Quantification of two viruses in technical preparations of Orgyia pseudotsugata baculovirus by means of buoyant density centrifugation of viral deoxyribonucleic acid.

A reliable method was developed for the quantitative determination of two nuclear polyhcdrosis viruses present in commercially prepared viral insecticides used against Orgyia pseudotsugata. Deoxyribonucleic acids, from nuclear polyhedrosis bundle virus and nuclear polyhedrosis single-rod virus, were separated on CsCl gradients according to their respective buoyant densities, 1.715 and 1.704 g/ml. The proportions of the two viruses were quantified by measuring the relative absorbance at 254 nm of their deoxyribonucleic acid peaks.     

Identification of two nuclear polyhedrosis viruses from the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae)

Two nuclear polyhedrosis viruses from the cabbage moth Mamestra brassicae found in two geographical areas in Europe have been characterized and compared. These two virus isolates have similar biological activities and have the same host range. The two M. brassicae nuclear polyhedrosis viruses can be distinguished by restriction endonuclease analysis of their DNA. They appear to be distinct but related virus strains.     

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.     

Enhancement in activity of homologous and heterologous baculoviruses infectious to beet armyworm (Lepidoptera: Noctuidae) by an optical brightener

The nuclear polyhedrosis virus (NPV) from the beet armyworm, Spodoptera exigua (Hübner) (SeMNPV), was the most active virus tested against the beet armyworm (LC50 = 4.1 PIBs/mm2), followed by nuclear polyhedrosis viruses from the alfalfa looper, Autographa californica (Speyer) (AcMNPV; LC50 = 92.6 PIBs/mm2), and the celery looper, Anagrapha falcifera (Kirby) (AfMNPV; LC50 = 195.7 PIBs/mm2). In the case of the nuclear polyhedrosis virus from the bollworm, Helicoverpa armigera (Hübner), LC50s could only be obtained for five/six replicates, whereas LC50s could only be obtained for two/six replicates for the nuclear polyhedrosis virus from the wax moth, Galleria mellonella (L.) (GmMNPV). When an optical brightener Tinopal LPW was added to virus suspensions, LC50 values were reduced by 130-fold for both SeMNPV and AcMNPV and by 300-fold for AfMNPV. The addition of Tinopal LPW greatly increased the activities of HaMNPV and GmMNPV. In terms of speed of kill, Tinopal LPW reduced the LT50s for all nuclear polyhedrosis viruses by 30-40%.     

Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses.

Monoclonal antibodies were produced to polyhedrins from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) and single-capsid nuclear polyhedrosis virus (OpSNPV). Although the polyhedrins are closely related, antibodies were selected which allowed differentiation between the two viruses. In an indirect enzyme-linked immunosorbent assay, purified OpMNPV and OpSNPV polyhedrins could be detected by specific monoclonal antibodies at concentrations as low as 2 and 5 ng/ml, respectively. The antibodies were also capable of identifying their homologous polyhedrin in extracts of infected insects. These antibodies would be useful for monitoring production of the viral insecticide, TM Biocontrol-1, which by license must contain only OpMNPV, and to confirm that insect mortality after aerial spraying with this insecticide is attributable to OpMNPV infection.     

Characterization of Nuclear Polyhedrosis Virus DNAs

The nuclear polyhedrosis virus DNAs characterized and compared in this study consist of the singly-enveloped nucleocapsids (SNPV) of Trichoplusia ni and the bundles of nucleocapsids common to a single envelope (MNPV) from Spodoptera frugiperda and Rachiplusia ou. The SNPV and MNPV DNAs are very similar in hydrodynamic properties and molecular weights. In addition, the NPV DNAs are similar in size to those extracted from the granulosis viruses that infect T. ni and S. frugiperda. As isolated from purified virus or directly from occluded virus, the nuclear polyhedrosis virus DNAs consist of a mixture of about 20 to 30% double-stranded covalently closed molecules and approximately 60% relaxed circles, with less than 10% in linear duplex form. The molecular weights of all nuclear polyhedrosis virus DNAs as compared in this study are slightly smaller than those of T4 bacteriophage DNA and perhaps slightly smaller than those of the granulosis virus DNAs. The best estimates of these molecular weights by neutral sucrose sedimentation for the nuclear polyhedrosis viruses range from 90 to 100 x 10(6) relative to a size of 108 x 10(6) for T4 DNA. The base compositions of the nuclear polyhedrosis viruses that infect T. ni and S. frugiperda are compared with the respective insect host DNAs.     

Review on microbial control of insect pests in forests in Japan

In Japan, two different serious defoliators were controlled by viruses:Dendrolimus spectabilis using cytoplasmic polyhedrosis virus andLymantria fumida using cytoplasmic and nuclear polyhedrosis viruses. For controllingD. spectabilis, good results were obtained when the spraying was done against old larvae of intermediate population density. AgainstL. fumida, a mixed suspension of native nuclear and cytoplasmic polyhedrosis viruses was sprayed at the early stage. The epizootic was initiated earlier than being supposed, and the population collapsed.     

Conservation of genome organization in two multicapsid nuclear polyhedrosis viruses.

The genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata was mapped by examining overlapping HindIII fragments from cosmid clones which had been constructed from partial HindIII digests of viral DNA. Five OpMNPV cosmid clones containing fragments encompassing the entire OpMNPV genome were hydridized to blots of DNA from the multicapsid nuclear polyhedrosis virus of Autographa californica. The hybridization pattern indicated that the genomes of these viruses are similarly organized.     

木毒蛾复合病毒制剂研究

通过木毒蛾质型多角体病毒、核型多角体病毒和颗粒体病毒,对木毒蛾幼虫的复合感染试验,以及三种病毒最佳配比的筛选试验,发现木毒蛾质型多角体病毒、核多角体病毒和颗粒体病毒,按6：3：1混合后防治木毒蛾效果最佳。同时通过添加适量936乳油和人工大田繁殖木毒蛾病毒,较好地解决了病毒制剂的室温保存问题和毒源生产问题。复合病毒制剂的林间大面积防治试验结果表明,该制剂防治效果好,配制容易,成本低廉,不污染环境,是一种优良的生物制剂。     

Isolation of A Cytoplasmic Polyhedrosis Virus by Physical and Immunological Techniques

Procedures are described for the separation and purification of cytoplasmic polyhedrosis viruses obtained from multiply infected Heliothis armigera larvae. Separation was achieved by differential centrifugation and density gradient zone electrophoresis followed by complexing with nuclear polyhedrosis virus specific antibody. The yield of cytoplasmic polyhedrosis virus was increased by passage in larvae reared on synthetic media.     

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.     

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.     

18. Isolation of a Cytoplasmic Polyhedrosis Virus by Physical and Immunological Techniques

ABSTRACT

Procedures are described for the separation and purification of cytoplasmic polyhedrosis viruses obtained from multiply infected Heliothis armigera larvae. Separation was achieved by differential centrifugation and density gradient zone electrophoresis followed by complexing with nuclear polyhedrosis virus specific antibody. The yield of cytoplasmic polyhedrosis virus was increased by passage in larvae reared on synthetic media.     

Persistence of occluded viruses in the nests of the paper waspPolistes hebraeus (Hym.: Vespidae)

Bioassays were used to study the infectivity of cytoplasmic polyhedrosis viruses (CPVs), nuclear polyhedrosis viruses (NPVs) and entomopoxviruses (EPVs) contained in 16 nests of the paper waspPolistes hebraues F. in Réunion Island. Several virosis were propagated from 6 nest contents in 5 Lepidoptera:Catopsilia thauruma Saalmüller,Catopsilia florella F.,Callixena versicolora Mabille,Polydesma umbricola Boisduval andEagris sabadius Boisduval. Each of the previous species supported the development of one or two virosis and therefore, wasp nests must be considered as accumulating centres where infectivity of occluded entomoviruses is preserved. The accurate origin of the virosis propagated through bioassays was searched for using ecological investigations on similar natural diseases, REN analysis and a cross-transmission test onSpodoptera mauritia Boisduval. The nuclear polyhedrosis and one cytoplasmic polyhedrosis appearing in bioassays can be assigned respectively to viruses produced byC. thauruma andC. versicolora; the other virosis must be considered as developing in alternate or substitution hosts. Wasp nests could therefore be used to detect the presence of specific viruses in an environment and to collect new virus isolates.     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVLl393,我们得到了表达载体pVLl393-hFSHβ,然后与BaculoGold^TM线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGoL一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

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.     

Comparative studies on the nuclear polyhedrosis viruses of Lymantria monacha and L. dispar

Immunodiffusion and tube precipitation tests, polyacrylamide gel electrophoresis of virus polypeptides, and cross-transmission experiments suggest that two nuclear polyhedrosis viruses, one from Lymantria monacha and one from L. dispar, are partially related to each other, but not identical. The virus particle proteins seem to be more specific than the polyhedron proteins.     

Fossil evidence of insect pathogens

The present report describes fossil evidence of insect pathogens, heretofore, almost non-existent, from six samples of amber ranging in age from 15 to 100 million years. They include a cytoplasmic polyhedrosis virus and trypanosomatid infection in an adult biting midge (Diptera: Ceratopogonidae), and a nuclear polyhedrosis virus in an adult sand fly (Diptera: Phlebotomidae), both from Early Cretaceous Burmese amber, several types of fungal thalli on the cuticle of an adult mosquito (Culicidae: Diptera), as well as a fungal growth on the prothorax of a fungus gnat (Mycetophilidae: Diptera) in Dominican amber and large tumors in the body cavity of a caterpillar (Lepidoptera) in Mexican amber. These discoveries suggest that insect polyhedrosis viruses were present 100 million years ago and present the possibility that vertebrate arboviruses (especially those in the family Reoviridae) could have evolved from cytoplasmic polyhedrosis viruses infecting biting insects. The flagellates in the Early Cretaceous biting midge represent the first fossil record of monogenetic trypanosomatid infections of arthropods.