Quantitative analysis of cytoplasmic actin gene promoter and nuclear polyhedrosis virus immediate-early promoter activities in various tissues of silkworm Bombyx mori using recombinant Autographa californica nuclear polyhedrosis virus as vector

Cassettes harboring luciferase reporter driven by Bombyx mori cytoplasmic actin gene promoter (A3) (671 bp) and B. mori nuclear polyhedrosis virus immediate-early promoter (IE-1) (580 bp) were transferred to the bacmid AcΔEGT to generate the recombinant Autographa californica nuclear polyhedrosis viruses, AcNPVA3Luc and AcNPVIELuc, respectively. Recombinant baculoviruses were injected into the hemocoele of newly ecdysed 5th instar larvae. The activities of the A3 and IE-1 promoters in various tissues were measured by luciferase activity assay and normalized by the copy number of recombinant virus. Results showed that the activity of the A3 promoter was approximately 10-fold higher than the IE-1 promoter. The promoter activities of A3 and IE-1 were highest in the silk gland, followed by fat body, middle gut, Malpighian tubule, and hemocyte. In silk gland, activity of the two promoters was highest in posterior silk gland, followed by middle and anterior silk glands. The difference in promoter activities reflects the growth speed of tissue in silkworm larvae. The activity of the A3 promoter remained unchanged and was not inhibited significantly by viral factors at least 3–4 d post injection of rAcNPV.     

牛痘病毒感染人巨噬细胞基因表达谱的RNA-Seq分析

[目的]利用RNA-Seq,分析人巨噬细胞在牛痘病毒(VACV)感染前后基因表达的变化,探索牛痘病毒与宿主细胞相互作用的机制.[方法]用牛痘病毒感染人巨噬细胞,采用RNA-Seq比较感染组和对照组的差异表达基因,并进行KEGG、GO以及STRING网络分析.[结果]感染组与对照组相比,筛选出显著性差异表达基因4796个...     

Isolation and reconstitution of the RNA replicase of the cytoplasmic polyhedrosis virus of silkworm,Bombyx mori

Summary After irradiation of the virus particles of CPV, the RNA replicase associated with the virion was isolated in the form of a genome-replicase complex with DEAE-Sephadex A-25 chromatography. This complex was then treated with Triton X-100 and purified by phosphocellulose column chromatography. The RNA replicase reconstituted with the doublestranded RNA of CPV showed both the enzyme activity of RNA polymerase and methyltransferase. The single-stranded RNA could not serve as the template for the RNA replicase. The role of the RNA replicase of CPV is discussed.     

昆虫质型多角体病毒的研究进展

质型多角体病毒(Cytoplasmic polyhedrosis virus,CPV)隶属呼肠孤病毒科Reoviridae质型多角体病毒属Cypovirus,通常基因组由10个节段双链RNA构成。RNA分子量为3～27u。根据病毒基因组dsRNA片段在聚丙烯酰胺或琼脂糖凝胶中电泳图谱的差异,目前CPV已被分为19个电泳型。不同于呼肠孤病毒科其它成员,CPV为单层衣壳,而不是常见的双层衣壳结构,衣壳蛋白主要由衣壳蛋白、大突起蛋白及塔式突起蛋白组成。大部分质型多角体病毒引起昆虫慢性疾病,造成寄主死亡或适应性降低。随着RNA病毒基因序列测定技术的成熟,质型多角体病毒的序列测定方面取得较大进展,目前GenBank核苷酸序列数据库中已经公布了家蚕Bombyx mori CPV电泳型1两个株系(H株和I株)、舞毒蛾Lymantria dispar CPV电泳型1、舞毒蛾CPV电泳型14及粉纹夜蛾Trichoplusia ni CPV电泳型全基因组序列,为该病毒的进化与起源的研究提供更多的遗传信息。本文从结构功能、侵染特点、基因组特点及应用前景等方面综述了昆虫质型多角体病毒的研究进展。     

Virus-specific RNA synthesis in the midgut of silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

Virus-specific RNA synthesis in the midgut of silkworm infected with cytoplasmic-polyhedrosis virus was investigated under the condition inhibiting host RNA synthesis by actinomycin D injection. Two species of virus-induced RNA were formed; one was sensitive to ribonuclease (RNase) but the other was resistant. The resistant RNA had a sedimentation coefficient of 15 S and was considered as viral progeny with doublestranded RNA. The sensitive RNA, presumably single-stranded RNA, consisted of two classes with 15 S and 22 S sedimentation coefficients. Annealing the single-stranded RNA with heat-denatured CPV-RNA indicated that the single-stranded RNA was transcribed from viral genome RNA. The function of 22 S and 15 S single-stranded RNAs was discussed from the viewpoint of virus multiplication.     

Quantitative analysis of the metabolism in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

The rate of oxygen uptake and the amount of several main cell constituents were determined in NPV-infected and uninfected isolated pupal abdomens of the silkworm, Bombyx mori. The oxygen uptake in uninfected isolated abdomens increases for the first 2 days due to the effect of water injection, but thereafter it remains almost unchanged at a relatively low level. When isolated abdomens are infected with an NPV, the oxygen uptake increases markedly from 3 days postinfection onward, being about three times that of uninfected isolated abdomens at late stages of infection. The amounts of cell constituents analyzed in the present study change little in uninfected isolated abdomens throughout the experiment. Infection of NPV leads to a marked accumulation of both DNA and RNA, and a loss of glycogen. The amount of protein, lipid, and sugar are scarcely affected by NPV infection. These results indicate that the activity of metabolism in uninfected isolated abdomens is not only low but also stable, and that infection of NPV results in an activation of host cell metabolism. It is considered that such isolated pupal abdomens provide an excellent opportunity to analyze both the process of NPV replication and the alteration of host cell metabolism resulting from NPV infection.     

Difference of proteins from inclusion bodies formed in the nucleus and cytoplasm of the cytoplasmic polyhedrosis virus-infected midgut in the silkworm,Bombyx mori

Strains of cytoplasmic polyhedrosis virus (CPV) of the silkworm Bombyx mori typically form proteinaceous inclusion bodies (IBs) which occlude many virions and are formed in the cytoplasm of the midgut epithelium. In contrast, an unusual strain of CPV termed “A” strain produces IBs containing no virions in the nuclei of the epithelial cells. In this case although the viruses multiply in the cytoplasm, few IBs are formed in the cytoplasm. To clarify why the A strain forms IBs in the nucleus, the structural differences on the IB proteins (IBPs) from A and a typical (H) strain were investigated. Analyses by SDS-PAGE showed A strain IBP had slightly lower electrophoretic mobility than these of H strain. When these IBPs were partially digested with Staphylococcus aureus V8 protease, one of the digested products between the two strains showed different electrophoretic mobility. Amino acid sequence analyses of peptides produced with lysylendopeptidase from IBP of A strain indicated that a histidine residue of H strain was replaced by a tyrosine residue. The carboxyl terminal regions of the two IBPs were also different; IBP of A strain was -Leu-Leu-Val-COOH, but the terminal residue of H strain could not be determined by the same method. These differences of amino acid sequence of IBPs between A and H strains may be responsible for the partitioning of them; i.e., in the case of A strain IBP may be transportable into the nucleus by the specific signal of amino acid sequence.     

Highly infectious free virions in the hemolymph of the silkworm (Bombyx mori) infected with a nuclear polyhedrosis virus

Density gradient centrifugation and electron microscope studies on the free virions of a nuclear polyhedrosis virus in the infected hemolymph of the silkworm showed that the free virus particle was a virion without an envelope. Only the nucleocapsid was observed in the highly infectious fraction of the infected hemolymph. The unenveloped virion in the hemolymph was nearly a hundred times more infectious than the enveloped virion, liberated from the polyhedra, when hemocoelic inoculation was employed. It was estimated that more than 80% of the infectivitiy in the infected hemolymph was due to the unenveloped virion.     

Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility

Digital gene expression (DGE) was performed to investigate the gene expression profiles of 4008 and p50 silkworm strains at 48 h after oral infection with BmCPV. 3,668,437 clean tags were identified in the BmCPV-infected p50 silkworms and 3,540,790 clean tags in the control p50. By contrast, 4,498,263 clean tags were identified in the BmCPV-infected 4008 silkworms and 4,164,250 clean tags in the control 4008. A total of 691 differentially expressed genes were detected in the infected 4008 DGE library and 185 were detected in the infected p50 DGE library, respectively. The expression profiles identified some important differentially expressed genes involved in signal transduction, enzyme activity and apoptotic changes, some of which were verified using quantitative real-time PCR (qRT-PCR). These results provide important clues on the molecular mechanism of BmCPV invasion and resistance mechanism of silkworms against BmCPV infection.     

Oxygen uptake in tobacco budworm larvae (Heliothis virescens) infected with cytoplasmic polyhedrosis virus

We measured oxygen uptake in 3- to 13-day-old Heliothis virescens (tobacco budworm) larvae. The “test” group was infected with cytoplasmic polyhedrosis virus (CPV) while the “control” group was not. The healthy budworms had an average oxygen uptake of 7.64 μl of O₂/mg body weight, while those infected with CPV had an average uptake of 47.11 μl of O₂/mg body weight/hr. The weights of larvae from the two groups were likewise recorded. Budworms from the “control” group showed an average weight of 186.9 mg, while larvae infected with CPV had an average weight of 18.3 mg.