Phylogenetic Analysis of Orgyia pseudotsugata Single-nucleocapsid Nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata （Lepidoptera： Lymantriidae） is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus （SNPV） isolated from O. pseudotsugata larvae in Canada （OpSNPV） was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group Ⅱ NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses.     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene.B.mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo.The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E.coli DH10B.Recombinant bacmids were screened by kanamycin resistance,PCR and restriction enzyme(REN)digestion.One of the bacmid colonies,BmBacJS13,which had similar REN profiles to that of wild-type BmNPV,was selected for further research.To investigate the infectivity of BmBacJS13,the polyhedrin gene was introduced into the bacmid and the resultant recombinant(BmBacJS13-ph)was transfected to BmN cells.The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells.Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV.Bio-assays indicated that BmBacJS13-ph was also infectious to B.mori larvae.     

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.     

Listeria monocytogenes Mutants Carrying Newcastle Disease Virus F Gene Fused to its actA and plcB： In vitro Expression and Immunogenicity in Chickens

Recombinant Listeria monocytogenes mutants carrying Newcastle disease virus (NDV) fusionprotein gene F were constructed by homologous recombination.NDV F or its truncated fragment Fa wasused as the model heterologous gene to be integrated into actA or plcB downstream of their signal sequences.Correct orientation of the inserted genes was verified by polymerase chain reaction amplification of F or Fa.The inserted F and Fa were expressed in the two recombinants Lm-AactA-F and Lm-AplcB-Fa as shown bysodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Both recombinantsexhibited reduced virulence to embryonated eggs and mice by about 1.5-2.5 logs as compared with theparent wild strain 10403S. They were also less invasive than strain 10403S (P<0.05). Chickens receiving therecombinant strains orally or intraperitoneally were partially protected from virulent NDV challenge possiblydue to enhancement of non-specific immunity because the antibody titers against the homologous virus strainor the recombinant truncated fusion protein were marginal. Further research is needed in other animal modelsto see if the low antibody response results from insufficient expression of the heterologous genes as a resultof failure of L. monocytogenes or its recombinants to persist or replicate in chickens.     

一种改良的对虾白斑综合征病毒的提纯技术

The envelope proteins of White spot syndrome virus (WSSV) are very fragile and easy to be destroyed during purification. It was difficult to obtain a large quantity of intact virions by routine sucrose gradient centrifugation. After modifying the sucrose gradient by adding citrate sodium, we can obtain a large quantity of intact virions and nucleocapsids. This purified virions and nucleocapsids were subsequently used for analyzing viral structural proteins and DNA extraction. The result showed that this modified techniaue is very efficient for virus purification.     

油桐尺蠖核型多角体病毒多角体蛋白的理化性质及二级结构预测

由SDS及梯度胶电泳测得油桐尺蠖核型多角体病毒（BsNPV）多角体蛋白天然状态及亚基分子量分别为363kD与31.5kD,从而推断此蛋白为十二聚体,亚基间无二硫键作用．BsNPV多角体蛋白的远紫外圆二色谱显示,它的二级结构含有31．7％的α螺旋,23．8％的β折叠及44．5％的无规卷曲,与二级结构预测结果相符．通过荧光光谱实验推知,BsNPV多角体蛋白的表面疏水性弱,其色氨酸残基位于蛋白疏水核内部．     

Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra.

We compared the DNA sequence of the Autographa californica nuclear polyhedrosis virus polyhedrin gene with that of the polyhedrin gene from a morphology mutant called M5. A single point mutation was found at the BamHI restriction site within the polyhedrin coding sequence. This point mutation caused a substitution of leucine for proline at amino acid 58 in the M5 polyhedrin. This point mutation was shown to be responsible for both the appearance of cubic polyhedra and the altered mobility of the polypeptide on sodium dodecyl sulfate-polyacrylamide gels by transferring the M5 polyhedrin gene to the wild-type virus by cotransfection. Recombinants were found which assembled cubic polyhedra in infected cells, had the BamHI restriction site missing, and had an altered mobility of their polyhedrin polypeptide. Computed-predicted secondary-structure analysis indicated that the amino acid at position 58 could be critical to the proper folding of polyhedrin.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus

To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus.     

Alkaline protease associated with virus particles of a nuclear polyhedrosis virus: assay, purification, and properties

Proteolytic activity was detected within polyhedra of the nuclear polyhedrosis virus of Spodoptera littoralis. The enzyme activity was detected by its ability to degrade the major structural polypeptide of polyhedra (polyhedrin). A quantitative assessment of activity was made by a radioassay technique using ³H-labeled polyhedrin as the substrate. Of the structural components of polyhedra, virus particles showed the greatest specific proteolytic activity. Preparations of purified nucleocapsids were inactive. The virus particle enzyme displayed a temperature optimum for proteolysis of 30 to 40°C and a pH optimum of 9.6. Its activity was inhibited by H²⁺ and Cu²⁺, but not by 2-mercaptoethanol. The enzyme was purified from detergent-treated virus particles by affinity column chromatography, using polyhedrin linked to cyanogen bromide-activated Sepharose. Three major envelope polypeptides (L107, L85, and L71) bound to the column at 4°C, but after incubation at 31°C, polypeptide L71 alone was eluted. The fractions containing this protein exhibited a specific enzyme activity more than 80-fold greater than that present in polyhedra. The possible significance of the alkaline protease, and other proteins with affinity for polyhedrin, is discussed.     

Crystal lattice as biological phenotype for insect viruses

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting viruses.     

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体,与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN,经200μg/ml zeocin抗生素筛选一个月,成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒,其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染,结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主,却并不在宿主体内形成包涵体,从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题,通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。