Mixtures of Insect-Pathogenic Viruses in a Single Virion: towards the Development of Custom-Designed Insecticides.

Alphabaculoviruses (Baculoviridae) are pathogenic DNA viruses of Lepidoptera that have applications as the basis for biological insecticides and expression vectors in biotechnological processes. These viruses have a characteristic physical structure that facilitates the transmission of groups of genomes. We demonstrate that coinfection of a susceptible insect by two different alphabaculovirus species results in the production of mixed-virus occlusion bodies containing the parental viruses. This occurred between closely related and phylogenetically more distant alphabaculoviruses. Approximately half the virions present in proteinaceous viral occlusion bodies produced following coinfection of insects with a mixture of two alphabaculoviruses contained both viruses, indicating that the viruses coinfected and replicated in a single cell and were coenveloped within the same virion. This observation was confirmed by endpoint dilution assay. Moreover, both viruses persisted in the mixed-virus population by coinfection of insects during several rounds of insect-to-insect transmission. Coinfection by viruses that differed in genome size had unexpected results on the length of viral nucleocapsids, which differed from those of both parental viruses. These results have unique implications for the development of alphabaculoviruses as biological control agents of insect pests.IMPORTANCE Alphabaculoviruses are used as biological insecticides and expression vectors in biotechnology and medical applications. We demonstrate that in caterpillars infected with particular mixtures of viruses, the genomes of different baculovirus species can be enveloped together within individual virions and occluded within proteinaceous occlusion bodies. This results in the transmission of mixed-virus populations to the caterpillar stages of moth species. Once established, mixed-virus populations persist by coinfection of insect cells during several rounds of insect-to-insect transmission. Mixed-virus production technology opens the way to the development of custom-designed insecticides for control of different combinations of caterpillar pest species.

Estimation of water quality by UV/Vis spectrometry in the framework of treated wastewater reuse.

The aim of this study is to investigate the potential of ultraviolet/visible (UV/Vis) spectrometry as a complementary method for routine monitoring of reclaimed water production. Robustness of the models and compliance of their sensitivity with current quality limits are investigated. The following indicators are studied: total suspended solids (TSS), turbidity, chemical oxygen demand (COD) and nitrate. Partial least squares regression (PLSR) is used to find linear correlations between absorbances and indicators of interest. Artificial samples are made by simulating a sludge leak on the wastewater treatment plant and added to the original dataset, then divided into calibration and prediction datasets. The models are built on the calibration set, and then tested on the prediction set. The best models are developed with: PLSR for COD (Rpred2 = 0.80), TSS (Rpred2 = 0.86) and turbidity (Rpred2 = 0.96), and with a simple linear regression from absorbance at 208 nm (Rpred2 = 0.95) for nitrate concentration. The input of artificial data significantly enhances the robustness of the models. The sensitivity of the UV/Vis spectrometry monitoring system developed is compatible with quality requirements of reclaimed water production processes.

Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization.

UNLABELLED: 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. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

The "11K" gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus.

The "11K" gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and sf95) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair bacmids were generated from a bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were ∼15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and sf95 resulted in a slower speed-of-kill by 9h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect.

Analagous population structures for two alphabaculoviruses highlight a functional role for deletion mutants.

A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large-deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origins of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. The homology of nucleotides in the deleted regions was as high as 79%, similar to those of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that independently generates and sustains deletion mutants within each virus population. Functional analyses using different proportions of complete and deletion genotypes were performed with the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete/deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but there were no significant changes in the speed of kill. In contrast, OB yields increased only in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most extensively deleted SeMNPV genotype and contribute toward the elucidation of the role of such mutants in baculovirus populations.

Development of a viral biopesticide for the control of the Guatemala potato tuber moth Tecia solanivora.

The Guatemala potato tuber moth Tecia solanivora (Povolny) (Lep. Gelechiidae) is an invasive species from Mesoamerica that has considerably extended its distribution area in recent decades. While this species is considered to be a major potato pest in Venezuela, Colombia, and Ecuador, currently no specific control methods are available for farmers. To address this issue we developed a biopesticide formulation to be used in integrated pest management of T. solanivora, following three steps. First, search for entomopathogenic viruses were carried out through extensive bioprospections in 12 countries worldwide. As a result, new Phthorimaea operculella granulovirus (PhopGV) isolates were found in T. solanivora and five other gelechid species. Second, twenty PhopGV isolates, including both previously known and newly found isolates, were genetically and/or biologically characterized in order to choose the best candidate for a biopesticide formulation. Sequence data were obtained for the ecdysteroid UDP-glucosyltransferase (egt) gene, a single copy gene known to play a role in pathogenicity. Three different sizes (1086, 1305 and 1353 bp) of egt were found among the virus isolates analyzed. Unexpectedly, no obvious correlation between egt size and pathogenicity was found. Bioassays on T. solanivora neonates showed a maximum of a 14-fold difference in pathogenicity among the eight PhopGV isolates tested. The most pathogenic PhopGV isolate, JLZ9f, had a medium lethal concentration (LC(50)) of 10 viral occlusion bodies per square mm of consumed tuber skin. Third, we tested biopesticide dust formulations by mixing a dry carrier (calcium carbonate) with different adjuvants (magnesium chloride or an optical brightener or soya lecithin) and different specific amounts of JLZ9f. During laboratory experiments, satisfactory control of the pest (>98% larva mortality compared to untreated control) was achieved with a formulation containing 10 macerated JLZ9f-dead T. solanivora larvae per kg of calcium carbonate mixed with 50 mL/kg of soya lecithin. The final product provides an interesting alternative to chemical pesticides for Andean farmers affected by this potato pest.

Deletion of egt is responsible for the fast-killing phenotype of natural deletion genotypes in a Spodoptera frugiperda multiple nucleopolyhedrovirus population.

A Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, includes fast-killing genotypes with deletions in the egt region. Four bacmid based recombinants were constructed to determine the role of egt in this phenotype. SfdelF bacmid encompassed the deletion found in the NIC-F genotype. Sfdel3AP2 bacmid was constructed using the deletion reported in SfMNPV-3AP2 (Missouri, fast-killing isolate), whereas Sfdelegt and Sfdel27 bacmids lacked the single genes egt and the adjacent sf27 gene, respectively. No significant differences were observed in occlusion body (OB) concentration-mortality metrics (LC(50) values) among the viruses. Larvae infected by NIC-B (a natural genotype with the largest genome), Sfbac (a bacmid with NIC-B genome) and Sfdel27 survived significantly longer than insects infected by NIC-F, SfdelF, SfMNPV-3AP2, Sfdel3AP2 or Sfdelegt. Fast-killing viruses produced ∼6-13-fold fewer OBs/larva compared to other viruses tested. We conclude that deletion/disruption of egt is responsible for the fast-killing phenotypes of naturally-occurring genotypes in SfMNPV populations from Missouri and Nicaragua.

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.

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.

Experimental mixtures of Phthorimaea operculella granulovirus isolates provide high biological efficacy on both Phthorimaea operculella and Tecia solanivora (Lepidoptera: Gelechiidae).

The Guatemalan potato moth Tecia solanivora (Povolny) recently invaded part of South America, colonizing zones where Phthorimaea operculella (Zeller), another potato moth species belonging to the same group, was previously established. T. solanivora is now the major insect pest of potato in this area encompassing Venezuela, Colombia and Ecuador. P. operculella granulovirus (PhopGV) (Betabaculovirus) is a biocontrol agent to be considered for the simultaneous management of these two potato pests, instead of classical chemical insecticides. In a previous work, five PhopGV isolates were isolated in Colombia from T. solanivora and were tested against larvae of the same species showing variable efficacies. Infections with mixtures of different genotypes of Baculoviruses had been carried out in a wide range of species and several showed interesting results. In the present study, the effect of sequential passages of PhopGV in P. operculella and T. solanivora larvae was analyzed through biological assays. Three different mixtures containing a Peruvian PhopGV isolate (Peru) adapted to P. operculella and a Colombian PhopGV isolate (VG003) adapted to T. solanivora were tested. A preliminary analysis of the correlation between the genotypic marker egt gene and the level of pathogenicity after a variable number of replication cycles was made. Mixtures of virus isolates showed a higher efficacy in both hosts compared to individual PhopGV isolates. This higher pathogenicity was maintained through passages. In P. operculella the mixtures were between 2.8 and 23.6-fold (from 7.15 OB/mm(2) to 0.10 OB/mm(2)) more pathogenic than isolate Peru applied alone. In T. solanivora they were between 2.3 and 4.9-fold (from 12.29 OB/mm(2) to 1.25 OB/mm(2)) more pathogenic than isolate VG003 alone. Viral biopesticide containing a mixture of selected genotypes active against each hosts seemed suitable for the development of a biopesticide aimed to simultaneously control P. operculella and T. solanivora.

Estrogenic and anti-estrogenic activity of 23 commercial textile dyes.

The presence of dyes in wastewater effluent of textile industry is well documented. In contrast, the endocrine disrupting effects of these dyes and wastewater effluent have been poorly investigated. Herein, we studied twenty-three commercial dyes, usually used in the textile industry, and extracts of blue jean textile wastewater samples were evaluated for their agonistic and antagonistic estrogen activity. Total estrogenic and anti-estrogenic activities were measured using the Yeast Estrogen Screen bioassay (YES) that evaluates estrogen receptor binding-dependent transcriptional and translational activities. The estrogenic potencies of the dyes and wastewater samples were evaluated by dose-response curves and compared to the dose-response curve of 17ß-estradiol (E2), the reference compound. The dose-dependent anti-estrogenic activities of the dyes and wastewater samples were normalized to the known antagonistic effect of 4-hydroxytamoxifen (4-OHT) on the induction of the lac Z reporter gene by E2. About half azo textile dyes have anti-estrogenic activity with the most active being Blue HFRL. Most azo dyes however have no or weak estrogenic activity. E2/dye or E2/waste water ER competitive binding assays show activity of Blue HFRL, benzopurpurine 4B, Everzol Navy Blue FBN, direct red 89 BNL 200% and waste water samples indicating a mechanism of action common to E2. Our results indicate that several textile dyes are potential endocrine disrupting agents. The presence of some of these dyes in textile industry wastewater may thus impact the aquatic ecosystem.

Coocclusion of Helicoverpa armigera Single Nucleopolyhedrovirus (HearSNPV) and Helicoverpa armigera Multiple Nucleopolyhedrovirus (HearMNPV): Pathogenicity and Stability in Homologous and Heterologous Hosts.

Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) is a virulent pathogen of lepidopterans in the genera Heliothis and Helicoverpa, whereas Helicoverpa armigera multiple nucleopolyhedrovirus (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 pathogenicity, 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 quantified 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 experiment 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 disparity 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 infectivity 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.

Sequence comparison between three geographically distinct Spodoptera frugiperda multiple nucleopolyhedrovirus isolates: Detecting positively selected genes.

The complete genomic sequence of a Nicaraguan plaque purified Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genotype SfMNPV-B was determined and compared to previously sequenced isolates from United States (SfMNPV-3AP2) and Brazil (SfMNPV-19). The genome of SfMNPV-B (132,954bp) was 1623bp and 389bp larger than that of SfMNPV-3AP2 and SfMNPV-19, respectively. Genome size differences were mainly due to a deletion located in the SfMNPV-3AP2 egt region and small deletions and point mutations in SfMNPV-19. Nucleotide sequences were strongly conserved (99.35% identity) and a high degree of predicted amino acid sequence identity was observed. A total of 145 open reading frames (ORFs) were identified in SfMNPV-B, two of them (sf39a and sf110a) had not been previously identified in the SfMNPV-3AP2 and SfMNPV-19 genomes and one (sf57a) was absent in both these genomes. In addition, sf6 was not previously identified in the SfMNPV-19 genome. In contrast, SfMNPV-B and SfMNPV-19 both lacked sf129 that had been reported in SfMNPV-3AP2. In an effort to identify genes potentially involved in virulence or in determining population adaptations, selection pressure analysis was performed. Three ORFs were identified undergoing positive selection: sf49 (pif-3), sf57 (odv-e66b) and sf122 (unknown function). Strong selection for ODV envelope protein genes indicates that the initial infection process in the insect midgut is one critical point at which adaptation acts during the transmission of these viruses in geographically distant populations. The function of ORF sf122 is being examined.

Nucleopolyhedrovirus Coocclusion Technology: A New Concept in the Development of Biological Insecticides.

Nucleopolyhedroviruses (NPV, Baculoviridae) that infect lepidopteran pests have an established record as safe and effective biological insecticides. Here, we describe a new approach for the development of NPV-based insecticides. This technology takes advantage of the unique way in which these viruses are transmitted as collective infectious units, and the genotypic diversity present in natural virus populations. A ten-step procedure is described involving genotypic variant selection, mixing, coinfection and intraspecific coocclusion of variants within viral occlusion bodies. Using two examples, we demonstrate how this approach can be used to produce highly pathogenic virus preparations for pest control. As restricted host range limits the uptake of NPV-based insecticides, this technology has recently been adapted to produce custom-designed interspecific mixtures of viruses that can be applied to control complexes of lepidopteran pests on particular crops, as long as a shared host species is available for virus production. This approach to the development of NPV-based insecticides has the potential to be applied across a broad range of NPV-pest pathosystems.

CpGV-M Replication in Type I Resistant Insects: Helper Virus and Order of Ingestion Are Important.

The genetic diversity of baculoviruses provides a sustainable agronomic solution when resistance to biopesticides seems to be on the rise. This genetic diversity promotes insect infection by several genotypes (i.e., multiple infections) that are more likely to kill the host. However, the mechanism and regulation of these virus interactions are still poorly understood. In this article, we focused on baculoviruses infecting the codling moth, Cydia pomonella: two Cydia pomonella granulovirus genotypes, CpGV-M and CpGV-R5, and Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). The influence of the order of ingestion of the virus genotypes, the existence of an ingestion delay between the genotypes and the specificity of each genotype involved in the success of multiple infection were studied in the case of Cydia pomonella resistance. To obtain a multiple infection in resistant insects, the order of ingestion is a key factor, but the delay for ingestion of the second virus is not. CrpeNPV cannot substitute CpGV-R5 to allow replication of CpGV-M.

Mining Impacts Assessment Using the LCA Methodology: Case Study of Afema Gold Mine in Ivory Coast.

Environmental impact assessment studies are mandatory for major industrial or infrastructure projects in most countries. These studies are usually limited to on-site impacts during exploitation but do not consider indirect impacts generated off-site or those concerning other steps of the project, including dismantling. National regulations in various countries have recently begun to include these neglected impacts to obtain a better appreciation of project trade-offs. Several scientists have highlighted the substantial potential of using the life cycle assessment methodology to increase the level of detail and completeness of environmental impact assessment (EIA) studies. Even if mining activities are known to produce significant local impacts, their consequences outside an extraction site have not yet been well documented. The implementation of the life cycle assessment (LCA) methodology in the EIA procedure has been carried out in a Au mining project by separating on-site and off-site impacts during the entire life cycle of the mine from prospection to site restoration following the end of exploitation. Mining projects occur over large time periods and require diverse materials and processes. The main difficulty of such analysis is the data collection that needs to be extrapolated for some of the activities. Even with these limitations, the Afema case study highlighted the significant share of off-site impacts (from a spatial perspective) and the major contribution of the exploitation phase of the mine (from a temporal perspective). Operating activities, especially excavation, ore, and waste rock transportation, blasting, ore processing, and tailing treatments, are the main impacts produced during the exploitation phase and are involved in climate change, particulate matter formation, and land destruction. Therefore, this standardized LCA method should be recommended by the regulatory authorities for use in EIA procedures. Integr Environ Assess Manag 2021;17:465-479. © 2020 SETAC.

Mixed genotype transmission bodies and virions contribute to the maintenance of diversity in an insect virus.

An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission.

Mixtures of complete and pif1- and pif2-deficient genotypes are required for increased potency of an insect nucleopolyhedrovirus.

The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-B Delta 16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-B Delta pifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-B Delta 16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-B Delta 16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-B Delta pifs were almost identical to the results observed with SfNIC-B Delta 16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-B Delta 16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of approximately 85% SfNIC-B plus approximately 15% SfNIC-B Delta 16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNIC-B Delta pifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1- and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.

Densovirus infectious pathway requires clathrin-mediated endocytosis followed by trafficking to the nucleus.

Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.

Stability of a Spodoptera frugiperda nucleopolyhedrovirus deletion recombinant during serial passage in insects.

The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.

Genetic and biological analysis of Colombian Phthorimaea operculella granulovirus isolated from Tecia solanivora (Lepidoptera: Gelechiidae).

Tecia solanivora (Lepidoptera: Gelechiidae) is an invasive potato pest of the north of South America that recently colonized zones where Phthorimaea operculella (Lepidoptera: Gelechiidae), a taxonomically related insect, was established. Nowadays, both species can be found in most areas in different proportions. The Phthorimaea operculella granulovirus (PhopGV) was found to efficiently control P. operculella and was used as a biopesticide in storage conditions. However, no appropriate biological control methods exist for T. solanivora, and the use of granulovirus isolates would provide a solution. The Colombian Corporation for Agricultural Research (CORPOICA) carried out several T. solanivora larva samplings in Colombia with the aim of finding potential isolates. Five geographical granulovirus isolates from T. solanivora (VG001, VG002, VG003, VG004, and VG005) were found, and molecular analysis by REN profiles shows three different genotypic variants in Colombia. Analysis of their genomes revealed their relatedness to PhopGV. Two isolates exhibited submolar bands in their REN patterns, suggesting a mixture of viral genotypes. These data were confirmed by PCR amplification and sequencing of particular regions of the viral genomes. Their biological activity was assayed on both hosts, T. solanivora and P. operculella. A significantly higher pathogenicity in both hosts was observed with isolates VG001 and VG005 than with isolate VG003 or a Peruvian isolate (from P. operculella) used as a reference in the bioassay. Based on their molecular and biological activity characteristics, VG001 and VG005 isolates should be selected for further analysis in order to establish their potential as biological control agents.