家蚕对马尾松毛虫质型多角体病毒的敏感性

用虫体克隆技术,对马尾松毛虫质型多角体病毒湖南株（DpCPV-HN）进行了分离纯化,鉴定为质型多角体病毒1型。以家蚕春蕾×镇珠杂种F₁代及自交的F₂代4或5日龄幼虫进行毒力测定,以纯化的家蚕质型多角体病毒对F₁代幼虫的毒力测定为对照。结果表明：家蚕品种春蕾×镇珠对家蚕质型多角体病毒敏感,马尾松毛虫质型多角体病毒湖南株能引起其感染发病;马尾松毛虫质型多角体病毒湖南株感染家蚕品种春蕾×镇珠F₁代幼虫和F₂代幼虫28天后的半致死剂量（LD₅₀）分别为885个和18个CPB(质多角体),前者为后者的49倍。马尾松毛虫质型多角体病毒湖南株感染后的家蚕,其结茧率、化蛹率、羽化率、全茧量、茧层量和单蛾产卵数均有所下降,全茧量、茧层量、茧层率和单蛾产卵数与病毒感染剂量之间无显著关联。     

Polyhedrin gene of Bombyx mori nuclear polyhedrosis virus.

A portion of the genome of the nuclear polyhedrosis virus of Bombyx mori has been cloned. This part of the viral genome contains the gene encoding the viral occlusion body protein, polyhedrin. The polyhedrin gene has been sequenced in its entirety together with some of its 5' and 3' flanking sequences. The primary structure of polyhedrin predicted from the nucleotide sequence of the gene was found to be somewhat different from the one reported previously for the authentic protein (E. A. Kozlov, T. L. Levitina, N. M. Gusak, and S. B. Serebryani, Bioorg. Khim., 7:1008-1015, 1981; S. B. Serebryani, T. L. Levitina, M. L. Kautsman, Y. L. Radavski, N. M. Gusak, M. N. Ovander, N. V. Sucharenko, and E. A. Kozlov, J. Invertebr. Pathol., 30:442-443, 1977). Comparison of the primary structures of the polyhedrin of the nuclear polyhedrosis virus of B. mori with that of Autographa californica suggests that considerable selective pressure has been exercised at the protein level during evolution. Nucleotide sequence comparisons of the two structural genes reveal that the coding sequences have diverged significantly through the accumulation of silent and replacement substitutions. In contrast, a remarkable degree of sequence conservation was found to exist in the domains corresponding to the 5' and 3' noncoding regions of the polyhedrin mRNAs.     

RNP-complex stoichiometry of Bombyx mori nuclear polyhedrosis virus polyhedra

By gel-filtration through Sephacryl S-300 it was shown that RNP A complex present in polyhedra of Bombyx mori nuclear polyhedrosis virus has molecular weight (M(w)) about 700 kDa. It was shown that RNP A with M(w) 788 kDa is composed of two polyhedrin 13S-associates with M(w) 342 kDa, two p14 polypeptide with M(w) 14 kDa, two 21 kDa small non-coded RNAs and two 17 kDa small non-coded RNAs. The model of RNP A formation from components making it is proposed. The complex role in the course of polyhedron formation and its role in the course of infection are discussed.     

Transgenic breeding of anti-Bombyx mori L. nuclear polyhedrosis virus silkworm Bombyx mori

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.     

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.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Silkworm salivary glands are not susceptible to Bombyx mori nuclear polyhedrosis virus

A nuclear polyhedrosis virus isolated from infected Bombyx mori, BmNPV, was used to inoculate silkworms to determine salivary gland cell susceptibility. The salivary gland was removed from infected silkworms at different times post-inoculation and examined by light microscopy. The salivary gland cells did not exhibit any signs of BmNPV infection; however, fat body and tracheal cells, used as positive controls, showed characteristic cytopathological changes caused by BmNPV infection, which confirmed inoculum viability. The morphological distribution of tracheal branches and the basal lamina, which serves as a barrier to viral penetration, are apparently involved in this resistance to infection.     

Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus.

Homologous regions (hrs) (hr1,hr2-left,hr2-right,hr3,hr4-left,hr 4-right, and hr5) similar to those found in the Autographa californica nuclear polyhedrosis virus (AcNPV) genome were found in the Bombyx mori NPV (BmNPV) genome. The BmNPV hrs contained two to eight repeats of a homologous nucleotide sequence which were on average about 75 bp long. All of these homologous sequence repeats contained a 26-bp-long palindrome motif with an EcoRI or EcoRI-like site at its core. The consensus sequence of the BmNPV hrs showed 95% conservation with respect to those found in AcNPV. Nucleotide sequence analysis indicated that hr2-left and hr2-right of BmNPV evolved from an ancestor similar to hr2 of AcNPV by inversion, cleavage, and ligation. The polarities of the BmNPV and AcNPV hrs were conserved except for that of hr4-left. Within hr4-right of BmNPV, four repeats of a previously underscribed palindrome motif were found. Bmhr5D, a BmNPV mutant which lacked hr5, replicated at a rate similar to that of wild-type BmNPV in BmN cells and silkworm larvae, indicating that hr5 was not essential for viral replication. After ten passages of Bmhr5D in BmN cells, no detectable changes in its genome were observed by restriction endonuclease analysis. The evolution and divergence of the BmNPV genome are also discussed.     

Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus.

The mechanisms of host specificity of nuclear polyhedrosis viruses (NPVs) (Baculoviridae) were analyzed after coinfection of Bombyx mori NPV (BmNPV) and one of four distinct groups of Spodoptera litura NPV (SlNPV), including an Autographa californica NPV (AcNPV) variant (S. Maeda, Y. Mukohara, and A. Kondo, J. Gen. Virol. 71:2631-2639, 1990), into various lepidopteran cell lines. Replication of BmNPV in nonpermissive cells (TN-386, SF-21, and CLS-79) was induced by coinfection with AcNPV but not with the other three SlNPV groups. These induced progeny NPVs were plaque purified in BmN cells, which are susceptible to only BmNPV, and characterized. Most of these isolates did not replicate in the cell lines in which they were produced, indicating the existence of a helper function of AcNPV for BmNPV replication in nonpermissive cells. Some of these isolates, however, were able to replicate in cell lines nonpermissive to BmNPV, indicating the appearance of a new virus with wider host specificity. DNA restriction endonuclease analysis showed that the isolates exhibiting wider host range were recombinant viruses between the parents, AcNPV and BmNPV, resulting from various types of crossovers of relatively large areas of their genomes. Expansion of host range was also observed in larvae.     

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus.     

Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene.     

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.     

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析.结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%.RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定.利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp protease proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性.     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析。结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%。RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定。利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp prote-ase proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性。