Spodoptera Exigua Multiple Nucleopolyhedrovirus Research Articles

Insecticidal Properties and Microbial Contaminants in a Spodoptera exigua Multiple Nucleopolyhedrovirus (Baculoviridae) Formulation Stored at Different Temperatures

The Spodoptera exigua (Hübner) multiple nucleopolyhedrovirus (SeMNPV) is currently being tested as a biological insecticide for use in greenhouse crops in southern Spain. We performed a study in which semipurified SeMNPV occlusion bodies (OBs) were formulated in phosphate-buffered saline, pH 6.5, with 5% (vol:vol) glycerol and 0.15% (wt:vol) sorbic acid, and they were stored at −20, 4, or 25°C during 18 mo. Initial aerobic counts (±SE) averaged 1.4 (±0.17) × 107 colony-forming units/ml after 17-h incubation at 37°C. Aerobic counts of microorganisms that contaminated OB formulations stored at 25°C decreased markedly over the period of the study, whereas only small decreases were observed in counts from OBs stored at 4 or −20°C. The principal microbial contaminants of OB suspensions were Enterococcus spp., Enterobacteriaceae, and yeasts. Potential human pathogens (Salmonella, Shigella, and Vibrio species) were not detected, and populations of Staphylococcus aureus and Bacillus cereus were extremely low. Compared with newly formulated OBs, the estimated LD50 values of OBs stored at 25°C increased by > 16,666-fold over the 18 mo of storage, whereas LD50 values were not greatly affected by storage at 4 or −20°C. Significant changes over time in OB concentrations were only observed in the 25°C treatment. Complete degradation of viral DNA was observed at 25°C but not in refrigerated or frozen OBs. We conclude that OB formulation with bacteriostatic or antioxidant additives, together with storage and distribution in refrigerated conditions, will likely result in an SeMNPV biopesticide shelf life that exceeds 18 mo.

GP64 of group I nucleopolyhedroviruses cannot readily rescue infectivity of group II f-null nucleopolyhedroviruses

The genus Nucleopolyhedrovirus (NPV) of the family Baculoviridae can be subdivided phylogenetically into two groups. The same division can be made on the basis of their budded virus (BV) envelope fusion protein. Group I NPVs are characterized by the presence of a GP64-like major envelope fusion protein, which is involved in viral attachment and the fusion of virus and cell membrane, and is required for budding of progeny nucleocapsids. Group II NPVs have an envelope fusion protein unrelated to GP64, named F. In contrast to GP64, F proteins are found in all baculoviruses, but they are not functional as envelope fusion proteins in group I NPVs. Autographa californica multiple NPV (AcMNPV) lacking GP64 can be pseudotyped by the F protein of Spodoptera exigua multiple NPV (SeMNPV), suggesting that F proteins are functionally analogous to GP64. GP64 homologues are thought to have been acquired by group I NPVs during evolution, thereby giving these viruses a selective advantage and obviating the need for a functional F protein. To address this supposition experimentally, attempts were made to pseudotype a group II NPV, SeMNPV, with GP64. Transfection of an f-null SeMNPV bacmid into Se301 cells did not result in the production of infectious BVs. This defect was rescued by insertion of SeMNPV f, but not by insertion of AcMNPV gp64. This suggests that the functional analogy between GP64 and F is not readily reciprocal and that F proteins from group II NPVs may provide additional functions in BV formation that are lacking in the GP64 type of fusion protein.

Abundance and genetic structure of nucleopolyhedrovirus populations in greenhouse substrate reservoirs

The abundance and genetic structure of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) occlusion body (OB) populations in greenhouse substrates in southern Spain was determined during an 18 month period over an area of 426 km 2 . To detect the presence of OBs, substrate samples were incorporated into a semi-synthetic diet and fed to first instar S. exigua. SeMNPV OBs were detected in 34% of substrate samples (N = 267). The prevalence of substrates positive for OBs did not differ significantly between samples from different crops or from four different geographical zones. Seasonally, Spring and Summer samples had significantly greater OB concentrations than samples taken in Autumn and Winter, concurrent with changes in the host insect population. Restriction fragment length polymorphism analysis revealed at least nine different variants. Substrate pH was identified as a major factor influencing the abundance and genetic composition of the soil OB population. Certain variants were more prevalent in substrates of more alkaline pH suggesting genotype-specific differences in survival outside the host. The prevalence of mixed variant infections was inversely related to an increasing substrate pH. Greenhouse substrates of southern Spain represent an abundant and diverse reservoir for the survival of SeMNPV. Studies are required to determine the degree to which agropractices that conserve soil OB populations would favor long-term microbial control of lepidopteran pests in greenhouse crops. � 2007 Elsevier Inc. All rights reserved.

Efficacy of Spodoptera exigua multiple nucleopolyhedrovirus as a biological insecticide for beet armyworm control in greenhouses of southern Spain

Chemical control measures targeted at Spodoptera exigua in greenhouse sweet pepper crops in Spain have resulted in pest resistance to virtually all commercially available insecticidal products. A multicapsid nucleopolyhedrovirus (SeMNPV), isolated from diseased S. exigua in Spain, was produced in laboratory reared larvae, tested for insecticidal activity in a laboratory bioassay, and was then applied in eleven commercial greenhouses planted with sweet pepper. Virus occlusion bodies (OBs) were applied on two occasions, at an interval of ∼7 days, at a rate of 5×108 OBs/L of spray in a volume of ∼600 L/ha, depending on crop phenology and greenhouse area. The percentage of plants showing recent (<48 h old) feeding damage fell dramatically in greenhouses with high infestations of S. exigua; the same pattern was observed, although less dramatically, in greenhouses with low infestations. Average mortality of larvae collected from treated plants at 4 days after each application, and reared in the laboratory until death, was high (70–89%) and was not significantly affected by the degree of crop infestation. In a separate trial, the rate of acquisition of infection was examined in larvae that fed on plants treated with 1×108 or 5×108 OBs/L of spray. Of the 27 and 60% of larvae, respectively, that acquired infection in the 48 h period after spraying, about half became infected in the first 6 h post-application, irrespective of application rate. Acquisition of infection proceeded more slowly during the night-time compared to the daytime period, underlining the advantages of early morning applications of the virus. We conclude that the Spanish SeMNPV isolate merits registration as a biological insecticide for use in greenhouse crops in this region.

Group I but not Group II NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.

Efficacy of optical brightener formulations of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) as a biological insecticide in greenhouses in southern Spain

Abstract The efficacy of optical brightener formulations of a native Spanish isolate of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) was determined for control of S. exigua on greenhouse grown sweet pepper (Capsicum annuum) in Almeria, Spain. In laboratory bioassays involving diet surface contamination, the 50% lethal concentration in fourth instars was reduced 25-fold and 5731-fold, compared to SeMNPV occlusion bodies (OBs) alone, or in mixtures with 0.1% and 1% of the stilbene-derived optical brightener leucophor AP, respectively. The efficacy of spray applications of 1 × 1012 OBs/Ha SeMNPV alone, or in mixtures with 0.1% leucophor AP, was tested in a greenhouse planted with sweet pepper. The prevalence of virus infection in larvae collected at intervals post-application and reared on artificial diet in the laboratory was very high (62–97% mortality). Compared to the treatment involving SeMNPV OBs alone, OBs applied in mixtures with leucophor AP resulted in a significant increase in the prevalence of infection in larvae collected at 2 days post-application, but not in insects collected subsequently. In contrast, a chemical insecticide treatment, lufenuron, performed poorly (

Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotis segetum: collinearity with Spodoptera exigua multiple NPV

The genome sequence of a Polish isolate of Agrotis segetum nucleopolyhedrovirus (AgseNPV-A) was determined and analysed. The circular genome is composed of 147,544 bp and has a G+C content of 45.7 mol%. It contains 153 putative, non-overlapping open reading frames (ORFs) encoding predicted proteins of more than 50 aa, together making up 89.8 % of the genome. The remaining 10.2 % of the DNA constitutes non-coding regions and homologous-repeat regions. One hundred and forty-three AgseNPV-A ORFs are homologues of previously reported baculovirus gene sequences. There are ten unique ORFs and they account for 3 % of the genome in total. All 62 lepidopteran baculovirus genes, including the 29 core baculovirus genes, were found in the AgseNPV-A genome. The gene content and gene order of AgseNPV-A are most similar to those of Spodoptera exigua (Se) multiple NPV and their shared homologous genes are 100 % collinear. Three putative enhancin genes were identified in the AgseNPV-A genome. In phylogenetic analysis, the AgseNPV-A enhancins form a cluster separated from enhancins of the Mamestra species NPVs.

Application of the PCR–RFLP method for the rapid differentiation of Spodoptera exigua nucleopolyhedrovirus genotypes

Quality control during mass production of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), and studies on environmental fate following the use of this virus as a biological pesticide, would be facilitated by a rapid method for the detection and identification of isolates. A molecular biology tool was developed that combined the polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) to differentiate SeMNPV isolates. Oligonucleotide primers were designed to amplify five variable SeMNPV genomic regions (V01, V02, V03, V04, V05). Four wild-type SeMNPV strains isolated from the United States (US2) and Spain (SP1, SP2, and SP3), and a laboratory cloned genotype (US1A), were analyzed with 36 different primer-endonuclease combinations. BglII digestion of the variable region 1 (V01) amplicon was the only combination that differentiated each of the five virus isolates tested, although genetic heterogeneity limited the discriminatory power of the technique. Six novel SeMNPV isolates originating from greenhouse soils in southern Spain were successfully identified using this method. As judged by sequence analysis, the V01 region, which comprises the homologous region 1 ( hr1), is the most variable genomic region among the genotypes present in the Spanish isolates. This method constitutes a useful tool for processing large number of environmental samples and could be used to address environmental biosafety concerns.

Identification and characterization of a major early-transcribed gene of Trichoplusia ni single nucleocapsid nucleopolyhedrovirus using the baculovirus expression system.

An early transcribed gene (me-53) of a South Africa strain of Trichoplusia ni single nucleocapsid nucleopolyhedrovirus (TnSNPV) was sequenced and identified. It has an open reading frame of 1146 nucleotides that encodes a protein of 382 amino acids with a molecular mass of 45.2 kDa. The deduced protein sequence alignment of 13 baculovirus ME-53s indicated that the TnSNPV ME-53 shares the highest homologies with NPV subgroup II-A Spodoptera exigua multiple and Mamestra configurata (Maco) nucleopolyhedrovirus ME-53s. The zinc finger-like motifs at the C-termini of ME-53s are highly conserved with similar patterns of cysteine positions. Upon introduction of the gene and a green fluorescent protein reporter gene into the baculovirus expression vector system, the transcriptional analysis of me-53 in two cell lines infected with the Autographa californica nucleopolyhedrovirus (AcMNPV) recombinant revealed that an early TnSNPV me-53 transcript can be detected by 1 h postinfection (hpi) until 12 hpi and a late one from 18 hpi up to 48 hpi, while early and late transcripts of the AcMNPV me-53 of the recombinant can be detected at 3 and 24 hpi, respectively. This suggested that the early and late promoters of both AcMNPV and TnSNPV me-53s were recognized in recombinant virus-infected cells. The regulatory elements of the TnSNPV me-53 promoter were also analyzed.

Characteristics of the transactivator gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus.

The genomic position and nucleotide sequence of the immediate early gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) were determined. The SeMNPV ie1 gene had the potential to encode a protein of 714 amino acids with a predicted molecular mass of 82.0 kDa, representing the largest baculovirus IE1 known to date. The similarity of SeMNPV IE1 with IE1 proteins from other baculoviruses was restricted to the basic C-terminal two-thirds of the protein, which is involved in DNA binding. A single ie1 transcript of 2.5 kb was detected at 1 h p.i., peaking at 8 h p.i. and disappearing at 24 h p.i. From 8 h p.i. onwards a 1.7 kb transcript was detected, which became more abundant late in infection. Primer extension analysis revealed the use of several start sites early and late in infection. One of these was located in the promoter motif CAGT located at position -15 relative to the translational start codon, most intensively used at 8 h p.i. Four kb upstream of the SeMNPV ie1 gene, ORF xd244 was identified with homology to the used left ie0 exon of AcMNPV, OpMNPV and LdMNPV. Phylogenetic analysis indicated that the SeMNPV IE1 protein shared the most recent ancestor with its HzSNPV and LdMNPV homologues, although their IE1 proteins had diverged considerably from each other and from other baculoviruses.