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.     

Construction of a Genetic Map of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus by Marker Rescue of Temperature-Sensitive Mutants

Mutations of seven temperature-sensitive mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (NPV) were mapped with respect to the physical restriction map of the A. californica NPV DNA by marker rescue. DNAs from two distantly related NPVs of the multiply embedded type and two NPVs of the singly embedded type were unable to rescue two A. californica NPV mutants.     

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.     

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.     

Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus

Twelve singly embedded isolates (SEV) and two multiply embedded isolates (MEV) of nuclear polyhedrosis viruses from Heliothis larvae were compared by time-mortality assays in neonate H. zea larvae. The isolates could be separated into six groups based on differences in the 50% survival time (ST₅₀) values. Isolates with identical restriction endonuclease (REN) profiles did not differ significantly in their ST₅₀ values, whereas isolates with several different REN cleavage sites also had significantly different ST₅₀ values. With the exception of one isolate from India, the singly embedded isolates acted faster than the multiply embedded isolates.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

Identification and localization of reiterated sequences in the Choristoneura fumiferana MNPV genome.

The genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) contained reiterated sequences interdispersed in four locations. These regions, termed RS, were found in EcoRI fragments A, F, E and B. The sequences were identified by hybridization of the fragment EcoRI-A to a Southern blot of EcoRI-digested viral DNA. Further confirmation and more precise localization of the RS sequences was obtained by hybridization of nick-translated 32P-labeled EcoRI-E fragment to Southern blots of viral DNA digested with EcoRI, BamHI, XbaI and Bg/II. Hybridization of 32P-labeled EcoRI-E to HindIII blots of viral DNA revealed the presence of a 'ladder' consisting of eight fragments. The three fragments of the ladder with the lowest sizes represented the HindIII fragments, O, PQ and R. The other five fragments were submolar in amount, in that they could not be seen in ethidium bromide-stained gels and probably represented minor virus variants that arose after passage of virus in larvae. Each variant was distinguished from the others by an additional insertion of 210 bp into the EcoRI-B fragment of the genome.     

Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus.

A nuclear polyhedrosis virus (MNPV) isolated from a lepidopteran (Noctuidae) insect, Autographa californica, was cloned by successive plaque purification using virions containing only one nucleocapsid per envelope as inoculum. The ability to clone the virus by this method was demonstrated by the isolation of nondefective, genotypic variants of the virus with similar but not identical restriction endonuclease fragment patterns. Five distinct variants were identified by genotypic analysis with HindIII, EcoRI, SalI, and Bam HI restriction endonucleases. The characteristic genotype of each variant was maintained upon passage in insect larvae. The isolation of these virus variants demonstrates (i) the heterogeneity of the uncloned virus preparation and (ii) the ability to clone MNPVs by plaque purification of media-derived nonoccluded virions. The A. californica MNPV is being considered for commercial use as a pesticide in the United States, and the cloning of the virus, in view of the heterogeneity detected, may be advisable. The cloning and genotype analyses are also significant with regard to understanding the genetic nature of multiply embedded NPVs (those NPVs containing more than one nucleocapsid per envelope in the occluded form of the virus) and indicate that further genetic analysis of these viruses is possible.     

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%.     

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.     

Restriction Maps of Five Autographa californica MNPV Variants, Trichoplusia ni MNPV, and Galleria mellonella MNPV DNAs with Endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI

The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) genomes were determined and compared to the endonuclease cleavage maps of AcMNPV E2 DNA. The viral structural polypeptides of AcMNPV variants S3, E2, S1, M3, and R9 were the same when analyzed by polyacrylamide gel electrophoresis. The major structural polypeptides of GmMNPV and TnMNPV had the same pattern in polyacrylamide gels as did AcMNPV structural polypeptides. GmMNPV and TnMNPV had several minor structural protein differences as compared with AcMNPV. AcMNPV variants, TnMNPV, and GmMNPV were distinct but with very similar genomes and protein structures.     

美国白蛾核型多角体病毒超氧化物歧化酶基因的序列分析和表达

根据测序结果 ,HcNPVsod的核苷酸序列与BmNPVsod的完全一致 ,与AcNPVsod的核苷酸序列相比 ,同源性达到 97 2 % ;推测HcNPVsod编码 1 51个氨基酸 ,与BmNPVsod的完全一致 ,与AcNPVsod编码的氨基酸相比 ,有三个氨基酸的差别。按基酸序列分析表明 ,HcNPVSOD蛋白中含有对SOD结构和活性必需的氨基酸残基 ,在HcNPVsod中均是保守的。SOD活性测定表明酶活为 1 47 0 9U/mL菌液     

Analysis of deoxyribonucleic acid from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Deoxyribonucleic acid (DNA) from isolates of five nuclear polyhedrosis viruses (NPV) from lepidopterous hosts of the noctuid subfamily Plusiinae was analyzed by ion-exchange and paper chromatography. Viruses and production hosts were: Trichoplusia ni singly embedded virion type (SEV) from T. ni, Pseudoplusia includens SEV from P. includens, T. ni multiply embedded virion type (MEV) from T. ni, Autographa californica MEV from A. californica, A. californica MEV from T. ni, and Rachiplusia ou MEV from R. ou. Neither uracil nor 5-methyl cytosine was detected in the DNAs. Adenine:thymine (A:T) and guanine:cytosine (G:C) ratios were nearly constant for all the NPVs. AT:GC ratios for the SEVs were 1.60 and 1.57 and were clearly separable from those of the MEVs which ranged from 1.32 to 1.38. No differences in DNA composition within SEV or MEV groups were apparent.     

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。     

Biochemical comparison of virion proteins from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

The virions of six isolates of five nuclear polyhedrosis viruses (NPV) infecting plusiine larvae (Lepidoptera: Noctuidae) were reproducibly separated by sucrose density gradient centrifugation and fractionation. Purity of the preparations was established by electron microscopy. Virion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); each produced 12 distinct polypeptides ranging from 10,300 to 82,900 mw. Qualitative and quantitative differences were found between most of the polypeptide patterns. The singly embedded viron (SEV)-type isolates had two major components with mw in the range of 32,900–35,200; multiply embedded virion (MEV)-type isolates had a major component of ca. 12,500 mw. SEV isolates showed almost no within-group differences, while minor differences were found among the MEV banding patterns in both intensity and presence of certain bands. Capsids and envelopes from MEV had two to four polypeptides with mw between 10,800 and 26,900. The presence of more than one polypeptide and electron microscopy of sample composition suggested that the capsid and envelope are composed of several distinct proteins.     

Studies on the pathology of a baculovirus in Aedes triseriatus

The pathology of a Baculovirus (BV) in Aedes triseriatus was studied. The virus infected the cardia, gastric caeca, and the entire stomach of larval midgut epithelium. The progression of the disease was similar to that of other Baculoviruses of the nuclear polyhedrosis virus (NPV) type. Rodshaped nucleocapsids were formed within a Feulgen-positive virogenic stroma and along the nuclear envelope. These nucleocapsids were enveloped by a membranous material and occluded randomly in small irregular and polyhedral proteinic inclusions. The disease differed from other BVs of the NPV type in that the small proteinic inclusions gradually coalesced as they grew, forming large fusiform inclusions.     

Comparison of the Structure of C- and N-Polyhedrins from Two Occluded Viruses Pathogenic for Orgyia pseudotsugata

C-and N-polyhedrins from a cytoplasmic polyhedrosis virus (a double-stranded RNA virus) and a nuclear polyhedrosis virus (a DNA virus), respectively, of Orgyia pseudotsugata were compared. Although both polyhedrins appear to stabilize their respective virions and have similar molecular weights, they differed in amino acid composition, tryptic peptide elution profiles from a cation-exchange resin, and N-terminal amino acid sequence and showed no antigenic relatedness. This suggests that these two proteins originated independently of one another.     

Physical Map of the DNA Genome of Autographa californica Nuclear Polyhedrosis Virus

A physical map of the 88 × 10⁶ dalton, circular DNA genome of Autographa californica nuclear polyhedrosis virus was constructed. The complete order of BamHI and XmaI restriction enzyme sites was determined. The EcoRI and HindIII fragments were partially ordered, and their general locations, relative to the BamHI and XmaI maps, were determined. Alterations in the restriction endonuclease fragment patterns of natural genotypic variants of A. californica nuclear polyhedrosis virus, including Trichoplusia ni MEV nuclear polyhedrosis virus, were located on the physical map. Alterations were found throughout the A. californica nuclear polyhedrosis virus DNA genome.