Serological relationships among plusiinae baculoviruses

Antisera were produced against nucleocapsids, NP-40 detergent soluble proteins, or polyhedral protein of the multiply embedded nuclear polyhedrosis virus (MNPV) of Autographa californica, nucleocapsids of Trichoplusia ni singly embedded virus (SNPV), and polyhedral protein of Lymantria dispar MNPV. Antigens consisting of nucleocapsids, NP-40 soluble proteins, and polyhedral protein were prepared from A. californica MNPV, T. ni MNPV, L. dispar MNPV, Rachiplusia ou MNPV, T. ni SNPV, and Pseudoplusia includens SNPV. Radial immunodiffusion patterns formed with Plusiinae nucleocapsid antigens and antiserum to nucleocapsids of A. californica MNPV or T. ni SNPV revealed a distinction between multiply and singly embedded viruses. The same alignment of Plusiinae viruses was observed in reactions between A. californica NP-40 soluble protein antiserum and the NP-40 soluble protein fractions from the Plusiinae NPVs. There were no reactions between the Plusiinae SNPV nucleocapsid antigens and the A. californica MNPV nucleocapsid antiserum. However, there were faint precipitin bands between MNPV nucleocapsid antigens and T. ni SNPV nucleocapsid antiserum. Each of the polyhedral protein fractions from the Plusiinae formed a single precipitin band with the antiserum to polyhedral protein of either A. californica or L. dispar. The precipitin bands formed with the A. californica antiserum by polyhedral proteins of T. ni SNPV, P. includens SNPV, and R. ou MNPV were confluent, and shared partial identity with those formed by A. californica MNPV and T. ni MNPV. All precipitin bands formed by Plusiinae polyhedral proteins against antiserum to L. dispar polyhedral protein were confluent, and shared partial identity with that formed by L. dispar polyhedral protein.     

Polypeptide analysis of genotypic variants of occluded Heliothis spp. baculoviruses

The structural polypeptides of 12 baculovirus isolates which included nuclear polyhedrosis viruses (NPVs) and granulosis viruses (GVs) obtained from four different species of the insect genus Heliothis collected in different geographical regions of the world were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The matrix proteins were compared according to their molecular weights and peptide profiles produced after limited proteolysis. Examination of the matrix and virion polypeptide profiles revealed three major polypeptide phenotypes which corresponded to the three baculovirus morphological groups; singly embedded nuclear polyhedrosis viruses (SNPVs), multiply embedded nuclear polyhedrosis viruses (MNPVs), and granulosis viruses (GVs). Enveloped nucleocapsid polypeptide profiles of isolates within each NPV phenotype differed in only one polypeptide whereas the two GV isolates differed by as many as five polypeptides. Nucleocapsid polypeptide profiles of isolates within each of the NPV subgroups were identical while those profiles from the GV nucleocapsids differed slightly in molecular weight of one polypeptide.     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.     

Application of a novel radioimmunoassay to identify baculovirus structural proteins that share interspecies antigenic determinants

Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120, 1979) and Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with ¹²⁵I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin (the occlusion body protein from NPVs) recognized antigenic determinants on all the polyhedrins and granulins (occlusion body protein from GVs) that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures.     

Characterization of an extremely basic protein derived from granulosis virus nucleocapsids

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex.     

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.     

混合感染后杆状病毒间的增效作用——病毒蛋白及核酸的初步分析

AsNPV+HasNPV、AsNPV+HaNPV、AsNPV+PsNPV分别感染烟青虫、棉铃虫和粘虫幼虫,对分离到的核型多角体病毒(AsNPV+HasNPV)-Helicoverpa assulta、(AsNPV+HaNPV)- H.Armigera和(AsNPV+PsNPV)-Pseudaletia separata,经电镜观察,多角体蛋白及病毒粒子蛋白SDS-PAGE电泳,病毒核酸的限制性内切酶酶解分析等研究,证明各病毒的多角体形态不规则,大小差异极大,病毒粒子为杆状,(AsNPV+HasNPV)-H.Assulta 和(AsNPV+HaNPV)-H.Armigcra病毒粒子有单粒和多粒包埋类型, (AsNPV+PsNPV)-P.Separata为多粒包埋型。各病毒的多角体蛋白基本上只有一种多肽,分子量为25 000道尔顿左右。(AsNPV+HasNPV)-H.Assulta、(AsNPV+HaNPV)-H.armigera和(AsNPV+PsNPV)-P.Separata的病毒粒子分别有10、14、5条多肽,分子量大小在13 500～98 000,13 000～88 000,18 500～52 000道尔顿之间。病毒核酸经EcoRI,HindIII,HindIII+BamHI酶解,其DNA的酶切位点,大小及DNA的总分子量与AsNPV和原寄主的Has-NPV,HaNPV和PsNPVDNA的酶切图谱存在一定差异,混合病毒侵染昆虫后新复制的病毒核酸发生一定的变化,从而导致病毒蛋白和病毒形态的变化。混合感染后AsNPV对Has-NPV、HaNPV和PsNPV的侵染有明显的增效作用,其机理有待深入研究。     

Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line.     

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.     

Host range and virulence of five baculoviruses from lepidopterous hosts

The host range and virulence of five insect baculoviruses (two multiply-enveloped nuclear polyhedrosis viruses (MNPVs) from Agrotis segetum and Mamestra brassicae; one singly-enveloped NPV from Plusia gamma and two granulosis viruses (GVs) from A. segetum and Pieris brassicae) were studied for seven lepidopterous pests of temperate agriculture (A. segetum, Agrotis exclamationis, Lacanobia oleracea, M. brassicae, Noctua pronuba, P. gamma and Pieris rapae). None of the viruses killed all species but M. brassicae MNPV failed to infect only P. rapae. The other viruses were restricted to the homologous host, or members of its genus or subfamily. In all examples except A. segetum GV, the median lethal dose for the most susceptible host, was less than 22 virus inclusion bodies and median lethal times for all infections ranged from 5·5 to 16·6 days. The low susceptibility of A. segetum and other noctuids to GV infections is discussed in relation to the structure of inclusion bodies and the nature of the infectious unit in baculoviruses.     

Comparative histopathology of three ascovirus isolates in larval noctuids

The histopathology caused by three ascoviruses isolated respectively from Heliothis zea, Spodoptera frugiperda, and Trichoplusia ni was studied in the host species from which the viruses were isolated originally, or in the case of the H. zea isolate, in H. virescens. In all three isolates, infected cells and virion-containing vesicles in the hemolymph were observed by 3 days postinoculation. The isolates from H. zea and T. ni exhibited relatively broad tissue tropisms infecting the tracheal matrix, epidermis, and connective tissue, and in the latter isolate, the fat body. Although considered variants of the same virus, these isolates varied slightly in their tissue tropism in that the isolate from H. zea replicated more extensively in the epidermis. The isolate from T. ni replicated in the fat body during early stages of disease, but not in advanced stages, and was not as common in the epidermis as the isolate from H. zea. The isolate from S. frugiperda replicated only in the fat body and completely destroyed this tissue by 12 days postinoculation. The progression of disease within the tissues was correlated with a rise in the concentration of virion-containing vesicles in the hemolymph of all three isolates. Vesicle concentrations rose from 10(7) on day 2 to 10(8)/ml of hemolymph on day 9, plateaued for 5-7 days, and then declined gradually through day 20. These results indicate that the chronic nature of the diseases caused by ascoviruses is due to the limited degree to which they infect major tissues (isolates from T. ni and H. zea) or the infection of tissues less essential to the maintenance of larval life (isolate from S. frugiperda).     

Effect of two granulosis viruses on the activity of the gypsy moth (Lepidoptera: Lymantriidae) nuclear polyhedrosis virus

Two granulosis viruses (GV) were tested as enhancers for the gypsy moth nuclear polyhedrosis virus (LdMNPV). Helicoverpa armigera (Hübner) CV (HaGV) had no detrimental effect upon larval growth and development, but in combination with LdMNPV it reduced both the LC50 and the LT50 for the NPV. In addition, the combination also adversely affected the growth and development of gypsy moth larvae. The LC50 of LdMNPV was reduced by as much as 300-fold (HaGV at 10(-2) dilution) and the LT50 was reduced by as much as 18% (HaGV at 10(-2) dilution). Spodoptera frugiperda (J. E. Smith) GV reduced the LC50 of LdMNPV by as much as 13-fold but had no effect upon the LT50.     

Restriction endonuclease analysis to distinguish two closely related nuclear polyhedrosis viruses: Autographa californica MNPV and Trichoplusia ni MNPV.

Restriction endonuclease fragment patterns of the deoxyribonucleic acid genomes of Autographa californica nuclear polyhedrosis virus (multiply embedded type) and Trichoplusia ni nuclear polyhedrosis virus (multiply embedded type) demonstrate that the two viruses are distinct but closely related variants.     

Restriction endonuclease analysis to distinguish two closely related nuclear polyhedrosis viruses: Autographa californica MNPV and Trichoplusia ni MNPV.

Restriction endonuclease fragment patterns of the deoxyribonucleic acid genomes of Autographa californica nuclear polyhedrosis virus (multiply embedded type) and Trichoplusia ni nuclear polyhedrosis virus (multiply embedded type) demonstrate that the two viruses are distinct but closely related variants.     

昆虫病毒增效剂研究进展

概述了昆虫病毒增效剂方面的研究进展,重点述及生物增效剂病毒增强素及化学增效剂荧光增白剂的增效活性特点和增效机理,以及病毒增强素分子生物学方面研究进展。在颗粒体病毒、核型多角体病毒、昆虫痘病毒中均发现了病毒增强素,它是一种金属蛋白酶。已克隆多个病毒增效基因,并构建了含增效基因的重组病毒和转基因作物。二苯乙烯类荧光增白剂对病毒具有增效作用。已明确病毒增强素和荧光增白剂增效作用与昆虫围食膜的破坏有关,其他增效机理有待进一步研究。还就增效剂的应用前景进行了展望。     

Comparison of biophysical and morphological properties of occluded and extracellular nonoccluded baculovirus from in vivo and in vitro host systems.

Electron microscopic examination and buoyant density profiles of nonoccluded Rachiplusia ou and Autographa californica nuclear polyhedrosis viruses purified from both infectious insect hemolymph and cell culture medium revealed that the viruses are enveloped, single nucleocapsids. The envelopes exhibited variation in the amount and degree of fit with regard to the nucleocapsids. This was determined by: (i) electron microscopic observations of virus budding from the surface of infected cells; (ii) electron microscopic observations of negatively stained preparations of pelleted, highly purified, nonoccluded enveloped particles; and (iii) the resolution and density distributions of nonoccluded virus in sucrose gradients after centrifugation to equilibrium; all were compared with virus extracted from polyhedra. Peplomers, ovserved on the surface of enveloped nucleocapsids of nonoccluded virus, are not associated with polyhedra-derived virus. Density gradient analysis indicated that virus from insect hemolymph and culture medium exhibited similar densities of approximately 1.17 to 1.18 g/ml. This is significantly different from the buoyant density of an alkali-liberated, enveloped single nucleocapsid (1.20 g/ml). Results of this study show that the nonoccluded forms of two nuclear polyhedrosis viruses from two different sources, hemolymph and cell culture, are similar with regard to several morphological and biophysical characteristics but are quite different from the alkali-liberated, polyhedra-derived form of the virus.     

In vivo and in vitro analyses of recombinant baculoviruses lacking a functional cg30 gene.

The cg30 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes two sequence motifs, a zinc finger-like motif and a leucine zipper, found in other polypeptides known to be involved in gene regulation. To gain insight into the function of the cg30 product, CG30, we constructed and characterized recombinant viruses lacking a functional cg30 gene. We found that cg30 mutants had no striking phenotype in cell lines derived from Spodoptera frugiperda or Trichoplusia ni or in T. ni larvae. Although cg30 is known to be transcribed as an early monocistronic RNA and as the second cistron of an abundant late bicistronic RNA, production of a CG30-beta-galactosidase fusion protein was observed mainly at early times postinfection. Viruses containing cg30 had a subtle growth advantage over those lacking cg30 after several viral passages in cell culture. We employed transient expression assays to determine whether cg30 and pe-38, an AcMNPV gene that encodes a polypeptide with zinc finger-like and leucine zipper motifs similar to those of cg30, have redundant functions. Although pe-38 may have a role in AcMNPV gene expression, there was no indication that cg30 and pe-38 are functionally redundant.     

粉纹夜蛾核型多角体病毒诱导的胸苷激酶特性研究

粉纹夜蛾(Trichoplusia ni)核型多角体病毒(TnNPV)感染草地贪夜蛾(Spodop-tera frugiperda)细胞后能诱导提高胸苷激酶的活性。不论是正常或感染细胞中的胸苷激酶都可将脱氧胞苷磷酸化,酶活最适pH及Mg~( )离子浓度值基本相同,DEAE-纤维素和Cibacron blue-sepharose柱层析所得酶活性图谱亦相似。TnNPV在胸苷激酶缺陷型的草地贪夜蛾细胞中能正常复制,但被感染细胞不具有胸苷激酶活性。由此可以确定,经TnNPV感染诱导后,活性有所提高的胸苷激酶不是病毒基因编码的。     

Mode of transmission of nuclear-polyhedrosis virus to progeny of adult Heliothis zea

Late second-instar Heliothis armigera larvae were infected with a granulosis and a nuclear polyhedrosis virus, and all the externally visible symptoms for each virus are described. The effects of the virus infections on the feeding habits of the insects are also described, and it was found that a granulosis infection can prolong the larval period by up to 100%. The larvae continue feeding during this prolonged larval period, and can reach almost double the size and mass of normal larvae.It was further found that each of the viruses displays a distinct set of symptoms which could indicate beyond any doubt which of the two viruses induced death in the host.