Expression of the Cydia pomonella granulovirus iap3 gene.

The IAP3 protein of Cydia pomonella granulovirus (CpGV) was the first identified member of the baculovirus IAP family of proteins, which have been shown to block apoptosis in diverse systems. However, little is known of the expression and subcellular localisation of CpGV IAP3 during a viral infection. This study examined IAP3 in cells infected by CpGV and in cells infected by an Autographa californica nucleopolyhedrovirus (AcMNPV) recombinant that carried the CpGV iap3 gene. The levels of iap3 specific transcripts were monitored and production of the protein was assessed using an IAP3-specific antiserum. The data showed that iap3 is expressed during both early and late phases of infection, with a switch occurring from distal early transcription start sites to proximal late start sites. Protein levels are highest after DNA replication. IAP3 is localised exclusively in the cytoplasm. Subcellular fractionation experiments demonstrated that the protein is present in both soluble and membrane-bound cytosolic fractions. The membrane-bound fraction includes IAP3 that is associated with the mitochondria. However, the data do not support the hypothesis that release of cytochrome C from the mitochondria is involved in baculovirus-induced apoptosis.

Biological and biochemical relationships between the nucleopolyhedroviruses of Mamestra brassicae and Heliothis armigera.

The multiply embedded nucleopolyhedroviruses (MNPV) originally isolated from Mamestra brassicae (German and Dutch isolates) and Heliothis armigera have been studied comparatively to establish their relatedness, both in terms of biological activity and genomic homology. All three viral isolates replicated in M. brassicae, H. armigera, Heliothis zea, and Heliothis virescens, resulting in each case in progeny virus that was essentially similar to the inoculum. Dose-mortality studies carried out on M. brassicae and H. armigera indicate that these viruses do not differ significantly with respect to their virulence to these insects. The same studies also clearly indicate that the susceptibility of M. brassicae and H. armigera larvae to viral infection differs significantly with increasing larval age. The increase in LD(50) values from L1 to L4 is, in fact, over 40,000-fold for M. brassicae, while it is only 1300-fold for H. armigera. The results of the present study also confirm that all three isolates are genetically closely related. Due to their high degree of homology and almost identical biological activity, it is suggested that these isolates should be considered variants of a single virus species.

A comparison of six cypovirus isolates by cross-hybridisation of their dsRNA genome segments.

Genetic relationships between the genome segments of six cypovirus (CPV) isolates were analysed by RNA cross-hybridisation. These included three type 1 viruses and single isolates of types 2, 5 and 12, which collectively are identical to those previously compared by serology and electrophoresis [Mertens et al. (1989), J Gen Virol 70: 173-185]. Since only genome segment 10 of three cypovirus types and segments 8 and 9 of a single virus strain (of type 1) have currently been sequenced, this initial study provides some additional information on sequence variation/similarity in each of the ten genome segments. The RNA of the type 1 viruses showed high levels of cross-hybridisation. Significant but much lower levels of cross-hybridisation were detected between type 1 and the related type 12 CPV. However, only very low levels of cross-hybridisation were detected between the other pairs of viruses. Apart from evidence of a slightly higher level of sequence similarity between the largest segments, the RNA sequence appeared to vary uniformly across the whole genome. There was no evidence for any type specific RNA sequences restricted to individual genome segment(s). The sequence variation, reflected in the levels of RNA sequence similarity and cross hybridisation, correlates well with serological data, showing large differences between CPV types and supports the continued use of electropherotype as one of the 'species parameters' for the classification of cypoviruses.

Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes.

A 3.2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpGV) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs). ORF7L is predicted to encode a 66.7 kDa protein (594 amino acid residues) that is 57% identical (amino acid sequence) to the chiA gene (ORF126) of Autographa californica nucleopolyhedrovirus (AcMNPV), encoding a chitinase. ORF8R is 333 amino acids in length and shows high similarity (between 64% and 67%) with baculovirus cathepsins. The partial ORF, ORF5L, is related to AcMNPV ORF145 of unknown function. Phylogenetic trees were constructed for both chitinase and cathepsin sequences from baculoviruses and other species. In both cases, the baculovirus sequences were monophyletic but with a deep division between the GVs and NPVs, suggesting both genes were present in an ancestral virus prior to the separation of the two genera. However, these studies did not provide definitive evidence for the origin of either protein in baculoviruses. To investigate CpGV cathepsin function, a rescue experiment was performed using a Bombyx mori NPV (BmNPV) mutant (BmCysPD) which lacks a functional cathepsin (cath) gene. Larvae infected with BmCysPD-Cp.cat, a BmCysPD derivative carrying CpGV cath, showed similar symptoms to wild-type BmNPV infected insects, confirming that CpGV cath encodes a functional cathepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infected cells showed that CpGV cath transcription was initiated from a consensus late transcription motif (ATAAG) within the CpGV sequences, indicating that a CpGV late promoter motif was recognized in this NPV system.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis.

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Sinceper os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes ( cry1Ab and cry1Ac ) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus

Comprehensive physical map of the Cydia pomonella granulovirus genome and sequence analysis of the granulin gene region.

A cloned strain of Cydia pomonella granulovirus, CpGV-M1, was obtained using successive rounds of an in vivo limiting dilution method. A detailed physical map of the genome was constructed using 11 restriction enzymes. The region containing the granulin gene and an open reading frame immediately upstream of the granulin gene was sequenced. This region showed a high degree of homology to the equivalent region from Cryptophlebia leucotreta granulovirus with 98% amino acid identity for the granulins and 68% identity for the putative polypeptides encoded by the upstream ORFs. These latter polypeptides contained two zinc finger-like motifs and showed a low degree of homology to ME53 from Autographa californica nucleopolyhedrovirus (AcMNPV). Evidence is presented for a similar upstream ORF in Artogeia rapae GV also. Hybridization studies showed that the CpGV genome had a similar overall organization to the Artogeia rapae GV genome. Hybridization between CpGV and AcMNPV was limited to fragments spanning about 15% of each genome suggesting that very few genes are highly conserved between GVs and NPVs.

Complete sequence and transposon mutagenesis of the BamHI J fragment of Cydia pomonella granulosis virus.

The BamHI J fragment of Cydia pomonella granulosis virus was subcloned and subjected to transposon mutagenesis in Escherichia coli using a Tn3 derivative. After screening by restriction endonuclease digestion and polymerase chain reaction (PCR), 44 clones were selected representing insertions every 100 to 300 bp. The complete sequence was compiled and the transposon insertion sites mapped precisely by sequencing. Analysis of the sequence revealed the presence of 7 potential open reading frames (ORFs). The BamHI J fragment was already known to encode IAP and OPDV-E6. Three other ORFs encode products similar to known proteins, viz. an Autographa californica nuclear polyhedrosis virus 8.6 kDa protein, a Lymantria dispar nuclear polyhedrosis virus 34 kDa protein, and vertebrate reovirus omega-1 proteins. The ORF with similarity to omega-1 is also similar to baculovirus p10 proteins. In both cases, the similarity occurs in regions likely to form a coiled-coil structure.

Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins.

Expression of full-length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp. kurstaki in a baculovirus and pathogenicity of the recombinant viruses.

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.

Replication of Cydia pomonella granulosis virus in cell cultures.

Several primary cell lines that support the complete replication of Cydia pomonella granulosis virus have been established from one culture of C. pomonella embryonic cells. Virus passaged three times in cells and once in larvae showed no change in restriction enzyme fragment patterns. Stages in virus replication observed by electron microscopy resembled those from in vivo studies. Cell lines that were maintained at or below 21 degrees C retained susceptibility to virus over a period of 4 years whereas the same cell lines maintained at 27 degrees C gradually lost their susceptibility and eventually could not be infected at all.

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.

Characterization of new baculovirus genotypes arising from inoculation of Pieris brassicae with granulosis viruses.

Previous studies have shown that of 15 Artogeia (Pieris) rapae granulosis virus isolates (ArGV1 to ArGV15) only two, ArGV1 and ArGV2, gave a normal dose-mortality response in larvae from an established colony of Pieris brassicae. We report here that at extremely high doses, approaching 10000 times the LD50 for ArGV1 and ArGV2, three other ArGV isolates caused low and irregular levels of mortality in P. brassicae. At similar doses Agrotis segetum GV caused 43% mortality in one infection, but no deaths ensued from other inoculations with this virus. Restriction endonuclease analysis of viral DNA recovered from individual larval cadavers revealed that, in most cases, progeny virus differed from the inoculum and consisted either of ArGV1 or of novel genotypes explicable as recombinants between genomes of the inoculum and of ArGV1. Field-collected P. brassicae inoculated with ArGV8 yielded a similar range of progeny genotypes. Physical maps were constructed for two such recombinants, based on comparative restriction analysis with reference to the published map of ArGV1 and to those of ArGV5 and ArGV8, which are presented. Replication of the inoculum genotype was observed in only two infections. The origin of ArGV1 DNA appearing among progeny from these infections and the relevance of our results to identifying ArGV DNA sequences that modulate pathogenicity for P. brassicae are discussed.

Cytoplasmic polyhedrosis virus classification by electropherotype; validation by serological analyses and agarose gel electrophoresis.

Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris-HCl/Tris-glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris-acetate-EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates.

In vivo isolation of baculovirus genotypes.

Restriction endonuclease profiles of a wild-type granulosis virus from Artogeia rapae (ArGV3) and a nuclear polyhedrosis virus from Lymantria dispar (LdMNPV) indicated that the preparations were genotypically heterogeneous. Eight constituent genotypes were isolated from ArGV3 by low mortality dose infections of late instar A. rapae and analysis of progeny viral DNA recovered from individual cadavers. Three distinct genotypes were isolated from LdMNPV by the same approach, using L. dispar larvae. These results show that larval mortality can occur as a consequence of productive infection by a single virus particle. Comparative physical mapping of the eight ArGV3 genotypes suggested that their diversity may be partly attributable to recombination during natural coinfection.

Properties of a novel DNA virus from the tsetse fly, Glossina pallidipes.

Virus particles were isolated from hypertrophied salivary glands of the tsetse fly, Glossina pallidipes collected near Mombasa, Kenya. Purified virus particles were rod-shaped, 57 nm wide by 700 to 1300 nm long. Particle lengths fell into two size classes, with 'short' particles averaging 869 nm and 'long' particles 1175 nm. The virus particles morphologically resembled elongated baculovirus nucleocapsids although, unlike baculoviruses, no fully enveloped virions were found in purified preparations. The particles contained double-stranded DNA which appeared to be linear when analysed by electrophoresis in agarose gels, ethidium bromide-caesium chloride gradient centrifugation or electron microscopy (EM). There was some evidence for the DNA being heterogeneous in size from EM studies and from the observation that restriction enzyme analysis failed to provide a clear profile of DNA fragments. Protein from purified virions contained at least 12 polypeptides with a major component of 39 000 mol. wt. These results suggest that the virus cannot be placed in any of the existing taxonomic groupings of DNA viruses.

A comparison of the granulosis viruses from Pieris brassicae and Pieris rapae.

The granulosis viruses of Pieris brassicae and P. rapae have been compared biochemically and biologically. No differences were found between virus capsules when examined by immunodiffusion, ELISA, or SDS-polyacrylamide gel electrophoresis. The virus particles were identical by immunodiffusion but distinguishable by ELISA. Comparison of virus particle polypeptides on SDS-polyacrylamide gels indicated small differences in molecular weight in three of the polypeptides of the virus envelope but no difference between the nucleocapsids. There were several differences between the DNA fragments produced by digestion with EcoRI, BamH1, and HindIII restriction endonucleases from which the homology between the two DNAs was calculated to be 97.7%. The small structural difference between the two viruses is associated with a very large difference in their virulence for P. brassicae; the LD50 with P. rapae GV being at least 1000 times greater than with P. brassicae GV. P. rapae was much more susceptible to either virus than P. brassicae and in this case the LD50 values were not significantly different for the two viruses.

Protection of mice against cancer by immunization with membranes but not purified virions from virus infected cancer cells.

The life span of C57/Bl mice inoculated with Lewis lung carcinoma cells was prolonged if the mice were pre-immunized with membranes from these cells infected in vitro with influenza virus. Likewise, BALB/c mice were protected against the malignant tumour WEHI-11 by prior immunization with extracts of cultured WEHI-11 cells which had been infected with influenza virus or Semiliki Forest virus (SFV). Partially purified SFV grown in WEHI-11 cells also protected mice from cancer grafts but neither highly purified SFV nor the glycoprotein from the envelope of this virus protected the mice. It is concluded that SFV-induced immunopotentiation against cancer is not due to covalent linkage of tumour specific transplantation antigen (TSTA) to viral envelope protein but more probably is due to the apposition of viral glycoprotein and cellular TSTA in the plasma membrane of the cancer cell.