Open Access
Complete genome sequence of five Chrysodeixis chalcites nucleopolyhedrovirus genotypes from a Canary Islands isolate
(American Society for Microbiology, 2013-10-24) Bernal Rodríguez, Alexandra; Williams, Trevor; Muñoz Labiano, Delia; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
The Chrysodeixis chalcites single nucleopolyhedrovirus (ChchSNPV) infects and kills C. chalcites larvae, an important pest of banana crops in the Canary Islands. Five genotypes present in the most prevalent and widespread isolate in the Canary Islands were sequenced, providing genetic data relevant to the genotypic and phenotypic diversity of this virus.
Open Access
The sf32 unique gene of spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a non-essential gene that could be involved in nucleocapsid organization in occlusion-derived virions
(Public Library of Science, 2013) Beperet Arive, Inés; Barrera Cubillos, Gloria Patricia; Simón de Goñi, Oihane; Williams, Trevor; López Ferber, Miguel; Gasmi, Laila; Herrero, Salvador; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
A recombinant virus lacking the sf32 gene (Sf32null), unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac). Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs) of Sf32null contained 62% more genomic DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration), speed-of-kill or budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity) to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion, resulting in increased numbers of nucleocapsids within ODVs.
Open Access
Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses
(Elsevier, 2012-10-21) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Williams, Trevor; López Ferber, Miguel; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A–I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366–27,225) and 60 bp (119,759–119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.
Open Access
Coocclusion of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV): pathogenicity and stability in homologous and heterologous hosts
(MDPI, 2022) Arrizubieta Celaya, Maite; Simón de Goñi, Oihane; Ricarte Bermejo, Adriana; López Ferber, Miguel; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) is a virulent pathogen of lepidopterans in the genera Heliothis and Helicoverpa, whereas Helicoverpa armigera multiple nu-cleopolyhedrovirus (HearSNPV) is a different virus species with a broader host range. This study aimed to examine the consequences of coocclusion of HearSNPV and HearMNPV on the patho-genicity, stability and host range of mixed-virus occlusion bodies (OBs). HearSNPV OBs were approximately 6-fold more pathogenic than HearMNPV OBs, showed faster killing by approximately 13 h, and were approximately 45% more productive in terms of OB production per larva. For coocclusion, H. armigera larvae were first inoculated with HearMNPV OBs and subsequently inoculated with HearSNPV OBs at intervals of 0-72 h after the initial inoculation. When the interval between inoculations was 12-24 h, OBs collected from virus-killed insects were found to comprise 41¿57% of HearSNPV genomes, but the prevalence of HearSNPV genomes was greatly reduced (3- 4%) at later time points. Quantitative PCR (qPCR) analysis revealed the presence of HearSNPV genomes in a small fraction of multinucleocapsid ODVs representing 0.47¿0.88% of the genomes quan-tified in ODV samples, indicating that both viruses had replicated in coinfected host cells. End-point dilution assays on ODVs from cooccluded mixed-virus OBs confirmed the presence of both viruses in 41.9¿55.6% of wells that were predicted to have been infected by a single ODV. A control exper-iment indicated that this result was unlikely to be due to the adhesion of HearSNPV ODVs to HearMNPV ODVs or accidental contamination during ODV band extraction. Therefore, the dispar-ity between the qPCR and end-point dilution estimates of the prevalence of mixed-virus ODVs likely reflected virus-specific differences in replication efficiency in cell culture and the higher in-fectivity of pseudotyped ODVs that were produced in coinfected parental cells. Bioassays on H. armigera, Spodoptera frugiperda and Mamestra brassicae larvae revealed that mixed-virus OBs were capable of infecting heterologous hosts, but relative potency values largely reflected the proportion of HearMNPV present in each mixed-virus preparation. The cooccluded mixtures were unstable in serial passage; HearSNPV rapidly dominated during passage in H. armigera whereas HearMNPV rapidly dominated during passage in the heterologous hosts. We conclude that mixed-virus coocclusion technology may be useful for producing precise mixtures of viruses with host range properties suitable for the control of complexes of lepidopteran pests in particular crops, although this requires validation by field testing.
Open Access
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
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.
Open Access
Determinant factors in the production of a co-occluded binary mixture of Helicoverpa armigera alphabaculovirus (HearNPV) genotypes with desirable insecticidal characteristics
(Public Library of Science, 2016) Arrizubieta Celaya, Maite; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIQ14065:RI1
A co-occluded binary mixture of Helicoverpa armigera nucleopolyhedrovirus genotypes HearSP1B and HearLB6 at a 1:1 ratio (HearSP1B+HearLB6) was selected for the development of a virus-based biological insecticide, which requires an efficient large-scale production system. In vivo production systems require optimization studies in each host-virus pathosystem. In the present study, the effects of larval instar, rearing density, timing of inoculation, inoculum concentration and temperature on the production of HearSP1B+HearLB6 in its homologous host were evaluated. The high prevalence of cannibalism in infected larvae (40–87%) indicated that insects require individual rearing to avoid major losses in OB production. The OB production of recently molted fifth instars (7.0 x 109 OBs/larva), combined with a high prevalence of mortality (85.7%), resulted in the highest overall OB yield (6.0 x 1011 OBs/100 inoculated larvae), compared to those of third or fourth instars. However, as inoculum concentration did not influence final OB yield, the lowest concentration, LC80 (5.5 x 106 OBs/ml), was selected. Incubation temperature did not significantly influence OB yield, although larvae maintained at 30°C died 13 and 34 hours earlier than those incubated at 26°C and 23°C, respectively. We conclude that the efficient production of HearSP1B+HearLB6 OBs involves inoculation of recently molted fifth instars with a LC80 concentration of OBs followed by individual rearing at 30°C.
Open Access
Effects of several UV-protective substances on the persistence of the insecticidal activity of the Alphabaculovirus of Chrysodeixis chalcites (ChchNPV-TF1) on banana (Musa acuminata, Musaceae, Colla) under laboratory and open-field conditions
(Public Library of Science, 2021) Çakmak, Taylan; Simón de Goñi, Oihane; Kaydan, Mehmet Bora; Tange, Denis Achiri; González-Rodríguez, Agueda María; Piedra-Buena Díaz, Ana; Caballero Murillo, Primitivo; Hernández Suárez, Estrella; Institute for Multidisciplinary Research in Applied Biology - IMAB
Alphabaculovirus of Chrysodeixis chalcites (ChchNPV-TF1) has been investigated as a useful bioinsecticide against C. chalcites (Esper) (Lepidoptera: Noctuidae) in banana crops. This study investigated the effects of several substances on the persistence of ChchNPV-TF1 under field conditions in the Canary Islands. Natural photoprotective substances, such as moringa, cacao, green tea, benzopurpurine, charcoal, iron dioxide, benzimidazole, kaolinite, and bentonite, were first evaluated under laboratory conditions using a Crosslinker as UV light source at 200 J/cm(2). The photoprotective substances were divided into three groups: low protection (0-8%; kaolinite), intermediate protection (48-62%; green tea, moringa, bentonite and cacao) and high protection (87-100%; charcoal, iron ioxide). Benzopurpurine and benzimidazole did not provide any photoprotective effects. Two of the substances that yielded the best results, 1% cacao and 1% charcoal, were selected for the open-field experiment in a banana plantation. The persistence of ChchNPV-TF1 OBs (occlusion bodies) on leaf surfaces with sunlight exposure was analysed by comparing the initial mortality of 2(nd) instar C. chalcites larvae with the mortality observed at various intervals postapplication. The mortality rates decreased over time in all treatments and were always higher in the UV-protective substance-treated parcels. The 1% charcoal treatment exhibited the highest protection in both the laboratory and field experiments. No specific interference of UV-protective substances on the maximum photochemical efficiency of banana plants was observed under field conditions.
Open Access
Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization
(American Society for Microbiology, 2014) Beperet Arive, Inés; Irons, Sarah L.; Simón de Goñi, Oihane; King, Linda A.; Williams, Trevor; Possee, Robert D.; López Ferber, Miguel; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses—AcMNPV and SfMNPV—but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses.
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.