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Pieris rapae granulovirus (PiraGV) is highly pathogenic to the cabbage butterfly (P. rapae), an important pest of cultivated cabbages and mustard crops. It therefore holds significant promise towards exploitation as a potent bio-control agent in the field controlling the pest population. Whole-genome elucidation of the Korean isolate of the granulovirus (PiraGV-K), reported the presence of a granulin gene corresponding to ORF 1 in its genome. Comprehensive studies towards functional characterization of the gene, established that it is composed of 744 nucleotides and encodes a peptide of 247 amino acid residues. It possessed significant homology with AoGV and ClanGV with 87% identity at amino acid level. Multiple alignment data suggests that the C-terminus region of the gene had three different conserved regions. Time-course studies conducted in PiraGV-K infected P. rapae larvae revealed a significant upsurge of the transcript (134-fold) at 4 days post infection followed by a significant decline at the most advanced stages of infection. Anti-PiraGV-K granulin antibody was produced and western blot conducted with the infected larvae further confirmed the induction pattern with a protein of 30 kDa. Immunofluorescent staining showed a granulin-specific signal in fat body and integument of the infected larvae. Granulin-specific signals were noticed 2 days post infection with the eventual systemic spread of infection to the associated tracheal matrix witnessed at 4 days post infection. Immunogold labeling and electron microscopic studies further proved the cytopathological effects as the presence of numerous membrane-bound vesicles with nucleocapsids and abruption of intercellular junctions in fat body and hypertrophied cells in the integument., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tea production in North-East India hit a record loss due to the widespread severe outbreak of a mixed brood of three species of looper caterpillar pests of geometrid moths (Lepidoptera) in 2008-2010. In addition to Buzura suppressaria, two newly recorded geometrids, viz., Hyposidra infixaria and Hyposidra talaca have caused widespread severe damage in recent years. In the present study we report the nucleopolyhedroviruses (NPV) isolated from the tea looper caterpillar from North-East India. We identified and characterized the NPV by cloning and sequencing a partial segment of polyhedrin gene of virus infected larvae of B. suppressaria, H. talaca and H. infixaria. A comparison of deduced amino acids of polyhedrin gene among H. talaca, H. infixaria and B. suppressaria showed that same strain was found to infect all the three loopers in India, which show high sequence identity with B. suppressaria Chinese isolates. Based on the polyhedrin sequence homology, it is predicted that a variant of B. suppressaria Chinese isolate of NPV found to infect H. talaca, H. infixaria and B. suppressaria in India., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81+/-2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [(35)S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.

A baculovirus has been isolated from infected larvae of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae) in Taiwan. Ultrastructural observation revealed that this virus is a multiple nucleopolyhedrovirus (MNPV), and the name L. xylina MNPV (LyxyMNPV) was proposed. Restriction endonuclease (BamHI, EcoRI, and EcoRV) profiles of LyxyMNPV genome differed from those of other known NPVs. The size of the LyxyMNPV genome was estimated to be 154+/-1.26 kbp (mean+/-SE). The polyhedrin gene is located in the BamHI-D, EcoRI-C, and EcoRV-K fragments of LyxyMNPV genome. The gene organization of the LyxyMNPV EcoRV-K fragment and the phylogenetic analysis based on the polyhedrin gene sequences showed that LyxyMNPV is closely related to the L. dispar MNPV (LdMNPV). Furthermore, a rapid assay method was developed to distinguish LyxyMNPV from LdMNPV based on PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis of polyhedrin genes.

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.

Baculovirus occlusion bodies, large proteinaceous structures which contain virions, have recently been engineered to incorporate foreign proteins. The major constituent protein of occlusion bodies from the baculovirus Autographa californica nucleopolyhedrovirus is polyhedrin, and assembly of recombinant occlusion bodies which incorporate a foreign protein depends on an interaction between native polyhedrin and a polyhedrin-foreign protein fusion. This technology has now been applied to the generation of a recombinant baculovirus (ColorBtrus) that produces occlusion bodies incorporating the Bacillus thuringiensis (Bt) insecticidal Cry1Ac toxin protein. ColorBtrus coexpresses native polyhedrin and a fusion protein in which polyhedrin is fused to the Bt toxin, which is in turn fused to green fluorescent protein (GFP). Analysis of ColorBtrus occlusion bodies confirmed that they include both Bt toxin and GFP, yet still incorporate virions. Bioassay of ColorBtrus demonstrated that its speed of action and pathogenicity are strikingly enhanced compared to wild-type virus. ColorBtrus represents a novel, powerful biological insecticide that combines positive attributes of both Bt toxin and baculovirus based systems.

The bean shoot borer, Epinotia aporema, is a major pest of soybeans in Argentina. Larvae of this pest are attacked by a granulovirus (EpapGV) that is the most important cause of sporadic epizootics in E. aporema populations. We studied the pathology of this virus in last-instar larvae using light and electron microscopy, and evaluated the effect of the disease on larval growth and development. EpapGV caused a polyorganotropic infection. No nucleocapsids were observed in the nuclei of infected cells prior to nuclear membrane disruption. Nevertheless, granulin was detected in the nucleus by immuno-gold staining, indicating that late gene expression occurred prior to nuclear membrane disruption. Establishment of the virogenic stroma led to complexes of continuous parallel convoluted membranous sheets. Nucleocapsids were enveloped in these areas to form virions, which were then occluded. Apparently as part of the cell-to-cell spread of infection, nucleocapsids were observed enclosed in large numbers within membrane-bound vesicles located between the cells and basal lamina. Larvae infected by EpapGV suffered a retardation of development and typically failed to pupate, but exhibited a weight increase greater than that of healthy E. aporema.

A baculovirus has been isolated from larvae of Lonomia obliqua, a Saturniidae of medical importance due to a potent toxin found in their spines. Electron Microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra of approximately 1 microm in diameter containing multiple nucleocapsids per envelope. This baculovirus was thus named Lonomia obliqua multicapsid nucleopolyhedrovirus (LoobMNPV). Restriction endonuclease profiles of viral DNA digested with three restriction enzymes were obtained and the genome size was estimated to be 95.52 +/- 2.3 kbp. The polyhedrin gene of LoobMNPV was identified and its DNA sequence was determined. Phylogenetic analysis of the polyhedrin gene showed that the LoobMNPV polyhedrin belongs to group I NPV and that it is closely related to the polyhedrin of the NPV of Amsacta albistriga., (Copyright 2002 Elsevier Science B.V.)

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect., (Copyright 2000 Academic Press.)

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from lepidopteran hosts have been previously identified. To reveal more detailed relationships, a series of DNA polymerase nucleotide sequences from the taxa MbMNPV, SeMNPV, HzSNPV, HearNPV, SpltNPV, BusuNPV, and OranNPV have been determined using a polymerase chain reaction (PCR)-based approach. This technique enabled gene sequence determination using microliter samples of NPV-infected insect cadavers. Polyhedrin genes from HearNPV, OranNPV, SeMNPV, and SpltNPV were also isolated and sequenced using a similar approach. These sequences, together with other database entries, were aligned for positional homology of peptide sequences. Phylogenetic analysis of DNA polymerase molecular sequence alignments supports LdMNPV as a taxon of Group II and three Group II subclades, designated A, B, and C. Comparison of DNA polymerase trees with those estimated from occlusion protein molecular sequences enabled identification of three subclades of Group II. These are Subgroup II-A [MbMNPV, LeseNPV, MacoNPV, PaflNPV, SeMNPV, SpltNPV (India isolate), SfMNPV]; Subgroup II-B [SpliNPV, SpltNPV (Japan isolate), SpltNPV (Queensland isolate), and possibly HzSNPV, HearNPV, and ManeNPV], and Subgroup II-C [OpSNPV, OranNPV (S-type), BusuNPV (S-type), and possibly EcobNPV (S-type)]. Notably, all Subgroup II-A taxa are from noctuid hosts. Correlations of virus and host evolution within Group II taxa are discussed. The methods and data developed in this study will allow rapid sequencing of NPV DNA polymerase genes., (Copyright 1999 Academic Press.)

In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.

The granulosis virus isolated from Achaea janata is composed of capsules (463 +/- 25 x 280 +/- 22 nm) which contain single virus particles. The virus particles (274 +/- 9 x 95 +/- 4 nm) contain nucleocapsids (297 +/- 7 x 52 +/- 2 nm) within an envelope. The empty capsids measured 265 +/- 9 x 63 +/- 3 nm. SDS-PAGE electrophoresis was carried out and the granulin polypeptide (28.9 +/- 0.5 kDa) was found to contain two other associated polypeptides of molecular weight 58.2 +/- 2.3 and 55.2 +/- 1.3 kDa, respectively. Protein gel electrophoresis of granulin gave degradation products from 27.2 to 15.4 kDa, when protease associated with capsules was not inhibited. The virus particles were found to contain 16 polypeptides with molecular weights ranging from 106.4 +/- 2.1 to 15. 5 +/- 0.8 kDa and 12 of these polypeptides were contained in the nucleocapsids. The major polypeptide of the capsids had molecular weight of 34.9 +/- 0.3 kDa. Restriction profiles of viral DNA with seven enzymes were obtained and the average molecular weight was found to be approximately 92 +/- 7 kb. Southern hybridization of the restriction profiles of the viral DNA with SalI fragment from pUCTnGV (containing granulin gene) was performed and hybridization was found on single discrete fragments with molecular weights ranging from 12.0 to 2.7 kb. The GC content of AjGV DNA was found to be 65 +/- 1% and the genome size as determined from reassociation kinetics was 92 kb., (Copyright 1998 Academic Press.)

The Perina nuda nucleopolyhedrovirus (PenuNPV) polyhedrin gene was located in EcoRI-G (6.3 kilobase pairs; kbp) and PstI-G (4.3 kbp) fragments of its genomic DNA. A portion of 1333 nucleotides (nt) containing this gene was sequenced. An open reading frame of 735 nt encoded a 245-amino-acid-long polyhedrin. A conserved TAAG motif which is associated with transcriptional start sites was identified 51 nt upstream of the translation initiation codon of PenuNPV polyhedrin gene. A putative polyadenylation signal, AATAAA, was found 116 nt downstream of the termination codon (TAA). Comparison of the amino acid sequences of PenuNPV polyhedrin with those of other NPVs showed that PenuNPV polyhedrin was most closely related to Orgyia pseudotsugata multiple NPV (OpMNPV) polyhedrin.

Both wild-type and recombinant baculoviruses are becoming more attractive for the control of insect pests. Thus, there is an increased incentive to address and resolve logistical problems associated with large-scale production of these viruses. In this study, we have compared the potential of two insect cell lines, Tn5B1-4 and Sf21, for the production of polyhedra and compared the efficacy of both cell culture-derived and host-derived viruses by bioassay. The efficacy of both wild-type AcMNPV and AcAaIT, a recombinant baculovirus expressing an insect-specific scorpion toxin, were compared. Yields of polyhedra from Tn5B1-4 were sixfold higher than those from the cell line Sf21. Morphological analysis of polyhedra derived from cell culture showed greater variability in size relative to host-derived polyhedra. The maximum size of cell culture-derived polyhedra was over 1.5 times larger than that of insect-derived polyhedra. The efficacy of AcMNPV and AcAaIT derived from cell culture, or from amplification in larvae of Trichoplusia ni or Heliothis virescens, was compared by bioassay in H. virescens. There was a significant difference between the slopes for lethal time data for host-derived and cell culture-derived wild-type virus. Mortality occurred at a faster rate following infection with host-derived virus. No significant difference was seen for the recombinant virus AcAaIT. Lethal doses of cell- and host-derived polyhedra were not significantly different. The reasons for and implications of this for pest control are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

The phylogenetic relationship of several baculovirus polyhedrin genes was investigated. Alignments of 18 polyhedrin gene amino acid sequences from which 12 were also available as DNA sequences were constructed based on positional homology and trees were calculated using character state and distance methods. Our results indicate that the Hymenopteran NsSNPV diverged from the Lepidopteran baculovirus before the separation of the NPV from the GV and subsequent radiation of these Lepidopteran groups. Moreover, this analysis indicates that lepidopteran NPV are clustered in at least two groups with distinct evolutionary rates. Group I includes the AcMNPV, BmMNPV, BmSNPV, OpMNPV, AgMNPV, and possibly the GmMNPV and AsMNPV. Group II includes the MbMNPV, PfMNPV, SfMNPV, SeMNPV, and possibly the OpSNPV. This grouping is also supported by a tree constructed from sequence data from the 5' untranslated leader region of the polyhedrin gene. Furthermore, the polyhedrin-based phylogeny was analyzed for its congruence to the genomic organization and other biological characteristics of the baculoviruses. This study shows that the SNPV and MNPV morphotypes have to be taken with caution as classificatory traits since they appear to be unpolarized. In addition, the comparison of the baculovirus phylogeny with the Lepidoptera phylogeny confirms the assumption that the evolutionary history of the baculoviruses is not entirely in agreement with the taxonomic organization of the Lepidoptera, implying that the common designation of baculoviruses based on the hosts may not be adequate for baculovirus classification since it does not reflect the genetic relatedness of the viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Full-length and truncated forms of the crystal protein gene cryIA(b) derived from Bacillus thuringiensis subsp. kurstaki HD-1 and full-length cryIA(c) gene of B. thuringiensis subsp. kurstaki HD-73 were introduced into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus, in place of the polyhedrin gene. All gene constructs were expressed at high levels in insect cells and insects upon infection with the recombinant viruses. The protein products were shown to be biologically and immunologically similar to the natural crystal protein. The expressed proteins formed crystals (in insects) up to 10 times bigger (in length) than their bacterial counterpart. The LT50 values for recombinant viruses were not significantly shorter than wild-type virus.

The polyhedrin gene of the nuclear polyhedrosis virus of the gypsy moth (Lymantria dispar) (LdMNPV) was cloned and sequenced. A polyhedrin open reading frame of 735 nucleotides (nt) was identified which can code for a protein of 245 amino acids that demonstrates a high degree of similarity to other polyhedrins. The protein predicted from the nucleotide sequence shows differences in several regions to that previously sequenced from the LdMNPV polyhedrin protein. The consensus sequence AATAAGTATTTT found at the mRNA start site of baculovirus hyperexpressed genes was located 55 nt upstream from the translational start site.