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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 provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.

Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.

The polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus was analysed with respect to which sequences are required upstream of the mRNA transcription initiation (CAP) site for efficient promoter activity. Insertions (8, 95 and 785 nucleotides) were made in this region at an EcoR V site between the CAAT- and TATA-like boxes. When these mutations were introduced into the virus they did not affect the activity of the polyhedrin promoter as judged by expression of the beta-galactosidase (lacZ) gene inserted in lieu of the polyhedrin coding sequences. Deletions were made in the promoter which progressively removed sequences upstream from the CAP site. Removal of the TATA motif did not affect lacZ gene expression. A sequence 69 nucleotides upstream to the normal position of the polyhedrin ATG translation initiation codon was sufficient for maximum promoter activity but this was reduced by 90% when only 56 nucleotides upstream remained. The normal CAP site was utilized by each deletion mutant. Promoter activity was undetectable when the CAP site was deleted. The results are discussed in relation to other eukaryotic promoters.

The prevalence of pathogens in wild populations has often been estimated by the appearance of overt symptoms in the host, and this is typically used as the sole gauge of the impact of the pathogen on host dynamics. However, the development of molecular methods has increased the sensitivity with which we can detect asymptomatic infections. Baculoviruses are insect pathogens that, like many microparasites, are usually only found when their hosts reach outbreak densities, when a disease epizootic occurs. Conventional wisdom is that the long-term persistence of baculoviruses relies on their survival in the external environment in the form of occlusion bodies. These are proteinaceous matrices in which the virus particles are embedded, and which provide a degree of protection from UV irradiation. However, Mamestra brassicae has also been shown to harbour a persistent, non-lethal baculovirus infection (M. brassicae nucleopolyhedrovirus) in laboratory culture, which may represent another putative persistence mechanism. Here, we present evidence that such covert infections are also present and frequent in natural populations of the moth. The persistent infections were triggered into the lethal overt state by exposure to another baculovirus, and two closely related but different baculoviruses were subsequently identified as persistent infections within the populations sampled. These results have broad-ranging implications for our understanding of host pathogen interactions in the field, in the use of pathogens as biocontrol agents, and in the evolution of virulence.

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.