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PubMed1996

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human beta 1,2-N-acetylglucosaminyltransferase I.

Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I.

Animals↗

PubMed1996

Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda.

Glycosylasparaginase (EC 3.5.1.26) from Sf9 cells (Spodoptera frugiperda) was purified to homogeneity with a specific activity of 2.1 unit/mg. The enzyme is composed of two non-identical alpha/beta subunits joined by strong non-covalent forces and has one glycosylation site located in the alpha subunit. Molecular masses of the subunits were determined to be 28 kDa and 17 kDa by SDS-PAGE. Native enzyme existed in quaternary structures of either heterodimer (alpha beta) or heterotetramer (alpha 2 beta 2). These forms exhibited different ionic characteristics during DE52 anion exchange chromatography, and their molecular masses were determined to be 47 kDa and 101 kDa by gel filtration. The enzyme was thermostable, requiring 65-70 degrees C to be denatured, and it had a broad pH optimum from 4-10.5 with a pKa around 6. SDS easily inactivated the enzyme. The K(m) of glycosylasparaginase for its normal substrate GlcNAc-Asn was 0.88 mM. The enzyme also exhibited asparaginase activity with a K(m) of 3.0 mM for asparagine. N-terminal amino acids of the denatured subunits were sequenced and degenerate primers were designed for cloning its cDNA using PCR and 5' and 3' RACE. Glycosylasparaginase cDNAs from bovine and rat were also cloned using similar strategies, and primary structures of glycosylasparaginases from six species (human, bovine, rat, mouse, Sf9 cells and Flavobacterium) have been compared and related to a recent crystal structure of the human enzyme.

Amino Acid Sequence↗

PubMed1999

Selection of a nucleopolyhedrovirus for control of Spodoptera frugiperda (Lepidoptera: Noctuidae): structural, genetic, and biological comparison of four isolates from the Americas.

Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is the principal pest of maize in tropical and subtropical regions of the Americas. Larvae of this species are susceptible to a nucleopolyhedrovirus (NPV) which has attracted interest as a potential biocontrol agent. Four strains of NPV isolated from infected S. frugiperda larvae in the United States, Nicaragua, and Argentina were subjected to a structural, genetic, and biological comparison to select a candidate isolate for use in biocontrol experiments in Mexico and Honduras. All isolates had an occlusion body polyhedrin protein of 32 kDa, but the virions of each isolate differed subtly in the pattern and abundance of certain structural polypeptides revealed by SDS-PAGE analysis. Restriction endonuclease analysis of viral DNA confirmed that these isolates were strains of a single virus species but showed that they were not genetically homogeneous; each isolate could be differentiated from the others using common restriction enzymes. Droplet feeding bioassays indicated that an isolate from Nicaragua (Sf-NIC) and an isolate from the United States (Sf-US) had the highest infectivity when tested against 2nd instars originating from a Honduran S. frugiperda colony. No significant differences were detected in the speed of kill of Sf-NIC (102.7 h), Sf-US (102.3 h) and Sf-AR (103.4 h), whereas that of Sf-2 (97.3 h) was significantly shorter. Additional bioassays of the Sf-NIC isolate against 2nd to 6th instars demonstrated that LC50 values increased with larval stage from 2.03 x 10(5) OBs/ml for 2nd instars to 1.84 x 10(8) OBs/ml for 5th instars. The concentration required to elicit a lethal infection of 6th instars was so high that a reliable estimate of LC50 could not be obtained. The mean time to death for each stage challenged with the Sf-NIC isolate increased with instar from an average of 102.7 h in 2nd instars to 136.9 h in 5th instars.

Analysis of Variance↗

PubMed2003

Effect of tinopal LPW on the insecticidal properties and genetic stability of the nucleopolyhedrovirus of Spodoptera exigua (Lepidoptera: Noctuidae).

The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.

Animals↗

PubMed2004

Ultralow rates of spinosad in phagostimulant granules provide control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize.

Field trails in 2002 and 2003 were performed to determine the efficacy of maize flour-based granular formulations with ultralow rates of the naturally derived insecticide spinosad (0.1, 0.3, and 1.0 g [AI]/ha), for control of Spodoptera frugiperda (J.E. Smith) in maize, Zea mays L., in southern Mexico. Spinosad formulations were compared with a chemical standard, a commercial granular formulation of chlorpyrifos (150 g [AI]/ha). In both years, application of spinosad resulted in excellent levels of control, indicated by the number of living S. frugiperda larvae recovered from experimental plots. The efficacy of spinosad applied at 0.3 and 1.0 g (AI)/ha was very similar to that of chlorpyrifos. Natural reinfestation caused S. frugiperda numbers in insecticide treated plots to return to values similar to the control treatmentby 10-15d postapplication. Many spinosad-intoxicated larvae collected in the field died later in the laboratory in 2002, but not in 2003. Percentage mortality due to parasitoid emergence did not differ in any treatment in either field trial. The number of parasitoids that emerged from S. frugiperda collected in each treatment was significantly reduced after application of spinosad (all rates) or chlorpyrifos due to a reduction in the number of host larvae. Parasitoid numbers returned to control values by 9-15 d postapplication in all treatments. The most prevalent parasitoid was the braconid Chelonus insularis Cresson, which represented approximately 80% of emerging parasitoids in both years. We conclude that appropriate formulation technology can greatly enhance the performance of this naturally derived, biorational insecticide.

Animals↗

PubMed2004

Formulation with an optical brightener does not increase probability of developing resistance to Spodoptera frugiperda nucleopolyhedrovirus in the laboratory.

Stilbene-derived optical brighteners can markedly enhance the insecticidal activity of certain baculoviruses. We evaluated the influence of an optical brightener on the rate at which Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) developed resistance to nucleopolyhedrovirus (SfMNPV). Two laboratory colonies of S. frugiperda were inoculated with an LC50 of SfMNPV, in the absence or presence of the optical brightener Tinopal LPW (0.1%), over a period of two and 11 generations, in the first and second experiment, respectively. Compared with the initial susceptibility of the insect colony, resistance ratios of 11- and 12-fold were observed after two generations of treatment with SfMNPV + Tinopal LPW and SfMNPV alone. Similar, but variable degrees of resistance were observed in the long-term experiment with resistance ratios of 8- to 35-fold after seven to 11 generations. The presence of Tinopal LPW alone, or in mixtures with SfMNPV, did not cause any systematic change in insect resistance in either experiment. At the end of the long-term experiment, debilitating effects on pupal weight, adult fecundity, and longevity were observed in the insects exposed to Tinopal LPW alone or in mixtures with SfMNPV, but the pattern of such effects among treatments differed in each generation. We conclude that optical brighteners are unlikely to affect the rate of development of resistance to nucleopolyhedroviruses applied as biological insecticides.

Animals↗

PubMed2004

Effects of two biorational insecticides, spinosad and methoxyfenozide, on Spodoptera littoralis (Lepidoptera: Noctuidae) under laboratory conditions.

The toxicity of two biorational insecticides, spinosad (Tracer) and methoxyfenozide (RH-2485), was tested against eggs, larvae, and pupae of the noctuid Spodoptera littoralis (Boisduval). In the first experiment, filter paper circles containing egg masses of two different age classes, young (<24 h old) and old (24-48 h old), were dipped in different concentrations of each insecticide diluted in either water or acetone. No ovicidal activity was recorded when insecticides were diluted in water. In contrast, when insecticides were diluted in acetone, both egg age classes generally showed a concentration-dependent response for both compounds. Mortality of larvae that hatched from both egg age classes was significantly increased, compared with control larvae, at all concentrations of both insecticides when diluted in water or acetone alike. The prevalence of mortality was similar with each insecticide. In the second experiment, third instars of S. littoralis were fed semisynthetic diet containing different concentrations of both insecticides. According to LC50 values, no significant differences were observed between spinosad (2.11 mg [AI]/kg diet) and methoxyfenozide (3.98 mg [AI]/kg diet) after 48 h of treatment, based on the overlap of 95% CL. Toxic effects on the mortality of pupae, adult emergence, and the prevalence of deformed adults after topical application on young pupae also were examined. Only methoxyfenozide caused pupal mortality and deformed adults. Our results suggest that spinosad and methoxyfenozide are potentially potent compounds for control of S. littoralis.

Animals↗

PubMed2026

Bacterially produced dsRNA targeting SePGRP-LB reduces population fitness of Spodoptera exigua (Lepidoptera: Noctuidae) and increases its susceptibility to SeMNPV.

The beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), is an important agricultural pest, and S. exigua multiple nucleopolyhedrovirus (SeMNPV) is a host-specific biological control agent. However, baculovirus efficacy can be limited by host antiviral responses. S. exigua peptidoglycan recognition protein LB (SePGRP-LB) has been identified as an antiviral immune factor, suggesting that its suppression may increase larval susceptibility to SeMNPV. In this study, bacterially produced double-stranded RNA targeting SePGRP-LB (bac-dsPGRP-LB) was orally delivered to larvae to induce RNA interference. Feeding bac-dsPGRP-LB reduced SePGRP-LB transcript levels by 24.0% to 65.7% over 7 d. SePGRP-LB knockdown prolonged fifth-instar larval development, reduced female pupal weight, shortened male adult longevity and the oviposition period, and decreased fecundity by approximately 51%. Life table analysis further showed significant reductions in the intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R0) following bac-dsPGRP-LB treatment. During SeMNPV infection, co-feeding with bac-dsPGRP-LB significantly suppressed SePGRP-LB expression, increased the SeMNPV genomic load, and reduced larval survival compared with the SeMNPV + bac-dsGFP treatment. These findings identify SePGRP-LB as a promising RNAi target for simultaneously reducing S. exigua fitness and enhancing its susceptibility to SeMNPV under laboratory conditions.

SePGRP-LB↗

PubMed2005

Influence of diet on the larval beet armyworm, Spodoptera exigua, glucose oxidase activity.

Many researchers investigating plant-insect interactions maintain their insect colonies on artificial diet and assume that salivary enzymes and elicitors remain representative of natural situations. These salivary elicitors, such as the enzyme glucose oxidase (GOX), play important roles in influencing plant defense responses. In fact, GOX has been implicated in suppressing induced nicotine-production in tobacco plants (Musser et al., 2002). In this study, we investigated the effect of artificial or plant diet on the GOX activity in caterpillars of the beet armyworm, Spodoptera exigua. In the later developmental stages, whole body GOX of S. exigua caterpillars reared on a wheat germ-based artificial diet is over ten times higher than when insects were fed plants of the legume, Medicago trunctula. Labial salivary GOX accounted for most of this whole body activity in 4th instar caterpillars (57.8%), with the remaining activity present in the carcass. Hemolymph GOX levels were below the detection limits of the o-dianisidine-peroxidase assay used to measure activity. Labial salivary GOX activity was significantly higher in 4th larval instars reared on artificial diet compared with plant-fed caterpillars (U/mg per pair labial salivary gland, p = 0.0062), suggesting that diet effects GOX activity. When 4th instar plant-fed caterpillars were transferred to artificial diet, increased labial salivary GOX activity is closely correlated with the amount of time spent feeding on artificial diet. This study shows that the labial salivary GOX activity of S. exigua caterpillars is dependent on diet and developmental stage and that caution must be exercised in the design of plant-insect experiments.

Animals↗

PubMed1995

Nucleotide sequence and transcriptional analysis of the gp41 gene of Spodoptera frugiperda nuclear polyhedrosis virus.

The Spodoptera frugiperda multiple nucleocapsid nuclear polyhedrosis virus (SfMNPV) gp41 structural protein gene was located in the 1.9 kbp EcoRI-S fragment and sequenced. An open reading frame (ORF) of 999 nucleotides was detected that encoded a protein of 332 amino acids. The gp41 gene transcript was detected after 12 h post-infection (p.i.) and remained detectable at 48 h p.i. Two major mRNAs, about 1.6 and 2.8 kb in length, were determined by Northern blot analysis. Primer extension analysis demonstrated that the gp41 gene promoter region contains three transcription start sites. Two of the gp41 gene transcription start sites were located at -42 and -41 nucleotides from the ATG translation start codon within a consensus late transcription start site (TAAG) and another transcription start site was located at -140 nucleotides from the ATG translation start codon for which no consensus motif has been determined. Comparison of SfMNPV gp41 nucleotide and amino acid sequences with the gp41 genes from Autographa californica, Bombyx mori, and Helicoverpa zea NPVs showed 60% homology of nucleotide sequences and 70% similarity of amino acid sequences.

Amino Acid Sequence↗

PubMed1996

Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo.

The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in Se-UCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNA from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, Bg/II, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12.9 and 32.3.

Animals↗

PubMed1996

Baculovirus infection of Spodoptera exigua larvae: lacZ expression driven by promoters of early genes pe38 and me53 in larval tissue.

To follow the progression of infection of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) within tissues of its larval host, we have constructed AcMNPV recombinants carrying lacZ reporter genes under the control of the early virus promoters pe38 and me53, in addition to the authentic genes. The early promoter-lacZ gene cassettes were located upstream of the very late polyhedrin gene. In infected insect cell lines, pe38 transcription is initiated at an early promoter, while me53 transcripts start from both early and late sites. Transcriptional mapping of the duplicated me53 and pe38 promoters driving lacZ expression showed that they initiated at the same start sites as in the authentic genes. Expression of lacZ by these recombinants was compared to a recombinant driving beta-glucuronidase expression from the very late p10 promoter and lacZ expression from the constitutive heat shock protein 70 promoter of Drosophila melanogaster. After infection of Spodoptera exigua larvae with the different recombinants, we followed reporter gene expression and polyhedron formation in different tissues using immunohistochemistry and electron microscopy. LacZ expression, indicative of early viral transcriptional activity, was detected in nearly all larval tissues during the course of infection. In most tissues these early events were followed by pathophysiological changes associated with late and very late gene expression. However, p10 transcription and polyhedron formation were not observed in midgut goblet cells, Malpighian tubules and salivary glands. These results suggest that expression of early virus genes, such as me53 and pe38, is not restricted to larval tissues that are permissive for AcMNPV replication.

Animals↗

PubMed1996

Sequence and transcriptional analysis of the ubiquitin gene cluster in the genome of Spodoptera exigua nucleopolyhedrovirus.

The nucleotide sequence of a 1200 bp DNA fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was determined. This sequence contained a cluster of two open reding frames (ORFs), one coding for a viral ubiquitin (v-ubi) and another with homology to orf2 of Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. The vubi ORF is 240 nucleotides (nt) long, potentially encoding a protein of 80 amino acids with a predicted molecular mass of 9.4 kDa. The amino acid sequence of the v-ubi gene in SeMNPV has 75% and 81.6% identity with the v-ubi gene of AcMNPV and OpMNPV and approximately 84% with cellular ubiquitins. Northern blot analysis revealed three major small transcripts late in infection, of about 690, 550 and 400 nt long. Primer extension analysis showed that transcription started from within two consensus late promoter elements (TAAG), located at positions -6 and -30. The start site at position -4/-5 precedes the shortest leader reported to date for a baculovirus gene. The other ORF, xb187, was identified in the opposite orientation immediately upstream of the v-ubi gene. This ORF potentially encodes a 22 kDa protein with unknown function and about 60% amino acid similarity to the products of the orf2 genes of AcMNPV and OpMNPV. The SeMNPV xb187 ORF is transcribed late in infection via two transcripts, 1.2 kb and 770 nt long. The v-ubi-xb187 gene cluster is located at map unit (m.u.) 89 on the genome of SeMNPV. This is different from the position of an identical cluster in the AcMNPV and OpMNPV genomes, located at relative m.u. 20.

Amino Acid Sequence↗

PubMed1997

Characterization of a putative Spodoptera exigua multicapsid nucleopolyhedrovirus helicase gene.

Putative baculovirus helicases have been implicated as playing an important role in viral DNA replication and host specificity. The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) helicase is therefore of interest since the virus only infects the beet army worm. Sequence analysis of the SeMNPV lef5-p39 (mu 46.5-55.1) region, which is collinear with the 39K-lef5 area in Autographa californica MNPV (AcMNPV), revealed an open reading frame (ORF) of 3666 bp potentially encoding a protein with a molecular mass of 143 kDa. This protein had considerable amino acid sequence similarity (58%) to AcMNPV p143, including seven conserved motifs characteristic of helicases. In cultured insect cells, this SeMNPV ORF is expressed from 4 to 12 h postinfection and its major transcript of 4 kb starts 11 to 12 nt upstream of the putative translational initiation site (ATG). To study their possible role in the specificity of baculovirus DNA replication, the putative AcMNPV and SeMNPV helicase genes were tested for their ability to replicate homologous regions (hrs; putative origins of DNA replication) in a transient DNA replication assay in insect cells. All viral cis- and trans-acting factors were provided as plasmids using either Achr2 or Sehr1 as the DNA replication origin. SeMNPV p143 could not substitute for AcMNPV p143 in the transient assays supplemented with either hr. Similar results were obtained when the SeMNPV and AcMNPV ie1 genes were exchanged. None of the essential AcMNPV trans-acting factors could be complemented by SeMNPV infections to support DNA replication of hrs. These data suggest a specific interaction between baculovirus DNA replication factors to form the replisome and/or between the replisome and the origin of DNA replication.

Amino Acid Sequence↗

PubMed1997

Transcriptional analysis and promoter activity of the Spodoptera littoralis multicapsid nucleopolyhedrovirus ecdysteroid UDP-glucosyltransferase gene.

The ecdysteroid UDP-glucosyltransferase gene (egt) of Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) is a homologue of the Autographa californica MNPV (AcMNPV) egt gene, which has been found to block insect moulting. Infection of larvae with an egt-deleted AcMNPV resulted in enhanced mortality as compared to infection with the wild-type virus. Consequently, deletion of an egt gene has been proposed as a tempting approach for enhancing the insecticidal properties of baculoviruses. In a previous report we described the mapping and sequencing of the SpliMNPV egt gene. Here we use time-course Northern blot and biochemical analyses to show the production of egt transcripts and protein. The SpliMNPV egt transcription start sites were mapped to 22 and 25 nucleotides downstream of the TATA box by primer extension. Transient expression assays of chimeric egt promoter-chloramphenicol acetyltransferase (cat) reporter gene constructs revealed low promoter activity that was transactivated by AcMNPV immediate-early viral protein IE-1.

Animals↗

PubMed1998

Specificity of multiple homologous genomic regions in Spodoptera exigua nucleopolyhedrovirus DNA replication.

The region upstream of the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) ubiquitin gene contains four near-identical 68-bp-long palindromic repeats. This region, named Sehr6 and located at map unit (m.u.) 88 of the SeMNPV genome on pSeEcoRI-2.2, showed structural homology to previously identified homologous regions (hrs) in a number of other baculoviruses. Hrs function as enhancers of transcription and as putative origins (oris) of baculovirus DNA replication. Five additional hrs (Sehr1-Sehr5) were identified on the SeMNPV genome by Southern blot hybridization with an 18-bp-long oligonucleotide complementary to a sequence conserved within the arms of the four palindromic repeats of Sehr6. Sehr1-Sehr6 were dispersed on the SeMNPV genome at m.u. 8.0, 30.0, 38.5, 51.0, 77.0 and 88.0, respectively. Sequence analysis of these hrs confirmed the presence of palindromic repeats, highly similar to those found in pSeEcoRI-2.2. The number of palindromes varied from one (Sehr4) to nine (Sehr1) per hr. The Sehrs are all present in non-coding regions of the SeMNPV genome and also contain multiple putative transcription recognition sequences. Plasmids containing either of the Sehrs replicated in an SeMNPV-dependent DNA replication assay. The Sehrs were unable to replicate in an AcMNPV-dependent DNA replication assay. This was in contrast to the previously observed SeMNPV non-hr type ori, which replicated in the presence of both AcMNPV and SeMNPV. These data suggest that the replication of SeMNPV and the role of hrs in this process is highly specific.

Animals↗

PubMed1999

Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome.

The nucleotide sequence of the DNA genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, was determined and analysed. The genome contains 135611 bp and has a G+C content of 44 mol%. Computer-assisted analysis revealed 139 ORFs of 150 nucleotides or larger; 103 have homologues in Autographa californica MNPV (AcMNPV) and a further 16 have homologues in other baculoviruses. Twenty ORFs are unique to SeMNPV. Major differences in SeMNPV gene content and arrangement were found compared with the group I NPVs AcMNPV, Bombyx mori (Bm) NPV and Orgyia pseudotsugata (Op) MNPV and the group II NPV Lymantria dispar (Ld) MNPV. Eighty-five ORFs were conserved among all five baculoviruses and are considered as candidate core baculovirus genes. Two putative p26 and odv-e66 homologues were identified in SeMNPV, each of which appeared to have been acquired independently and not by gene duplication. The SeMNPV genome lacks homologues of the major budded virus glycoprotein gene gp64, the immediate-early transactivator ie-2 and bro (baculovirus repeat ORF) genes that are found in AcMNPV, BmNPV, OpMNPV and LdMNPV. Gene parity analysis of baculovirus genomes suggests that SeMNPV and LdMNPV have a recent common ancestor and that they are more distantly related to the group I baculoviruses AcMNPV, BmNPV and OpMNPV. The orientation of the SeMNPV genome is reversed compared with the genomes of AcMNPV, BmNPV, OpMNPV and LdMNPV. However, the gene order in the 'central' part of baculovirus genomes is highly conserved and appears to be a key feature in the alignment of baculovirus genomes.

Animals↗

PubMed2000

Isolation of a Spodoptera exigua baculovirus recombinant with a 10.6 kbp genome deletion that retains biological activity.

When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13.7 and 21.6 map units of SeMNPV and including ecdysteroid UDP glucosyl transferase, gp37, chitinase and cathepsin genes, as well as several genes unique to SeMNPV. The result indicated, however, that these genes are dispensable for virus replication both in vitro and in vivo. A mutant with a similar deletion was identified by PCR in the parental wild-type SeMNPV isolate, suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses. The generation of recombinants in vivo, combined with the alternate cloning between insects and insect cells, is likely to be applicable to many baculovirus species in order to obtain biologically active recombinants.

Animals↗