Genome Sequence of a Bombyx mori Nucleopolyhedrovirus Strain with Cubic Occlusion Bodies

Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical species of Baculoviridae. The complete genome sequence of a BmNPV strain with cubic occlusion bodies is reported here. The genome of this strain consists of 127,465 nucleotides with a G+C content of 40.36% and is 97.3% and 97.5% identical to those of BmNPV strain T3 and Bombyx mandarina NPV S1, respectively. Despite the abnormal polyhedra it forms, the polyhedrin gene of the BmNPV cubic strain is 100% identical to those of the other two strains. Baculovirus repeated ORFs and homologous repeat regions cause the major differences in genome size of these BmNPV isolates.     

Genomic Sequence Analysis of Bombyx mori Nucleopolyhedrovirus Isolated from Yunnan Sericulture Region,China

The blood pathogens of grasserie caused by Bombyx mori nucleopolyhedrovirus BmNPV have a serious impact on the sericulture industry. To understand the genetic status of BmNPV endemic strains in the Yunnan sericulture region, the structure and complete genome sequence of BmNPV isolated from Baoshan city of Yunnan Province were described and compared to known strains. The BmNPV-Baoshan isolate was a nucleopolyhedrovirus parasitized in silkworm larvae. Its genome has 128, 452 bp with a G + C content of 40.4%. Phylogenetic analysis clustered the virus with China BmNPV isolates; BmNPV-Baoshan was closely related to BomaNPV-S1 (both strains originated from the same ancestor). BmNPV-Baoshan strain has bro-b gene deletion, hr1 missing 4 repeat units of 30-bp palindrome structure compared to BmNPV-T3 strain. The aim of this study was to elucidate the evolution of the virus further and provide insights for the protection of virus-induced hematologic sepsis.     

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often eoevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Acl6 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac 16 interacts with baculoviral and cellular proteins. Bm8/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group INPVs.     

Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"L33180","term_id":"3745835","term_text":"L33180"}}L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

Nucleopolyhedroviruses (NPVs) have large (80- to 180-kb) circular double-stranded DNA (dsDNA) genomes, which replicate in nuclei of infected cells. Despite the widespread use of NPVs for the expression of foreign genes and their potential for pest control, little is known about the mechanism of their replication and the properties of their replication factors. The most widely studied baculovirus, Autographa californica multicapsid NPV (AcMNPV), has the potential to encode about 150 proteins (3), including factors required for virus DNA replication. The products of nine viral genes (ie-1, ie-2, lef-1, lef-2, lef-3, dnahel, dnapol, p35, and lef-7 or pe-38) are necessary and sufficient for efficient replication of transfected plasmid DNAs containing a putative baculovirus replication origin (16, 22). It is likely that DNA polymerase and DNA helicase, which are encoded by the viral genes dnapol and dnahel, respectively (20, 35), form a core of the virus DNA replication machinery. The roles of other factors are less obvious. Single-stranded DNA binding (SSB) protein function was proposed for the protein LEF-3, which binds specifically single-stranded DNA (ssDNA) (10, 14). However, direct proof for the SSB function of LEF-3 in viral DNA replication is lacking. In addition, SSB function was also suggested for LEF-7 on the basis of its predicted amino acid sequence (22). It was recently demonstrated that LEF-1 forms a complex with LEF-2 and may serve as a DNA primase (9). The function of IE-1, IE-2, and PE-38 may result from their ability to activate in trans expression of other genes required for virus replication. The transactivator IE-1 may also participate in the initiation of DNA replication, due to its ability to bind putative replication origins (7, 13, 17, 33). P35 is an inhibitor of apoptosis and may not be involved directly in DNA replication. Its stimulatory effect in the transient-replication assay may result from inhibition of virus-induced apoptosis in cells transfected with the replication genes. Several genes required for DNA replication (six essential and three stimulatory) were also identified in the genome of Orgyia pseudotsugata NPV (1). Homology of these genes to those required for replication of AcMNPV suggests similar replication mechanisms for the two viruses. The genome organization of the Bombyx mori NPV (BmNPV) closely resembles that of AcMNPV. Nineteen homologs of the AcMNPV late expression factor genes (lef genes) were identified in BmNPV (12). At least three of these, ie-2, lef7, and p35, are not essential for virus DNA replication as demonstrated by deletion analysis (12). Because the daughter DNA molecules synthesized under control of the nine essential viral genes appear to be synthesized as concatemers (16, 22, 31, 32), factors required for maturation of nascent DNA and its further processing are still unknown. Although the nine AcMNPV factors were sufficient for efficient DNA replication in Sf cells, an additional viral gene, designated hcf-1, was essential for replication in TN-368 cells (21), indicating dependence of the transient assay on host cell-specific factors. Few proteins involved in NPV DNA replication have been purified from infected cells and characterized in cell-free systems. Among them are AcMNPV DNA polymerase (28, 37), BmNPV DNA polymerase (27), AcMNPV DNA helicase (19), and AcMNPV LEF-3 (10, 14). Isolation of other replication proteins of NPVs is still anticipated.In this report we describe the purification of a viral DNA-binding protein (designated DBP) from BmNPV-infected cells. DBP binds preferentially to ssDNA and is capable of unwinding duplex DNA. The BmNPV open reading frame (ORF) encoding DBP (dbp gene) is a homolog of AcMNPV ORF25, whose product has not been identified so far.     

Propagation of Bombyx mori Nucleopolyhedrovirus in nonpermissive insect cell lines

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via beta-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.     

V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus

Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.     

家蚕核型多角体病毒广东株bro基因家族分析

对BmNPV广东株进行了空斑纯化,并对该基因组的bro基因家族进行克隆,获得4个bro基因序列(bro-a、bc、、d),与GenBank数据库中BmNPVbro基因序列及本实验室测定的重庆株的bro基因序列进行比较分析,结果表明广东株BmNPVbro基因存在插入及缺失,其氨基酸的改变主要发生在对应蛋白的N端部分;同时进行的bro基因的系统发生分析表明,广东株bro基因分别位于3个亚组中,广东株bro-d与日本T3、重庆CQ1株bro-d以及法国SC7株bro-Ⅲ属于亚组A,广东株bro-a、c与T3、CQ1株bro-a、c以及SC7株bro-Ⅱ属于亚组B,广东株bro-b与T3、CQ1株bro-b、e以及SC7株bro-Ⅰ属于亚组C,bro基因进化与地理位置的相关性不明显。根据bro基因在四个不同株系基因组中的位置特征,进一步支持了Kang等的观点:bro-d是BmNPV生存所必需的,bro-a和bro-c相互间功能互补。同时推测:SC7株的3个bro基因可能是BmNPVbro家族出现的最简约形式。     

零背景转座技术高效构建重组家蚕杆状病毒表达系统

摘要：【目的】获得零转座背景的基于家蚕核型多角体病毒(Bombyx mori Nucleopolyhedrovirus, BmNPV) Bac-to-Bac 系统,为高效经济构建重组BmNPV在家蚕体内表达目标蛋白提供新系统。【方法】利用R6Kγ作为复制子构建新的条件复制型杆状病毒转移载体pRADM,同时封闭BmNPV-Bacmid（BmBacmid）宿主菌（Escherichia coli BmDH10Bac）的Tn7转座受体位点attTn7,获得新的封闭型宿主菌E.coli BmDH10Bac△Tn7。【结果】由于pRADM无法在宿主菌E.coli BmDH10Bac中复制,封闭了attTn7位点的宿主菌也不能再和BmBacmid竞争与转移载体的重组,显著提高了转座效率。封闭宿主菌的attTn7位点,能使转座效率提高近4倍,使用条件复制型转座载体pRADM时,转座效率提高近10倍。而用pRADM转座E.coli BmDH10Bac△Tn7时,转座阳性率为100％。避免了获得重组病毒DNA的鉴定程序,缩短了获得重组蛋白所需时间。用携带红色荧光蛋白基因DsRed的重组质粒pRADM-Red转座E.coli BmDH10Bac△Tn7,获得重组BmBacmid转染BmN细胞,红色荧光蛋白在细胞中得到高效表达。【结论】结果表明pRADM和E.coli BmDH10Bac△Tn7是一种零背景高效构建重组BmNPV的新系统。     

利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒(BmNPV)bacmid在大肠杆菌BW25113中快速地敲除BmNPVorf60基因。从大肠杆菌BmDH10Bac中提取BmNPVbacmid,将其电转化到含有质粒pKD46(能表达Red重组酶)的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物(5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因(cat)的首尾序列),以pKD3质粒(含cat)为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPVbacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPVorf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

家蚕核型多角体病毒BmNPV囊膜蛋白P74膜外区克隆和原核表达

通过PCR扩增家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)囊膜蛋白p74基因膜外区片段,对切胶纯化得到的DNA目的片段与原核表达载体pET28a进行连接,通过不同浓度的IPTG对含有pET28a-p74重组质粒的大肠杆菌BL21(DE3)进行诱导,对诱导产物进行SDS-PAGE电泳.结果表明p74基因膜外区获得了表达;通过His单抗对原核表达产物进行Western blotting分析,其结果证实诱导蛋白带为融合有组氧酸的目的蛋白.对割胶获得的P74蛋白和免疫佐剂进行克分研磨,以研磨后的匀浆液对昆明小鼠进行皮下多点注射,通过收获的抗血清对BmNPV ODV病毒粒子进行Westem blotting分析,检测到一条分子量大小为74 kD的特异杂交带,表明获得的多抗可用于P74蛋白功能的进一步研究.     

Complete Genome Sequence of Brucella canis Strain 118, a Strain Isolated from Canine

Brucella canis infects several species of animals, and canine is the preferred host. Genome sequences of strains from different hosts are valuable for comparative analysis of host adaptation and microevolution. Here, we report the genome sequence of Brucella canis strain 118, a strain isolated from canine.     

A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.     

A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.     

Draft Genome Sequence of Cecembia lonarensis Strain LW9T,Isolated from Lonar Lake,a Haloalkaline Lake in India

The draft genome sequence (4.84 Mb) of Cecembia lonarensis strain LW9^T, isolated from a water sample (4.5-m depth) from Lonar Lake, a meteorite-created haloalkaline lake in India, is reported. The enzymes produced by these microorganisms need to be stable under alkaline conditions prevailing in its habitat. Such enzymes would be of immense importance for enzymatic processes operating at high pH.     

Genome Sequence of a Novel Actinophage PIS136 Isolated from a Strain of Saccharomonospora sp

A wide-host-range bacteriophage (phage) PIS136 was isolated from PA136, a strain of Saccharomonospora belonging to the group actinomycetes. Here, we present the genome sequence of the PIS136 phage, which is 94,870 bp long and contains 132 putative coding sequences and one tRNA gene. An IS element-like region with two genes for putative transposases was identified in the genome. The presence of IS element-like sequences suggests that PIS136 is still under active evolution.     

家蚕肠道纤维素酶菌株的筛选及产酶条件的优化研究

从5龄家蚕肠道分离筛选得到一株产纤维素酶菌株BMC-2,以羧甲基纤维素酶(CMCase)比活力为响应值,通过Plackett-Burman试验设计、最陡爬坡试验和Box-Behnken试验设计对菌株BMC-2产纤维素酶发酵条件进行优化,结果表明,发酵时间、发酵温度、培养基初始pH和转速对CMCase比活力具有显著影响,其影响程度由大到小依次为发酵时间、培养基初始pH、发酵温度、转速.确定菌株BMC-2产纤维素酶最优发酵条件为:发酵时间94.35 h,发酵温度30.3℃,培养基初始pH 7.01,转速179 r/min.在此条件下, CMCase比活力理论值为25.801 U/mg,验证值为25.526 U/mg,较产酶条件优化前提高了1倍,预测模型可靠性高,可应用于菌株BMC-2产纤维素酶条件的优化.