Group Ⅰ but not Group Ⅱ NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group Ⅰ and Group Ⅱ based on the phylogenetic analysis. It has been reported that Group Ⅰ NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group Ⅱ NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group Ⅱ had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.     

Group I but not Group II NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus(NPV) is divided into Group I and Group II based on the phy-logenetic analysis.It has been reported that Group I NPVs such as Autographa californica multiple NPV(AcMNPV) can transduce mammalian cells,while Group II NPVs such as Helicoverpa armigera single NPV(HaSNPV) cannot.Here we report that AcMNPV was capable of stimulating antiviral ac-tivity in human hepatoma cells(SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus(VSV) replication.In contrast,the HaSNPV and the Spodoptera exigua multiple NPV(SeMNPV) of group II had no inhibitory effect on VSV.Recombinant AcMNPV was shown to induce interferons al-pha/beta even in the absence of transgene expression in human SMMC-7721 cells,while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.     

Group I but not Group II NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.     

A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.     

A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.     

Properties of a unique mutant of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus that exhibits a partial many polyhedra and few polyhedra phenotype on extended serial passaging in suspension cell cultures

Summary Serial passaging of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) in H. zea (Hz-AM1) insect cell cultures results in rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. On serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type virus. However, subsequent passaging of ppC19 resulted in a steep decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus at high passage numbers. Genomic deoxyribonucleic acid profiling of the latter suggested that defective interfering particles (DIPs) were implicated in this phenomenon and represented another undesirable mutation during serial passaging of HaSNPV. Hence, a strategy to isolate HaSNPV clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPs, could prove commercially invaluable.     

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.     

Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in Trichoplusia ni Indicates High Multiplicity of Cellular Infection

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.     

AcMNPVP35抑制HaSNPV诱导的Tn-Hi5细胞凋亡

苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nuclear polyhedrosis virus,AcMNPV)能够抑制棉铃虫核多角体病毒(Helicoverpa armigera Nucleopoly hedrovirus,HaSNPV)诱导的Tn Hi5 细胞凋亡,并能辅助HaSNPV在Tn Hi5细胞中复制,产生具有感染能力的子代病毒。瞬时表达实验证明,在Tn Hi5细胞中,p35具有明显抑制凋亡的能力,但是不能辅助HaSNPV在Tn Hi5细胞中的复制;进一步构建超表达p35 的重组病毒:vHap35,发现vHap35能够抑制Tn Hi5细胞凋亡,但是不能产生具有感染力的病毒粒子。电镜观察发现感染重组病毒的部分细胞中存在单粒包埋的病毒粒子(ODV)。     

Systematic differences in the population density of green spruce aphid, Elatobium abietinum in a provenance trial of Sitka spruce, Picea sitchensis

Quantitative bioassay techniques were used to measure the susceptibility of Heliothis armigera to three nuclear polyhedrosis viruses (NPVs): H. armigera singly-enveloped NPV (HaSNPV), H. zea SNPV (HzSNPV) and H. armigera multiply-enveloped NPV (HaMNPV). Viruses were identified by EcoRI restriction endonuclease analysis. Electrophoretic profiles of DNA fragments revealed that the HaSNPV isolate was a previously undescribed genotypic variant. Bioassays with neonate and 6-day-old larvae measured small but significant differences in virulence between the three viruses. HzSNPV was the most virulent for neonate larvae with a median lethal dose (LD₅₀) of five polyhedra. HaMNPV was least virulent for 6-day-old larvae, with a LD₅₀ of 1400 polyhedra compared with 640–670 polyhedra for HaSNPV and HzSNPV. In addition, the median lethal time (LT₅₀) for infection with HaMNPV in neonate larvae was approximately 1·7 days longer than for the other viruses. Although they varied in virulence, each of the three viruses was sufficiently virulent to have considerable potential as a microbial control agent of H. armigera.     

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.     

Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus,is a viral pathogenicity factor

Viral envelope fusion proteins are important structural proteins that mediate viral entry and may affect or determine the host range of a virus. The acquisition, exchange, and evolution of such envelope proteins may dramatically affect the success and evolutionary divergence of viruses. In the family Baculoviridae, two very different envelope fusion proteins have been identified. Budded virions of group I nucleopolyhedroviruses (NPVs) such as the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), contain the essential GP64 envelope fusion protein. In contrast group II NPVs and granuloviruses have no gp64 gene but instead encode a different envelope protein called F. F proteins from group II NPVs can functionally substitute for GP64 in gp64null AcMNPV viruses, indicating that GP64 and these F proteins serve a similar functional role. Interestingly, AcMNPV (and other gp64-containing group I NPVs) also contain an F gene homolog (Ac23) but the AcMNPV F homolog cannot compensate for the loss of gp64. In the present study, we show that Ac23 is expressed and is found in budded virions. To examine the function of F protein homologs from the gp64-containing baculoviruses, we generated an Ac23null AcMNPV genome by homologous recombination in E. coli. We found that Ac23 was not required for viral replication or pathogenesis in cell culture or infected animals. However, Ac23 accelerated the mortality of infected insect hosts by approximately 28% or 26 h. Thus, Ac23 represents an important viral pathogenicity factor in larvae infected with AcMNPV.     

Infectivity and mode of action of Baculoviruses

The genus Baculovirus contains three subgroups of viral types: (1) nuclear polyhedrosis viruses (NPVs), (2) granulosis viruses (GVs), and (3) nonoccluded baculoviruses. While little information is available for viruses from the third subgroup, several aspects of the infectivity and mode of action of NPVs and GVs have been studied. The most common route of entry of a virus into an insect host is per os, and both virus types enter midgut cells (primary site of infection) by membrane fusion. However, two distinct mechanisms of virus uncoating occur among the baculoviruses: NPVs uncoat within the nucleus, whereas GVs uncoat at the nuclear pore complex. Baculoviruses of subgroup 3 appear to uncoat by either mechanism. In addition to replicating within the nucleus, NPV inoculum virus may pass through the intestinal epithelium immediately after ingestion, thereby establishing a systemic infection of the hemocoel prior to virus replication in midgut cells. The GVs do not appear to pass through midgut cells as rapidly as NPVs and in general, the developmental cycle of GVs is longer than that of NPVs. NPVs have been grown in cell culture while GVs have not.     

与宿主昆虫液化相关的杆状病毒基因及其蛋白

昆虫被杆状病毒感染后会发生液化现象,这有利于病毒向周围环境扩散。目前在杆状病毒苜蓿银纹夜蛾核型多角体病毒NPV和GV中,发现与昆虫宿主液化相关的基因有组织蛋白酶基因V-cath基因和几丁质酶基因。V-cath基因表达产物在苜蓿银纹夜蛾多角体病毒(AcMNPV)中能特异性降解昆虫细胞内的肌动蛋白。几丁质酶不仅参与了虫体体表面几丁质的降解,同时还参与V-CATH蛋白前体的加工过程,起分子伴侣的作用。对家蚕核型多角体病毒(BmNPV)的研究表明其FP25K基因表达产物通过影响组织蛋白酶的释放与分泌而参与虫体液化。简要综述了此3种基因及其表达产物的结构、功能与特性,并讨论了它们在生产上的应用前景。     

Functional expression of lepidopteran-selective neurotoxin in baculovirus: potential for effective pest management

Recombinant baculovirus expressing insect-selective neurotoxins derived from venomous animals are considered as an attractive alternative to chemical insecticides for efficient insect control agents. Recently we identified and characterized a novel lepidopteran-selective toxin, Buthus tamulus insect-selective toxin (ButaIT), having 37 amino acids and eight half cysteine residues from the venom of the South Indian red scorpion, Mesobuthus tamulus. The synthetic toxin gene containing the ButaIT sequence in frame to the bombyxin signal sequence was engineered into a polyhedrin positive Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the control of the p10 promoter. Toxin expression in the haemolymph of infected larvae of Heliothis virescens and also in an insect cell culture system was confirmed by western blot analysis using antibody raised against the GST-ButaIT fusion protein. The recombinant NPV (ButaIT-NPV) showed enhanced insecticidal activity on the larvae of Heliothis virescens as evidenced by a significant reduction in median survival time (ST50) and also a greater reduction in feeding damage as compared to the wild-type AcMNPV.     

Cellular VPS4 is required for efficient entry and egress of budded virions of Autographa californica multiple nucleopolyhedrovirus

Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells.     

A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.     

Increase of host cell longevity by the expression of 30K protein originating from silkworm hemolymph in an insect cell–baculovirus system

A recombinant baculovirus was constructed by the homologous recombination between wild-type AcMNPV DNA and a baculovirus transfer vector containing a gene coding for the 30K protein originating from silkworm hemolymph. The 30K protein was successfully expressed in Sf9 cells infected with the recombinant baculovirus (AcMNPV/30K). To investigate the effect produced by the expression of the 30K protein, host cell viability after infection was compared with that of Sf9 cells infected with AcMNPV/β-gal. The viability of the cells infected with AcMNPV/β-gal began to decrease exponentially 3 days after infection, whereas that of the cells infected with AcMNPV/30K remained at a high level until 5 days after infection. This indicates that the 30K protein increases cell longevity after viral infection. This increased cell longevity is considered to be due to the inhibition of host cell apoptosis induced by a baculovirus, and the extent of apoptosis was measured by the flow cytometric method. The percentage of the sub-G1 fraction, which represents the extent of apoptosis, was decreased by the expression of the 30K protein. This indicates that the expression of the 30K protein in insect cells increases host cell longevity by inhibiting apoptosis.     

Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been developed as a commercial biopesticide to control the cotton bollworm, H. armigera, in China. The major limitation to a broader application of this virus has been the relative long time to incapacitate the target insect. Two HaSNPV recombinants with improved insecticidal properties were released in bollworm-infested cotton. One recombinant (HaCXW1) lacked the ecdysteroid UDP-glucosyltransferase (egt) gene and in another recombinant (HaCXW2), an insect-selective scorpion toxin (AaIT) gene replaced the egt gene. In a cotton field situation H. armigera larvae treated with either HaCXW1 or HaCXW2 were killed faster than larvae in HaSNPV-wt treated plots. Second instar H. armigera larvae, which were collected from HaCXW1 and HaCXW2 treated plots and further reared on artificial diet, showed reduced ST(50) values of 15.3 and 26.3%, respectively, as compared to larvae collected from HaSNPV-wt treated plots. The reduction in consumed leaf area of field collected larvae infected with HaCXW1 and HaCXW2 was approximated 50 and 63%, respectively, as compared to HaSNPV-wt infected larvae at 108 h after treatment. These results suggest that in a cotton field situation the recombinants will be more effective control agents of the cotton bollworm than wild-type HaSNPV.