Studies On Proliferation And Structural Protein Of Bombyx Mori Densovirus (China Isolate)

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate) ,named BmDNV-3, is a kind of bidensovirus. Similar to the typical phenomenon in the host of silkworm that was infected by the BmDNV-1(Ina Isolate), the BmDNV-3 also infected the columnar cells of midgut epithelium and caused flacherie disease of silkworm. 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(VDl,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 was collected at 9 time points. The silkworm cytoplasm actin A3 was used to be normalized gene, so the number of cells in collected tissue could be determined. The special primers according VD1 or VD2 were devised respectively and fluorescence quantitative PCR amplification was used to detect the copies of VD1 or VD2 in collected tissue at different time points. On the other hand, we identified the structural protein of BmDNV-3 through Gel electrophoresis and mass spectrometry analysis. The result shows that:1. VD1 and VD2 of BmDNV-3 were replicated with synchronization .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 time point were almost at the equal level respectively, so that the VD1 and VD2 were replicated with synchronization. This synchronization may be having relationship with their common terminal sequence and encoding DNA polymerase by itself.2. BmDNV-3 could enter the columnar cells of midgut epithelium in the resistant silkworm strain and it could replicate slowly. In the susceptible silkworm strain the virus amplified and in the stationary phase (96 hours post inoculation) the copies of virus were about 20,000. In the resistant silkworm strain the virus amplified and in the stationary phase the copies of virus were about 150~200.The result showed that BmDNV-3 could enter the columnar cells of midgut epithelium in the resistant silkworm strain and the virus were replicated at a very low level. It couldn't affect the normal physiological activities of host Cells .So we predicted 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.3 The number of VD1 and VD2 in the mature virus was different. Through the fluorescence real-time PCR the Ct value of VD1 and VD2 in the inoculation were 9.38 and 9.67. By the standard curve and calculation with dilution the copies of VD1 and VD2 in the inoculation were VD1 5.04×10~6 copies/μL, VD2 6.43×10~6 copies/μL .So in the mature virus, VD1: VD2=1: 1.28.4. We used a new kind of method, that was inoculating the silkworm strain determinate dose of BmDNV-3 by oral ingestion. By the new method, the silkworm can uptake enough virus in 1 minute and in normal way it needs 24 hours. The new method cut the time in dilution. All the virus could enter the body of silkworm, infect the columnar cells of midgut epithelium, express the gene and replication in the same time. The precise analysis on the virus replication in the cell at the level of individual was possible by the new method.5 By Gel electrophoresis, Bm-DNV-3 had 6 structural protein,they were VP1(48kDa),VP2(50kDa ),VP3(53kDa ),VP4(55 kDa),VP5 (72 kDa) and VP6 (97 kDa) . VP1, VP2 and VP3 were encoded by VD1-ORF3.We conferred that VP4 was Glucosidase- Bombyx mori, VP5 was Arylphorin. - Bombyx mori and vp6 had relationship with DNA-directed RNA polymerase.