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

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

Timely trigger of caterpillar zombie behaviour: temporal requirements for light in baculovirus-induced tree-top disease.

Host behavioural manipulation is a common strategy used by parasites to enhance their survival and/or transmission. Baculoviruses induce hyperactivity and tree-top disease (pre-death climbing behaviour) in their caterpillar hosts. However, little is known about the underlying mechanisms of this behavioural manipulation. A previous study showed that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induced tree-top disease at 3 days post infection in third instar S. exigua larvae and that light plays a key role in triggering this behaviour. Here we investigated the temporal requirements for the presence of light to trigger this behaviour and found that light from above was needed between 43 and 50 h post infection to induce tree-top disease. Infected larvae that were not exposed to light from above in this period finally died at low positions. Exposure to light prior to this period did not affect the final positions where larvae died. Overall we conclude that light in a particular time frame is needed to trigger SeMNPV-induced tree-top disease in S. exigua larvae.

Identification of Spodoptera exigua nucleopolyhedrovirus genes involved in pathogenicity and virulence.

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.

Natural populations of Spodoptera exigua are infected by multiple viruses that are transmitted to their offspring.

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.

Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

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.

A proctolin-like peptide is regulated after baculovirus infection and mediates in caterpillar locomotion and digestion.

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.

Antiviral Treatment Reveals a Cooperative Pathogenicity of Baculovirus and Iflavirus in Spodoptera exigua, a Lepidopteran Insect.

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.

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.

Iflavirus Covert Infection Increases Susceptibility to Nucleopolyhedrovirus Disease in Spodoptera exigua.

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.

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

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.

Baculovirus PTP2 Functions as a Pro-Apoptotic Protein.

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.

Genetic and Biological Characterization of Four Nucleopolyhedrovirus Isolates Collected in Mexico for the Control of Spodoptera exigua (Lepidoptera: Noctuidae).

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.

In vitro production of Spodoptera exigua multiple nucleopolyhedrovirus with enhanced insecticidal activity using a genotypically defined virus inoculum.

Defective virus accumulations during baculovirus passages in insect cell culture are impediments to large scale baculovirus production. A genotypically defined virus inoculum comprises of stable genotypes was proposed for production of a Thailand isolated SeMNPV in Se-UCR1 insect cells. Targeted genotypes were from wild-type SeMNPV containing naturally mixed genotypes. Plaque assays, PCR screening and XbaI restriction analysis were employed for genotype purification, genotype selection and genome analysis, respectively. A selective marker was pif2 encoded per os infection factor which predominantly deleted, along with the adjacent pif1, in defective viruses. A purified, genetically stable pif2+ (and pif1+) genotype, namely SeThpif2+, was the first tryout. SeThpif2+ occlusion bodies (OBs) possessed insecticidal activity but at lower level than the wild-type. When the SeThpif2+ was co-infected with another purified, genetically stable pif1- (and pif2-) genotype, SeThpif2-, at ratio of 3:1, respectively, mixed genotypes OBs had 2.8 times greater insecticidal activity than the SeThpif2+ alone. Dilution of deleterious PIF1 of SeThpif2+ by the pif1 deletion genotypes, SeThpif2-, was the key for this enhanced activity. A promising approach was described for SeMNPV production in vitro using the virus inoculum whose genotypes compositions were designed to mimic virus interactions in the wild-type, to generate per oral infective baculovirus.

De novo transcriptome analysis of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) genes in latently infected Se301 cells.

Cells of the P8-Se301-C1 strain are Spodoptera exigua cell clones that each harbor a partial version of the S. exigua multiple nucleopolyhedrovirus (SeMNPV) genome and which are resistant to homologous SeMNPV infections. The cells produce no viral progeny, suggesting that the infection is a latent-like viral infection. To investigate the SeMNPV genes harbored in the P8-Se301-C1 cells, the de novo transcriptomes of P8-Se301-C1 cells and S. exigua Se301 cells were analyzed and compared. A total of 54,569,296 reads were obtained from the P8-Se301-C1 cells that yielded 112,565 final unigenes with a mean length of 1,093 nt. A total of 56,865,504 reads were obtained from the Se301 cells that yielded 102,996 final unigenes with a mean length of 1,082 nt. Ten SeMNPV gene transcripts (se5, se7, se8, se12, se43, se45, se89, se90, se124, and se126) were detected in the P8-Se301-C1 cells by RNA-Seq but not in the Se301 cells, which was verified by RTPCR. 5'/3' RACE analyses showed that the 3'- or 5'-end sequences of the viral transcripts are aligned to the host gene sequences in P8-Se301-C1 cells, suggesting that the SeMNPV genes may integrate into and be transcribed with the host genes in the P8-Se301-C1 cells. Furthermore, six additional viral gene transcripts, se11, se42, se44, se88, se91, and se127 (incorporated into chimeric fusion transcripts in the P8-Se301-C1 cells), were detected in the RACE analyses. Taken together, sixteen SeMNPV transcripts were identified in the P8-Se301-C1 cell strain. This study provides information to develop the understanding of baculovirus latent infections and superinfection exclusion.

Parasitic Manipulation of Host Behaviour: Baculovirus SeMNPV EGT Facilitates Tree-Top Disease in Spodoptera exigua Larvae by Extending the Time to Death.

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.

Localization and Functional Analysis of SeMNPV IE1 in Mammalian Cells / 中国病毒学·英文版

In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), belonging to group Ⅱ nucleopolyhedrovirus, was studied in mammalian cells.We amplified the SeMNPV ie1 gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn't influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.

The Function of SeMNPV IAP3 in Mammalian Cells / 中国病毒学·英文版

The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I/Bam H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.