠的初始量（接种后2h）基本相等，每个细胞约为6～10拷贝数。在敏感性宿主体内病毒感染过程表现为潜伏期，指数增长期，平台期。从接种后2h到12h为病毒潜伏期；12h到36h为指数增长期，倍增时间为1.71h，大约扩增15次；36h到96h为平台期，进入平台期病毒的拷贝数达到20万个。在抗性宿主体内病毒处于一种极低水平的增殖，从添毒后2h的6～10拷贝数到96h的150～200拷贝数, 病毒复制倍增时间分别为3h和12h，大约扩增5次。推测家蚕对浓核病毒中国株的抗病性，只是一种慢性的带毒不发病的表现。

Abstract:

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3（Zhenjiang isolate）,named BmDNV-3，is a kind of bidensovirus. The most obvious characteristic in the genome of BmDNV-3 is that it has 2 sets of DNA molecular (VD1, VD2),and each of them is encapsidated respectively in the form of single-stranded liner DNA (+VD1,-VD1,+VD2,-VD2) in equal percentage. So the BmDNV-3 has 4 kinds of virions. Furthermore the sequence of BmDNV-3 is able to encode DNA polymerase itself. Some strains of silkworm revealed complete resistance to BmDNV-3, so they didn't fall sick. To investigate the difference in the process of infection and replication between the 2 virions(VD1,VD2) of this bidensovirus, and the difference of the increment in the resistant or susceptible host, the 5th instar larvae of the susceptible silkworm strain (HUABA 35 )and the resistant silkworm strain(QIUFENG d) were inoculated determinate dose of BmDNV-3 by oral ingestion. Then the midgut were collected at 9 timepoints. The silkworm cytoplasm actin A3 was used to be normalized gene, so the number of cells in collected tissue could be determined. The result shows that whatever in the susceptible silkworm strain or in the resistant one, the copies of VD1 and VD2 in the genome of BmDNV-3 collected at the different timepoint were almost at the equal level respectively, so that the VD1 and VD2 were replicated with synchronization. The process of infection in the susceptible silkworm strain was devided into 3 partitions, latent period(2～12 hours post inoculation), exponential phase (12～36 hours post inoculation)and stationary phase (36～96 hours post inoculation and there are about 2×10⁵ copies per cell). In the resistant silkworm strain, the virus were replicated at a very low level, that was from 6～10 copies 2 hours post inoculation to 150～200 copies 96 hours post inoculation (about 20 times). So we predict that the resistance in some of the silkworm strains from BmDNV-3 was a kind of chronic representation that the host carried virus without being caused flacherie.