Williams, Trevor

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https://academica-e.unavarra.es/handle/2454/50181

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Williams

First Name

Trevor

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Producción Agraria

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1173239

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2621

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Open Access
Bacmid expression of granulovirus enhancin En3 accumulates in cell soluble fraction to potentiate nucleopolyhedrovirus infection
(MDPI, 2021) Ricarte Bermejo, Adriana; Simón de Goñi, Oihane; Fernández González, Ana Beatriz; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy.
Open Access
A qPCR assay for the quantification of selected genotypic variants of spodoptera frugiperda multiple nucleopolyhedrovirus (Baculoviridae)
(MDPI, 2024-05-20) Molina-Ruiz, Cindy S.; Zamora-Briseño, Jesús Alejandro; Simón de Goñi, Oihane; Lasa, Rodrigo; Williams, Trevor; Institute for Multidisciplinary Research in Applied Biology - IMAB
Alphabaculoviruses are lethal dsDNA viruses of Lepidoptera that have high genetic diversity and are transmitted in aggregates within proteinaceous occlusion bodies. This mode of transmission has implications for their efficacy as biological insecticides. A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV-NIC) comprising nine genotypic variants has been the subject of considerable study due to the influence of variant interactions on the insecticidal properties of mixed-variant occlusion bodies. As part of a systematic study on the replication and transmission of variant mixtures, a tool for the accurate quantification of a selection of genotypic variants was developed based on the quantitative PCR technique (qPCR). First, primer pairs were designed around a region of high variability in four variants named SfNic-A, SfNic-B, SfNic-C and SfNic-E to produce amplicons of 103–150 bp. Then, using cloned purified amplicons as standards, amplification was demonstrated over a dynamic range of 108–101 copies of each target. The assay was efficient (mean ± SD: 98.5 ± 0.8%), reproducible, as shown by low inter- and intra-assay coefficients of variation (<5%), and specific to the target variants (99.7–100% specificity across variants). The quantification method was validated on mixtures of genotype-specific amplicons and demonstrated accurate quantification. Finally, mixtures of the four variants were quantified based on mixtures of budded virions and mixtures of DNA extracted from occlusion-derived virions. In both cases, mixed-variant preparations compared favorably to total viral genome numbers by quantification of the polyhedrin (polh) gene that is present in all variants. This technique should prove invaluable in elucidating the influence of variant diversity on the transmission and insecticidal characteristics of this pathogen.
Open Access
Use of biocides to minimize microbial contamination in Spodoptera exigua multiple nucleopolyhedrovirus preparations
(Elsevier, 2020-08-13) Presa-Parra, Ehdibaldo; Lasa, Rodrigo; Reverchon, Frédérique; Simón de Goñi, Oihane; Williams, Trevor; Institute for Multidisciplinary Research in Applied Biology - IMAB
The presence of contaminant microbes in baculovirus-based insecticides is regulated by phytosanitary product registration authorities. We aimed to determine whether the abundance of microbes in suspensions of Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies (OBs) could be reduced by treatment with a range of biocidal compounds. The diversity of contaminant bacteria was determined by next-generation sequencing of the 16S rRNA gene. Overall, 97.9% of sequences detected were Gammaproteobacteria (mostly Pseudomonas spp. and Enterobacteriaceae) and 2.1% were Firmicutes (mostly Enterococcus spp.). Colloidal silver, benzalkonium chloride and chlorhexidine digluconate were identified as highly effective biocides. Incubation of OB suspensions with high concentrations of colloidal silver (450 mg/l) or benzalkonium chloride (6000 mg/l) resulted in marked reductions in colony forming unit counts over a 180 day period at 4° or 25 °C. Benzalkonium chloride and colloidal silver treatments, at either 4 or 25 °C, did not affect the insecticidal activity of OBs over an 80 day period. However, OB activity decreased following 180 days of treatment by benzalkonium chloride at either 4 or 25 °C, or by colloidal silver at 25 °C, but not at 4 °C. Counts of OBs revealed a significant decrease in OB numbers in benzalkonium chloride-treated suspensions after 180 days at both temperatures, whereas colloidal silver-treated OBs were not affected. Benzalkonium chloride also caused aggregation of OBs at the concentration tested. We conclude that biocidal compounds can markedly reduce the abundance of contaminant microorganisms in OB suspensions, and can be accompanied by reductions in OB infectivity and OB numbers in some circumstances. Future studies should focus on lower concentrations of biocides that do not affect OBs in long-term storage.
Open Access
Remarkably efficient production of a highly insecticidal Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) isolate in its homologous host
(Wiley, 2018-01-03) Bernal Rodríguez, Alexandra; Simón de Goñi, Oihane; Williams, Trevor; Muñoz Labiano, Delia; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Background: a Chrysodeixis chalcites nucleopolyhedrovirus from the Canary Islands (ChchNPV-TF1) has proved to be effective for control of Chrysodeixis chalcites on banana crops. Commercialization of this virus as a bioinsecticide requires an efficient production system. Results: the sixth instar (L6) was the most suitable for virus production, producing 1.80 × 1011 occlusion bodies (OB)/larva and showed a lower prevalence of cannibalism (5.4%) than fourth (L4) or fifth (L5) instars. Inoculation of L6 at 24 h post molting produced six times more OB (5.72 × 1011 OB/larva) than recently molted L6 larvae (1.00 × 1011 OB/larva). No significant differences were recorded in mean time to death (165–175 h) or OB production per larva (3.75 × 1011 to 5.97 × 1011) or per mg larval weight (1.30 × 1011 to 2.11 × 109), in larvae inoculated with a range of inoculum concentrations (LC50–LC90). Groups of infected L6 larvae reared at a density of 150 larvae/container produced a greater total number of OBs (8.07 × 1013 OB/container) than lower densities (25, 50 and 100 OB/container), and a similar number to containers with 200 inoculated larvae (8.43 × 1013 OB/container). Conclusion: the processes described here allow efficient production of sufficient OBs to treat ∼ 40 ha of banana crops using the insects from a single container.
Open Access
Lacanobia oleracea nucleopolyhedrovirus (LaolNPV): a new European species of alphabaculovirus with a narrow host range
(Public Library of Science, 2017) Simón de Goñi, Oihane; Erlandson, Martin A.; Frayssinet, Marie; Williams, Trevor; Theilmann, David A.; Volkoff, Anne Nathalie; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIQ1406-RI1
During an insect sampling program in alfalfa crops near Montpellier, France in 2011, Lacanobia oleracea larvae were collected that died due to nucleopolyhedrovirus infection (LaolNPV). This virus was subjected to molecular and biological characterization. The virus was a multiple nucleocapsid NPV that showed similar restriction profiles to Mamestra configurata NPV-A (MacoNPV-A) but with significant differences. Polypeptide analysis demonstrated similar proteins in occlusion bodies and occlusion derived virions, to those observed in NPVs from Mamestra spp. Terminal sequencing revealed that the genome organization shared similarity with that of MacoNPV-A. The most homologous virus was MacoNPV-A 90/2 isolate (95.63% identity and 96.47% similarity), followed by MacoNPV-A 90/4 strain (95.37% and 96.26%), MacoNPV-B (89.21% and 93.53%) and M. brassicae MNPV (89.42% and 93.74%). Phylogenetic analysis performed with lef-8, lef-9, polh and a concatenated set of genes showed that LaolNPV and the Mamestra spp. NPVs clustered together with HaMNPV, but with a closer genetic distance to MacoNPV-A strains. The Kimura 2-parameter (K-2-P) distances of the complete genes were greater than 0.05 between LaolNPV and the MbMNPV/MacoNPV-B/HaMNPV complex, which indicates that LaolNPV is a distinct species. K-2-P distances were in the range 0.015±0.050 for comparisons of LaolNPV with MacoNPV-A strains, such that additional biological characteristics should be evaluated to determine species status. While MacoNPV-A was pathogenic to seven lepidopteran species tested, LaolNPV was only pathogenic to Chrysodeixis chalcites. Given these findings, Lacanobia oleracea nucleopolyhedrovirus should be considered as a new species in the Alphabaculovirus genus.