Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.     

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.     

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.     

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm,Bombyx mori

Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.     

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.     

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。     

Photoreception in decapitated larvae of silkworm Bombyx mori

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.     

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.     

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.     

Characterization and purification of the biologically active v-sis protein generated in silkworm larvae with the Bombyx mori nuclear polyhedrosis virus vector system

The v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The biologically active v-sis protein was expressed in silkworm larvae using the Bombyx mori nuclear polyhedrosis virus vector system. The v-sis protein purified from infected silkworm larvae is a 30 kDa disulfide-linked homodimer. Mitogenic activity of the v-sis protein was comparable to that of PDGF and inhibited by the pretreatment with anti-PDGF antibody. These results show that the recombinant v-sis protein is biologically and antigenically related to PDGF.     

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.     

Sterol composition in larvae of the silkworm, Bombyx mori

Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.     

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.