The Studies Of Molecular Epidemiology And Host Specificity Of Bombyx Mori Nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a severe pathogen that lethally infects the Bombyx mori silkworm. BmNPV disease is a common but quite destructive viral disease in sericulture. Guangxi is located in southern China, where the subtropical climate is perfect for mulberry cultivation and silkworm husbandry, and the sericulture industry in Guangxi has been developed extensively during the past few years. However, due to the high raising density and long production period, the prevalence of silkworm diseases especially viral epidemics are likely to happen. The BmNPV epidemic takes place almost every year in Guangxi, and recently becomes more serious resulting from the large-scale development of sericulture and the accumulation of the pathogen in the silkworm-raising regions in Guangxi. This intractable disease has been caused considerable economic losses in Guangxi sericulture industry. In order to keep sustainable development of sericulture industry in Guangxi, it is essential to perform an investigation on the epidemic condition of BmNPV in Guangxi and make a better understanding of the BmNPV transmission, which will help to devise strategies for preventing this epidemic disease to spread widely in the silkworm-raising regions.In this study,45wild BmNPV isolates were collected from different silkworm-raising regions in China’s Guangxi Zhuang Autonomous Region. Two highly expressed very late genes from each isolate, polh and p10, were sequenced and subjected to phylogenetic analysis. The polh gene was found to be highly conserved, while the p10gene was more variable frequently harboring point mutations and displaying variations in codon use without obvious codon bias. The BmNPV isolates from Guangxi were separated into three main clades, Ⅰ, Ⅱ and Ⅲ,according to the p10gene phylogenetic tree. The geographical distribution of clade Ⅰ isolates in Guangxi showed a concentrated pattern and that of clade Ⅱ isolates showed a connected pattern. Local transmission of this pathogen clearly occurred in the silkworm-raising regions in Guangxi. And clade Ⅲ isolates were irregularly scattered throughout Guangxi, which suggested long-distance transmission may happen. This study may provide some data on BmNPV transmission in the silkworm-raising regions and be helpful in devising strategies for the prevention and control of BmNPV disease.There is quite a rich resource of silkworm production in Guangxi. Silkworm serves as an ideal bioreactor for abundantly producing recombinant protein by using a baculovirus expression system. Rabies virus glycoprotein (RVG) is the major protective antigen which can induce neutralizing antibody against lethal rabies virus infection. In this study, the complete open reading frame (ORF) of RVG gene was cloned and inserted into a modified bacmid/BmNPV deleting both chitinase and cysteine-protease genes. A mammalian cell-activate promoter cytomegalovirus immediate-early promoter-enhancer (CMV-IE) was also constructed into the bacmid. The recombinant baculovirus was generated by syringe injection of the silkworm larvae hemocoel with the recombinant bacmid and Cellfectin Reagent mixture. Results showed that recombinant RVG was highly expressed in the tissues of recombinant baculovirus infected silkworm larvae. The recombinant RVG was displayed on the membrane surface and its molecular weight was slightly smaller than that of the natural RVG expressed in mammalian cells. But the reacting patterns of anti-RVG monoclonal antibodies with the recombinant RVG were almost the same as those with the natural RVG, suggested that the structure and function of the recombinant RVG were intact.Studies on BmNPV host specificity have shown that this virus possess relatively narrow host ranges both in vivo and in vitro. Spodoptera frugiperda cell line Sf9was classically considered to be nonpermissive for BmNPV proliferation. In this study, two recombinant BmNPV carrying exogenous reporter gene, the recombinant rBm-PCMV-RVG carrying RVG gene and CMV-IE promoter sequence and the recombinant rBm-GFP carrying green fluorescent protein (GFP) gene, were constructed and showed infectivity in Sf9cells. There was no obvious cytopathic effect in the Sf9cells infected with recombinant BmNPV, showing an inapparent and mild pattern of infection. Low-pH-triggered envelope fusion was detected in the infected Sf9cells. When inoculating Sf9cells with rBm-GFP, virus budding was confirmed by the formation of GFP fluorescent focus and the increase of budded virion yield in Sf9cells. However, the viral replication only occured with high inoculation dose and showed a pattern different from that in permissive BmN cells. To the contrary, low inoculation dose led to abortive infection. The rBm-GFP after serial passages in Sf9cells showed more efficient replication than the original virus without serial passage.According to the sequences of Bombyx mori silkworm antiviral protein genes Bmlipase-1(lipase) and BmSP-2(serine protease) published in Genbank, specific primers were designed to clone the complete ORF of the two genes from Guangxi excellent silkworm race "Liangguang NO.2". The cloned products were sequenced and analyzed with the homologs derived from other silkworm species. The results show that Bmlipase-1is composed of885bp nucleotides coding294amino acids and BmSP-2is composed of855bp nucleotides coding284amino acids. Sequence analysis reveal that the two genes from "Liangguang NO.2" shared more than92%homology with those from different Bombyx mori races even Bombyx mandarina and Samia cynthia ricini, especially for Bmlipase-1which reached more than99%homology. Amino acid residues for the lipase active site of Bmlipase-1and serine protease catalytic triad of BmSP-2are identical among these gene sequences. The data above suggested that these two antiviral genes are highly conserved during silkworm species genetic evolution and may play important roles in immune defence as well as food digestion. Then the cloned genes were ligated into protokaryon expression vector pET32a(+) and expressed in E.coli BL21. Fused proteins were detected by SDS-PAGE and Western blot. According to the result, the protein mass of expressed Bmlipase-1is about47kD and BmSP-2is about42kD, which consisted with the anticipation.