含外源类蜗牛毒素基因的AcMNPV的虫体感染性研究

类蜗牛毒素基因(conotoxinlike,ctl)是在一些杆状病毒基因组中存在的与蜗牛毒素类似的一类基因,其功能尚不清楚。本文利用苜蓿银纹夜蛾核多角病毒(AcMNPV)bacmid表达系统构建了含油桐尺蠖核多角体病毒(BusuNPV)ctl基因的重组病毒AcBac-ph-ctl。在细胞水平上对ctl基因的RT-PCR分析表明,该基因转录出mRNA。在甜菜夜蛾体内进行了生物活性测定,结果表明AcBac-ph-ctl与对照野生型AcMNPV的LC50,ST50无显著性差异,表明在此系统中,外源的CTL无杀虫增效性能。     

Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.     

Greenhouse Evaluation of Dose– and Time–Mortality Relationships of Two Nucleopolyhedroviruses for the Control of Beet Armyworm, Spodoptera exigua, on Chrysanthemum

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV) against synchronized populations of S. exigua larvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exigua target stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exigua were controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigua larvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigua larvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.     

A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV)

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.     

Enhancement in activity of homologous and heterologous baculoviruses infectious to beet armyworm (Lepidoptera: Noctuidae) by an optical brightener

The nuclear polyhedrosis virus (NPV) from the beet armyworm, Spodoptera exigua (Hübner) (SeMNPV), was the most active virus tested against the beet armyworm (LC50 = 4.1 PIBs/mm2), followed by nuclear polyhedrosis viruses from the alfalfa looper, Autographa californica (Speyer) (AcMNPV; LC50 = 92.6 PIBs/mm2), and the celery looper, Anagrapha falcifera (Kirby) (AfMNPV; LC50 = 195.7 PIBs/mm2). In the case of the nuclear polyhedrosis virus from the bollworm, Helicoverpa armigera (Hübner), LC50s could only be obtained for five/six replicates, whereas LC50s could only be obtained for two/six replicates for the nuclear polyhedrosis virus from the wax moth, Galleria mellonella (L.) (GmMNPV). When an optical brightener Tinopal LPW was added to virus suspensions, LC50 values were reduced by 130-fold for both SeMNPV and AcMNPV and by 300-fold for AfMNPV. The addition of Tinopal LPW greatly increased the activities of HaMNPV and GmMNPV. In terms of speed of kill, Tinopal LPW reduced the LT50s for all nuclear polyhedrosis viruses by 30-40%.     

Localization and functional analysis of SeMNPV IE1 in mammalian cells

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.     

Homologous and nonhomologous recombination resulting in deletion: effects of p53 status, microhomology, and repetitive DNA length and orientation

Repetitive DNA elements frequently are precursors to chromosomal deletions in prokaryotes and lower eukaryotes. However, little is known about the relationship between repeated sequences and deletion formation in mammalian cells. We have created a novel integrated plasmid-based recombination assay to investigate repeated sequence instability in human cells. In a control cell line, the presence of direct or inverted repeats did not appreciably influence the very low deletion frequencies (2 x 10(-7) to 9 x 10(-7)) in the region containing the repeat. Similar to what has been observed in lower eukaryotes, the majority of deletions resulted from the loss of the largest direct repeat present in the system along with the intervening sequence. Interestingly, in closely related cell lines that possess a mutant p53 gene, deletion frequencies in the control and direct-repeat plasmids were 40 to 300 times higher than in their wild-type counterparts. However, mutant p53 cells did not preferentially utilize the largest available homology in the formation of the deletion. Surprisingly, inverted repeats were approximately 10,000 times more unstable in all mutant p53 cells than in wild-type cells. Finally, several deletion junctions were marked by the addition of novel bases that were homologous to one of the preexisting DNA ends. Contrary to our expectations, only 6% of deletions in all cell lines could be classified as arising from nonhomologous recombination.     

Deletion of the baculovirus ecdysteroid UDP-glucosyltransferase gene induces early degeneration of Malpighian tubules in infected insects.

Deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the Autographa californica nuclear polyhedrosis virus (AcNPV) genome increases the speed of killing of this virus (D. R. O'Reilly and L. K. Miller, Bio/Technology 9:1086-1089, 1991). Second-instar Spodoptera exigua larvae are killed more rapidly by the egt deletion mutant of AcNPV than by wild-type AcNPV. Unlike wild-type AcNPV-infected larvae, larvae infected with an egt deletion mutant molt and resume feeding as mock-infected larvae do. Wild-type AcNPV and egt deletion mutant recombinants marked with a lacZ gene were used to study their pathogenesis in insects. Histopathological investigation revealed that early degeneration of the Malpighian tubules, not the molting per se, may be the cause of this increased speed of killing by AcNPV.     

Validation of a Comprehensive Process-Based Model for the Biological Control of Beet Armyworm,Spodoptera exigua,with Baculoviruses in Greenhouses

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.     

Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.     

The Polyhedral Membrane does not Protect Polyhedra of AcMNPV Against Inactivation on Greenhouse Chrysanthemum

Polyhedral inactivation of wild-type AcMNPV and an AcMNPV mutant lacking the gene for the polyhedral membrane protein (AcMNPV-Delta pp34) was studied on greenhouse chrysanthemum. It was hypothesized that polyhedra without a polyhedral membrane might be more susceptible to inactivation on plants. The density of infectious polyhedra of both viruses on the leaf surface decreased in time in a near-exponential fashion. The inactivation curves suggested the presence of two distinct fractions of polyhedra with differences in persistence. One fraction of polyhedra is not inactivated at all, whereas the other fraction is inactivated in an exponential fashion. Relative inactivation rates of the inactivated polyhedra fraction for wild-type AcMNPV and AcMNPV-Delta pp34 were 0.16 and 0.13 per day, respectively, which is not significantly different. After 28 days on leaves in a greenhouse, both viruses still showed residual infectivity. The fraction of residual infectious polyhedra were not significantly different and amounted to approximately 20% of the original density for both wild-type AcMNPV and AcMNPV-Delta pp34. Therefore, the polyhedral membrane does not protect polyhedra against inactivation on greenhouse chrysanthemum.     

甜菜夜蛾核多角体病毒sod基因的克隆及原核表达

甜菜夜蛾核型多角体病毒中国株(Spotoptera exigua MNPV—Z)超氧化物歧化酶基因(sod)业已被克隆及在大肠杆菌中进行了表达,证明了SeMNPV—Z的sod基因产物确有SOD活性,其活力单位约为291．19U／mL培养液。DNA测序结果表明SeMNPV-Z的sod基因编码151个氨基酸,与人的sodl基因的核苷酸的同源性为50％,与LdNPV、HaSNPV、HcNPV、AcNPV和BmNPV的sod基因的同源性分别为64％、63％、63％、65％、63％。     

甜菜夜蛾核多角体病毒BAC-TO-BAC外源基因表达系统的建立

用直接克隆法将miniF-lacZ-attFn7-kan 片段插入甜菜夜蛾核多角体病毒（Spodoptera exigua multicapsid nucleopolyhedrovirus, SeMNPV）〖JP〗美国分离株（SeUS1）基因组的多角体蛋白基因框内,miniF是大肠杆菌F因子复制子,携带miniF的重组病毒能够在大肠杆菌中低拷贝稳定复制,称为bacmid。由于SeUS1由不同的SeMNPV基因型组成,每个bacmid携带了一种病毒基因型,所有bacmid构成了SeUS1分离株的BAC文库。REN对111个bacmid分析表明,SeUS1分离株中除了包含具有完整SeMNPV遗传信息的基因型外,还包括不同类型的缺失基因型。将具有完整SeMNPV基因组的基因型SeBAC10转染昆虫细胞,可产生子代病毒,故SeBAC10是一种在真核细胞和原核细胞中均能复制的穿梭质粒。因为SeBAC10中多角体蛋白基因(Seph)被插入失活,将Seph作为报告基因通过位点特异性重组方式插入位于LacZ框内转座子Tn7的附着靶位点attTn7,得到重组SeBAC10 (即SeBAC10ph)转染甜菜夜蛾培养细胞Se301后,细胞出现典型的病理变化,核中出现多角体,证明SeMNPV BAC-TO-BAC外源基因表达载体系统构建成功。     

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.     

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.     

Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni

A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.     

Effect of tinopal LPW on the insecticidal properties and genetic stability of the nucleopolyhedrovirus of Spodoptera exigua (Lepidoptera: Noctuidae)

The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.     

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.     

The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.     

Vertical transmission of TnSNPV,TnCPV, AcMNPV,and possibly recombinant NPV in Trichoplusia ni

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels.