Entry into midgut epithelial cells is a key step in the selection of genotypes in a nucleopolyhedrovirus

An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes (e.g., SfNIC-C), that lack pif1 and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype (SfNIC-B) in coinfected cells. To determine whether selection for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum and budded virus (BV) samples taken from larvae inoculated with OBs comprising 10% SfNIC-B + 90% SfNIC-C indicating that a marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures of 50% SfNIC-B + 50% SfNIC-C or those comprising 10% SfNIC-B + 90% SfNIC-C as most of the OBs in these mixtures originated from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely modulated by changes in MOI during the infection period.     

Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses

The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A-I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366-27,225) and 60 bp (119,759-119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.     

Defective or effective? Mutualistic interactions between virus genotypes

Defective viruses lack genes essential for survival but they can co-infect with complete virus genotypes and use gene products from the complete genotype for their replication and transmission. As such, they are detrimental to the fitness of complete genotypes. Here, we describe a mutualistic interaction between genotypes of an insect baculovirus (nucleopolyhedrovirus of Spodoptera frugiperda (Lepidoptera)) that increases the pathogenicity of the viral population. Mixtures of a complete genotype able to be transmitted orally and a deletion mutant unable to be transmitted orally resulted in a phenotype of increased pathogenicity. Because the infectiousness of mixed genotype infections was greater than that of single genotype infections, we predict that the transmissibility of mixed genotype occlusion bodies will be greater than that of any of their single genotype components. Such interactions will be subject to frequency-dependent selection and will influence the impact of these viruses on insect population dynamics and their efficacy as biological insecticides.     

Functional importance of deletion mutant genotypes in an insect nucleopolyhedrovirus population

A Nicaraguan isolate of a nucleopolyhedrovirus (SfNIC) that attacks the fall armyworm, Spodoptera frugiperda, survives as a mixture of nine genotypes (SfNIC A to I) that all present genomic deletions, except variant B (complete genotype). Sequencing of cloned restriction fragments revealed that genotypic variants lack between 5 and 16 of the open reading frames present in a contiguous sequence of 18 kb of the SfNIC genome. The absence of oral infectivity of SfNIC-C and -D variants is related to the deletion of the pif and/or pif-2 gene, while that of SfNIC-G remains unexplained. The presence of open reading frame 10, homolog of Se030, also appeared to influence pathogenicity in certain variants. Previous studies demonstrated a significant positive interaction between genotypes B and C. We compared the median lethal concentration of single genotypes (A, B, C, D, and F) and co-occluded genotype mixtures (B+A, B+D, B+F, A+C, and F+C in a 3:1 ratio). Mixtures B+A and B+D showed increased pathogenicity, although only B+D restored the activity of the mixture to that of the natural population. Mixtures of two deletion variants (A+C and F+C) did not show interactions in pathogenicity. We conclude that minority genotypes have an important influence on the overall pathogenicity of the population. These results clearly demonstrate the value of retaining genotypic diversity in virus-based bioinsecticides.     

Deletion Genotypes Reduce Occlusion Body Potency but Increase Occlusion Body Production in a Colombian Spodoptera frugiperda Nucleopolyhedrovirus Population

A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.     

草地贪夜蛾核型多角体病毒对我国草地贪夜蛾不同地理种群毒力的比较分析

草地贪夜蛾Spodoptera frugiperda(J. E. Smith)入侵我国多个地区,逐渐形成地理种群。在草地贪夜蛾核型多角体病毒Spodoptera frugiperda multiple nucleopolyhedrovirus(SfMNPV)生物农药的生产过程中发现,不同地理来源的宿主对SfMNPV的敏感性和产量存在明显差异,显著影响了SfMNPV的生产效率。为解析其敏感性差异及其产生的原因,本研究首先对云南德宏、广东广州、广西钦州、西藏林芝4个地区草地贪夜蛾种群基因型进行了鉴定,采用生物测定法测试SfMNPV对4龄幼虫的口服毒力,然后,通过向幼虫体内注射草地贪夜蛾核多角体病毒出芽型病毒粒子(budded virions, BVs)的方式,越过口服感染中肠的过程,分析敏感性差异发生的阶段。最后比较了高敏感和低敏感种群中肠肠液pH,并基于16S rDNA测序测定了肠道菌群组成。结果表明,广西种群属于纯合玉米型,其余种群为带有水稻型COI标记的杂合玉米型。广西种群对SfMNPV的口服敏感性最低,西藏种群的敏感性最高,但两者注射BV后死亡率差异无统计学意义,暗示病毒敏感性差异...     

Optical brightener Tinopal C1101 as an ultraviolet protectant for a nucleopolyhedrovirus

Baculoviruses are arthropod-specific viruses currently used as biological insecticides in several countries of the world. One important factor limiting the efficacy of these bioinsecticides is related to the inactivation of the virus by solar ultraviolet (UV) radiation. This has motivated studies focused on the use of optical brightener compounds that can protect the virus from UV inactivation. The effects of the optical brightener Tinopal C1101 (ethenedyl benzenesulfonic derivative) on the persistance of a nucleopolyhedrovirus (SfMNPV) was quantified in third instar Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), the principal pest of maize in the Americas. For this, laboratory studies were performed to determine the relationship between virus concentrations and radiation-caused inactivation of SfMNPV alone or in mixtures with 1.25 or 0.1% (vol/vol) Tinopal C1101 using a UV system [radiation was provided by a 15 W UV tube (emission maxima at 312 nm)]. SfMNPV alone was more sensitive to UV than in mixtures with 1.25 or 0.1% Tinopal C1101. For instance, at concentrations of 2.22 x 10(8) and 2.75 x 10(6) occlusion bodies (OBs)/ml, SfMNPV alone-caused mortality was reduced from 88 and 44% (without UV radiation) to 0%, in both cases, after 30 and 15 min exposure to the UV tube, respectively. In contrast, the incorporation of 1.25% Tinopal C1101 into the SfMNPV inoculum at 2.22 x 10(8) and 2.75 x 10(6) OBs/ml caused a mortality of 86.6 and 96.6% after 240 min, respectively. UV-caused inactivation of SfMNPV was directly related to the optical brightener concentration. We conclude that optical brighteners are likely to represent valuable components of nucleopolyhedrovirus formulations.     

Functional Importance of Deletion Mutant Genotypes in an Insect Nucleopolyhedrovirus Population

A Nicaraguan isolate of a nucleopolyhedrovirus (SfNIC) that attacks the fall armyworm, Spodoptera frugiperda, survives as a mixture of nine genotypes (SfNIC A to I) that all present genomic deletions, except variant B (complete genotype). Sequencing of cloned restriction fragments revealed that genotypic variants lack between 5 and 16 of the open reading frames present in a contiguous sequence of 18 kb of the SfNIC genome. The absence of oral infectivity of SfNIC-C and -D variants is related to the deletion of the pif and/or pif-2 gene, while that of SfNIC-G remains unexplained. The presence of open reading frame 10, homolog of Se030, also appeared to influence pathogenicity in certain variants. Previous studies demonstrated a significant positive interaction between genotypes B and C. We compared the median lethal concentration of single genotypes (A, B, C, D, and F) and co-occluded genotype mixtures (B+A, B+D, B+F, A+C, and F+C in a 3:1 ratio). Mixtures B+A and B+D showed increased pathogenicity, although only B+D restored the activity of the mixture to that of the natural population. Mixtures of two deletion variants (A+C and F+C) did not show interactions in pathogenicity. We conclude that minority genotypes have an important influence on the overall pathogenicity of the population. These results clearly demonstrate the value of retaining genotypic diversity in virus-based bioinsecticides.     

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

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

Effects of a nucleopolyhedrovirus in mixtures with azadirachtin on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) larvae and viral occlusion body production

Abstract

The combined and alone effects of azadirachtin (AZA) and Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) on the mortality of S. frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) were evaluated in the laboratory. For this, diet surface contamination bioassays were performed on S. frugiperda in the third instar. LC₅₀ values for SfMNPV alone were determined to be 430 and 373 viral occlusion bodies (OBs)/mm² at 192 and 216 h after treatment, respectively. LC₅₀ values for AZA alone were estimated for two periods of continuous exposure (4 or 5 days). In this case, LC₅₀ values were 45.5 and 16.8 mg L^?1 at 216 h after treatment (4 or 5 days of larval exposition to insecticide, respectively). We observed that although the interaction of AZA with SfMNPV increased viral pathogenicity, such improvements were of greater magnitude and more consistent at the lower OB concentration used (177 OBs/mm²). Application of SfMNPV (430 OBs/mm²), AZA (26.4 mg L^?1) or SfMNPV–AZA mixtures resulted in a significant reduction in the mean weight of larvae treated in the third instar across the experiment, by 23–41%, 17–95% and 26–97%, respectively, compared to control. The duration of larval development during the third and fourth instars increased significantly in larvae exposed to SfMNPV–AZA mixtures and AZA alone compared to SfMNPV alone and control treatments. The yield of OBs/mg weight of larvae treated with SfMNPV alone was 1.8-fold higher than OB yields from insects inoculated with SfMNPV–AZA mixtures. We conclude that AZA + SfMNPV mixtures are unlikely to be useful for the mass production of this virus and laboratory observations on the value of AZA + SfMNPV mixtures as a potentiator of biological insecticides require validation in field studies under commercial growing conditions.     

A Chrysodeixis chalcites Single-Nucleocapsid Nucleopolyhedrovirus Population from the Canary Islands Is Genotypically Structured To Maximize Survival

A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus wild-type isolate from the Canary Islands, Spain, named ChchSNPV-TF1 (ChchTF1-wt), appears to have great potential as the basis for a biological insecticide for control of the pest. An improved understanding of the genotypic structure of this wild-type strain population should facilitate the selection of genotypes for inclusion in a bioinsecticidal product. Eight genetically distinct genotypes were cloned in vitro: ChchTF1-A to ChchTF1-H. Quantitative real-time PCR (qPCR) analysis confirmed that ChchTF1-A accounted for 36% of the genotypes in the wild-type population. In bioassays, ChchTF1-wt occlusion bodies (OBs) were significantly more pathogenic than any of the component single-genotype OBs, indicating that genotype interactions were likely responsible for the pathogenicity phenotype of wild-type OBs. However, the wild-type population was slower killing and produced higher OB yields than any of the single genotypes alone. These results strongly suggested that the ChchTF1-wt population is structured to maximize its transmission efficiency. Experimental OB mixtures and cooccluded genotype mixtures containing the most abundant and the rarest genotypes, at frequencies similar to those at which they were isolated, revealed a mutualistic interaction that restored the pathogenicity of OBs. In OB and cooccluded mixtures containing only the most abundant genotypes, ChchTF1-ABC, OB pathogenicity was even greater than that of wild-type OBs. The ChchTF1-ABC cooccluded mixture killed larvae 33 h faster than the wild-type population and remained genotypically and biologically stable throughout five successive passages in vivo. In conclusion, the ChchTF1-ABC mixture shows great potential as the active ingredient of a bioinsecticide to control C. chalcites in the Canary Islands.     

Mixtures of Complete and pif1- and pif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-BΔ16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-BΔpifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-BΔ16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-BΔ16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-BΔpifs were almost identical to the results observed with SfNIC-BΔ16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-BΔ16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of ∼85% SfNIC-B plus ∼15% SfNIC-BΔ16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNIC-BΔpifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1- and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.When hosts are infected by multiple genotypes of a pathogen, competition between genotypes of low relatedness may favor rapid exploitation of host resources, resulting in an increase in the virulence of the infection, reflected in the degree of damage inflicted on the host (12). Because individual self-interest prevails under such conditions, a rapidly replicating genotype will quickly use up host resources for the production of progeny particles, thereby penalizing a more prudent coinfecting genotype, an interaction known as the “tragedy of the commons” (33, 44, 49). In contrast, when relatedness between genotypes is high, cooperative exploitation of host resources is favored because the rate of exploitation of host resources is often determined by the production of intracellular products by the infecting group, an interaction known as “collective action” (2). Each of these models entail different temptations to cheat or defect from the common goal, by excessive greed in the acquisition of public goods (intracellular products) in the case of the tragedy model, and by overly frugal contribution to the pool of public goods in the case of the collective action model (3). In the case of viruses, such game theory approaches to social dilemmas have provided unique insights into the role of cooperation and defection in the evolution of virulence (2, 11, 24, 44), pathogenesis (45), disease management, and the development of potential therapeutic agents (7, 15, 26).Coinfection by multiple genotypes is a common characteristic of many host-parasite systems, especially insect viruses (5, 6, 14, 19-21). When multiple virus particles infect individual host cells, deletion mutants can arise that have lost genes that are essential for transmission or replication (35). These defective particles survive by sequestering the gene products of complete genotypes in coinfected cells. Recently, we demonstrated that deletion genotypes were prevalent in a genotypically diverse population of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) originally isolated in Nicaragua (SfNIC). The most abundant deletion genotype, named SfNIC-C, comprised a 16.4-kb deletion compared to the complete genotype, named SfNIC-B (40). This deletion included two genes, pif1 and pif2, that encode peroral infection factors that are essential for per os infection (9, 25, 32, 36), which is the usual route of transmission of these viruses. SfNIC-C survives by complementation with PIF-encoding genotypes in the population, a process that requires that complete and deletion genotypes replicate simultaneously in the same cells. The persistence of deletion genotypes at a high frequency in the population signifies that multiple infection of cells is likely to be a common event. However, pif1- and pif2-defective genotypes can replicate autonomously in cell culture or in larvae, if injected. Remarkably, the progeny viruses from insects that had been simultaneously inoculated with a mixture of complete and deletion genotypes, in a ratio similar to that found in nature (∼3:1), were ∼2.5 times more pathogenic, as indicated by concentration-mortality metrics, than the complete genotype alone (28, 38, 39). Whether the increased pathogenicity of mixed genotype inocula was specifically due to the deletion has remained unclear and identifying the gene(s) involved represents a key to our understanding of baculovirus infection strategies.The structure of NPV is complex (46). The multiple NPVs consist of single genomes of double-stranded DNA inside individual nucleocapsids. Groups of approximately one to seven nucleocapsids are then wrapped by an envelope to form occlusion-derived virions (ODVs) that are in turn occluded by a protein matrix to form occlusion bodies (OBs) with approximately 20 to 80 virions within each OB. When consumed by a susceptible insect larva, the OBs dissolve in the alkaline insect midgut releasing numerous virions. The ODV membrane fuses with the membranes of midgut epithelial cells, a process that requires the presence of PIFs in the virion membrane (25, 31). After membrane fusion, the package of nucleocapsids is delivered into the host cell and nucleocapsids migrate to the nucleus, where they uncoat and commence replication. Initially, progeny nucleocapsids migrate from the nucleus and bud from the cell as individual virions, each containing a single genome that spread within the host to initiate secondary infections. It is estimated that each cell of the insect is infected by ∼4 budded virions (4, 38). Later, nucleocapsids are retained in the nucleus, wrapped into ODVs and occluded into OBs. After death, the tegument of the insect breaks down and OBs are released onto plant surfaces for transmission to other susceptible insects. It is clear that both the physical structure and the replication cycle of these viruses foster a high prevalence of infection by multiple genotypes.In the present study we examine the genetic basis for increased pathogenicity and test the hypothesis that coinfection of cells by complete and deleted genotypes results in OBs with increased potency compared to those of the complete genotype alone. Here, we use the term potency to mean the quantity of OBs required to produce a certain prevalence of host mortality, such as the 50% lethal concentration, compared to a standard reference, which in this case are OBs of the complete genotype B. As such, potency is a comparative measure of virus insecticidal activity. We demonstrate that the deletion of the pif1 and pif2 region is necessary and sufficient to explain this increased potency. Finally, we examine the dynamics of mixed infection in serial passage in the natural host and demonstrate that previous findings, in which the ratios of genotypes in experimental mixtures converged to a common equilibrium, were determined by the influence that pif1- and pif2-deficient genotypes exert on OB potency, thereby indirectly influencing the probability of virus transmission.     

Persistence and Effects of Parasitic Genotypes in a Mixed Population of the Spodoptera exigua Nucleopolyhedrovirus

Spod-X, a commercialized bioinsecticide for the control of the pest Spodoptera exigua, is based on a nucleopolyhedrovirus (NPV) of S. exigua (SeMNPV) isolated in Florida (US2wt). This field isolate is made up of at least seven genotypic variants, of which two (US2C and US2E) have defective genomes and act as parasites, reducing the pathogenicity of the viral population. Upon co-infection of US2wt and a Spanish field isolate of the same virus (SP2wt), persistence of the defective variants (US2C, US2E) in the viral progeny was observed. This persistence diluted the presence of intact, self-infectious genotypes in the progeny, decreasing the pathogenicity of these viral inocula. Further passages of viral occlusion bodies produced after the co-infection revealed that the parasite US2C continued replicating and constituted up to 30% of the viral progeny in some samples. In addition, the presence of US2C within SP2wt significantly decreased the pathogenicity of contaminated inocula by 3.6-fold. The use of foreign virus field isolates containing defective genomes and their possible impact on the biological activity of native NPV populations may be a cause for concern where these viruses are used as agents for biological control.     

Microencapsulated Spodoptera frugiperda nucleopolyhedrovirus: insecticidal activity and effect on arthropod populations in maize

Excessive use of chemical insecticides to control Spodoptera frugiperda in maize (Zea mays L.) crops in Colombia has resulted in pest resistance and environmental contamination. A Colombian multicapsid nucleopolyhedrovirus (SfMNPV) was produced in laboratory reared larvae and formulated as a wettable powder by microencapsulating the viral occlusion bodies (OBs) with a methacrylic acid polymer. Microencapsulated OBs were tested for insecticidal activity in laboratory, greenhouse and field bioassays, and were then sprayed onto a commercial crop where their effect on arthropod populations was estimated. In laboratory bioassay, microencapsulated and non-microencapsulated OBs caused a larval mortality of 98 and 96%, respectively. The percent of plants showing recent feeding damage in a greenhouse experiment oscillated between 2.5 and 7.5% when OBs were applied compared to 70% observed in the control. In two field trials, the highest dose of 1.5×10¹² OBs/ha (1500 g/ha) was selected as the recommended rate for field application in maize crops, with efficacies higher than 70%. Total arthropod populations were negatively affected by chemical pesticides; whereas the OBs formulation did not affect the diversity of these organisms. Our results indicate that microencapsulated SfMNPV has the potential to be commercialised as an alternative to substitute or complement chemical insecticides in fall armyworm integrated management programmes.     

Population genetic structure determines speed of kill and occlusion body production in Spodoptera frugiperda multiple nucleopolyhedrovirus

A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfNIC) survives as a complex mixture of genotypes (named A to I). The speed of kill, time-mortality distribution, and occlusion body (OB) production of single genotypes (A, B and F) and co-occluded mixtures of genotypes, in a 75% + 25% ratio, were compared to determine the contribution of each genotype to the transmissibility of the viral population. Pure genotypes differed markedly in their speed of kill in second instar S. frugiperda. The speed of kill of SfNIC was attenuated compared to that of the dominant genotype B, indicating that interactions involving two or more genotypes likely determine host killing traits in the virus population. Genotypes A, F and defective genotype C, had no significant effects on the distribution of insect deaths over time when present as minority components in mixtures comprising 75% of genotype B. Similarly, the mortality pattern over time of insects infected by genotype F, the fastest-killing genotype tested, was not affected by the presence of genotypes A or C. Semi-quantitative PCR studies indicated that the genetic composition did not differ significantly between SfNIC-infected insects that died soon (67 h) or late (139 h) after inoculation, suggesting that stability in genotypic composition is important for virus survival. Median OB production per insect was correlated with mean time to death so that attenuated speed of kill of SfNIC resulted in high OB yields. We conclude that (i) minority genotypes play a functional role in determining the timing of mortality of infected hosts and (ii) the genotypic structure of the virus population is stably maintained to maximize the likelihood of survival.     

Recombination in the genesis and evolution of hepatitis B virus genotypes

Hepatitis B virus (HBV) infection is widely distributed in both human and ape populations throughout the world and is a major cause of human morbidity and mortality. HBV variants are currently classified into the human genotypes A to H and species-associated chimpanzee and gibbon/orangutan groups. To examine the role of recombination in the evolution of HBV, large-scale data retrieval and automated phylogenetic analysis (TreeOrder scanning) were carried out on all available published complete genome sequences of HBV. We detected a total of 24 phylogenetically independent potential recombinants (different genotype combinations or distinct breakpoints), eight of which were previously undescribed. Instances of intergenotype recombination were observed in all human and ape HBV variants, including evidence for a novel gibbon/genotype C recombinant among HBV variants from Vietnam. By recording sequence positions in trees generated from sequential fragments across the genome, violations of phylogeny between trees also provided evidence for frequent intragenotype recombination between members of genotypes A, D, F/H, and gibbon variants but not in B, C, or the Asian B/C recombinant group. In many cases, favored positions for both inter- and intragenotype recombination matched positions of phylogenetic reorganization between the human and ape genotypes, such as the end of the surface gene and the core gene, where sequence relationships between genotypes changed in the TreeOrder scan. These findings provide evidence for the occurrence of past, extensive recombination events in the evolutionary history of the currently classified genotypes of HBV and potentially in changes in its global epidemiology and associations with human disease.     

A Novel Binary Mixture of Helicoverpa armigera Single Nucleopolyhedrovirus Genotypic Variants Has Improved Insecticidal Characteristics for Control of Cotton Bollworms

The genotypic diversity of two Spanish isolates of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) was evaluated with the aim of identifying mixtures of genotypes with improved insecticidal characteristics for control of the cotton bollworm. Two genotypic variants, HearSP1A and HearSP1B, were cloned in vitro from the most pathogenic wild-type isolate of the Iberian Peninsula, HearSNPV-SP1 (HearSP1-wt). Similarly, six genotypic variants (HearLB1 to -6) were obtained by endpoint dilution from larvae collected from cotton crops in southern Spain that died from virus disease during laboratory rearing. Variants differed significantly in their insecticidal properties, pathogenicity, speed of kill, and occlusion body (OB) production (OBs/larva). HearSP1B was ∼3-fold more pathogenic than HearSP1-wt and the other variants. HearLB1, HearLB2, HeaLB5, and HearLB6 were the fastest-killing variants. Moreover, although highly virulent, HearLB1, HearLB4, and HearLB5 produced more OBs/larva than did the other variants. The co-occluded HearSP1B:LB6 mixture at a 1:1 proportion was 1.7- to 2.8-fold more pathogenic than any single variant and other mixtures tested and also killed larvae as fast as the most virulent genotypes. Serial passage resulted in modified proportions of the component variants of the HearSP1B:LB6 co-occluded mixture, suggesting that transmissibility could be further improved by this process. We conclude that the improved insecticidal phenotype of the HearSP1B:LB6 co-occluded mixture underlines the utility of the genotypic variant dissection and reassociation approach for the development of effective virus-based insecticides.