Egt Gene Research Articles

Isolation and Characterization of a Baculovirus Associated with the Insect Parasitoid Wasp,Cotesiamarginiventris, or Its Host,Trichoplusia ni

A multiple nucleopolyhedrovirus (MNPV) was isolated from Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) larvae that had been stung by the parasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). The wild type virus was plaque purified by infecting a Heliothis subflexa (BCIRL- HsAM1) cell line and isolating several clones. The mean estimated genomic size of this virus based on PstI, BstEII, StyI, HindIII restriction profiles was estimated to be 106 ± 2.5 kbp (mean±SE). A clone designated as TnMNPV/CmBCL9 was used in bioassays against several lepidopteran pests and in comparative studies with the baculoviruses AcMNPV, AgMNPV, AfMNPV, PxMNPV and HzSNPV of Autographa califomica, Anticarsia gemmatalis, Anagrapha falcifera, Plutella xylostella, and Helicoverpa zea, respectively. Infectivity studies showed that TnMNPV/CmBCL9 was highly infectious for Heliothis subflexa and T. ni, with an LC50 value 0.07 occlusion bodies/mm2 in both species and also infectious for H. zea and Heliothis virescens with LC50 values of 0.22 and 0.27 occlusion bodies/mm2, respectively. Restriction endonuclease analysis of the isolate and selected baculoviruses revealed profiles that were very similar to AfMNPV but different from the restriction endonuclease profiles of the other baculoviruses. Hybridization studies suggest that the TnMNPV/CmBCL9 was closely related to AfMNPV and AcMNPV-HPP. Further support for this comes from a phylogenetic analysis employing a split-graphs network, comparing the polh, egt, and p10 genes from TnMNPV/CmBCL9 with those from other baculoviruses and suggests that this virus is closely related to the AcMNPV variants, AfMNPV and RoMNPV of Rachiplusia ou.

Genomic sequence analysis of a fast-killing isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus

Six clones of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were plaque-purified from field isolates collected in Missouri, USA. In bioassays, four of the plaque-purified isolates killed neonate S. frugiperda larvae more rapidly than the field isolates from which they were derived, with LT(50) values (mean time to kill 50 % of the test larvae) ranging from 34.4 to 49.7 h post-infection. The complete genomic sequence of one of these isolates, SfMNPV-3AP2, was determined and analysed. The SfMNPV-3AP2 genome was 131 330 bp with a G+C content of 40.2 %. A total of 144 open reading frames (ORFs) was identified and examined, including the set of 62 genes in common among lepidopteran nucleopolyhedrovirus genomes. Comparisons of ORF content, order and predicted amino acid sequences with other nucleopolyhedoviruses indicated that SfMNPV is part of a cluster of viruses within NPV group II that includes NPVs isolated from Spodoptera, Agrotis and Mamestra host species. SfMNPV-3AP2 shared a high degree of nucleotide sequence similarity with partial sequences from other SfMNPV isolates. Comparison of the SfMNPV-3AP2 genome sequence with a partial sequence from a Brazilian isolate of SfMNPV revealed that SfMNPV-3AP2 contained a deletion that removed parts of ORF sf27 and the gene encoding ecdysteroid UDP-glucosyltransferase (egt). An examination of the egt region in the other isolates revealed that the other five SfMNPV clones also contained deletions of varying length in this region. Variant genotypes with deletions extending around the egt gene have been reported previously from a Nicaraguan field isolate of SfMNPV, suggesting that the presence of such variants is a common feature of SfMNPV populations.

Structural and ultrastructural alterations of Malpighian tubules of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae infected with different Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) recombinant viruses

Malpighian tubules constitute the main excretion organ of insects. Infection by egt − recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt − , and used another two recombinants (one egt − ) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt − viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.

In vivo characterization of a group II nucleopolyhedrovirus isolated from Mamestra brassicae (Lepidoptera: Noctuidae) in Japan

A Japanese isolate of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) was identified phylogenetically as a group II nucleopolyhedrovirus (NPV) that is related closely to other NPVs isolated from Mamestra spp. based on nucleotide sequence data of its polh, egt and lef-3 genes. The multiplication of MabrNPV in M. brassicae larvae was characterized following inoculation at various doses and in combination with the fluorescent brightener Tinopal by measuring temporal changes in the concentrations of its viral DNA using real-time quantitative PCR. The growth curves of budded-virus replication were analysed by fitting the data of viral DNA concentration in the host haemolymph to a modified Gompertz model. When fifth-instar larvae were inoculated with an LD95 equivalent dose of MabrNPV and Tinopal, the time lag between the onset of primary and secondary infection was estimated to be 25 h. Another 65 h was required to reach a plateau titre equivalent to a level of 10(9) virions ml(-1) in the haemolymph. All larvae died during the sixth instar following this inoculation regime. In contrast, following inoculation with a 1000-fold higher dose of MabrNPV and Tinopal, the time lag between the onset of primary and secondary infection was only 20 h. Subsequently, the same plateau titre was reached after a further 20 h. Following this inoculation regime, most larvae died during the fifth instar. Quantification of viral DNA by real-time quantitative PCR and application of the Gompertz model are valuable for the characterization of baculovirus replication in vivo.

Production of polyhedral inclusion bodies from Helicoverpa armigera larvae infected with wild-type and recombinant HaSNPV

We report on the yield of Polyhedral Inclusion Bodies (PIBs) from first to fifth instar Helicoverpa armigera larvae inoculated with wild-type Helicoverpa armigera nucleopolyhedrovirus (HaSNPV-WT) or with one of two HaSNPV recombinants, one in which the ecdysteroid UDP-glucosyltransferase (egt) gene was deleted (HaSNPV-EGTD) and a second in which the egt gene was replaced by a scorpion toxin (AaIT) gene (HaSNPV-AaIT). A significant linear relationship between the logarithm of cadaver weight and the logarithm of the number of PIBs per cadaver was observed for all three virus types. The increase of the number of PIBs with larval weight was significantly greater for HaSNPV-WT than for the recombinant viruses. For each of the three HaSNPVs, PIB yield per cadaver was significantly affected by larval instar at death and by time to death, with later instars and longer surviving larvae producing a greater number of PIBs. As both recombinants caused host larvae to die at earlier instars than HaSNPV-WT, their virus yields were significantly reduced. Virus yield per larva, inoculated with HaSNPV-AaIT in the first, second, third, fourth or fifth larval stage was 23, 32, 41, 44 and 47% of the yield of HaSNPV-WT, respectively. For HaSNPV-EGTD, virus yield per larva inoculated in first through fifth instar, respectively, was 41, 55, 63, 54 and 82% of the yield of HaSNPV-WT. These results provide a basis for optimizing the production regime of recombinant HaSNPVs in larvae and for modeling the behavior of these viruses in agro-ecosystems

The Ecdysteroid UDP-Glucosyltransferase Gene Promoter from Autographa californica Multicapsid Nucleopolyhedrovirus

The ecdysteroid UDP-glucosyltransferase (egt) gene promoter fragments of different lengths were generated from the genomic DNA of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) by PCR. After being purified and enzymatic digestion, they were cloned into the pGEM-3Z vector for construction of reporter plasmids pAcegt542-luc, pAcegt309-luc and pAcegt159-luc with the luciferase gene driven by the AcMNPV egt promoter. The results of transient expression in the Spodoptera frugiperda cell line-21 (Sf21) showed that the transcriptional activity of the AcMNPV egt promoter required the transactivation of viral factor(s). The expression of luciferase gene driven by the AcMNPV egt promoter was first detected at 24 h post infection. The egt promoter from the Bombyx mori nucleopolyhedrovirus (BmNPV), closely related to AcMNPV, revealed similar properties to that of the AcMNPV egt promoter. When BmNPV homologous region 3 was subcloned downstream the luciferase gene, the luciferase activity of the reporter plasmid was enhanced by over 1000-fold, but the property of the promoter was not changed. As a substrate of ecdysteroid UDP-glucosyltransferase (EGT), foreign insect ecdysone showed negative effects on egt promoter transcriptional activity. Ecdysone of 1.0-2.0 microg/ml significantly down-regulated the promoter activity. Promoter activities of different lengths showed that an AcMNPV egt promoter fragment of 159 bp has the basal transcriptional activity but it was almost abolished only about 0.2% of that of 309 bp and 542 bp, respectively, and the nucleotide sequence from - 159 to - 309 nt upstream the translation initiation site includes the main cis-acting elements interacting with viral factors.

Microparasite manipulation of an insect: the influence of theegtgene on the interaction between a baculovirus and its lepidopteran host

SummaryParasites and pathogens manipulate their hosts in a variety of ways that are thought to enhance their fitness. However, it is rare to be able to link such phenotypic changes to specific genes.Here the effect of a single pathogen gene is examined. The ecdysteroid UDP‐glucosyltransferase (egt) gene of insect baculoviruses produces an enzyme that interferes with host moulting.The effect of theegtgene was examined by comparing two baculoviruses that differed only in the expression of this gene. All three fitness traits examined – pathogenicity, infection duration and pathogen productivity – were affected by deletion of the gene.Trichoplusia nilarvae in all five instars died earlier when infected with theegt‐minus virus compared with those infected by the wild‐typeAutographa californicanucleopolyhedrovirus.Unexpectedly, theegt‐minus virus was more pathogenic to final instar larvae than the wild‐type virus. Virus genotype and dose both influenced insect development.Wild‐type infected insects had a significantly higher yield of virus at death, cadaver weight and yield of virus per unit weight than those infected with theegt‐minus virus.The size of the virus challenge had a major influence on the outcome of the interaction. The consequences of these data for pathogen fitness are discussed.

Field inactivation of wild-type and genetically modified Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus in cotton

Cotton bollworm (Helicoverpa armigera) is a serious pest on cotton in China. A specific baculovirus, H. armigera nucleopolyhedroviruses (HaSNPV) is used as a commercial biopesticide to control this pest. To improve the pesticidal properties, HaSNPV has been genetically engineered by both deleting the ecdysteroid UDP-glucosyltransferase (egt) gene from its genome (recombinant HaSNPV-EGTD) and incorporating an insect-selective toxin gene from the scorpion Androctonus australis (AaIT) (recombinant HaSNPV-AaIT). In the field, there was no significant difference among the inactivation rates of the two recombinant HaSNPVs and their parent wild-type, HaSNPV-WT. The inactivation rate of these viruses was significantly different in different years. The average half-life of HaSNPV was 0.57, 0.90 and 0.39 days in 2000, 2001 and 2002, respectively. Inactivation rates correlated well with solar radiation over these years.

Biological activity and field efficacy of a genetically modified Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus expressing an insect-selective toxin from a chimeric promoter

Abstract A recombinant baculovirus (HaSNPV-AaIT) with improved insecticidal properties was constructed for the control of the cotton bollworm (Helicoverpa armigera). A chimeric promoter of the p6.9 and polyhedrin gene of H. armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) was used to drive the expression of an insect-selective scorpion toxin (AaIT) at the egt gene locus of HaSNPV. This chimeric promoter, denoted ph-p69p, was constructed by directional insertion of the p6.9 promoter downstream of the polyhedrin promoter. Laboratory bioassays indicate that the infectivity (LD50s) of this recombinant is unchanged, compared to one wild-type clone of HaSNPV (HaSNPV-WT), and an egt-deletion mutant (HaSNPV-EGTD). The median survival times (ST50s) of 1st to 5th instar H. armigera larvae were reduced 17–34% after infection with HaSNPV-AaIT in comparison to HaSNPV-WT. The median times of feeding cessation (FT50s) were 30–43% shorter for HaSNPV-AaIT than for HaSNPV-WT in the 3rd to 5th instar of this species. This virus acts also quicker than HaSNPV-EGTD. Field trials at two research sites in 2000 indicate that the number of larvae and the proportion of damaged squares, flowers, and bolls was significantly lower in cotton plots treated with HaSNPV-AaIT than in plots treated with HaSNPV-WT or HaSNPV-EGTD. When HaSNPV-AaIT was applied to control infestations of bollworm over an entire cotton season, yield of cotton lint in plots treated by this recombinant was 22.1% higher than that in HaSNPV-WT treated plots in 2001 (p

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase ( egt ) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus ( egt −) AgMNPV and its virulence towards its insect host. We have inserted an hsp70 - lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ- lacZ ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ- lacZ compared to the wild-type virus. A mean of 20.4×10 8 OBs/g/larva and 40.7×10 8 OBs/g/larva was produced from vAgEGTΔ- lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC 50 ) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ- lacZ . The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ- lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.

Sequence analysis of a 5.1 kbp region of the Spodoptera frugiperda multicapsid nucleopolyhedrovirus genome that comprises a functional ecdysteroid UDP-glucosyltransferase (egt) gene.

The ecdysteroid UDP-glucosyltransferase gene from the Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) was identified using degenerate primers whose sequence were derived from conserved regions of the EGT proteins encoded by other baculoviruses. Analysis of the gene sequence revealed the presence of an open reading frame (ORF) with potential to encode a polypeptide of 525 amino acids. Promoter sequences typical of baculovirus genes were found in the 5' region of this ORF. A polyadenylation signal was identified downstream the translation stop codon. A transient expression assay showed that the product of this ORF was able to conjugate glucose from UDP-glucose with ecdysone confirming that the gene identified was indeed the SfMNPV egt gene. The SfMNPV egt gene and the sequences of other baculovirus egt genes were used to infer a phylogenetic tree. The nucleotide sequence of the entire BamHI fragment that contains the SfMNPV egt gene was determined. Search of the available sequence databases suggested that, besides the egt gene, this region contains 5 ORFs similar to the baculovirus genes gp37 (fusolin), to ptp2 and to ORFs 28, 29, and 30 of Spodoptera exigua multicapsid nucleopolyhedrovirus. Both the phylogenetic analysis of the egt genes and the gene order of the region that flanks the egt gene indicated that SfMNPV is closely related to the baculoviruses that infects S. exigua and Mamestra configurata.

Functional characterization of the ecdysteroid UDP-glucosyl transferase gene of Helicoverpa armigera single-enveloped nucleopolyhedrovirus isolated in South Africa.

The ecdysteroid UDP-glucosyltransferase (egt) gene of a single enveloped nucleopolyhedrovirus was located using an Hz-SNPV gene-specific probe. This SNPV was found infecting a colony of Helicoverpa armigera (HaSNPV) in the Western Cape region of South Africa. The open reading frame of the HaSNPV-SA egt is 1.548 nucleotides long and encodes a predicted protein of 516 amino acids with a Mr of 58,897-kDa. The 5'-noncoding region contained an early transcription initiation motif (CAGT) and a baculovirus late transcription motif (ATAAG). A transcription enhancer sequence (GATA) was also identified. Two possible TATA boxes together with an AT rich region were also recognized. A putative signal peptide of 20 residues was present at the N-terminus of the predicted EGT sequence. A polyadenylation signal (AATAAA) was found downstream of the translation stop codon. Five Helicoverpa NPV EGT's that have an extremely high degree of nucleotide and amino acid sequence homology were used in this study. Single nucleotide polymorphisms (SNPs) within the gene were tabulated. The Helicoverpa NPV egts seem to be closely related to the egt genes of Mamestra configurata NPV (MacoNPV), Buzura suppressaria NPV (BusuSNPV) and Spodoptera exigua NPV (SeMNPV) with amino acid identities of approximately 50%. The Helicoverpa NPV EGTs show ten conserved motifs with other EGTs. A phylogenetic tree of 27 baculovirus EGTs and a human UDP-glucoronosyltransferase was constructed using Neighbour-joining within CLUSTAL X. That a secreted and active EGT is encoded by HaSNPV-SA was confirmed by assay of infected cell culture medium.

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.

Granulovirus prevents pupation and retards development of Adoxophyes honmai larvae

Abstract Larvae of Adoxophyes honmai (Lepidoptera: Tortricidae) infected with granulovirus (AdhoGV) do not pupate; instead, they undergo prolonged larval development and die during the final stadium. Non‐infected larvae, however, pupate after five larval stadia. Insect metamorphosis is regulated by fluctuations of ecdysteroid and Juvenile Hormone (JH). JH esterase activity and titres of ecdysteroid must be measured to understand fully the interaction of an insect virus and its host. JH esterase activity is consistently low in AdhoGV‐infected larvae, which suggests that JH in AdhoGV‐infected larvae is not degraded during the final stadium. The ecdysteroid titre in non‐infected larvae showed a large peak in the final stadium before pupation, whereas that in AdhoGV‐infected larvae increased from day 2 to day 5 in the final stadium, and then remained at a high level until death. Furthermore, an ecdysteroid UDP‐glucosyltransferase (EGT) assay showed that this activity occurs in haemolymph from AdhoGV‐infected larvae, but not in haemolymph of non‐infected larvae. PCR and sequencing analysis revealed that the AdhoGV genome contains an egt gene, which encodes a protein of 445 amino acids, located approximately 1 kbp upstream from the granulin gene. These results suggest that AdhoGV‐infected larvae are prevented from pupating because JHE activity is suppressed and EGT expression inactivates ecdysteroid in the haemolymph.

Identification and characterization of the ecdysteroid UDP-glycosyltransferase gene of Epinotia aporema granulovirus.

The ecdysteroid UDP-glycosyltransferase (egt) gene of Epinotia aporema granulovirus (EpapGV) was cloned sequenced and its biological activity was assessed. It encodes a protein of 446 amino acids. Direct evidence that the cloned gene encodes an active EGT protein was obtained by transient expression assays in insect cells. The upstream untranslated region of the egt gene exhibits several consensus early promoter elements. Accordingly, the gene is expressed early upon infection of Epinotia aporema larvae and the EGT activity remains high until later times post infection. Sequence analyses indicate the presence of clusters of amino acid residues conserved among all the baculoviral EGTs, although their relation with proper protein folding, ligand binding and catalytic activity remain to be assessed. Phylogenetic trees consistently cluster the granulovirus EGTs separating them clearly from the nucleopolyhedroviruses.

Phylogenetic analysis of conserved genes within the ecdysteroid UDP-glucosyltransferase gene region of the slow-killing Adoxophyes orana granulovirus

A physical map of the genome of Adoxophyes orana granulovirus (AoGV) was constructed for the restriction enzymes BamHI, BglII, EcoRI, PstI and SacI using restriction endonuclease analysis and DNA hybridization techniques. This enabled the size of the AoGV genome to be estimated at 100.9 kbp. A plasmid library covering 99.9% of the AoGV genome was constructed using five restriction enzymes. The ecdysteroid UDP-glucosyltransferase gene (egt) was located by hybridization with the egt gene of Cydia pomonella granulovirus. The sequence of 6000 bp of the egt region is presented and compared to the equivalent area in other GVs. Database searches showed that this region contained eight open reading frames (ORFs) similar to the baculovirus genes egt, granulin, pk-1, me53 and four ORFs of Xestia c-nigrum granulovirus (ORF 178, ORF 2, ORF 7 and ORF 8). The egt gene was shown to encode an active EGT using an EGT assay. Phylogenetic trees of the granulovirus genes egt, granulin, pk-1 and me53 were constructed using maximum parsimony and distance analyses. These analyses indicated that AoGV genes may be more closely related to other tortricid-infecting GVs than to GVs that infect other lepidopteran families.

Identification and in vivo characterization of the Epiphyas postvittana nucleopolyhedrovirus Ecdysteroid UDP-glucosyltransferase.

The genome of Epiphyas postvittana Nucleopolyhedrovirus (EppoMNPV) contains an ecdysteroid UDP-glucosyltransferase (egt) gene. The egt gene was completely sequenced and surrounding open reading frames identified. EppoMNPV egt is 1479 nucleotides in length encoding a protein with a predicted molecular mass of 55 kDa. Analysis of upstream sequence revealed dual TATA boxes and two CGT upstream activating region motifs. Mapping of the 5' terminus of the egt transcript identified a major transcript produced from an adenine residue 29 nucleotides downstream from the distal TATA box. No transcript was detected from a late promoter motif (GTAAG). Characterization of egt transcripts showed that poly-adenylation occurs at the 3' terminus. EppoMNPV egt transcripts were first detected in infected Epiphyas postvittana larvae by Northern hybridization at 9 h post-infection (hpi) and EGT enzyme activity was detected at 9 hpi in haemolymph from infected larvae. EppoMNPV EGT can conjugate the sugars from both UDP-glucose and UDP-galactose to ecdysone in vitro. Localization assays performed using radiolabelled ecdysone demonstrated that the conjugation of glucose or galactose, from the respective UDP-sugar, led to the prevention of the uptake of ecdysone by SF-21 cells. We propose that EGT functions to prevent the uptake of ecdysone or 20-hydroxyecdysone by metabolite and target cells respectively.

Characterization of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus.

The Anticarsia gemmatalis nucelopolyhedrovirus (AgMNPV) egt gene was cloned, sequenced and its expression characterized by RT-PCR and western blot analysis. Sequence analysis of the gene indicated the presence of an open reading frame (ORF) of 1482 nucleotides, which codes for a polypeptide of 494 amino acids. ATATA box and a conserved regulatory sequence (CATT) found in other baculovirus early genes were present in the promoter region of the egt gene. A poly-A consensus sequence was present in the 3' untranslated region (3'-UTR) of the gene. Homology comparisons showed that the EGT protein of AgMNPV is most closely related (95.9% amino acid sequence identity) to the EGT from the Choristoneura fumiferana DEF nucleopolyhedrovirus (CfDEF). Transcriptional analysis of the AgMNPV egt gene showed that egt-specific transcripts can be detected both early and late in infection. The EGT protein was detected, by western blot analysis, in the intra- (from 12 to 48 h post-infection) and extra-cellular (from 12 to 96 h post-infection) fractions of infected insect cells. The AgMNPV Bgl II-F fragment, which has homology to the AcMNPV ie-1 gene, was cloned and used to cotransfect SF21 cells with the cloned AgMNPV egt gene. EGT activity was observed, suggesting that AgMNPV ie-1 can transactivate egt expression.

Sublethal Nucleopolyhedrovirus Infection Effects on Female Pupal Weight, Egg Mass Size, and Vertical Transmission in Gypsy Moth (Lepidoptera: Lymantriidae)

Gypsy moth females that survived inoculation with Lymantria dispar (L.) nucleopolyhedrovirus (LdNPV) as fifth instars were smaller as pupae and laid fewer eggs as adults. Treatment with both wild type virus containing the egt gene and a genetically manipulated, egt- virus, lacking this gene, reduced pupal mass to a similar degree. Sublethal infection with wild type virus reduced the masses of surviving pupae at 20, 25, and 28°C. A relationship between virus dose (5,000, 50,000 and 500,000 occlusion bodies per larva), mass of pupae, and egg mass size only occurred in one experiment in which larvae were reared at 25°C and inoculated 5 d after molt to the fifth instar. Vertical transmission of overt infection occurred in two of 13 egg masses (15%) produced by females inoculated with virus as larvae. The five larvae infected with virus were ≈0.5% of the larvae tested. Whether sublethal effects of LdNPV infection occur in field populations of gypsy moth remains to be tested.

Age-Related Effects of the Autographa californica Multiple Nucleopolyhedrovirus egt Gene in the Cabbage Looper (Trichoplusia ni)

Abstract The effects of deleting the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egt gene on speed of kill and virus productivity were compared in second and fourth instar Trichoplusia ni (Hubner) larvae. Time to death was significantly reduced in larvae infected with an egt deletion mutant compared to insects infected with the wild-type virus. Moreover, time to death was reduced by the same proportion (11%) in second and fourth instar larvae. Virus yield was also significantly lower in fourth instar larvae infected with the deletion mutant but no difference was apparent in second instar larvae. A comparison of cadaver weights showed that insects infected with the deletion mutant were lighter than those infected with the wild-type virus, suggesting that the decrease in virus yield resulted from a reduction in larval growth. An analysis of yield per unit body weight showed no evidence for differences in replication rate in the two viruses. To determine whether differences in larval growth rate were related to differences in feeding activity, frass production was monitored in fourth instar larvae. Larvae infected with the deletion mutant produced less frass than larvae infected with the wild-type virus. Whereas the average rate of feeding for fourth instars did not differ between virus treatments, the rate peaked and declined earlier for larvae infected with the deletion mutant than for those infected with wild-type virus, suggesting enhanced early feeding in the absence of egt expression.