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Although the Baculoviridae are a large and diverse family of viruses, they are united by a number of shared features that form the basis for their unique life cycle. These include the mechanism of cell entry, genome replication and processing, and late and very late gene transcription. In this review, the molecular systems that are conserved within the Baculoviridae and that are responsible these processes are described.
We present evidence that a newly discovered mosquito virus from Culex nigripalpus is an unusual member of the family BACULOVIRIDAE: Development of this virus was restricted to nuclei of midgut epithelial cells in the gastric caeca and posterior stomach. The globular occlusion bodies were not enveloped, measured around 400 nm in diameter, occurred exclusively in nuclei of infected cells and typically contained four, sometimes up to eight, virions. The developmental sequence involved two virion phenotypes: an occluded form (ODV) that initiated infection in the midgut epithelial cells, and a budded form that spread the infection in the midgut. Each ODV contained one rod-shaped enveloped nucleocapsid (40x200 nm). The double-stranded DNA genome was approximately 105-110 kbp with an estimated GC content of 52%. We have sequenced approximately one-third of the genome and detected 96 putative ORFs of 50 amino acids or more including several genes considered to be unique to baculoviruses. Phylogenetic analysis of the amino acid sequences of DNApol and p74 placed this virus in a separate clade from the genera NUCLEOPOLYHEDROVIRUS: and GRANULOVIRUS: We provisionally assign this virus in the genus NUCLEOPOLYHEDROVIRUS:, henceforth abbreviated as CuniNPV (for Culex nigripalpus nucleopolyhedrovirus), and suggest that, awaiting additional data to clarify its taxonomic status, it may be a member of a new genus within the family BACULOVIRIDAE:
We report the complete sequence of a large rod-shaped DNA virus, called the Hz-1 virus. This virus persistently infects the Heliothis zea cell lines. The Hz-1 virus has a double-stranded circular DNA genome of 228,089 bp encoding 154 open reading frames (ORFs) and also expresses a persistence-associated transcript 1, PAT1. The G+C content of the Hz-1 virus genome is 41.8%, with a gene density of one gene per 1.47 kb. Sequence analysis revealed that a 9.6-kb region at 43.6 to 47.8 map units harbors five cellular genes encoding proteins with homology to dUTP pyrophosphatase, matrix metalloproteinase, deoxynucleoside kinase, glycine hydroxymethyltransferase, and ribonucleotide reductase large subunit. Other cellular homologs were also detected dispersed in the viral genome. Several baculovirus homologs were detected in the Hz-1 virus genome. These include PxOrf-70, PxOrf-29, AcOrf-81, AcOrf-96, AcOrf-22, VLF-1, RNA polymerase LEF-8 (orf50), and two structural proteins, p74 and p91. The Hz-1 virus p74 homolog shows high structural conservation with a double transmembrane domain at its C terminus. Phylogenetic analysis of the p74 revealed that the Hz-1 virus is evolutionarily distant from the baculoviruses. Another distinctive feature of the Hz-1 virus genome is a gene that is involved in insect development. However, the remainder of the ORFs (81%) encoded proteins that bear no homology to any known proteins. In conclusion, the sequence differences between the Hz-1 virus and the baculoviruses outnumber the similarities and suggest that the Hz-1 virus may form a new family of viruses distantly related to the Baculoviridae:
The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.
Baculovirus chitinases and other family 18 glycohydrolases have been shown to possess both exo- and endochitinase activities when assayed against fluorescent chito-oligosaccharides. Homology modelling of the chitinase of Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) against Serratia marcescens chitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and an alpha/beta TIM barrel catalytic domain characteristic of a family 18 glycohydrolase. EppoNPV chitinase has many features in common with other baculovirus chitinases, including high amino acid identity, an N-terminal secretion signal and a functional C-terminal endoplasmic reticulum-retention sequence. EppoNPV chitinase displayed exo- and endochitinolytic activity against fluorescent chito-oligosaccharides, with K(m) values of 270+/-60 and 240+/-40 microM against 4MU-(GlcNAc)2 and 20+/-6 and 14+/-7 microM against 4MU-(GlcNAc)3 for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc)(2-6) chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc)2, indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidal beta-chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc)2 complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc)(3-6) substrates, but not (GlcNAc)2, acted as inhibitors of EppoNPV chitinase. Short-chain substrates are unlikely to interact with the aromatic residues of the PKD1 substrate-feeding mechanism and hence may not accurately reflect the activity of these enzymes against native substrates. Based upon these results, the chitinase of the baculovirus EppoNPV is an exochitinase.
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A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.
A quantitative bioassay method for the detection of Helicoverpa armigera (Lepidoptera: Noctuidae) singly encapsulated nucleopolyhedroviruses (HaSNPVs) in soil is described. Calibration curves used for estimating soil virus titres in environmental samples were generated by incorporating a sterilised soil into a semi-synthetic insect diet then inoculating known concentrations of an HaSNPV into the soil-diet mixture. Calibration curves were constructed for soil diets containing varying proportions of soil: 0, 1, 5, 10 and 25% soil (w/v). Their accuracy was assessed in a series of blind tests in which the actual soil virus concentration fell within the estimated mean 95% confidence region for each of three samples. The five soil-diet incorporation rates were compared in terms of larval survivorship and growth rate. There was no significant difference in larval survivorship after 10 days (i.e. for the duration of the bioassay period). The stage structure of bioassay larvae at 10 days and pupal weight at 20 days was significantly different for individuals reared on 25% soil-diet in terms of both a slower growth rate and a lower mean pupal weight compared to individuals reared on 10, 5 and 1% soil diets. This did not, however, appear to lead to greater variability in bioassay response at the high soil rate at 10 days. The level of sensitivity of virus detection achieved using this method was extremely good with the LC10 value for mid-first instar H. armigera larvae reared on the 25% soil-diet estimated at 26 polyhedral inclusion bodies (PIBs) per gram of soil. The suitability of using this approach for quantifying Helicoverpa NPVs in Australian soils was assessed by comparing percent bioassay infection across a range of five isolates known to be present in Australia. The effect of soil pH and soil management (cultivated versus non-cultivated) on percent bioassay infection was also examined. In both cases, no significant differences were observed. Finally, percent idopathic mortality, percent NPV infection and estimates of Helicoverpa SNPV concentration in a selection of samples from the Australian environment are presented.
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Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146 bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.
The first characterization of a nucleopolyhedrovirus (NPV Baculoviridae) isolated from the vapourer moth, Orgyia antiqua (Lepidoptera Lymatriidae), in the United Kingdom is presented. Transmission electron microscopy revealed that the virus nucleocapsid rods were singly enveloped in the polyhedron inclusion body (PIB) so that the virus is assigned to the SNPV subgenus of the Baculoviridae. Restriction endonuclease analyses of viral DNA indicated a genomic size of approximately 148 kb. Restriction profiles of O. antiqua SNPV closely resembled those of heterologous NPVs isolated from four different Orgyia species and was most similar to a North American SNPV isolate from the Douglas-fir tussock moth, O. pseudotsugata. In host range tests, O. antiqua SNPV was not infectious to 23 lepidopteran species representing four families. Heterologous Orgyia NPV isolates were permissive in O. antiqua larvae. Using a diet plug bioassay method, the median lethal dose response (LD(50)) for this virus in second and third instar O. antiqua larvae were estimated at 52 and 539 PIBs per larva, respectively.
Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).
Well over 100 isolates of granulosis viruses (GVs), genus Granulovirus (family Baculoviridae), have been reported, all from lepidopterous insects. Three types of GVs are recognized, those of Type 1, which attack the fat body, Type 2, which attack most tissues, and Type 3, which attack only the midgut epithelium. To determine whether a correlation exists between tissue tropism and lepidopteran family phylogeny, the granulin gene of the Harrisina brillians (HbGV), a virus that attacks the midgut epithelium of H. brillians (family Zygaenidae) was cloned, sequenced, characterized, and compared with granulin genes of GVs that attack species of Tortricidae, Pieridae, and Noctuidae. The HbGV granulin gene encoded a peptide of 248 amino acids with a predicted Mr of 29.6 kDa, and shared a significant level of homology with other granulin (81-95% identical and 90-98% similar) and polyhedrin (49-58% identical and 62-72% similar) proteins. Phylogenetic analyses based on granulin and polyhedrin genes as well as on their 5'-untranslated sequences (5'-UTSs) indicated that HbGV was more closely related to GVs isolated from the tortricids, Cryptophlebia leucotreta (ClGV), Cydia pomonella (CpGV) and Choristoneura fumiferana (CfGV) than to other GVs and NPVs. This analysis provides preliminary evidence for a correlation between GV tissue tropism and the phylogeny of lepidopteran families, suggesting that GVs attacking species of Tortricidae and Zygaenidae are ancestral to those attacking species of the family Noctudiae.
Biological control of agricultural pests has gained importance in recent years due to increased pressure to reduce the use of agrochemicals and their residues in the environment and food. Viruses of a few families are known to infect insects but only those belonging to the highly specialized family Baculoviridae have been used as biopesticides. They are safe to people and wildlife, their specificity is very narrow. Their application as bioinsecticides was limited until recently because of their slow killing action and technical difficulties for in vitro commercial production. Two approaches for the wider application of baculoviruses as biopesticides will be implemented in future. In countries where use of genetically modified organisms is restricted, the improvements will be mainly at the level of diagnostics, in vitro production and changes in biopesticide formulations. In the second approach, the killing activity of baculoviruses may be augmented by genetic modifications of the baculovirus genome with genes of another natural pathogen. It is expected that the baculoviruses improved by genetic modifications will be gradually introduced in countries which have fewer concerns towards genetically modified organisms.
The introduction of Oryctes virus into outbreak areas of the rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), has been a major success for "classical" biocontrol with a virus and has led to a dramatic reduction in palm damage in many areas of the Asia/Pacific region. In recent years, however, there have been new reports of high levels of rhinoceros beetle damage to palms. Damage has been especially intense in SE Asia following the introduction of no-burn polices for land clearance and replanting, but outbreaks have also been reported from some Pacific Islands where control seems to have diminished over time. SE Asian studies show that there is considerable genetic variation among endemic Oryctes virus isolates and studies in new island release areas have shown rapid evolution of the virus. The consequences of such genetic variation are in need of further study. Furthermore, the taxonomic position of the virus is unclear, with its removal from the Baculoviridae to an "unassigned' virus, reflecting its novel characteristics. Genomic sequencing could help resolve the taxonomy of the virus and provide a basis for studying strain variation. Oryctes virus has achieved wide success in the past without the benefit of molecular analysis and identification techniques. In order to fully take advantage of this unique pathogen for protection of palms, a renewed, coordinated effort centered on genetic selection and distribution of effective strains is required.
The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed.