Pathogen-Mediated Tritrophic Interactions: Baculovirus-Challenged Caterpillars Induce Higher Plant Defenses than Healthy Caterpillars.

Although the tritrophic interactions of plants, insect herbivores and their natural enemies have been intensely studied for several decades, the roles of entomopathogens in their indirect modulation of plant-insect relationships is still unclear. Here, we employed a sublethal dose of a baculovirus with a relatively broad host range (AcMNPV) to explore if feeding by baculovirus-challenged Helicoverpa zea caterpillars induces direct defenses in the tomato plant. We examined induction of plant defenses following feeding by H. zea, including tomato plants fed on by healthy caterpillars, AcMNPV-challenged caterpillars, or undamaged controls, and subsequently compared the transcript levels of defense related proteins (i.e., trypsin proteinase inhibitors, peroxidase and polyphenol oxidase) and other defense genes (i.e., proteinase inhibitor II and cysteine proteinase inhibitor) from these plants, in addition to comparing caterpillar relative growth rates. As a result, AcMNPV-challenged caterpillars induced the highest plant anti-herbivore defenses. We examined several elicitors and effectors in the secretions of these caterpillars (i.e., glucose oxidase, phospholipase C, and ATPase hydrolysis), which surprisingly did not differ between treatments. Hence, we suggest that the greater induction of plant defenses by the virus-challenged caterpillars may be due to differences in the amount of these secretions deposited during feeding or to some other unknown factor(s).

Baculovirus Per Os Infectivity Factor Complex: Components and Assembly.

Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

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.

Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity.

Self-biotinylation and site-specific double labeling of baculovirus using quantum dots for single-virus in-situ tracking.

Single-virus labeling and tracking represent a powerful tool to study virus-cell interactions. Using baculovirus as a model, here we developed a biochemical method for labeling both the viral envelope and the viral capsid of a virus. Viral envelope of the baculovirus AcMNPV was self-biotinylated and site-specifically conjugated with quantum dots (QDs) following one-step binding reaction, while the viral nucleocapsid was site-specifically labeled with green fluorescent protein (GFP) during viral replication. The established procedure of labeling did not affect viral infectivity, showing that the double-labeled virus retained functional structure and could be tracked for viral localization and movement in the host cells. The double-labeled virus also demonstrated the potential to be used for in-situ and real-time visualizing the internalization of a single viral particle into the host cells. Furthermore, the disassembly processes of the viral envelope and the viral nucleocapsid could be monitored for a long period of time (up to 2 h). Using the established method, several interaction details between the labeled baculoviruses and the host cells have been revealed. Given its advantages in high efficiency, high specificity, convenience and the maintenance of viral infectivity, the established approach provides a promising means for elucidating virus-cell interactions.

Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization.

Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.

High-level expression, purification and production of the fungal immunomodulatory protein-gts in baculovirus-infected insect larva.

Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 109 cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

Nuclear actin and lamins in viral infections.

Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.

Iron levels change in larval Heliothis virescens tissues following baculovirus infection.

Inductively coupled plasma mass spectrometry and (59)Fe radiotracers were used to investigate changes in levels of Fe in the tissues of 4th instar Heliothis virescens larvae following infection with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or with Autographa californica multiple nucleopolyhedrovirus. Baculovirus infection led to significant changes in hemolymph Fe levels late in infection. (24)Na radiotracer ingested by 4th instar larvae was rapidly cleared to nearly undetectable levels 6 h post-ingestion. In contrast, (59)Fe radiotracer fed to 4th instar larvae declined within the first few hours of ingestion and then remained constant at approximately 60% of the initial tracer activity. While Fe radiotracer levels among larval tissues changed, whole insect tracer levels did not decline from 6 to 60 h post-ingestion. Tissues from HzSNPV larvae had higher radiotracer levels in the hemolymph and midgut 36 and 60 h post-infection. The protein-bound/free ratio of (59)Fe was significantly higher in baculovirus infected hemolymph than in uninfected hemolymph at 60 h post-infection, indicating that Fe released from damaged cells is protein-bound. In both studies, hemolymph Fe levels were higher in HzSNPV infected larvae. This first study of tissue Fe levels during viral infection of an insect clearly demonstrates that Fe homeostasis is substantially disrupted.

Surface display of AcMNPV occlusion-derived P74 does not enhance oral infectivity of budded viruses.

Baculovirus occlusion-derived viruses (ODVs) and budded viruses (BVs) are morphologically and functionally distinct. ODVs are responsible for primary infection in insect hosts because of their high per os infectivity. On the contrary, BVs poorly infect endothelial gut cells, but propagate the infection in the tissues of insects with a high efficiency. P74 is one of the most important proteins from ODVs, and it participates in the attachment of this viral phenotype to endothelial cells in the midgut. We evaluated the possibility of pseudotyping BVs of Autographa californica multiple nucleopolyhedrovirus with two versions of P74 and its effect on their oral infectivity. Both recombinant BVs contained P74 and replicated similarly to wild-type viruses. Nevertheless, the presence of P74 on the BV's surface does not enhance the oral infectivity of this phenotype, suggesting that the presence of P74 in the membrane of budded virions interferes with their mechanism of infecting midgut cells.

Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.

A criação do bombyx mori (bicho-da-seda) e as principais doenças / Cría del bombyx mori (gusano de seda) y las principales enfermedades / Bombyx mori (silkworm) breeding and main diseases

Este artigo revisa os principais aspectos relacionados à sericicultura, que abrange a criação do Bombyx mori, popularmente conhecido como bicho-da-seda, que representa uma atividade desenvolvida principalmente nas pequenas propriedades rurais, onde predomina o trabalho familiar, representando uma alternativa importante para a melhoria da renda e, contribuindo desta forma para a diminuição do êxodo rural. Além dessas características, a sericicultura é uma atividade de baixo impacto ambiental, que contribui para o desenvolvimento sustentável do país. O presente trabalho tem por objetivo discutir a criação do bicho-da-seda e as principais doenças que acarretam perdas na produção, destacando que o melhoramento genético pode auxiliar na criação de híbridos mais resistentes à doenças e com melhor qualidade e quantidade de seda por casulo.

This article revises the main aspects related to sericulture which includes the breeding of Bombyx mori, popularly known as silkworm. It represents an activity developed mainly in small rural properties, where the family work prevails, representing an important alternative for the income improvement and, thus, contributing to the decrease of rural exodus. Besides, sericulture is an activity with low environmental impact which contributes to the sustainable development of the country. This study aims to discuss silkworm breeding and the main diseases that cause production losses, noting that genetic improvement can help breed more disease-resistant hybrids with better quality and quantity of silk by cocoon.

Este artículo revisa los principales aspectos relacionados con la sericultura, que abarca la cría del Bombyx mori, popularmente conocido como gusano de seda, que representa una actividad desarrollada principalmente en las pequeñas propiedades rurales, donde predomina el trabajo familiar, representando una alternativa importante para la mejora de renta y, contribuyendo así a reducir el éxodo rural. Además de esas características, la sericultura es una actividad de bajo impacto ambiental, que contribuye al desarrollo sostenible del país. Este trabajo pretende discutir la cría del gusano de seda y las principales enfermedades que causan pérdidas en la producción, señalando que el mejoramiento genético puede ayudar a crear híbridos más resistentes a las enfermedades y con mejor calidad y cantidad de seda por capullo.

[Expression and purification of EBV-LMP2 protein].

OBJECTIVE: To obtain a second Epstein-Barr virus membrane protein (LMP2) in insect cells. METHODS: The full length EBV-LMP2 gene was inserted into baculovirus expression transfer vector pFastBac HT B to obtain the recombinant baculoviruses Bac-LMP2. And generation of recombinant baculoviruses was followed by transfection of the recombinant Bac-LMP2 into insect cells, then the recombinant LMP2 protein was recognized by SDS-PAGE and western blot. The expressed LMP2 protein was purified by one step with Ni-NTA metal chelation chromatography. RESULTS: The expressed LMP2 protein was confirmed by SDS-PAGE and western blot. The purity of purified LMP2 protein is up to 86% by HPLC analysis. CONCLUSION: The EBV-LMP2 was expressed in insect cells, and the purified LMP2 protein was obtained.

Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice.

Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections.

Baculovirus infectivity and the actin cytoskeleton.

Baculovirus infection occurs when susceptible insect larvae ingest viral occlusions and occlusion-derived virus is released in the midgut lumen. Midgut columnar epithelial cells (the sole targets) are penetrated when the viral envelopes fuse with microvillar membranes; subsequently, nucleocapsids are transported basally through the microvilli toward the nucleus where replication ensues. Rapid infection of trachael cells (secondary targets) is under heavy selection because midgut cells are sloughed, an effective defense against systemic infection. The unique multiple nucleocapsid per virion trait acquired by some baculoviruses functions in countering this defense. Systemic infection is amplified after infected tracheal cells transmit infection to hemocytes. Tracheal cells serve as the conduit for virus spread through basal laminal barriers. Discordant susceptibilities to infection of midgut cells, tracheal cells and hemocytes may exist within an individual insect; in fully permissive hosts, all are highly susceptible. Viral manipulation of the actin cytoskeleton both during nucleocapsid transport and after viral gene expression is at the core of successful infection and replication. G-actin, normally cytoplasmic, is efficiently localized within the nucleus during early viral gene expression, and nuclear actin polymerizes during late gene expression, concurrent with shut down of host protein synthesis and early viral gene expression. Nuclear G-actin is now considered essential for cellular transcription and nuclear F-actin can affect transcription by binding chromatin-remodeling complexes. A new hypothesis is offered for how viral manipulation of actin influences timing of viral gene transcription, genome processing and packaging.

Virus-like particle and viral vector production using the baculovirus expression vector system/insect cell system: adeno-associated virus-based products.

The ability to make a large variety of virus-like particles (VLPs) has been successfully achieved in the baculovirus expression vector system (BEVS)/insect cell system. The production and scale-up of these particles, which are mostly sought as candidate vaccines, are currently being addressed. Furthermore, these VLPs are being investigated as delivery agents for use as therapeutics. Recently, adeno-associated viral (AAV) vectors, which can be potentially used for human gene therapy, have been produced in insect cells using three baculovirus vectors to supply the required genes. The use of host insect cells allows mass production of VLPs in a proven scaleable system. This chapter focuses on the methodology, based on the work done in our lab, for the production of AAV-like particles and vectors in a BEVS/insect cell system.

Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea.

* Botrytis cinerea is a necrotrophic fungus that causes grey mould on a wide range of food plants, especially grapevine, tomato, soft fruits and vegetables. This disease brings about important economic losses in both pre- and postharvest crops. Successful protection of host plants against this pathogen is severely hampered by a lack of resistance genes in the hosts and the considerable phenotypic diversity of the fungus. * The aim of this study was to test whether B. cinerea manipulates the immunity-signalling pathways in plants to restore its disease. * We showed that B. cinerea caused disease in Nicotiana benthamiana through the activation of two plant signalling genes, EDS1 and SGT1, which have been shown to be essential for resistance against biotrophic pathogens; and more interestingly, virus-induced gene silencing of these two plant signalling components enhanced N. benthamiana resistance to B. cinerea. Finally, plants expressing the baculovirus antiapoptotic protein p35 were more resistant to this necrotrophic pathogen than wild-type plants. * This work highlights a new strategy used by B. cinerea to establish disease. This information is important for the design of strategies to improve plant pathogen resistance.

Comparative studies of Bombyx mori nucleopolyhedrovirus chitinase and its host ortholog, BmChi-h.

Baculovirus-encoded chitinases (V-CHIAs) were first proposed to be acquired from a bacterium via horizontal gene transfer. However, we have recently reported that lepidopteran hosts also encode v-chiA orthologs. Here we describe comparative studies of Bombyx mori nucleopolyhedrovirus (BmNPV) chitinase and its host ortholog, BmChi-h. We constructed recombinant BmNPVs in which native and modified forms of BmChi-h were driven under the polyhedrin promoter and the authentic v-chiA was deleted. Western blot analysis indicated that BmCHI-h was rapidly secreted from virus-infected BmN cells whereas BmNPV CHIA was localized within the virus-infected cells; probably because of the presence of a C-terminal endoplasmic reticulum retention motif on BmNPV CHIA. Enzymological studies showed that BmNPV CHIA was able to retain much higher chitinolytic activity under alkaline conditions. For B. mori larvae infected with v-chiA-deleted BmNPV, the terminal liquefaction of dead larvae and the activation of baculovirus-encoded cysteine protease were not observed, and the introduction of BmChi-h did not rescue these defects. Our findings show that BmNPV chiA possesses unique features that are not shared by host orthologs, which may reflect functional specialization of baculovirus chitinases.

The incidence and use of Oryctes virus for control of rhinoceros beetle in oil palm plantations in Malaysia.

The rhinoceros beetle, Oryctes rhinoceros, has emerged as a serious pest of oil palm since the prohibition of burning as a method for maintaining estate hygiene in the 1990s. The abundance of beetles is surprising given that the Malay peninsula was the site of first discovery of the Oryctes virus, which has been used to effect good as a biological control agent in other regions. A survey of adult beetles was carried out throughout Malaysia using pheromone traps. Captured beetles were examined for presence of virus using both visual/microscopic examination and PCR detection methods. The survey indicated that Oryctes virus was common in Malaysia among the adult beetles. Viral DNA analysis was carried out after restriction with HindIII enzyme and indicated at least three distinct viral genotypes. Bioassays were used to compare the viral strains and demonstrate that one strain (type B) is the most virulent against both larvae and adults of the beetle. Virus type B has been cultured and released into healthy populations where another strain (type A) forms the natural background. Capture and examination of beetles from the release site and surrounding area has shown that the spread and persistence of the applied virus strain is accompanied by a reduction in palm frond damage.

Furin is involved in baculovirus envelope fusion protein activation.

The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology 275:30-41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F0) protein. BV produced in the presence of the inhibitor possesses the uncleaved F0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F0 is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F1 and against the putative N terminus (F2) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60- (F1) and 21-kDa (F2) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominantly as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.