Open Access
Efecto de las enhancinas sobre las propiedades insecticidas de los baculovirus
(2014) Aguirre Sánchez, Eduardo; Simón de Goñi, Oihane; Caballero Murillo, Primitivo; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
El objetivo del presente trabajo fue caracterizar molecularmente una enhancina (VEF, virus enhancing factor) localizada en el genoma de un granulovirus de Agrotis segetum (AgseGV) aislado en Badajoz y determinar su efecto sobre la actividad insecticida del Alphabaculovirus de Autographa californica (AcMNPV) para distintas especies de insectos susceptibles. La enhancina de AgseGV presentó un 99% de identidad con la enhancina de otro aislado del AgseGV de China (AY522332) mientras la identidad con las VEFs localizadas en el NPV de A. segetum AgseNPV (DQ123841) sólo fue del 24-28%. Las VEFs del AgseGV y del AgseNPV presentan una valina (HVMGH) y una alanina (HAISF), respectivamente, en el motivo de unión a zinc en vez del característico ácido glutámico (HEXXH) de las metaloproteasas lo que sugiere que carezcan de funcionalidad. Para determinar su posible funcionalidad se construyeron recombinantes mediante el sistema Bac-to-Bac con el AcMNPV, y las VEFs del AgseGV (BacENHAgseGV) y del AgseNPV (BacVEF1AgseNPV, BacVEF2AgseNPV y BacVEF3AgseNPV). La incorporación del gen de la enhancina de AgseGV en BacENHAgseGV se comprobó mediante PCR, análisis de restricción y secuenciación; sin embargo, no se detectó expresión génica en células Sf21 infectadas con el BacENHAgseGV. El análisis de los componentes proteicos de OBs recombinantes en geles de SDS-PAGE tampoco reveló la presencia de bandas del tamaño correspondiente al de las enhancinas (100-115 kDa). Este dato sugiere que no ha habido síntesis de la enhancina o que si ha habido síntesis no se ha incorporado a los OBs. Finalmente, se cuantificó la actividad insecticida de los recombinantes en larvas L2 y L4 de distintas especies de lepidópteros. En ninguna de las especies ensayadas se pudo apreciar una mejora de la actividad insecticida de los virus recombinantes, mientras que en el control positivo, Bacᴓ al que se añadió Tinopal UNPA-GX, si hubo una mejora significativa de la actividad biológica del virus para las larvas L4. En base a los resultados obtenidos, podríamos concluir que los baculovirus recombinantes no expresan las correspondientes enhancinas, lo cual sugiere la necesidad de abordar la construcción de nuevos recombinantes que contemplen la inclusión de los promotores nativos de los genes de las enhancinas que se quieran expresar
Open Access
Genetic diversity in Chrysodeixis includens nucleopolyhedrovirus and factors involved in its generation
(2021) Aguirre Sánchez, Eduardo; Caballero Murillo, Primitivo; Beperet Arive, Inés; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
En la presente tesis se ha examinado la variabilidad genética de varios aislados de campo del nucleopoliedrovirus de Chrysodeixis includens (ChinNPV). Los resultados obtenidos revelan la gran variabilidad presente en los aislados naturales de ChinNPV, confirma las diferencias existentes entre dos variantes genotípicas procedentes de esos aislados y, lo más importante, parecen identificar a la variante ChinNPV-K como generadora de variabilidad en esos aislados. En conclusión, hemos encontrado una gran variabilidad en aislados naturales de ChinNPV, con una sola variante que muestra un papel clave en la generación de esta diversidad de manera dependiente de la concentración.
Open Access
Synergy of lepidopteran nucleopolyhedroviruses AcMNPV and SpliNPV with insecticides
(MDPI, 2020) Dáder, Beatriz; Aguirre Sánchez, Eduardo; Caballero Murillo, Primitivo; Medina, Pilar; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The joint use of baculoviruses and synthetic insecticides for integrated pest management requires the study of the additive, synergistic or antagonistic effects among them on pest mortality. Droplet bioassays were conducted with Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) and seven insecticides (azadirachtin, Bacillus thuringiensis, cyantraniliprole, emamectin, metaflumizone, methoxyfenozide and spinetoram) on Spodoptera exigua and Spodoptera littoralis. The lethal concentrations LC50 and LC95 were calculated through probit regressions. Then, the sequential feeding of insecticides and nucleopolyhedroviruses was studied. Larvae were provided with the LC50 of one insecticide, followed by the LC50 of one nucleopolyhedrovirus 24 h later. The inverse order was also conducted. The insecticide LC50 and LC95 were higher for S. littoralis than for S. exigua. AcMNPV showed greater toxicity on S. exigua than SpliNPV on S. littoralis. Emamectin showed synergy with AcMNPV when the chemical was applied first, and metaflumizone and AcMNPV were synergistic regardless of the order of application, both from the first day of evaluation. SpliNPV was synergistic with azadirachtin and emamectin when it was applied first, but synergy was reached after 12–13 days. Excellent control is possible with the LC50 of azadirachtin, emamectin and metaflumizone in combination with nucleopolyhedroviruses, and merits further study as a means of controlling lepidopteran pests.
Open Access
Genetic variability of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and the insecticidal characteristics of selected genotypic variants
(MDPI, 2019) Aguirre Sánchez, Eduardo; Beperet Arive, Inés; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Genetic variation in baculoviruses is recognized as a key factor, not only due to the influence of such variation on pathogen transmission and virulence traits, but also because genetic variants can form the basis for novel biological insecticides. In this study, we examined the genetic variability of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) present in field isolates obtained from virus-killed larvae. Different ChinNPV strains were identified by restriction endonuclease analysis, from which genetic variants were isolated by plaque assay. Biological characterization studies were based on pathogenicity, median time to death (MTD), and viral occlusion body (OB) production (OBs/larva). Nine different isolates were obtained from eleven virus-killed larvae collected from fields of soybean in Mexico. An equimolar mixture of these isolates, named ChinNPV-Mex1, showed good insecticidal properties and yielded 23 genetic variants by plaque assay, one of which (ChinNPV-R) caused the highest mortality in second instars of C. includens. Five of these variants were selected: ChinNPV-F, ChinNPV-J, ChinNPV-K, ChinNPV-R, and ChinNPV-V. No differences in median time to death were found between them, while ChinNPV-F, ChinNPV-K, ChinNPV-R and ChinNPV-V were more productive than ChinNPV-J and the original mixture of field isolates ChinNPV-Mex1. These results demonstrate the high variability present in natural populations of this virus and support the use of these new genetic variants as promising active substances for baculovirus-based bioinsecticides.
Open Access
Generation of variability in Chrysodeixis includens nucleopolyhedrovirus (ChinNPV): the role of a single variant
(MDPI, 2021) Aguirre Sánchez, Eduardo; Beperet Arive, Inés; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
The mechanisms generating variability in viruses are diverse. Variability allows baculoviruses to evolve with their host and with changes in their environment. We examined the role of one genetic variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and its contribution to the variability of the virus under laboratory conditions. A mixture of natural isolates (ChinNPVMex1) contained two genetic variants that dominated over other variants in individual larvae that consumed high (ChinNPV-K) and low (ChinNPV-E) concentrations of inoculum. Studies on the ChinNPV-K variant indicated that it was capable of generating novel variation in a concentrationdependent manner. In cell culture, cells inoculated with high concentrations of ChinNPV-K produced OBs with the ChinNPV-K REN profile, whereas a high diversity of ChinNPV variants was recovered following plaque purification of low concentrations of ChinNPV-K virion inoculum. Interestingly, the ChinNPV-K variant could not be recovered from plaques derived from low concentration inocula originating from budded virions or occlusion-derived virions of ChinNPV-K. Genome sequencing revealed marked differences between ChinNPV-K and ChinNPV-E, with high variation in the ChinNPV-K genome, mostly due to single nucleotide polymorphisms. We conclude that ChinNPV-K is an unstable genetic variant that is responsible for generating much of the detected variability in the natural ChinNPV isolates used in this study.