Nuclear Polyhedrosis Virus Infection Research Articles

Field evaluation of multi-viral diseases tolerant bivoltine silkworm hybrid of Bombyx mori l. (Lepidoptera: Bombycidae)

Bombyx mori Densonucleosis virus (BmDV), Bombyx mori Infectious flacherie virus (BmIFV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) infection to silkworms causes major sericulture crop loss. The multi-viral diseases tolerant bivoltine silkworm double hybrid, RDIN1, developed through marker-assisted selection was field evaluated by rearing 20,000 dfls at different location and season. RDIN1, had better pupation (94.8%) compared to control, FC1 X FC2 (91.5%) at farmers’ level with 4 % significant improvement. Average Cocoon yield of RDIN1 (71.2%) was better compared to FC1 X FC2 (69.3%) in unfavourable conditions. Other parameters such as cocoon weight, shell weight, shell percentage, reelability, filament length, raw silk, denier, neatness had also significant difference between RDIN1 and FC1 x FC2. The RDIN1 is expected to reduce cocoon crop losses invariably due to viral infection and fetch more profit to sericulture farmers.

Effects of midgut bacteria inHyphantria cunea(Lepidoptera: Erebidae) on nuclear polyhedrosis virus andBacillus thuringiensis(Bacillales: Bacillaceae)

Hyphantria cunea Drury (Lepidoptera: Erebidae) is a quarantine pest in China that can cause damage to hundreds of plants. As biological control agents, Nuclear Polyhedrosis Virus (NPV) and Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt) are commonly used to inhibit the prevalence of H. cunea. To investigate the role of midgut bacteria in the infection of NPV and Bt in H. cunea, we performed a series of tests, including isolating the dominant culturable bacteria in the midgut, eliminating intestinal bacteria, and respectively inoculating the dominant strains with NPV and Bt for bioassay. Two dominant bacteria, Klebsiella oxytoca Lautrop (Enterobacterales: Enterobacteriaceae) and Enterococcus mundtii Collins (Lactobacillales: Enterococcaceae), in the midgut of H. cunea were identified, and a strain of H. cunea larvae without intestinal bacteria was successfully established. In the bioassays of entomopathogen infection, K. oxytoca showed significant synergistic effects with both NPV and Bt on the death of H. cunea. In contrast, E. mundtii played antagonistic effects. This phenomenon may be attributed to the differences in the physico-chemical properties of the two gut bacteria and the alkaline environment required for NPV and Bt to infect the host. It is worth noting that the enhanced insecticidal activity of K. oxytoca on NPV and Bt provides a reference for future biological control of H. cunea by intestinal bacteria.

Identification of key metabolic pathways reprogrammed by BmNPV in silkworm Bombyx mori

Elucidating the mechanism of infection of Bombyx mori nuclear polyhedrosis virus (BmNPV) and host antiviral response remains a major scientific task in sericulture. Virus invasion causes a series of antiviral immune responses in the host, and successful infection leads to massive changes in the host’s physiological and biochemical state. Current research mainly focuses on silkworm genes and proteins associated with viral infection and resistance, but little is known regarding the host metabolic pathways that the virus utilizes for optimal replication. In this work, key metabolites involved in viral infection were identified, including trehalose, riboflavin, tryptophan, tyrosine, and phenylalanine. The genes associated with metabolite biosynthesis and catabolism were analyzed, and their expression levels were found to be largely consistent with their respective metabolite levels before and after viral treatment in both strains. The screened metabolites were further investigated for their roles in viral replication using exogenous metabolite addition into the culture medium. The results showed that tryptophan effectively inhibited BmNPV replication, while glutamine promoted viral replication in a dose-dependent manner. Trehalose and riboflavin exhibited a complex effect on BmNPV replication. This study outlines the critical metabolites and metabolic pathways required for BmNPV to proliferate and infect the host, indicting the potential of metabolite-based treatment for viral inhibition.

Nuclear polyhedrosis virus‐induced proteomic changes in Antheraea mylitta

AbstractInnate immunity, the evolutionarily conserved defence system, has been extensively explored in insect models like Drosophila and mosquito species for their immune responses towards diverse pathogens. Viruses being obligate parasites take over cellular macromolecular machinery and routinely control intracellular signalling pathways through the direct or indirect control of kinases and phosphatases. Domestic silkworm, Bombyx mori is an economically important insect. Wild silkworm Antheraea mylitta produces a unique variety of silk in terms of its colour, lustre and strength called as Tasar silk. Both the silkworms vary in their immune response to nuclear polyhedrosis virus (NPV). NPV causes 80% of crop losses in B. mori, interestingly there are no documented reports of NPV infection in A. mylitta. To investigate the mechanism of A. mylitta's immune response to NPV infection, we analysed viral induced proteins through proteomic approach (SDS‐PAGE and LC–MS/MS). We report the expression of a myotubularin‐related protein (MTMR6) of 23 kDa at 72 h post infection (hpi) indicating its role in host immunity as a phosphatase enzyme. And also, a differentially expressed serpin‐like protein of 67 kDa was upregulated in a significant way from 72 hpi further strengthening the immune response by suppressing the viral replication through serine protease inhibitory activity. This observation was further confirmed through qPCR studies on virus proliferation using lef‐1 gene which clearly indicated a reduction in viral load from 72 hpi onwards in A. mylitta silkworms.

Antiviral activity of seselin from Aegle marmelos against nuclear polyhedrosis virus infection in the larvae of silkworm, Bombyx mori

Ethnopharmacological relevanceTraditional Indian medicine has utilized Aeglemarmelos (L.) Corr. commonly called as bael in several indigenous systems against various diseases. Bioactive components isolated from various plant parts of A. marmelos were used in ethno-medicine. More precisely they are known for its antiviral property against various human and animal viruses. Aim of the studyThe study was conducted to investigate the antiviral activity of A.marmelos against Bombyx mori nucleopolyhedrovirus (BmNPV). Materials and methodsAmong the various crude extracts tested, hexane extracts of leaves of A. marmelos with promising anti-BmNPV activity was subjected to bioactivity guided fractionation based on column chromatography. Out of 40 fractions obtained from the fractionation, fractions showing similar TLC profiles were pooled into 14 fractions. A fraction with potential activity was used to purify a molecule with anti-BmNPV activity. This molecule was characterized through structural and functional analyses. ResultsThe functionally and structurally characterized molecule in the fraction with prospective anti-BmNPV activity revealed a single crystal compound ‘seselin’ (8, 8-dimethyl pyrido oxazine-2-one). ConclusionIt is therefore understood that this seselin compound could be used as a natural medicine for the management of NPV infection in the silkworm larvae under commercial conditions after suitable field evaluations.

Effects of Bombyx mori nuclear polyhedrosis virus on serpin and antibacterial peptide expression in B. mori

The silkworm (Bombyx mori) is a typical and economically important lepidopteran species, and research has resulted in the development and accumulation of breeding lines. Studies of immune-related silkworm genes not only promote our understanding of silkworm immune response mechanisms, but they also inform insect immune molecular diversity research. Here, silkworm proteins were screened using proteomics after Bombyx mori nuclear polyhedrosis virus (BmNPV) infection, and 2368 silkworm proteins were identified, including six antimicrobial peptides and 12 serpins. The mRNA expression levels of these 18 proteins were examined at different times. The results indicated that attacin had the highest expression level, while serpin-5 and cecropin-D exhibited a negative regulatory correlation. These results provide a significant step toward a deeper understanding of B. mori immunoregulation.

Identification and Evaluation of Suitable Reference Genes for Normalization of MicroRNA Expression in Helicoverpa armigera (Lepidoptera: Noctuidae) Using Quantitative Real-Time PCR.

More and more studies have focused on microRNAs (miRNAs) expression in the pest Helicoverpa armigera (Lepidoptera: Noctuidae) recently. Quantitative real-time PCR (qRT-PCR) is being widely used in miRNA expression studies. Suitable reference genes are necessary for the correct analysis of results. In this study, 10 candidate genes of H. armigera were selected and analyzed for their expression stability under different biotic and abiotic conditions with 3 statistical methods, including geNorm, NormFinder, and Bestkeeper. Combination the best number of reference genes was calculated by geNorm. One target gene, let-7, was used to validate the selection of reference genes. The suitable candidate reference genes were shown as follows: miR-9 and U6 snRNA for developmental stages, miR-100 and U6 snRNA for larval tissues, miR-100 and miR-305 for adult tissues, miR-9 and miR-279 for parasitic treatment, miR-998 and U6 snRNA for nuclear polyhedrosis virus infection, miR-9 and U6 snRNA for insecticide treatment, miR-92a, miR-100, and miR-279 for temperature treatment, miR-92a, miR-305, and miR-998 for starvation treatment, miR-9 and miR-279 for light treatment, miR-305 and miR-998 for hormone treatment, and there was not one reference gene suitable for all samples. This study could promote future research on miRNAs expression in H. armigera with optimal reference genes under different experimental conditions.

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae)

BackgroundRecent studies have focused on determining functional genes and microRNAs in the pest Helicoverpa armigera (Lepidoptera: Noctuidae). Most of these studies used quantitative real-time PCR (qRT-PCR). Suitable reference genes are necessary to normalize gene expression data of qRT-PCR. However, a comprehensive study on the reference genes in H. armigera remains lacking. ResultsTwelve candidate reference genes of H. armigera were selected and evaluated for their expression stability under different biotic and abiotic conditions. The comprehensive stability ranking of candidate reference genes was recommended by RefFinder and the optimal number of reference genes was calculated by geNorm. Two target genes, thioredoxin (TRX) and Cu/Zn superoxide dismutase (SOD), were used to validate the selection of reference genes. Results showed that the most suitable candidate combinations of reference genes were as follows: 28S and RPS15 for developmental stages; RPS15 and RPL13 for larvae tissues; EF and RPL27 for adult tissues; GAPDH, RPL27, and β-TUB for nuclear polyhedrosis virus infection; RPS15 and RPL32 for insecticide treatment; RPS15 and RPL27 for temperature treatment; and RPL32, RPS15, and RPL27 for all samples. ConclusionThis study not only establishes an accurate method for normalizing qRT-PCR data in H. armigera but also serve as a reference for further study on gene transcription in H. armigera and other insects.

A suspended cell line from Trichoplusia ni (Lepidoptera): Characterization and expression of recombinant proteins

AbstractA suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. This cell line had characteristics distinct from the BTI‐Tn5Bl‐4 cell line (Tn5Bl‐4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late‐log phase in shake‐flask or T‐flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle‐shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average. Production of β‐galactosidase and secreted alkaline phosphatase was high with 3.97 ± 0.13 × 104IU/mL and 3.48 ± 0.40 IU/mL, respectively. This cell line may be applicable for studies of scale‐up production of viruses or baculovirus‐insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5Bl‐4.

Induction of Antitumor Acquired Immunity by Baculovirus Autographa californica Multiple Nuclear Polyhedrosis Virus Infection in Mice

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been studied as a gene therapy vector. Here, we demonstrated that AcMNPV induces antitumor acquired immunity. These results suggest that AcMNPV has the potential to be an efficient virus or tumor therapy agent which induces innate and acquired immunity.

Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp ( Penaeus monodon)

Heat shock cognate 70 (HSC70) functions as a molecular chaperon and plays an important role in protein folding. HSC70 cDNA of tiger shrimp ( Penaeus monodon) was cloned and characterized in our previous study. After shrimps were treated with the 1-hr heat shock, the HSC70 mRNA level in hemocytes increased (∼ 8 fold) using real-time quantitative PCR. An hsc70 clone was obtained from genomic library screening. The gene contains 2 exons separated by a 1557-bp intron. The 5′-flanking region sequence (∼ 1 kb) ahead of the hsc70 gene contains a putative core promoter region and transcription elements including perfect heat shock element (HSE), imperfect HSE, CAAT elements, SP1, NF-κB and GC box. In insect Sf21 cells, the region could drive expression of the enhanced green fluorescent protein (EGFP) and luciferase gene to verify its promoter function. In the luciferase assay system, the effects of serial deletions on the hsc70 promoter were elucidated. Autographa californica multiple nuclear polyhedrosis virus infection (MOI = 0.1) on Sf21 cells significantly increased the hsc70 promoter activity. In addition, the effects of amino acid analogs and arsenic acid incubation with the cells on the hsc70 promoter activity were examined.

Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2′-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV- gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

Utilization of the polymerase chain reaction in the diagnosis of nuclear polyhedrosis virus infections of gypsy moth (Lymantria dispar, Lep., Lymantriidae) populations

Abstract: Three specific DNA probes were used for the detection of the nuclear polyhedrosis (NPV) virus of Lymantria dispar (LdNPV) genome. Two of these probes, H2 and H3 were obtained by classical cloning method and one (TR6) by polymerase chain reaction (PCR). These probes, used individually or in a pool in the standard slot–blot hybridizations, were able to detect 109 genome copies. By performing 35 cycles of PCR amplification before hybridization with primers specific to LdNPV genome on DNA extracted from infected larvae, the sensitivity of the hybridization technique was increased, so that as little as 10 copies of the LdNPV genome could be detected. Using these methods, L. dispar naturally infected by LdNPV were identified among field populations in Canada and in the United States near the eastern Canadian border. Using a combination of PCR and hybridization, LdNPV contamination of egg masses were also detected. By disinfecting the eggs with sodium hypochlorite prior to PCR amplification and hybridization, it was also demonstrated that transmission of viral infection in the natural populations is mainly caused by external contamination of the egg and is unlikely to occur through the transovarial route.

Rapid assessment of Autographa californica nuclear polyhedrosis virus infection of Sf-9 insect cells by analyzing suppression of reagent-induced apoptosis

Spodoptera frugiperda Sf-9 insect cells that undergo apoptosis by the treatment of apoptosis-inducing reagents were individually determined as `comet cells' having a tail of fragmented DNA during single cell gel electrophoresis, the fragmented DNA being migrated from the cells under an electric field of the electrophoresis. However, the apoptosis induction of the cells infected with a recombinant strain of Autographa californica nuclear polyhedrosis virus (AcMNPV) was blocked, probably by an intrinsic anti-apoptotic p35 gene of the virus, because the virus-infected cells did not have a tail of fragmented DNA on a single cell gel electrophoresis. The virus-infected cells were individually discriminated from non-infected cells by determining the anti-apoptotic nature of the cells. At higher multiplicity of infection and under better aeration conditions of virus-infected cultures, the apoptosis-suppressive ratio, which represented a ratio of non-comet cells, was increased more rapidly. This apoptosis-suppressive behavior was a good benchmark for assessing successful infection of insect cells with AcMNPV during very early infectious period and forecasting the subsequent production of recombinant proteins.

Aetiology of melanized inclusions in the body cavity ofNeodiprion sertifer(Geoffroy) (Hym., Diprionidae) adults

Abstract:The aetiology of large melanized inclusions occurring in the haemocoel of adults of some diprionid sawflies is not fully understood. Their occurrence has been associated with nuclear polyhedrosis virus (NPV), but also with extreme temperatures, parasitism, and prolonged diapause. High frequencies of inclusions are found most often during NPV epizootics. In laboratory experiments withNeodiprion sertiferlarvae, ingestion of large doses of a common bacterium,Enterobacter cloacae, was shown to induce inclusions indistinguishable from those associated with NPV infection. It is argued that a bacterial aetiology is not contradicted by the other conditions reported to be associated with inclusions. The bacterial aetiology provides an explanation for the low frequencies of inclusions inN. sertiferpopulations with no NPV infection. It is proposed that the ability to form inclusions may have evolved as a defence against bacterial infection.

Peroral infection of nuclear polyhedrosis virus budded particles in the host, Bombyxmori L., enabled by an optical brightener, Tinopal UNPA-GX

Perorally inoculated budded particles of a nuclear polyhedrosis virus was used to infect Bombyx mori (BmNPV) (Lepidoptera; Bombycidae), aided by an optical brightener, Tinopal UNPA-GX (Tinopal). BmNPV budded particles not occluded in the occlusion body do not infect successfully the host, B. mori, when administered perorally. It was found that feeding the host Tinopal enabled perorally delivered BmNPV budded particles to infect the host. B. mori larvae ingesting BmNPV budded particles (1.3×10 6 TCID 50 units per larva) after they consumed an artificial diet containing 0.3% Tinopal died of the viral infection. Peroral administration of these particles to host larvae with 1% Tinopal also resulted in virus infection. Tinopal is a candidate for viral activity enhancing agent promoting viral insecticide infection in hosts. The results suggest that B. mori-BmNPV budded particles are convenient for detecting viral infection enhancement activity of a chemical of interest. Since recombinant baculovirus vectors are constructed by replacing the polyhedrin gene with the foreign gene of interest, they do not produce occlusion bodies, i.e. polyhedra. Budded particles of a baculovirus vector not occluded in polyhedra cannot infect their hosts when administered perorally. The peroral inoculation of BmNPV budded particles by Tinopal leads to industrial pharmaceutics production using a baculovirus vector for a huge number of insect hosts, i.e. an ‘insect factory’.

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection.

Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.

Effect of Deltamethrin Treatment on Lepidopteran Larvae Infected with Baculoviruses Expressing Insect-Selective Toxins μ-Aga-IV, As II, or Sh 1

Abstract Modification of sodium channels by pyrethroids is promoted by channel opening. The insect-specific neurotoxins μ-Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus all increase sodium channel opening and so we tested each of them in combination with deltamethrin for synergistic insecticidal activity. We found that As II increased the insecticidal activity of deltamethrin against the blowfly Lucilia sericata. We also examined the effect of deltamethrin on the infectivity and virulence of Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant toxin-expressing viruses in both dose-dependent and time-mortality studies. The recombinant viruses expressed chimeric synthetic genes for μ-Aga-IV, As II, or Sh I (genes mag4, sat2, and ssh1, respectively). When Trichoplusia ni larvae were infected with a LC 20 of the recombinant viruses and treated with a LC 20 of deltamethrin in diet incorporation bioassays, the doses were strictly additive. A cooperative decrease in mortality rate was observed in droplet feeding bioassays when a low dose (LC 20 ) of deltamethrin and high doses of toxin-expressing viruses were fed to larvae but this effect was host-dependent. In T. ni, only wild-type AcMNPV and a recombinant expressing mag4 under very late viral promoter control exhibited reduced times in controlling host feeding and mortality in the presence of deltamethrin. For Heliothis virescens, larvae infected with sat2 -expressing viruses displayed shorter feeding times in the presence of deltamethrin than those infected with virus alone but only at very high virus doses. H. virescens larvae infected with wild-type AcMNPV and a mag4 -expressing recombinant had shorter feeding times and also shorter effective and lethal times in the presence of deltamethrin. Our results indicate that coapplication of this pyrethroid will confer little or no advantage in field applications of recombinant baculovirus pesticides and that application of pyrethroids will probably have no adverse affect on recombinant virus efficacy unless it deters larval feeding and virus acquisition.

In vitro transcription of pe38/polyhedrin hybrid promoters reveals sequences essential for recognition by the baculovirus-induced RNA polymerase and for the strength of very late viral promoters.

In vitro transcription was used to analyze the promoter specificity of the alpha-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5' untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.

Identification of the early actin-rearrangement-inducing factor gene, arif-1, from Autographa californica multicapsid nuclear polyhedrosis virus.

In response to Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) infection, a sequential rearrangement of the actin cytoskeleton occurs. Previous studies suggest that the penetration of nucleocapsids induces early actin cables followed by further changes of the actin cytoskeleton which depend on early viral gene expression. By transfection of a plasmid library into Trichoplusia ni TN-368 cells, we have identified an early viral gene, designated arif-1, that is able to induce actin rearrangement. The determination of the nucleotide sequence of arif-1 revealed one open reading frame potentially encoding a gene product of 45 kDa with no significant sequence homology to known proteins. After expression of arif-1 in transfected cells, the induced actin rearrangement, visualized by fluorescence microscopy, was comparable to the changes of the actin cytoskeleton at 3 to 7 h postinfection. These changes are based on early viral gene expression during the infection cycle. A causal link between arif-1 expression and actin rearrangement in infected cells is suggested by infection studies with the AcMNPV/Spodoptera frugiperda MNPV hybrid, which carries a deletion in the arif-1 gene. In transfection experiments the presence of the known viral transactivator IE1 was required in addition to ARIF-1 to induce actin rearrangement. IE1 was needed for promoter activation of the arif-1 gene, since arif-1 expression under the control of the early pe38 promoter was sufficient to induce actin rearrangement in transfected cells. Primer extension analyses showed that the arif-1 gene is transcribed only during the early phase of AcMNPV infection in T. ni TN-368 cells. There was a delay of about 1 h compared to ie1 transcription, which is in agreement with the assumption that IE1 transactivates the arif-1 promoter during infection.