Open Access
Occlusion body pathogenicity, virulence and productivity traits vary with transmission strategy in a nucleopolyhedrovirus
(Elservier, 2011-10-25) Cabodevilla de Andrés, Oihana; Ibáñez Elosua, Itxaso; Simón de Goñi, Oihane; Murillo Pérez, Rosa; Caballero Murillo, Primitivo; Williams, Trevor; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The prevalence of sublethal infections of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) was quantified in natural populations of S. exigua in Almería, Spain, during 2006 and 2007. Of 1045 adults collected, 167 (16.1%) proved positive for viral polyhedrin gene transcripts by RT-PCR. The prevalence of covert infection varied significantly according to sex and sample date. Of 1660 progeny of field-collected insects, lethal disease was observed in 10¿33% of offspring of transcript-positive females and 9¿49% of offspring of transcript-negative females. Isolates associated with vertically transmitted infections were characterized by restriction endonuclease analysis using BglII or EcoRV and compared with isolates originating from greenhouse soil-substrate believed to be horizontally transmitted. Insects from a sublethally infected Almerian colony were between 2.3-fold and 4.6-fold more susceptible to infection than healthy insects from a Swiss colony, depending on isolate. Horizontally transmitted isolates were significantly more pathogenic than vertically transmitted isolates in insects from both colonies. Mean speed of kill in second instars (Swiss colony) varied between isolates by >20 h, whereas mean occlusion body (OB) production in fourth instars (Swiss colony) varied by 3.8-fold among isolates. Intriguingly, all three horizontally transmitted isolates were very similar in speed of kill and OB production, whereas all three vertically transmitted isolates differed significantly from one another in both variables, and also differed significantly from the group of horizontally transmitted isolates in speed of kill (one isolate) or both variables (two isolates). We conclude that key pathogenicity and virulence traits of SeMNPV isolates vary according to their principal transmission strategy.
Open Access
Spodoptera frugiperda multiple nucleopolyhedrovirus as a potential biological insecticide: genetic and phenotypic comparison of field isolates from Colombia
(Elsevier, 2011-04-24) Barrera Cubillos, Gloria Patricia; Simón de Goñi, Oihane; Villamizar, Laura; Williams, Trevor; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Thirty-eight isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), collected from infected larvae on pastures, maize, and sorghum plants in three different geographical regions of Colombia, were subjected to molecular characterization and were compared with a previously characterized Nicaraguan isolate (SfNIC). Restriction endonuclease analysis (REN) using six different enzymes showed two different patterns among Colombian isolates, one profile was particularly frequent (92%) and was named SfCOL. The physical map of SfCOL was constructed and the genome was estimated to be 133.9 kb, with few differences in terms of number and position of restriction sites between the genomes of SfNIC and SfCOL. The PstI-K and PstI-M fragments were characteristic of SfCOL. These fragments were sequenced to reveal the presence of seven complete and two partial ORFs. This region was collinear with SfMNPV sf20–sf27. However, two ORFs (4 and 5) had no homologies with SfMNPV ORFs, but were homologous with Spodoptera exigua MNPV (se21 and se22/se23) and Spodoptera litura NPV (splt20 and splt21). Biological characterization was performed against two different colonies of S. frugiperda, one originating from Colombia and one from Mexico. Occlusion bodies (OBs) of the SfCOL isolate were as potent (in terms of concentration–mortality metrics) as SfNIC OBs towards the Mexican insect colony. However, SfCOL OBs were 12 times more potent for the Colombian colony than SfNIC OBs and three times more potent for the Colombian colony than for the Mexican colony. SfCOL and SfNIC showed a slower speed of kill (by ∼50 h) in insects from the Colombian colony compared to the Mexican colony, which was correlated with a higher production of OBs/larvae. SfCOL is a new strain of SfMNPV that presents pathogenic characteristics that favor its development as the basis for a biopesticide product in Colombia.
Unknown
Expression of a peroral infection factor determines pathogenicity and population structure in an insect virus
(Public Library of Science, 2013) Simón de Goñi, Oihane; Williams, Trevor; Cerutti, Martine; Caballero Murillo, Primitivo; López Ferber, Miguel; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus is being studied as a possible biological insecticide. This virus exists as a mixture of complete and deletion genotypes; the latter depend on the former for the production of an essential per os transmission factor (pif1) in coinfected cells. We hypothesized that the virus population was structured to account for the prevalence of pif1 defector genotypes, so that increasing the abundance of pif1 produced by a cooperator genotype in infected cells would favor an increased prevalence of the defector genotype. We tested this hypothesis using recombinant viruses with pif1 expression reprogrammed at its native locus using two exogenous promoters (egt, p10) in the pif2/pif1 intergenic region. Reprogrammed viruses killed their hosts markedly faster than the wild-type and rescue viruses, possibly due to an earlier onset of systemic infection. Group success (transmission) depended on expression of pif1, but overexpression was prejudicial to group-specific transmissibility, both in terms of reduced pathogenicity and reduced production of virus progeny from each infected insect. The presence of pif1-overproducing genotypes in the population was predicted to favor a shift in the prevalence of defector genotypes lacking pif1-expressing capabilities, to compensate for the modification in pif1 availability at the population level. As a result, defectors increased the overall pathogenicity of the virus population by diluting pif1 produced by overexpressing genotypes. These results offer a new and unexpected perspective on cooperative behavior between viral genomes in response to the abundance of an essential public good that is detrimental in excess.
Open Access
Genetic structure of a Spodoptera frugiperda nucleopolyhedrovirus population: high prevalence of deletion genotypes
(American Society for Microbiology, 2004) Simón de Goñi, Oihane; Williams, Trevor; López Ferber, Miguel; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena
A Nicaraguan field isolate (SfNIC) of Spodoptera frugiperda nucleopolyhedrovirus was purified by plaque assay on Sf9 cells. Nine distinct genotypes, A to I, were identified by their restriction endonuclease profiles. Variant SfNIC-B was selected as the standard because its restriction profile corresponded to that of the wild-type isolate. Physical maps were generated for each of the variants. The differences between variants and the SfNIC-B standard were confined to the region between map units 9 and 32.5. This region included PstI-G, PstI-F, PstI-L, PstI-K and EcoRI-L fragments. Eight genotypes presented a deletion in their genome compared with SfNIC-B. Occlusion body-derived virions of SfNIC-C, -D and -G accounted for 41% of plaque-purified clones. These variants were not infectious per os but retained infectivity by injection into S. frugiperda larvae. Median 50% lethal concentration values for the other cloned genotypes were significantly higher than that of the wild type. The variants also differed in their speed of kill. Noninfectious variants SfNIC-C and -D lacked the pif and pif-2 genes. Infectivity was restored to these variants by plasmid rescue with a plasmid comprising both pif and pif-2. Transcription of an SfNIC-G gene was detected by reverse transcription-PCR in insects, but no fatal disease developed. Transcription was not detected in SfNIC-C or -D-inoculated larvae. We conclude that the SfNIC population presents high levels of genetic diversity, localized to a 17-kb region containing pif and pif-2, and that interactions among complete and deleted genotypic variants will likely influence the capacity of this virus to control insect pests.
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
Sf29 Gene of Spodoptera frugiperda multiple nucleopolyhedrovirus is a viral factor that determines the number of virions in occlusion bodies
(American Society for Microbiology, 2008) Simón de Goñi, Oihane; Williams, Trevor; Asensio, Aarón C.; Ros Terés, Sarhay; Gaya Cacho, Andrea; Caballero Murillo, Primitivo; Possee, Robert D.; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died 24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB.
Open Access
Selection of a nucleopolyhedrovirus isolate from Helicoverpa armigera as the basis for a biological insecticide
(Wiley, 2014-05-01) Arrizubieta Celaya, Maite; Williams, Trevor; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
Background: the cotton bollworm, Helicoverpa armigera, is an insect that causes damage in a wide range of crops in Spain.Seven isolates of H. armigera single nucleopolyhedrovirus (HearSNPV) from the Iberian Peninsula were subjected to molecularand biological characterization and compared with a Chinese genotype (HearSNPV-G4). Results: the estimated sizes of the Iberian genomes varied between 116.2 and 132.4 kb, compared to 131.4 kb of theHearSNPV-G4 reference genome. Phylogenetic analysis based on the lef-8, lef-9 and polh genes revealed that the Iberianstrains were more closely related to one another than to other HearSNPV isolates. Occlusion body (OB) concentration-mortalityresponses (LC 50 values) did not differ signiﬁcantly among Iberian isolates when tested against a Helicoverpa armigera colonyfrom Oxford (UK). Despite being the fastest killing isolate, HearSNPV-SP1 was as productive as isolates with lower virulence,with an average yield of 3.1 × 109 OBs larva−1 . OBs of HearSNPV-SP1 and HearSNPV-G4 were similarly pathogenic against arecently established colony from southern Spain, although HearSNPV-SP1 was faster killing than HearSNPV-G4 against a rangeof instars. Conclusion: the insecticidal properties of HearSNPV-SP1 mean that this strain is likely to prove useful as the basis for abiological insecticide for control of Helicoverpa armigera in Spain.
Unknown
Virus entry or the primary infection cycle are not the principal determinants of host specificity of Spodoptera spp. nucleopolyhedroviruses
(Microbiology Society, 2004-10-01) Simón de Goñi, Oihane; Williams, Trevor; López Ferber, Miguel; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena
The multicapsid nucleopolyhedroviruses (NPVs) of Spodoptera exigua (SeMNPV), Spodoptera frugiperda (SfMNPV), and Spodoptera littoralis (SpliNPV) are genetically similar (78% similarity) but differ in their degree of host specificity. Infection by each of the three NPVs in these three Spodoptera host species was determined by oral inoculation of larvae with occlusion bodies (OBs) or intrahaemocoelic injection with occlusion derived virions (ODVs). RT-PCR analysis of total RNA from inoculated insects, targeted at immediate early (ie-0), early (egt, DNA polymerase), late (chitinase) and very late genes (polyhedrin), indicated that each of the NPVs initiated an infection in all three host species tested. SpliMNPV produced a fatal NPV disease in both heterologous hosts, S. frugiperda and S. exigua, by oral inoculation or injection. SfMNPV was lethal to heterologous hosts, S. exigua and S. littoralis, but infected larvae did not melt and disintegrate, and progeny OBs were not observed. SeMNPV was able to replicate in heterologous hosts and all genes required for replication were present in the genome, as the virus primary infection cycle was observed. However, gene expression was significantly lower in heterologous hosts. SeMNPV pathogenesis in S. frugiperda and S. littoralis was blocked at the haemocoel transmission stage and very nearly cleared. SeMNPV mixtures with SpliMNPV or SfMNPV did not extend the host range of SeMNPV; in all cases, only the homologous virus was observed to proliferate. It is concluded that entry and the primary virus infection cycle are not the only, or the major determinants, for SeMNPV infection of heterologous Spodoptera species.
Open Access
Chrysodeixis chalcites, a pest of banana crops on the Canary Islands: incidence, economic losses and current control measures
(Elsevier, 2018-03-05) Fuentes Barrera, Ernesto Gabriel; Hernández Suárez, Estrella; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Chrysodeixis chalcites is an emergent pest in bananas (Musa acuminata Colla) grown on the Canary Islands. Feeding damage to leaves and fruit and the control measures targeted at this pest were evaluated over a two-year period (2013–2014). The prevalence of infestations (42–100%) on the islands was similar during the two years of the study. Mean foliar damage (1.5–7.3% depending on island) and fruit damage (1.0–5.7%) detected in field surveys varied significantly across islands, plantation aspect (north- or south-facing) and season. Fruit damage was not correlated with foliar damage (P > 0.05). The weight of C. chalcites damaged bananas varied significantly (0.2–4.2% of harvested fruit) across islands, particularly in the spring. Overall, 3155 tonnes of bananas/yr are likely discarded due to C. chalcites damage, representing 1.5% of annual production or 2.68 million €/yr. The most frequently used pesticide was indoxacarb, usually applied on three occasions per crop cycle, for which the cost of control measures would average 240 €/ha per crop cycle. The direct damage that C. chalcites causes to banana fruit results in significant economic losses in addition to the direct costs of pesticide based control measures. Effective and sustainable control strategies are required against this pest.
Open Access
A native variant of Chrysodeixis chalcites nucleopolyhedrovirus: the basis for a promising bioinsecticide for control of C. chalcites in Canary Islands' banana crops
(Elsevier, 2013-08-13) Bernal Rodríguez, Alexandra; Williams, Trevor; Hernández Suárez, Estrella; Carnero, Aurelio; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Chrysodeixis chalcites (Lepidoptera: Noctuidae) larvae cause up to 30% production loss in banana crops in the Canary Islands. Larvae of this species are susceptible to a nucleopolyhedrovirus (ChchNPV). This study aimed at evaluating the genetic diversity and bioinsecticidal activity of ChchNPV isolates collected from C. chalcites larvae in the Canary Islands. From a total 97 isolates collected in different banana greenhouses, restriction endonuclease analysis identified five genetic variants that differed slightly from ChchNPV isolates from Netherlands (ChchSNPV-NL) and Almería, Spain (ChchNPV-SP1). Physical maps revealed minimal differences at the genome level, mostly due to variation in the position/existence of restriction sites. ChchSNPV-TF1 was the most prevalent variant, representing 78% of isolates examined, and was isolated at all Canary Island sampling sites. This isolate was the most pathogenic isolate against C. chalcites second instars in terms of concentration-mortality metrics, compared to homologous variants or two heterologous viruses Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Anagrapha falcifera multiple nucleopolyhedrovirus (AnfaMNPV). ChchSNPV-TF1 was also one of the fastest killing variants although no differences were observed in occlusion body production among the different variants in second instars. We conclude that ChchSNPV-TF1 merits further evaluation as the basis for a biological insecticide for control of C. chalcites in banana crops in the Canary Islands.