Phenotypic and genotypic characterisation of persistent baculovirus infections in populations of the cabbage moth (Mamestra brassicae) within the British Isles.

The genotypic relatedness of persistent baculovirus infections within UK populations of Mamestra brassicae was assessed by sequencing amplified regions from polyhedrin and ie1. Most populations harboured Mamestra brassicae (Mb) nucleopolyhedrosis virus (NPV) which showed very little genotypic variation between populations. However, one population harboured a virus that closely resembled a baculovirus found previously only in Pine Beauty Moth (Panolis flammea) populations in Scotland. Persistent baculoviruses that had emerged spontaneously as lethal, overt infections from two of the insect populations were compared with the type strain of MbNPV and a mixture of P. flammea (Pafl) NPV strains, isolated from a single host, by bioassay in virus-free Spodoptera exigua larvae. Reactivated baculoviruses were as pathogenic as the stock virus and showed phenotypic characteristics closest to the type strain they most resembled genetically. Sequence data from the insect host cytochrome oxidase genes were compared and showed a high degree of sequence conservation between populations and it was not possible to determine whether the persistent baculovirus infections had arisen on many occasions or whether they represented a single initial infection that had spread with the host. However, the presence of two distinct virus genotypes in separate M. brassicae populations suggests multiple colonisations of the host are a possibility.

Host ecology determines the relative fitness of virus genotypes in mixed-genotype nucleopolyhedrovirus infections.

Mixed-genotype infections are common in many natural host-parasite interactions. Classical kin-selection models predict that single-genotype infections can exploit host resources prudently to maximize fitness, but that selection favours rapid exploitation when co-infecting genotypes share limited host resources. However, theory has outpaced evidence: we require empirical studies of pathogen genotypes that naturally co-infect hosts. Do genotypes actually compete within hosts? Can host ecology affect the outcome of co-infection? We posed both questions by comparing traits of infections in which two baculovirus genotypes were fed to hosts alongside inocula of the same or a different genotype. The host, Panolis flammea, is a herbivore of Pinus sylvestris and Pi. contorta. The pathogen, PfNPV (a nucleopolyhedrovirus), occurs naturally as mixtures of genotypes that differ, when isolated, in pathogenicity, speed of kill and yield. Single-genotype infection traits failed to predict the 'winning' genotypes in co-infections. Co-infections infected and caused lethal disease in more hosts, and produced high yields, relative to single-genotype infections. The need to share with nonkin did not cause fitness costs to either genotype. In fact, in hosts feeding on Pi. sylvestris, one genotype gained increased yields in mixed-genotype infections. These results are discussed in relation to theory surrounding adaptive responses to competition with nonkin for limited resources.

Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene.

We have constructed a transfer vector (pAgGal) containing the beta-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The beta-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay.

Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene.

The chitinase of Autographa californica nucleopolyhedrovirus (AcMNPV) is required for the characteristic liquefaction of baculovirus-infected insect larvae. Alignments of the putative active sites of a range of chitinases revealed two highly conserved residues, glutamate and aspartate, which have been proposed to constitute the catalytic residues of the active site. These residues were mutated in the AcMNPV chitinase. Three recombinant viruses were generated, AcchiA(D311G), AcchiA(E315G) and AcchiA(D311G E315G), which contained mutations at either the glutamate, the aspartate or both. It was demonstrated that chitinase protein production was unaffected by the mutation of these residues. However, mutation of both residues resulted in the attenuation of chitinolytic activity and the cessation of liquefaction of Trichoplusia ni larvae infected with AcchiA(D311G E315G). Mutagenesis of the glutamate residue led to a reduction in exochitinase activity and a delay in the appearance of endochitinase activity. In addition, larvae infected with this virus, AcchiA(E315G), liquefied more slowly than those larvae infected with wild-type AcMNPV. Mutagenesis of the aspartate residue resulted in a reduction of exochitinase activity but an unexpected enhancement of endochitinolytic activity. Liquefaction of AcchiA(D311G)-infected larvae was observed at the same time as that of AcMNPV-infected larvae.

In vitro host range of Autographa californica nucleopolyhedrovirus recombinants lacking functional p35, iap1 or iap2.

We have examined the host range in different insect cell lines of Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants lacking p35, iap1 or iap2. These genes encode, or are predicted to encode, anti-apoptotic proteins. Abrogation of p35 reduced the ability of AcMNPV to replicate in permissive cell lines derived from Spodoptera frugiperda insects by inducing apoptosis. In semi-permissive cell lines, such as Lymantria dispar and Spodoptera littoralis cells, we observed cytopathic effects after infection with AcMNPV but little virus production. Infection of these cells by AcMNPV lacking p35 resulted in apoptosis. However, p35-deficient viruses were still able to replicate normally in Trichoplusia ni, Mamestra brassicae and Panolis flammea cell lines. Disruption of AcMNPV iap1 and iap2 was found not to affect virus replication in any of the cell lines. It was also possible to disrupt both iap1 and iap2 in the same virus without loss of infectivity. A virus without iap1 and p35 demonstrated identical growth characteristics and host range to a virus lacking p35. We conclude that in cells which respond to AcMNPV infection by initiating programmed cell death, the p35 gene product alone is sufficient to inhibit apoptosis. Removal of iap1 or iap2 has no effect on virus replication, even in cell lines which do not undergo apoptosis in response to AcMNPV infection. Our results with two semi-permissive cell lines further indicate that whilst p35 is important in blocking block apoptosis, other factors are involved in restricting AcMNPV replication within these cells.

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum.

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.

The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes.

We examined the role of the Autographa californica nucleopolyhedrovirus (AcMNPV)-encoded chitinase in virus pathogenesis in Trichoplusia ni larvae. In conjunction with the AcMNPV-encoded cathepsin, it promotes liquefaction of the host in the latter stages of infection. Insects infected with virus mutants lacking either the chitinase A gene (chiA) or cathepsin gene (cath) remained intact several days after death. However, if both viruses were used to infect insects, liquefaction of the host was restored. Chitinase was readily detected in AcMNPV-infected insects using a chitinase-specific antibody, but it was absent from insects infected with a chiA deletion mutant (AcchiA-). The chitinase was also detected in polyhedra purified from AcMNPV-infected insects but not in those from AcchiA-. However, polyhedra derived from a virus lacking an intact chiA were no less effective in initiating an infection in second instar T. ni larvae than those of the unmodified AcMNPV. It was also demonstrated that the virus chitinase retained high levels of activity between pH 3.0 and 10.0. In contrast, chitinases isolated from Serratia marcescens, although active under acidic conditions, rapidly lost activity above pH 7.0 illustrating that despite 57% sequence identity, the two proteins have distinct enzymic activities.

Baculoviruses as expression vectors.

Recent advances in baculovirus expression vector technology include improvements to methods for the selection of recombinant viruses and further developments in virion display vectors. It is now also possible to modify the host cell glycosylation pathway to alter the structure of glycans added to the recombinant polypeptide. Baculovirus vectors also continue to be modified to facilitate gene expression in mammalian cells.

Evidence for the presence of a low-level, persistent baculovirus infection of Mamestra brassicae insects.

A laboratory culture of Mamestra brassicae insects (MbLC) harbours a latent or occult baculovirus that resembles M. brassicae multiple nucleocapsid nucleopolyhedrovirus (MbMNPV). Although conventional extraction techniques have failed to detect the presence of virus in MbLC, control virus-free insects (MbWS) died of an MbMNPV-like infection after being fed MbLC fat-body cells. This suggested that the MbLC cells harboured infectious MbMNPV, albeit at low levels. We have also demonstrated that fat-body cells from MbLC, but not from MbWS, contain mRNA specific for the polyhedrin gene and transcriptional factors that are capable of activating baculovirus late and very late gene promoters linked to a reporter gene encoding chloramphenicol acetyltransferase. Our data provide indirect evidence that the latent MbMNPV in the MbLC insects is maintained as a persistent infection, with the expression of viral genes at a low level.

Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains.

Manipulation of baculovirus vectors.

Baculovirus expression vectors provide an excellent system for the synthesis of recombinant proteins in insect cells. This article presents sufficient background information to allow the nonspecialist to understand the basic principles of the technology and the development of baculovirus expression vectors. A summary of the most commonly used plasmids and viruses is presented. Detailed techniques are described to enable recombinant baculoviruses to be constructed. These methods include the protocols required for propagating insect cells in culture and their subsequent infection with viruses.

An Autographa californica nucleopolyhedrovirus lef-2 mutant: consequences for DNA replication and very late gene expression.

In order to define factors involved in very late Autographa californica nucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1.lacZ) by 5'-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of the EcoRI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and in lef-2. Further mapping experiments demonstrated that only replacement of the point mutation in lef-2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that the lef-2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in the lef-2 gene of VLD1 did not have an effect on DNA replication. It is proposed that lef-2 may play a dual role, both in DNA replication and very late gene expression.

Identification and preliminary characterization of a chitinase gene in the Autographa californica nuclear polyhedrosis virus genome.

A functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.

Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector.

The scorpion, Androctonus australis Hector, produces an insect-specific toxin (AaHIT) encoded by the Scotox gene. To assess the toxicity of AaHIT for mosquitoes, we have taken a novel approach to express the Scotox gene in vivo. We have engineered a double subgenomic Sindbis (dsSIN) virus that contains the Scotox gene in the viral genome and intrathoracically inoculated the virus (TE/3'2J/Scotox) into mosquitoes (Aedes aegypti, Ae. triseriatus and Culex pipiens), houseflies (Musca domestica) and ticks (Dermacentor andersoni). Mosquitoes, which normally show no pathologic effects from Sindbis (SIN) virus infections, died 1-5 days after infection with TE/3'2J/Scotox virus. Neither flies nor ticks were killed. The mosquitocidal action of AaHIT in mosquitoes makes AaHIT a potential candidate for inclusion in molecular-based methods of mosquito control. The expression of an arthropod gene in vivo demonstrates the utility of dsSIN expression vectors for future use to examine and potentially disrupt endogenous gene functions in mosquitoes.

Quantification of latent Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects using a PCR-scintillation proximity assay.

A laboratory culture of Mamestra brassicae insects (MbLC) was found to harbour a latent baculovirus infection. The copy number of the occult MbNPV genome in both the MbLC larvae, and in a cell line derived from the fat body of MbLC was determined by the use of a rapid and convenient PCR-scintillation proximity assay (SPA). The SPA system relies on the use of fluomicrospheres (SPA beads) coated with acceptor molecules which are capable of binding radiolabelled ligands in solution. In the assay described, a biotinylated PCR primer is used and [3H]dNTPs are incorporated into the amplified DNA. The SPA beads are coated with streptavidin, and after binding the biotinylated primer, any amplified, radiolabelled DNA will activate the fluor. The amount of amplified DNA from the target sequence can then be directly quantified using a scintillation counter. The number of MbNPV genomes present in a persistently infected M. brassicae cell, as proposed by SPA, suggest between 13 and 20 copies of the viral genome may be present in individual fat body cells.