Viral Occlusion Research Articles

Baculoviruses in Integrated Pest Management of Fall Armyworm, (Spodoptera frugiperda) (Lepidoptera: Noctuidae): Structure, Classification and Application

Baculoviruses are crucial biological agents in integrated pest management (IPM), particularly for controlling lepidopteran pests in agriculture. These are highly specific, eco-friendly viruses characterized by rod-shaped nucleocapsids containing a protein-encased genome. The Baculoviridae family comprises four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus, each targeting specific insect orders. Nucleo polyhedron virus (NPVs) and granuloviruses (GVs) are extensively used in pest management due to their high virulence and specificity, ensuring safety for non-target organisms. Ascoviruses are dsDNA viruses that infect Spodoptera frugiperda larvae, transmitted primarily by parasitoids. Despite high virulence, their use in pest control is limited due to ineffective oral transmission. Combining ascoviruses with other pathogens, such as Bacillus thuringiensis kurstaki (Btk), may enhance their pest control potential. The infection process begins when insect larvae ingest viral occlusion bodies, leading to systemic infection and host death. Despite their narrow host range and slower action compared to chemical insecticides, baculoviruses are invaluable in sustainable agriculture. Their application has expanded beyond pest control to include gene therapy and vaccine production. This review examines the structure, classification, infection process, and current status of baculoviruses in controlling the fall armyworm, Spodoptera frugiperda, a significant agricultural pest. By exploring their potential as a cornerstone in biological pest management, we highlight the importance of baculoviruses in developing sustainable and environmentally friendly pest control strategies. The integration of baculoviruses into IPM programs offers a promising approach to reduce chemical pesticide use, preserve biodiversity, and enhance crop protection, ultimately contributing to more sustainable agricultural practices and food security.

Influence of delayed density and ultraviolet radiation on caterpillar baculovirus infection and mortality.

Infectious disease is an important potential driver of population cycles but must occur through delayed density-dependent infection and resulting fitness effects. Delayed density-dependent infection by baculoviruses can be caused by environmental persistence of viral occlusion bodies (OBs), which can be influenced by environmental factors. Specifically, ultraviolet radiation is potentially important in reducing the environmental persistence of viruses by inactivating OBs. Delayed density-dependent viral infection has rarely been observed empirically at the population level although theory predicts that it is necessary for pathogens to drive population cycles. Similarly, field studies have not examined the effects of ultraviolet radiation on viral infection rates in natural animal populations. We tested if viral infection is delayed density-dependent with the potential to drive cyclic dynamics and if ultraviolet radiation influences viral infection levels. We censused 18 Ranchman's tiger moth (Arctia virginalis) populations across 9° of latitude over 2 years and quantified the effects of direct and delayed density and ultraviolet radiation on proportion infected by baculovirus, infection severity and survival to adulthood. Caterpillars were collected from field populations and reared in the laboratory. Baculovirus has not previously been described infecting A. virginalis, and we used genetic methods to confirm the identity of the virus. We found that proportion infected, infection severity and survival to adulthood exhibited delayed density dependence. Ultraviolet radiation in the previous summer decreased infection severity, which increased caterpillar survival probability. Structural equation modelling indicated that the effect of lagged density on caterpillar survival was mediated through proportion infected and infection severity and was 2.5-fold stronger than the indirect effect of ultraviolet. We successfully amplified polh, lef-8 and lef-9 viral genes from caterpillars, and BLAST results confirmed that the virus was a nucleopolyhedrovirus. Our findings provide clear evidence that delayed density-dependent mortality can arise through viral infection rate and severity in insects, which supports the role of viral disease as a mechanism, among others, that may drive insect population cycles. Furthermore, our findings support predictions that ultraviolet radiation can modify viral disease dynamics in insect populations, most likely through attenuating viral persistence in the environment.

Functional analyses of inhibitor of apoptosis protein 1 (IAP1) of Antheraea pernyi multinucleocapsid nucleopolyhedrovirus (AnpeNPV) in viral replication and occlusion body production

Inhibitor of apoptosis protein 1 (IAP1) of Antheraea pernyi multinucleocapsid nucleopolyhedrovirus (AnpeNPV) belongs to the baculovirus IAP1 type. The function of AnpeNPV-IAP1 in viral replication and occlusion body (OB) production remains unknown. In this study, we demonstrated that AnpeNPV-iap1 is a late gene. AnpeNPV-IAP1 mainly localizes to the nuclear ring zone and exhibits dynamic distribution in the cytoplasm and the virogenic stroma during AnpeNPV infection. AnpeNPV-IAP1 impacted the expression of a variety of viral genes at the very late phase of infection in Tn-Hi5 cells. The deletion of AnpeNPV-iap1 caused decreased expression levels of polyhedrin, morphological changes to OBs and reduced OB production in A. pernyi pupae, along with a lengthening of the lethal time of A. pernyi larvae. These results suggest that AnpeNPV-iap1 is involved in regulating viral gene expression, OB production and morphogenesis in A. pernyi.

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.

Isolation of a Novel Alphabaculovirus (Baculoviridae) from Automeris liberia (Cramer, 1780) (Lepidoptera: Saturniidae) in African Oil Palms in Brazil.

A novel baculovirus observed to infect Automeris liberia(Cramer) (bullseye moth) is here described. Caterpillars of A. liberia with symptoms of viral infection were collected from African oil palm plantations in Tailândia, PA, Brazil. Macerated caterpillars were then offered to caterpillars of Automeris cinctistriga(Felder & Rogenhoper), leading to viral symptoms and death before pupation. A transmission electron microscope was used for virus ultrastructural identification. The presence of viral occlusion bodies (OBs) containing multiple nucleocapsids was observed and such features are compatible with Alphabaculovirus (Baculoviridae). Molecular detection by PCR with primers for polyhedrin gene (polh) and for late expression factor-8 gene (lef-8), confirmed that this isolate belonged to Alphabaculovirus genus. To our knowledge, this is the first record of a baculovirus isolated from or associated to Automeris. The name Automeris liberia nucleopolyhedrovirus (AuliNPV) is proposed for the new virus.

The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Bacmid Expression of Granulovirus Enhancin En3 Accumulates in Cell Soluble Fraction to Potentiate Nucleopolyhedrovirus Infection.

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.

Molecular characterization and biological activity of native Spodoptera littoralis nucleopolyhedrovirus isolate

BackgroundSpodoptera littoralis nucleopolyhedrovirus (SpliNPV) is considered a promising biocontrol agent that can be used for the effective control of the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), which is an economic pest on many cultivated plants and crops in tropical and sub-tropical regions. The genome of the SpliNPV-AN1956 isolate has been fully sequenced, providing a reference strain for comparison of new isolates. In this study, identification, biological activity, and genetic characterization of a field collected SpliNPV isolate were carried out.ResultsThe examination of viral occlusion bodies (OBs) by TEM showed a typical baculovirus OBs of type nucleopolyhedrovirus (NPV) with polyhedral structures. The phylogenetic analysis of the late expression factor- 5 (lef-5) gene as well as the restriction profile analysis confirmed the identity of SpliNPV as a variant isolate. Digestion with ScaI endonuclease showed that 4 fragments of 50, 35, 27, and 13 kb were detected but 3.2 kb was absent in SpliNPV-Cab3 pattern compared to the reference strain. Meanwhile, upon digestion with PstI and HindIII endonucleases, no differences were observed in both isolates’ pattern. Moreover, the virulence evaluation against S. littoralis 1st instar larvae indicated that LC50 value of SpliMNPV-Cab3 was higher than that estimated for the reference strain. Also, ST50 of SpliNPV-Cab3 (120 h) was significantly different with that of the reference strain (132 h).ConclusionThe host specificity based on virulence parameters make SpliNPV-Cab3 isolate a potential candidate to be involved in the integrated pest management strategies for the control of S. littoralis population with a prospect to biopesticide development.

Gypsy Moth Management with LdMNPV Baculovirus in Cork Oak Forest

(1) Research Highlights: Applications of a species-specific baculovirus is a promising method to control the gypsy moth and regulate its population dynamics in forest ecosystems. (2) Background and Objectives: Cork oak protection against the Lepidopteran defoliator Lymantria dispar requires an appropriate forest ecosystem management program, involving the application of eco-sustainable microbial products during population outbreaks. The species-specific multicapsid nucleopolyhedrovirus (LdMNPV), agent of natural epizootics in gypsy moth populations, represents an option that was investigated in a multi-year field study, involving viral applications either from the ground or by aerial treatment. (3) Materials and Methods: Efficacy trials against L. dispar populations were conducted in 2018 and 2019 in Sardinia, according to a randomized block design. Each year, two trials were conducted, applying a baculovirus commercial formulation with an atomizer from the ground and assessing the effects of different doses and application timing, respectively. An aerial application trial distributing LdMNPV at ultra-low volumes (2 L/ha) was also conducted in 2019 to assess the virus efficacy at a larger field scale. (4) Results: In both years, a significant increase in larval mortality was detected in plots treated with higher viral occlusion body (OB) doses and with an earlier application targeting younger larvae, in comparison with untreated controls. Due to an observed retrogradation phase of the target pest in 2019, no significant differences in larval density between areas treated from a helicopter and control were detected, but in the few weeks following application, a meaningful vitality decrease in larval samples from treated plots was observed. (5) Conclusions: Based on the results of this study, the use of LdMNPV in forest protection programs against gypsy moth can be worth consideration in multi-year integrated program strategies to modulate population dynamics.

Autographa Californica Multiple Nucleopolyhedrovirus orf13 Is Required for Efficient Nuclear Egress of Nucleocapsids.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.

Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells

Baculovirus-infected larvae release progeny viral occlusion bodies (OBs) to enable cyclical virus transmission to new hosts. The alphabaculovirus chitinase and cathepsin enzymes cause terminal liquefaction of host insect cadavers, aiding OB dispersal. The mechanism of cell lysis required to release the OBs is unclear but here we show Autographa californica multiple nucleopolyhedrovirus cathepsin protease activity is required for efficient release of the host tissue-degrading chitinase and cathepsin enzymes and critical for release of progeny OBs from virus-infected cells. Comparisons between viruses containing or lacking cathepsin indicate that cathepsin was necessary for OB release into cultured cell media or hemolymph of insects. In addition, pharmacological inhibition of cysteine protease activity in cells during infection blocked maturation of active cathepsin and OB release from infected cells. Together, these results suggest an important link between baculovirus-induced cell lysis, the concomitant maturation of cathepsin, and cellular release of chitinase, cathepsin and progeny OBs from cells.

The Role of Chrysoperla carnea (Steph.) (Neuroptera: Chrysopidae) as a Potential Dispersive Agent of Noctuid Baculoviruses.

Simple SummaryBaculoviruses (BV) infect several lepidopteran pests of economic importance, such as the beet armyworm Spodoptera exigua. The joint use of microbiological and macrobiological strategies may improve the efficacy of control. Laboratory bioassays were developed to evaluate the interactions between two BVs: the multiple nucleopolyhedroviruses of S. exigua (SeMNPV) and Autographa californica (AcMNPV), and the predator Chrysoperla carnea. The excretion products of the predator’s larvae (drops) and adults (meconia) were microscopically examined after the ingestion of BV-infected S. exigua larvae. For both types of excreta and BVs, viral occlusion bodies (OBs) (resistance forms) were observed. These OBs were infective to healthy S. exigua larvae when applied in water suspension and in direct deposition. The virulence of meconia was higher in suspensions (higher viral load), while larval drops were more virulent in direct application due to their liquid nature and their easiness of consumption. The fitness of C. carnea was slightly affected by the consumption of both BV-infected prey. No preference was shown between healthy and BV-infected S. exigua, and both were preferred vs. the aphid Macrosiphum euphorbiae. Our findings present C. carnea, and particularly its larvae, as a promissory candidate for BV dispersion in the field.Baculoviruses (BV) are highly effective against lepidopteran pests of economic importance such as Spodoptera exigua. The combined use of entomopathogens and macrobiological control agents requires the study of their relationships. Laboratory bioassays were developed to evaluate the interactions between the multiple nucleopolyhedroviruses of S. exigua (SeMNPV) and Autographa californica (AcMNPV), and the predator Chrysoperla carnea. The microscopic examination of predator’s excreta (larval drops and meconia) after the ingestion of BV-infected S. exigua revealed the presence of viral occlusion bodies (OBs). The reinfection of S. exigua larvae with BVs-contaminated excreta by using OBs water suspensions or by direct application both yielded high mortality values but different speed-of-kill results. Meconia killed before in suspensions due to their higher viral load and larval excretion drops did so in direct application due to their liquid nature and their easiness of consumption. The prey-mediated ingestion of SeMNPV and AcMNPV triggered slight effects in C. carnea, which were probably derived from the food nutritional quality. Chrysoperla carnea larvae did not discriminate between healthy and BV-infected S. exigua, while a preference was shown for S. exigua (healthy or infected) vs. Macrosiphum euphorbiae. Our findings present C. carnea, and particularly its larvae, as a promissory candidate for BV dispersion in the field.

Bombyx mori nucleopolyhedrovirus Bm46 is essential for efficient production of infectious BV and nucleocapsid morphogenesis

Bombyx mori nucleopolyhedrovirus (BmNPV) orf46 (Bm46), the orthologues of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac57, is a highly conserved gene in group Ⅰ and group Ⅱ nucleopolyhedroviruses (NPVs). However, its function in viral life cycle is unclear. Our results indicated that Bm46 transcript was detected from infected cells at 12 h post infection, while Bm46 protein was detectable from 24 to 72 h post infection. Upon the deletion of Bm46, fewer infectious BVs were produced by titer assays, but neither viral DNA synthesis nor occlusion bodies (OBs) production was affected. Electron microscopy revealed that Bm46 knockout interrupted nucleocapsid assembly and occlusion-derived virus (ODV) embedding, resulting in aberrant capsid-like tubular structures accumulated in the RZ (ring zone). Interestingly, this abnormally elongated capsid structures were consistent with the immunofluorescence microscopy results showing that VP39 assembled into long filaments and cables in the RZ. Moreover, DNA copies decreased by 30 % in occlusion bodies (OBs) produced by Bm46-knockout virus. qRT-PCR and Western blot analysis showed that the expression of VP39 was affected by Bm46 disruption. Taken together, our findings clearly pointed out that Bm46 played an important role in BV production and the proper formation of nucleocapsid morphogenesis.

Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis

The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpb/Vpa (formerly Vip1/Vip2) is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee.

Individual and combined impact of nuclear polyhedrosis virus and spinosad to control the tropical armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), in cotton in Pakistan

The tropical armyworm, Spodoptera litura Fabricius (Noctuidae; Lepidoptera), is among the most harmful pests causing economic loss in the quality and production of a variety of crops, particularly cotton. Entomopathogens play an important role in insect pest management. The nuclear polyhedrosis virus (NPV) isolate of S. litura (V-SpltNPV) was isolated from infected larvae in a cotton crop, and viral occlusion bodies were confirmed, using an inverted microscope. The pathogenicity of V-SpltNPV against 2nd, 3rd, and 4th larval instars of S. litura was evaluated at various concentrations (1 × 104 to 1 × 108 OBs/ml). Mortality rate was high (37.65–96.82%) in early instar larvae against tested concentrations. LC50 and LT50 values increased with increasing larval age. There was 689,865 times increase in LC50 value (1.35 × 102 OBs/ml) for 2nd instar larvae to LC50 value (6.90 × 105 OBs/ml) for 4th instar larvae. LT50 values enhanced from 4.99 days for 2nd instar larvae to 7.49 days for 4th instar larvae, due to a decrease in efficacy of NPVs with the increasing age of larvae. In a greenhouse experiment, a combined application of spinosad with V-SpltNPV (1 × 108 OBs/ml) caused (100%) mortality of 2nd instar larvae. A single application of V-SpltNPV (1 × 108 OBs/ml) resulted to mean mortality (52.63%) of tested larvae. The native isolate V-SpltNPV seems to have a potential to be used in integrated manner with other IPM tactics to significantly reduce the use of toxic chemical pesticides.

Role of Toll-receptors in the inhibition of BmNPV proliferation and their interplay with antimicrobial seroin proteins in Bombyx mori

The domesticated silkworm, Bombyx mori, is an economically important insect that majorly contributes in financially viable sericulture industry. The baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV) is a deadly virus that infects B. mori and inflicts heavy loss to silk production by causing high mortality of silkworms. A number of studies have been done to identify antiviral genes and pathways against BmNPV infection in B. mori . Toll-pathway is a well-known immune pathway responsible for combating bacterial and fungal infections in silkworm. However, the role of Toll-receptors in inhibiting BmNPV has not been explored. In the present study, we have attempted to examine the role of Toll-receptors in inhibiting BmNPV proliferation in B. mori larvae. The effect of Toll-receptors on viral proliferation was determined by quantifying viral DNA and counting viral occlusion bodies upon RNA interference-mediated knockdown of Toll-receptors in B. mori larvae. Knockdown of BmToll, BmToll-6, BmToll-7, BmToll-8 and BmToll-9 led to higher accumulation of BmNPV. Further, interplay between Toll-receptors and the antimicrobial proteins of B. mori, called seroins were studied using RNA interference. As a result, we found a downregulation of Seroin1 transcript level upon knockdown of BmToll , BmToll-7 , BmToll-8 , BmToll-10 , BmToll-11 and BmToL K2, whereas Seroin2 expression was found to decrease only upon BmToll-7 knockdown. Moreover, knockdown of seroins did not result in clearly interpretable expression patterns of Toll-receptors. These results suggest the involvement of B. mori Toll-pathway in defense against BmNPV infection and provides a new insights into the antiviral defense mechanism of B. mori. • BmNPV proliferation increases upon knockdown of B. mori Toll-receptors in larvae. • Knockdown of B. mori Toll-7 significantly enhances viral load. • Seroins are likely to function downstream of Toll-7-mediated signaling pathway in B. mori.

Genetic Variability of Chrysodeixis Includens Nucleopolyhedrovirus (ChinNPV) and the Insecticidal Characteristics of Selected Genotypic Variants.

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.

The response of newly established cell lines of Spodoptera littoralis to group I and group II baculoviruses.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV) belong to group I and group II nucleopolyhedroviruses, respectively and can replicate in a wide range of insect species. In this study, the ability of newly established S. littoralis cell lines to support replication of AcMNPV and SpliMNPV was examined. The microscopic observations showed that the S. littoralis cells infected with AcMNPV exhibited morphological changes such as cells breaking into small bodies and forming apoptosis-like bodies post-infection. Nuclear DNA fragmentation was observed in all AcMNPV-infected cell lines through DNA gel electrophoresis analysis. Therefore, the virus replication was unsuccessful in most of cells, which were able to abort the virus replication. On the other hand, cells that were infected with SpliMNPV did not show similar morphological changes and no small bodies were formed. In addition, SpliMNPV succeeded to infect the cells, replicate, and form viral occlusion bodies inside the infected cells. In suspension culture, S. littoralis cells, which were infected with AcMNPV, accumulated as composed balls in shaker flasks after infection overnight, with cell density decreasing dramatically. In contrast, there was no cell clumping seen in the infected cells with SpliMNPV and the uninfected cells. In conclusion, the newly established embryonic S. littoralis cells were highly susceptible to SpliMNPV, whereas the cells were non-permissive to AcMNPV, yet they still underwent programmed cell death.

Protein composition of the occlusion bodies of Epinotia aporema granulovirus.

Within family Baculoviridae, members of the Betabaculovirus genus are employed as biocontrol agents against lepidopteran pests, either alone or in combination with selected members of the Alphabaculovirus genus. Epinotia aporema granulovirus (EpapGV) is a fast killing betabaculovirus that infects the bean shoot borer (E. aporema) and is a promising biopesticide. Because occlusion bodies (OBs) play a key role in baculovirus horizontal transmission, we investigated the composition of EpapGV OBs. Using mass spectrometry-based proteomics we could identify 56 proteins that are included in the OBs during the final stages of larval infection. Our data provides experimental validation of several annotated hypothetical coding sequences. Proteogenomic mapping against genomic sequence detected a previously unannotated ac110-like core gene and a putative translation fusion product of ORFs epap48 and epap49. Comparative studies of the proteomes available for the family Baculoviridae highlight the conservation of core gene products as parts of the occluded virion. Two proteins specific for betabaculoviruses (Epap48 and Epap95) are incorporated into OBs. Moreover, quantification based on emPAI values showed that Epap95 is one of the most abundant components of EpapGV OBs.

The baculovirus Ac108 protein is a per os infectivity factor and a component of the ODV entry complex.

Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108), the stable core is still present but nevertheless fails to form an entry complex, affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles, the occlusion-derived viruses (ODVs), are embedded. The protein matrix dissolves in the alkaline environment of the insect's midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins, called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity, but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex, named the ODV entry complex. In this study, we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity, while production and infectivity of the BVs remains unaffected. Furthermore, repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant, we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex, where the formation was abolished in the absence of this protein. Based on these results, we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.