Characterization of a cytoplasmic polyhedrosis virus from Estigmene acrea (Lepidoptera)

A cytoplasmic polyhedrosis virus (CPV) containing a segmented double-stranded RNA genome was isolated from Estigmene acrea larvae by isopycnic centrifugation in a sucrose density gradient. Ten double-stranded RNA segments with molecular weights (MW) from 2.8 to 0.67 × 10⁶ were separated by agarose gel electrophoresis. A total of ten virus proteins ranging from 14,000 to 128,000 MW were detected after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A MW of 28,500 was determined for E. acrea CPV occlusion body protein.     

Electron microscope observations of Autographa californica (Noctuidae) nuclear polyhedrosis virus replication in the midgut of the saltmarsh caterpillar, Estigmene acrea (Arctiidae)

The nuclear polyhedrosis virus from Autographa californica was studied with the electron microscope in the midgut of the salt marsh caterpillar, Estigmene acrea. The results of the present study were compared with a previous study in which the same inoculum was fed to Spodoptera exigua. In Estigmene acrea polyhedra were produced, but virions were not occluded. Nonoccluded virions were found throughout the midgut cytoplasm and budding into the hemocoel. Within the cytoplasm, the rough endoplasmic reticulum was observed to contain paracrystalline proteinaceous bodies. Fibrous bodies and annulate lamellae were also found in the cytoplasm of infected cells.     

Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells

Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [³H]thymidine incorporation 12 hr after virus inoculation. Hybridization of ³²P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [su35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and virus-containing occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Comparison of nuclear polyhedrosis virus replication in five lepidopteran cell lines

Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides.     

Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication

The titer of Amsacta entomopoxvirus (EPV) protein detected in murine L-929 cells by enzyme-linked immunosorbent assay (ELISA) decreased to within preimmune serum levels by 24 hr after inoculation of the virus which indicates that Amsacta EPV structural protein biosynthesis does not occur in the vertebrate cell line. A viral-induced protein of approximately 100,000 M_r was detected by [³⁵S]methionine incorporation 4 hr after inoculation of Tn-368 cells with Amsacta EPV. Biosynthesis of protein which reacted with vaccina antiserum was detected in Estigmene acrea (BTI-EAA) cells by ELISA 10 hr after inoculation with 10 PFU of virus per cell. The amount of putative vaccinia structural protein detected in BTI-EAA cells increased approximately twofold by 70 hr after virus inoculation. No increase in vaccinia structural protein biosynthesis was detected in BTI-EAA cells inoculated with vaccinia virus previously inactivated by heat and UV light.     

Infectivity of nuclear polyhedrosis virus extracted with digestive juices

Autographa californica NPV, which had been obtained by dissolving polyhedra in the digestive juice of Estigmene acrea larvae, was infectious to a Trichoplusia ni cell line (TN-368). Virions thus botained were infective, and as few as 0.0025–0.005 polyhedral equivalents could infect newly transferred tissue culture cells. Activity decreased after 8 min of digestion.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Infectivity of a nuclear polyhedrosis virus from the alfalfa looper,Autographa californica,after passage through alternate hosts

A nuclear polyhedrosis virus isolated from the alfalfa looper, Autographa californica, was found to infect several species of caterpillars including the cabbage looper, Trichoplusia ni; the beet armyworm, Spodoptera exigua; and the saltmarsh caterpillar, Estigmene acrea. Studies were therefore conducted to determine the quantitative effects of passage through the alternate hosts, S. exigua and E. acrea, on the infectivity of this virus to newly hatched first-instar cabbage looper larvae. When 11 preparations of polyhedra obtained from a like number of primary passages through the original or alternate hosts were assayed and the mortality at 7-, 10-, and 14-day intervals were subjected to probit analysis, the LD₅₀s for the three intervals differed but those for the preparations at any given interval did not. Therefore, any of the three hosts could be used to propagate the virus, and whichever proves the easiest to rear and provides the highest yields of polyhedra can be selected.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

Tests for Transmission of Prunus Necrotic Ringspot and Two Nepoviruses by Criconemella xenoplax

In two of three trials, detectable color reactions in ELISA for Prunus necrotic ringspot virus (PNRSV) were observed for Criconemella xenoplax handpicked from the root zone of infected peach trees. Criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with PNRSV failed to transmit the virus to cucumber or peach seedlings. The nematode also failed to transmit tomato ringspot (TomRSV) or tobacco ringspot viruses between cucumbers, although Xiphinema americanum transmitted TomRSV under the same conditions. Plants of peach, cucumber, Chenopodium quinoa, and Catharanthus roseus were not infected by PNRSV when grown in soil containing C. xenoplax collected from root zones of PNRSV-infected trees. Shirofugen cherry scions budded on Mazzard cherry seedling rootstocks remained symptomless when transplanted into root zones of PNRSV-infected trees. Virus transmission was not detected by ELISA when C. xenoplax individuals were observed to feed on cucumber root explants that were infected with PNRSV and subsequently fed on roots of Prunus besseyi in agar cultures. Even if virus transmission by C. xenoplax occurs via contamination rather than by a specific mechanism, it must be rare.     

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [³H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.     

Mapping of BamHI and SmaI DNA restriction sites on the genome of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica

The DNA of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), has been analyzed with restriction endonucleases BamHI and SmaI. The molecular weight of the BamHI fragments, SmaI fragments, and BamHI + SmaI fragments has been determined. The molecular weight of AcNPV DNA is calculated to be about 82 million. A presumptive physical map of the BamHI and SmaI restriction sites on the AcNPV genome has been constructed.     

Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus

Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of SINPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of β-galactosidase (βGal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest βGal activity was obtained with S1AcNPV4.lacZ. Production of βGal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.     

Biosynthesis of sex pheromone components from linolenic acid in arctiid moths

Sex pheromone components of two species of arctiid moths, Estigmene acrea and Phragmatobia fuliginosa, were shown to be derived from linolenic acid. Female pupae were injected with radiolabeled malonic acid or an 18-, 20-, 21-, or 22-carbon triunsaturated fatty acid, and the pheromone components from emerged adults analyzed for radioactivity. The data support a biosynthetic pathway in which the 21-carbon pheromone component,(Z, Z)-3,6-cis-9,10-epoxyheneicosadiene, of these moths is produced by chain elongation of linolenic acid to docosatrienoic acid with subsequent reductive decarboxylation. The 18-carbon aldehyde components,(Z, Z)-9,12-octadecadienal and (Z, Z, Z)-9,12,15-octadecatrienal, of E. acrea are produced from linoeic and linolenic acids directly. No detectable amounts of intermediate 20-, 21-, or 22-carbon fatty acid precursors were found in the gland of E. acrea.     

侵染浙贝母3种病毒CP基因的原核表达、抗血清制备及其病毒检测

根据GenBank报道的浙贝母花叶病毒(Thunberg fritillary mosaic virus,TFMV)、浙贝母Y病毒(Fritillary virus Y,FVY)和百合斑驳病毒(Lily mottle virus,LMoV)序列设计引物,扩增其CP基因。将CP基因插入表达载体pSBET,转化大肠杆菌BL21(DE3)Plys E菌株,IPTG诱导表达。经12% SDS-PAGE和5%~20%梯度SDS-PAGE两次纯化CP,分别免疫小鼠获得抗CP血清。采用Western blot分析确定抗体的特异性及其之间的血亲学关系;采用ELISA分析确定抗体是否能与天然病毒粒子结合。采用Western blot、间接ELISA法和Dot-ELISA 法检测侵染浙贝母的3种病毒。结果表明,制备的抗体对CP有高度特异性,相互之间无交叉反应,且能与天然病毒离子结合。制备的抗血清可以用于检测3种病毒,其中间接ELISA法和Dot-ELISA法检测效果较好。     

Competition between Wild-Type and Recombinant Nucleopolyhedroviruses in a Greenhouse Microcosm

Wild-type Autographa californica nucleopolyhedrovirus (AcNPV or AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing a mutated juvenile hormone esterase (AcJHE.SG) were compared in their capability to produce epizootics in larvae of Trichoplusia ni infesting collards in a greenhouse microcosm. Larvae treated in four different ways were released into 1.8-m² microplots in week 1. The four treatments included (1) uninfected larvae (control), (2) 100% AcNPV.WT-infected larvae (WT), (3) 100% AcNPV.AaIT-infected larvae (AaIT), and (4) 1:1 ratio of AcNPV.WT-infected and AcNPV.AaIT-infected larvae (WT+AaIT). On a weekly basis, larvae were sampled and new, uninfected larvae were added to all plots. Sampled larvae were reared until death and then subjected individually to DNA–DNA dot-blot hybridization assay to determine the proportion of insects infected with each virus in each plot. The entire experiment was repeated with AcJHE.SG in the place of AcNPV.AaIT. Epizootics of AcNPV.WT lasted 8 weeks after a single viral release in the replicated greenhouse microplots. AcJHE.SG epizootics also lasted 8 weeks after viral release, but this virus and AcNPV.AaIT were both out-competed by AcNPV.WT. AcNPV.AaIT was no longer detected in the T. ni population by the fourth week after release. AcNPV.WT also increased to greater numbers in soil than AcNPV.AaIT or AcJHE.SG after 8 weeks. Thus, it was possible to induce 8-week epizootics of AcNPV.WT in replicated microplots under artificial greenhouse conditions, and the wild-type virus out-competed the recombinant virus for a niche in this greenhouse microcosm, which reduces the probability that the recombinant virus will persist in an agroecosystem.     

Mitotic responses of the anterior pericardial wall of Biomphalaria glabrata (Mollusca) subjected to challenge

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [³H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.     

Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.     

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.