Search

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation. [ABSTRACT FROM AUTHOR]

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Baculoviruses constitute a large group of invertebrate DNA viruses, predominantly infecting larvae of the insect order Lepidoptera. During a baculovirus infection, the virus spreads throughout the insect body producing a systemic infection in multiple larval tissues, included the central nervous system (CNS). As a main component of the CNS, neuropeptides are small protein‐like molecules functioning as neurohormones, neurotransmitters, or neuromodulators. These peptides are involved in regulating animal physiology and behavior and could be altered after baculovirus infection. In this study, we have investigated the effect of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) infection on expression of Spodoptera exigua neuropeptides and neuropeptide‐like genes. Expression of the gene encoding a polypeptide that resembles the well‐known insect neuropeptide proctolin and named as proctolin‐like peptide (PLP), was downregulated in the larval brain following infection and was chosen for further analysis. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) overexpressing the C‐terminal part of the PLP was generated and used in bioassays using S. exigua larvae to study its influence on the viral infection and insect behavior. AcMNPV‐PLP‐infected larvae showed less locomotion activity and a reduction in growth compared to larvae infected with wild type AcMNPV or mock‐infected larvae. These results are indicative of this new peptide as a neuromodulator that regulates visceral and skeletal muscle contractions and offers a novel effector involved in the behavioral changes during baculovirus infection. [ABSTRACT FROM AUTHOR]

Beet armyworm, Spodoptera exigua (Hubner), is an important pest for many crops. SeMNPV infected development, survival process, and population parameters of S. exigua were studied in present study. The results demonstated that relationship beween probit value of mortality (y) and the logarimic of SeMNPV concentration (x) followed the linear equation: y=0.455+0.9300x. At 27 cels. degree, the LC50 of the third-instar larvae of S. exigua was tested to be 77082 PIBs/ml. Pupa weight (y; mg) of S. exigua decreased significantly with the SeMNPV concentration (x; SeMNPV concentration (PIBs/ml)): y=106.038-1.1962 log(x) (r2=0.915, p=0.044). Fecundity (y; eggs/female) of S. exigua decreased significantly with the SeMNPV concentration: y=690.523-28.5209 log(x) (r2=0.997, p=0.001). Both net reproduction rate (R0) and population trend index (I) decreased with the SeMNPV concentration (x): R0=744.121-54.6707 log(x) (r2=0.983, p=0.009); I=354.259-24.4705log(x) (r2=0.987, p=0.006).

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.

Baculovirus-based biopesticides have been developed for an efficient control of lepidopteran pests; however, very few studies have assessed the effect of other viral microorganisms that might coexist with the applied baculovirus pesticide. Systematic studies are needed to predict the effect of such microorganisms on the baculovirus efficacy following application in the field. In this context, we examined the effect of a sublethal doses of the DNA virus Junonia coenia densovirus (JcDV) on the infectivity of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) when infecting the same host, Spodoptera exigua. In our experimental conditions, the infection timing was critical for the mixed infection outcome. The consecutive infections with SeMNPV and later with JcDV did not affect the larval mortality or the measured infection parameters compared with single infection with the baculovirus. However, when larvae were first infected with JcDV, a clear antagonism was detected between both viruses and resulted in a patent reduction in viral pathogenicity and deleterious effects observed in the SeMNPV progeny. On the other hand, the simultaneous infection with both viruses led to an additive effect of the mortalities caused by the two viruses. Our results reveal that temporal order of the infection with one or the other virus affects the outcome of mixed viral infections on the targeted pest and may influence the ecology of baculovirus and its use as biological insecticide. [ABSTRACT FROM AUTHOR]

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.

Naturally occurring covert infections in lepidopteran populations can involve multiple viruses with potentially different transmission strategies. In this study, we characterized covert infection by two RNA viruses, Spodoptera exigua iflavirus 1 (SeIV-1) and Spodoptera exigua iflavirus 2 (SeIV-2) (family Iflaviridae) that naturally infect populations of Spodoptera exigua, and examined their influence on susceptibility to patent disease by the nucleopolyhedrovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) (family Baculoviridae). The abundance of SeIV-1 genomes increased up to ten-thousand-fold across insect developmental stages after surface contamination of host eggs with a mixture of SeIV-1 and SeIV-2 particles, whereas the abundance of SeIV-2 remained constant across all developmental stages. Low levels of SeIV-2 infection were detected in all groups of insects, including those that hatched from surface-decontaminated egg masses. SeIV-1 infection resulted in reduced larval weight gain, and an unbalanced sex ratio, whereas larval developmental time, pupal weight, and adult emergence and fecundity were not significantly affected in infected adults. The inoculation of S. exigua egg masses with iflavirus, followed by a subsequent infection with SeMNPV, resulted in an additive effect on larval mortality. The 50% lethal concentration (LC50) of SeMNPV was reduced nearly 4-fold and the mean time to death was faster by 12 h in iflavirus-treated insects. These results suggest that inapparent iflavirus infections may be able to modulate the host response to a new pathogen, a finding that has particular relevance to the use of SeMNPV as the basis for biological pest control products.

Many parasites enhance their dispersal and transmission by manipulating host behaviour. One intriguing example concerns baculoviruses that induce hyperactivity and tree-top disease (i.e., climbing to elevated positions prior to death) in their caterpillar hosts. Little is known about the underlying mechanisms of such parasite-induced behavioural changes. Here, we studied the role of the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in tree-top disease in S. exigua larvae. Larvae infected with a mutant virus lacking the egt gene exhibited a shorter time to death and died before the induction of tree-top disease. Moreover, deletion of either the open reading frame or the ATG start codon of the egt gene prevented tree-top disease, indicating that the EGT protein is involved in this process. We hypothesize that SeMNPV EGT facilitates tree-top disease in S. exigua larvae by prolonging the larval time to death. Additionally, we discuss the role of egt in baculovirus-induced tree-top disease.

The biological activity of 4 Mexican isolates (SeSIN6, SeSIN8, SeSLP6, and SeSLP8) of the homologous multiple nucleopolyhedrovirus SeMNPV and their effects on the development of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), were studied. An exotic isolate of SeMNPV from the United States (SeUS2) was used as a reference. Early third-instar larvae were inoculated with 5 × 106 occlusion bodies per mL, resulting in 78% mortality for SeUS2 and approximately 90% mortality for each of the 4 Mexican isolates at 144 h post-inoculation. At 168 h postinoculation, 100% mortality was obtained in all cases. All of the isolates, including SeUS2, significantly reduced the body weights of S. exigua larvae compared with the control larvae; however, at 120 h post-inoculation, the differences among isolates were not significant. All of the isolates reduced the development time of third-instar S. exigua larvae (range: 1.2- to 1.6-fold) compared with the control larvae. An independent experiment was performed to determine production of occlusion bodies and mean time to death. Third-instar S. exigua larvae (15 h after molting) were inoculated with each isolate using the same concentration mentioned above. The occlusion body production rate was similar among all isolates. The isolates SeUS2, SeSLP6, and SeSLP8 yielded the fastest mortality (range: 187-191 h). Thus, the biological activities of the Mexican SeMNPV isolates were similar to the activity of the exotic isolate, indicating that these indigenous viruses are promising for the biological control of S. exigua in Mexico. [ABSTRACT FROM AUTHOR]

This study describes four multiple nucleocapsid nucleopolyhedrovirus isolates recovered from infected larvae of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on crops in two different geographical regions of Mexico. Molecular and biological characterization was compared with characterized S. exigua multiple nucleopolyhedrovirus (SeMNPV) isolates from the United States (SeUS1 and SeUS2) and Spain (SeSP2). Restriction endonuclease analysis of viral DNA confirmed that all Mexican isolates were SeMNPV isolates, but molecular differences between the Mexican and the reference isolates were detected using PCR combined with restriction fragment length polymorphism (RFLP). Amplification of the variable region V01 combined with RFLP distinguished the two Mexican isolates, SeSLP6 and SeSIN6. BglII digestions showed that the majority of the isolates contained submolar bands, indicating the presence of genetic heterogeneity. Amplification of the variable regions V04 and V05 distinguished between American and the Spanish isolates. Biological characterization was performed against two laboratory colonies of S. exigua, one from Mexico, and another from Switzerland. Insects from the Mexican colony were less susceptible to infection than insects from Se-Swiss colony. In the Se-Mex colony, SeSP2 was the most pathogenic isolate followed by SeSIN6, although their virulence was similar to most of the isolates tested. In Se-Swiss colony, similar LD50 values were observed for the five isolates, although the virulence was higher for the SeSLP6 isolate, which also had the highest OB (occlusion body) yield. We conclude that the Mexican isolates SeSIN6 and SeSLP6 possess insecticidal traits of value for the development of biopesticides for the control of populations of S. exigua. [ABSTRACT FROM AUTHOR]

Following the consumption of baculovirus occlusion bodies ( OBs), insects may succumb to lethal disease, but the survivors can harbour sublethal covert infections and may develop, reproduce and transmit the infection to their offspring. The use of different chemical and biological stressors was examined to determine whether they could be used to activate covert infections in populations of Spodoptera exigua larvae infected by the homologous nucleopolyhedrovirus (Se MNPV). Treatment of covertly infected S. exigua second instars with Tinopal UNPA- GX, hydroxylamine, paraquat, Bacillus thuringiensis var. kurstaki crystals, spores or mixtures of crystals + spores, or a heterologous nucleopolyhedrovirus ( Chrysodeixis chalcites SNPV) did not result in the activation of Se MNPV covert infections. Similarly, virus treatments involving permissive NPVs did not result in greater mortality in covertly infected insects compared with the virus-free controls. In contrast, 0.1% copper sulphate, 1% iron ( II) sulphate and 1 mg/l sodium selenite treatments resulted in 12-41% lethal polyhedrosis disease in covertly infected larvae. A greenhouse trial using copper sulphate and sodium selenite as activation factors applied to covertly infected S. exigua larvae on sweet pepper plants resulted in very low levels of Se MNPV activation (<3%). These results highlight the important roles of copper, iron and selenium in insect immunity and baculovirus-induced disease. However, these substances seem unlikely to prove useful for the activation of covert Se MNPV infections in S. exigua larvae under greenhouse conditions. [ABSTRACT FROM AUTHOR]

Previous studies identified distinct genotypes of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) that were associated with horizontal transmission (named HT-SeG25) or vertical transmission (named VT-SeAl1) in the host insect, S. exigua (Lepidoptera: Noctuidae). We examined the use of mixtures of occlusion bodies (OBs) of these genotypes as the basis for a virus preparation that could provide immediate pest control and establish a persistent sublethal infection in the survivors of an OB application for transgenerational pest suppression. Mixtures of HT-SeG25 + VT-SeAl1 comprising 25:75 or 75:25 % of each genotype, respectively, resulted in improved OB pathogenicity in terms of concentration-mortality metrics compared to OBs of VT-SeAl1 alone or similar values compared to OBs of the HT-SeG25 genotype alone. In contrast, no significant differences were observed in speed of kill or mean OB production per larva. Laboratory and greenhouse trials revealed that the prevalence of sublethal infection in adults that survived OB treatments in the larval stage increased with the proportion of VT-SeAl1 present in the inoculum, as determined by qPCR. Greenhouse trials indicated that the 75 % VT-SeAl1 + 25 % HT-SeG25 mixture was as effective as methoxyfenozide in preventing pest damage to pepper fruits. The potential contribution of vertically transmitted genotypes to transgenerational suppression of pest populations is discussed. [ABSTRACT FROM AUTHOR]

The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as immunological responses, which may affect virus transmission. Here we show that the SeMNPV-encoded protein tyrosine phosphatase 2 (PTP2) induces mild apoptosis in Spodoptera frugiperda (Sf) 21 cells upon transient expression. Transient expression of a catalytic-site mutant of ptp2 did not lead to apoptosis, indicating that the phosphatase activity of PTP2 is needed to induce apoptosis. We also found that the caspase level (indicator of apoptosis) was higher in cells transfected with the ptp2 gene than in cells transfected with the catalytic mutant. Adding a caspase inhibitor reduced the level of ptp2-induced apoptosis. Moreover, deletion of the ptp2 gene from the viral genome prevented the induction of apoptosis in S. exigua hemocytes. The virus titer and virulence indices (the viral infectivity and the time to death) were not affected by deletion of the ptp2 gene. However, the viral occlusion body yield from S. exigua larvae infected with the mutant virus lacking the ptp2 gene was much lower than the yield from larvae infected with the wild-type (WT) virus. We hypothesize that the observed pro-apoptotic effects of PTP2 are the result of PTP2-mediated immune suppression in larvae, which consequently leads to higher viral occlusion body yields.

Genome sequence analysis of seven different Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolates that differed in insecticidal phenotype permitted the identification of genes likely to be involved in pathogenicity of occlusion bodies (OBs) and speed of kill (virulence) of this virus: se4 ( hoar ), se5 (unknown function), se28 (unknown function), se76 ( cg30 ), se87 ( p26 ) and se129 ( p26 ). To study the role of these genes experimentally on the insecticidal phenotype, a bacmid-based recombination system was constructed to delete selected genes from a SeMNPV isolate, VT-SeAL1, designated as SeBacAL1. All of the knockout viruses were viable and the repair viruses behaved like the wild-type control, vSeBacAL1. Deletion of se4 , se5 , se76 and se129 resulted in decreased OB pathogenicity compared to vSeBacAL1 OBs. In contrast, deletion of se87 did not significantly affect OB pathogenicity, whereas deletion of se28 resulted in significantly increased OB pathogenicity. Deletion of se4 , se28 , se76, se87 and se129 did not affect speed of kill compared to the bacmid vSeBacAL1, whereas speed of kill was significantly extended following deletion of se5 and in the wild-type isolate (SeAL1), compared to that of the bacmid. Therefore, biological assays confirmed that several genes had effects on virus insecticidal phenotype. Se5 is an attractive candidate gene for further studies, as it affects both biological parameters of this important biocontrol virus. [ABSTRACT FROM AUTHOR]

Sublethal infections by baculoviruses (Baculoviridae) are believed to be common in Lepidoptera, including Spodoptera exigua . In addition, novel RNA viruses of the family Iflaviridae have been recently identified in a laboratory population of S. exigua ( S. exigua iflavirus-1: SeIV-1; S. exigua iflavirus-2: SeIV-2) that showed no overt signs of disease. We determined the prevalence of these viruses in wild populations and the prevalence of co-infection by the different viruses in shared hosts. Infection by S. exigua multiple nucleopolyhedrovirus (SeMNPV) and iflaviruses in S. exigua adults ( N = 130) from horticultural greenhouses in southern Spain was determined using qPCR and RT-PCR based techniques respectively. The offspring of these insects ( N = 200) was reared under laboratory conditions and analyzed to determine virus transmission. Overall, 54% of field-caught adults were infected by SeMNPV, 13.1% were infected by SeIV-1 and 7.7% were infected by SeIV-2. Multiple infections were also detected, with 8.4% of individuals harboring SeMNPV and one of the iflaviruses, whereas 2.3% of adults were infected by all three viruses. All the viruses were transmitted to offspring independently of whether the parental female harbored covert infections or not. Analysis of laboratory-reared insects in the adult stage revealed that SeIV-1 was significantly more prevalent than SeMNPV or SeIV-2, suggesting high transmissibility of SeIV-1. Mixed infection involving three viruses was identified in 6.5% of laboratory-reared offspring. We conclude that interspecific interactions between these viruses in co-infected individuals are to be likely frequent, both in the field, following applications of SeMNPV-based insecticides, or in laboratory colonies used for SeMNPV mass production. [ABSTRACT FROM AUTHOR]

The insecticidal characteristics of two Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) strains produced on two different S. exigua colonies were measured using the same two host colonies. These strains constitute the active ingredients of the biological insecticides Vir-ex and Spexit and were produced on insect colonies from Spain and Switzerland. Demographic characteristics of insects from each colony were examined before infection. Larval developmental time, larval survival, and adult sex ratio did not differ between the colonies, whereas mean pupal weight was significantly higher in the Spanish colony insects. After infection, susceptibility to virus occlusion bodies (OBs), time to death, larval weight at death, and total production of OBs/larva varied significantly depending on virus strain and the colony used. Vir-ex OBs produced in Spanish colony larvae had improved insecticidal characteristics in terms in lethal dose and speed of kill metrics than other strain-colony combinations. OB production was significantly higher in Spanish colony insects infected with Spexit compared with Vir-ex infected insects from the Swiss colony, with intermediate values for the other two strain-colony combinations. Virus strain and host colony origin were highly influential in determining the insecticidal characteristics of OBs and should be considered as key parameters that require optimization during the production of SeMNPV-based insecticides. [ABSTRACT FROM AUTHOR]

Abstract: The Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV), a pathogen of the Egyptian cotton leaf worm S. littoralis, was subjected to sequencing of its entire DNA genome and bioassay analysis comparing its virulence to that of other baculoviruses. The annotated SpliMNPV genome of 137,998bp was found to harbor 132 open reading frames and 15 homologous repeat regions. Four unique genes not present in SpltMNPV were identified, as were 14 genes that were absent or translocated by comparison. Bioassay analysis of experimentally infected Spodoptera frugiperda revealed an extended killing time for SpliMNPV as compared to S. frugiperda MNPV (SfMNPV), but a level of mortality similar to that caused by infection with SfMNPV and superior to that of Autographa californica MNPV (AcMNPV). Although extensive similarity was observed between the genome structure and predicted translation products of SpliMNPV and Spodoptera litura MNPV (SpltMNPV), genetic distances between isolates of SpliMNPV and SpltMNPV suggest that they are in fact different species of genus Alphabaculovirus. [Copyright &y& Elsevier]

Abstract: We have obtained and characterized the transcriptome of Spodoptera exigua larvae with special emphasis on pathogen-induced genes. In order to obtain a highly representative transcriptome, we have pooled RNA from diverse insect colonies, conditions and tissues. Sequenced cDNA included samples from 3 geographically different colonies. Enrichment of RNA from pathogen-related genes was accomplished by exposing larvae to different pathogenic and non-pathogenic microbial agents such as the bacteria Bacillus thuringiensis, Micrococcus luteus, and Escherichia coli, the yeast Saccharomyces cerevisiae, and the S. exigua nucleopolyhedrovirus (SeMNPV). In addition, to avoid the loss of tissue-specific genes we included cDNA from the midgut, fat body, hemocytes and integument derived from pathogen exposed insects. RNA obtained from the different types of samples was pooled, normalized and sequenced. Analysis of the sequences obtained using the Roche 454 FLX and Sanger methods has allowed the generation of the largest public set of ESTs from S. exigua, including a large group of immune genes, and the identification of an important number of SSR (simple sequence repeats) and SNVs (single nucleotide variants: SNPs and INDELs) with potential use as genetic markers. Moreover, data mining has allowed the discovery of novel RNA viruses with potential influence in the insect population dynamics and the larval interactions with the microbial pesticides that are currently in use for the biological control of this pest. [Copyright &y& Elsevier]

Abstract: Analysis of the transcriptome of Spodoptera exigua larvae revealed the presence of several ESTs with homology to virus of the order Picornavirales and with the highest similarity to Infectious flacherie virus (Iflaviridae) that infects Bombyx mori larvae. Iflaviridae is a recently defined family of insect-infecting viruses that consist of positive single strand RNA genomes translated into a single polyprotein of around 3000 amino acids long. Using the sequence information derived from the obtained ESTs, we have completed the genomic sequence of this virus. The novel S. exigua iflavirus (SeIV-1) has a genome of 10.3kb and codes for a 3222 aa polyprotein. Expression analysis has revealed the presence of the virus in all tissues tested and insect stages, being more abundant in the midgut of the larvae. High infectivity of this virus against S. exigua has been demonstrated after observing the presence of this virus in different colonies that were reared in the same chamber with the virus-infected colony, despite no evidence of pathological effects. Further study of viral covert infections of SeIV-1 could lead to a better understanding of its pathological effect as well as any possible interaction with other microbial pathogens used for the control of this pest. [Copyright &y& Elsevier]

Abstract: Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6–12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findings suggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems. [ABSTRACT FROM AUTHOR]

A low-cost simplified diet has been successfully developed for rearing Spodoptera exigua larvae under laboratory conditions. The cost of ingredients was lower than that of the standard diet based on a modified tobacco hornworm, Manduca sexta (L.), diet. The simplified diet fulfilled larval nutritional requirements without apparent adverse effects on the reproductive capacity of the insect. Survival, pupal sex ratio, and fecundity registered in insects that were reared on the simplified diet did not differ from those observed on the standard diet. The mean larval development period of insects that consumed the simplified diet was also similar to that of insects that consumed the standard diet, whereas weight of pupae and adult longevity were significantly higher in insects reared on simplified diet. Larvae consumed ≈11% more of the standard diet compared with the simplified diet and a corresponding increase was observed in the number of larvae that could be reared through to pupation on each liter of simplified diet. The production of S. exigua multiple nucleopolyhedrovirus (SeMNPV) occlusion bodies (OBs) in insects grown on each type of diet was also evaluated. Weights of larvae at inoculation and at death, OB yields and biological activity of OBs did not differ significantly for each type of diet. The simplified low-cost diet appears suitable for large-scale in vivo production of SeMNPV OBs. [ABSTRACT FROM AUTHOR]

Successive viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and SeIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into nucleocapsids is hypothesized. [Copyright &y& Elsevier]

Scenario studies were carried out with a process-based model for control of wild-type and genetically modified baculoviruses in populations of Spodoptera exigua in glasshouse chrysanthemum (BACSIM). These scenario studies were used to evaluate the effectiveness of different spraying regimes, concentrations, UV-protection agents, and mean time to kill insect. In simulations on concentration and timing of S. exigua multicapsid nucleopolyhedrovirus (SeMNPV), good control efficacy was obtained with SeMNPV concentrations of 1×107 polyhedra/m2 or higher. An early timing of virus applications, soon after egg deposition, is essential for effective control, especially at high temperatures, when the developmental rate of larvae is high and the window of opportunity for virus control is short. UV-protection agents contribute only marginally to effective biological control in the glasshouse because the decay rates of SeMNPV and Autographa californica multicapsid nucleopolyhedrovirus already ensure a period of exposure to active virus that is long enough for caterpillars to acquire a lethal concentration under practical conditions. The effect of genetic improvement towards shortening the survival time of infected larvae depends on the ability to time virus applications accurately and the mean time to kill the unmodified virus. Recombinant viruses with a lower mean time to kill are more effective than wild-type viruses when applied against second, third, and fourth instars because in these stages S. exigua infests a considerable number of plants and the rate of foliage consumption increases rapidly. When applied against first instars that infest only a small number of plants and have low consumption rates, recombinants will only be slightly more effective than wild-type viruses. In the case of an unmodified virus that already has a relatively high mean time to kill, a further reduction in the time to kill will not result in improved control. Thus, accurate monitoring followed by virus application is indispensable for adequate control and may even alleviate the need for fast killing viruses. [Copyright &y& Elsevier]

This paper describes the validation and sensitivity analysis of a process-based simulation model (BACSIM) for the control of beet armyworm, Spodoptera exigua, with baculoviruses. Model predictions are compared to results of independent greenhouse experiments in which second, third, or fourth instar larvae of S. exigua in chrysanthemum plots are treated with different concentrations of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and S. exigua MNPV (SeMNPV), two viruses with distinct differences in infectivity and mean time to kill. BACSIM provides robust predictions for the control of S. exigua populations in greenhouse chrysanthemum with both AcMNPV and SeMNPV. Mortality levels caused by AcMNPV and SeMNPV were generally predicted within a 25% margin of error compared to the observed values. None of the deviations was higher than 40%. All values of simulated foliage consumption, caused by S. exigua populations treated with AcMNPV or SeMNPV applications, fell within 95% confidence intervals of measurements. Simulated time to kill was, in general, lower than the measurements. This discrepancy may be caused by the behavior of S. exigua larvae which feed on the underside of chrysanthemum leaves where they are protected from polyhedra. This suggests that the larval foraging behavior may play an important role in the efficacy of baculovirus applications and should be further studied experimentally. This validated model can be used for the pretrial evaluation of the efficacy of genetically modified baculoviruses as biological control agents and for the optimization of spraying regimes in chrysanthemum cultivation. [Copyright &y& Elsevier]

We describe the development of a comprehensive process-based model simulating the epizootiology and agronomic efficacy of baculoviruses used for biological control of beet armyworm, Spodoptera exigua, in greenhouse chrysanthemum. The model is built to help understand, evaluate, and predict the effects of genetic modification, formulation, and application strategy of a baculovirus on its epizootiology and agronomic efficacy in a greenhouse cropping system. The model was constructed such that the impact of detailed aspects of the virus–host relationship, cropping system, spraying regime, and insect ecology on control could be evaluated. The resulting model is highly detailed, covers four spatial scales, from the leaf to the greenhouse, and addresses spatially explicit processes at the individual level and spatially averaged population processes. Submodels describe the crop production system and plant growth, the distribution and dispersal of insects within the crop canopy, spray deposition, uptake of virus, the probability of death and survival time of infected insects, and vertical and horizontal transmission. This paper describes the conceptual basis of the model and its parameterization by quantitative descriptions of model components. Model components are tested by comparison with dependent and independent experimental data. [Copyright &y& Elsevier]

One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID50 assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×106 TCID50/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×106 TCID50/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×106TCID50/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×106TCID50/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Bacterial diversity in SeMNPV OB suspensions characterized by next-generation sequencing. • Biocides screened for ability to reduce microbial contaminants in OB suspensions. • Benzalkonium chloride and colloidal silver were highly effective over 180 day period. • Benzalkonium chloride adversely affected insecticidal properties of OBs. The presence of contaminant microbes in baculovirus-based insecticides is regulated by phytosanitary product registration authorities. We aimed to determine whether the abundance of microbes in suspensions of Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies (OBs) could be reduced by treatment with a range of biocidal compounds. The diversity of contaminant bacteria was determined by next-generation sequencing of the 16S rRNA gene. Overall, 97.9% of sequences detected were Gammaproteobacteria (mostly Pseudomonas spp. and Enterobacteriaceae) and 2.1% were Firmicutes (mostly Enterococcus spp.). Colloidal silver, benzalkonium chloride and chlorhexidine digluconate were identified as highly effective biocides. Incubation of OB suspensions with high concentrations of colloidal silver (450 mg/l) or benzalkonium chloride (6000 mg/l) resulted in marked reductions in colony forming unit counts over a 180 day period at 4° or 25 °C. Benzalkonium chloride and colloidal silver treatments, at either 4 or 25 °C, did not affect the insecticidal activity of OBs over an 80 day period. However, OB activity decreased following 180 days of treatment by benzalkonium chloride at either 4 or 25 °C, or by colloidal silver at 25 °C, but not at 4 °C. Counts of OBs revealed a significant decrease in OB numbers in benzalkonium chloride-treated suspensions after 180 days at both temperatures, whereas colloidal silver-treated OBs were not affected. Benzalkonium chloride also caused aggregation of OBs at the concentration tested. We conclude that biocidal compounds can markedly reduce the abundance of contaminant microorganisms in OB suspensions, and can be accompanied by reductions in OB infectivity and OB numbers in some circumstances. Future studies should focus on lower concentrations of biocides that do not affect OBs in long-term storage. [ABSTRACT FROM AUTHOR]

Simple Summary: Baculoviruses (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. [ABSTRACT FROM AUTHOR]

Naturally occurring covert infections in lepidopteran populations can involve multiple viruses with potentially different transmission strategies. In this study, we characterized covert infection by two RNA viruses, Spodoptera exigua iflavirus 1 (SeIV-1) and Spodoptera exigua iflavirus 2 (SeIV-2) (family Iflaviridae) that naturally infect populations of Spodoptera exigua, and examined their influence on susceptibility to patent disease by the nucleopolyhedrovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) (family Baculoviridae). The abundance of SeIV-1 genomes increased up to ten-thousand-fold across insect developmental stages after surface contamination of host eggs with a mixture of SeIV-1 and SeIV-2 particles, whereas the abundance of SeIV-2 remained constant across all developmental stages. Low levels of SeIV-2 infection were detected in all groups of insects, including those that hatched from surface-decontaminated egg masses. SeIV-1 infection resulted in reduced larval weight gain, and an unbalanced sex ratio, whereas larval developmental time, pupal weight, and adult emergence and fecundity were not significantly affected in infected adults. The inoculation of S. exigua egg masses with iflavirus, followed by a subsequent infection with SeMNPV, resulted in an additive effect on larval mortality. The 50% lethal concentration (LC50) of SeMNPV was reduced nearly 4-fold and the mean time to death was faster by 12 h in iflavirus-treated insects. These results suggest that inapparent iflavirus infections may be able to modulate the host response to a new pathogen, a finding that has particular relevance to the use of SeMNPV as the basis for biological pest control products. [ABSTRACT FROM AUTHOR]

NPVThe beet armyworm, Spodoptera exigua , is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Doublestranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua .

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., (Copyright © 2021 American Society for Microbiology.)