Professor Linda King

Humanised antibodies targeting Crimean-Congo Haemorrhagic virus (CCHFV) are needed for the development and standardisation of serological assays. These assays are needed to address a shortfall in available tests that meet regulatory diagnostic standards and to aid surveillance activities to extend knowledge on the distribution of CCHFV. To generate a humanised monoclonal antibody against CCHFV, we have compared two methods: the traditional mouse hybridoma approach with subsequent sequencing and humanisation of antibodies verus a non-animal alternative using a human combinatorial antibody library (HuCAL). Our results demonstrated that the mouse hybridoma followed by humanisation protocol gave higher affinity antibodies. Whilst not yet able to demonstrate the generation of equivalent humanised antibodies without the use of animals, sequencing data enables the subsequent recombination production of antibodies, thus providing a reduction in future animal usage for this application. Ultimately, our report provides information on development of a humanised standardised control, which can form an important positive control component of serological assays against CCHFV.

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target coding region is placed in frame with the AUU then some product will comprise a chimeric molecule with part of the polyhedrin protein. In this study we showed that if AUU is placed in frame with a Strep tag and eGFP coding region we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues.

P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury. Gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet β cells (EndoC βH3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells.

African horse sickness is a severe, often fatal, arboviral disease of equids. The control of African horse sickness virus (AHSV) in endemic countries is based currently on the use of live attenuated vaccines despite some biosafety concerns derived from its biological properties. Thus, experimental vaccination platforms have been developed over the years in order to avoid the biosafety concerns associated with the use of attenuated vaccines. Various studies showed that baculovirus-expressed AHSV-VP2 or modified Vaccinia Ankara virus expressing AHSV-VP2 (MVA-VP2) induced virus neutralising antibodies and protective immunity in small animals and horses. AHSV is an antigenically diverse pathogen and immunity against AHS is serotype-specific. Therefore, AHS vaccines for use in endemic countries need to induce an immune response capable of protecting against all existing serotypes. For this reason, current live attenuated vaccines are administered as polyvalent preparations comprising combinations of AHSV attenuated strains of different serotypes. Previous studies have shown that it is possible to induce cross-reactive virus neutralising antibodies against different serotypes of AHSV by using polyvalent vaccines comprising combinations of either different serotype-specific VP2 proteins, or MVA-VP2 viruses. However, these strategies could be difficult to implement if induction of protective immunity is highly dependent on using a two-dose vaccination regime for each serotype the vaccine intends to protect against. In our study, we have tested the protective capacity of MVA-VP2 and baculovirus-expressed VP2 vaccines when a single dose was used. Groups of interferon alpha receptor knock-out mice were inoculated with either MVA-VP2 or baculovirus-expressed VP2 vaccines using one dose or the standard two-dose vaccination regime. After vaccination, all four vaccinated groups were challenged with AHSV and clinical responses, lethality and viraemia compared between the groups. Our results show that complete clinical protection was achieved after a single vaccination with either MVA-VP2 or baculovirus sub-unit VP2 vaccines. 

IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during the viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for survival of its progeny and therefore P10 presents an enigma. As P10 polymerises to form organised cytoskeletal structures that co-localise with the host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus, potentially, be responsible for its dissemination and survival.

 Incubation of HK-2 cells with antimycin A and 2-deoxyglucose resulted in a significant decrease in intracellular ATP levels; following reperfusion, ATP levels significantly increased overtime in cells overexpressing sod-2. In addition, lactate dehydrogenase (LDH) release declined over 72 h in BacMam-transduced injured cells. Ex vivo delivery of sod-2 significantly increased ATP levels in organs after 24 h of cold perfusion.
 

 In vitro and ex vivo results suggested that BacMam transduction successfully delivered sod-2, which reduced injury associated with I/R, by improving ATP cell content and decreasing LDH release with a subsequent increase in kidney tissue viability. These data provide further evidence for the potential application of BacMam as a gene delivery system for attenuating injury after cold preservation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in the nucleus of insect cells to produce nucleocapsids, which are transported from the nucleus to the plasma membrane for budding through GP64-enriched areas to form budded viruses. However, little is known about the anterograde trafficking of baculovirus nucleocapsids in insect cells. Preliminary confocal scanning laser microscopy studies showed that enhanced green fluorescent protein (EGFP)-tagged nucleocapsids and capsid proteins aligned and colocalized with the peripheral microtubules of virus-infected insect cells. A colchicine inhibition assay of virus-infected insect cells showed a significant reduction in budded virus production, providing further evidence for the involvement of microtubules and suggesting a possible role of kinesin in baculovirus anterograde trafficking. We investigated the interaction between AcMNPV nucleocapsids and kinesin-1 with fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) and show for the first time that AcMNPV capsid proteins VP39 and EXON0, but not Orf1629, interact with the tetratricopeptide repeat (TPR) domain of kinesin. The excited-state fluorescence lifetime of EGFP fused to VP39 or EXON0 was quenched from 2.4 +/- 1 ns to 2.1 +/- 1 ns by monomeric fluorescent protein (mDsRed) fused to TPR (mDsRed-TPR). However, the excited-state fluorescence lifetime of an EGFP fusion of Orf1629 remained unquenched by mDsRed-TPR. These data indicate that kinesin-1 plays an important role in the anterograde trafficking of baculovirus in insect cells.

Historically, it has been proved difficult to adapt the traditional baculovirus expression systems to an automated platform because of the complexity of the processes involved. One of the major bottlenecks is the selection of recombinant from parental viruses. We have developed a bacmid vector (flashBAC (TM)) that does not require any form of selection pressure to separate recombinant virus from nonrecombinant parental virus. The method relies on homologous recombination in insect cells between a transfer plasmid containing the gene of interest and a replication-deficient bacmid. The gene of interest replaces the bacterial replicon at the polyhedrin locus, simultaneously restoring a virus gene essential for replication, and as only recombinant virus can replicate, no further separation techniques are required. This chapter describes methods for producing and expression testing multiple recombinant baculoviruses on automated platforms using the flashBAC system.

Concerns over the safety of conventional viral vectors have limited the translation of gene transfer from an exciting experimental procedure to a successful clinical therapy in transplantation. Baculoviruses are insect viruses, but have the ability to enter mammalian cells and deliver potential therapeutic molecules with no evidence of viral replication. This study provides evidence of the ability of recombinant baculovirus to enter mammalian kidneys and livers during cold preservation. Six kidneys and six liver lobules retrieved from large pigs were perfused with University of Wisconsin (UW) solution containing a baculovirus tagged with green fluorescent protein and preserved for 8 h. In addition, six kidneys were perfused with UW containing a baculovirus expressing red fluorescent protein and preserved for 24 h. Green fluorescent virus particles were detected within transduced kidneys and livers after 8 h standard cold storage and red fluorescent protein mRNA was detected in kidneys after 24 h of cold preservation. There were no significant differences in tissue architecture, cell morphology or ATP content between experimental organs and their controls. Ex vivo transduction of organs with recombinant baculovirus during conventional cold preservation was demonstrated with no evidence of additional injury or reduction in cell viability.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is able to transduce a wide range of mammalian cells and shows preferential uptake in some, particularly liver and kidney cells. This suggests that the virus may be useful for delivery of protective genes for ameliorating the effects of ischaemia reperfusion injury (IRI) in solid organs during transplantation procedures. In this chapter we discuss the advantages of the baculovirus over other virus vectors for gene delivery in organ transplantation and describe some of the protective genes which may be used to ameliorate the effects of IRI. We then describe a method for concentrating baculovirus for use in an ex vivo transduction model. Data are also provided for the effects of virus transduction in vitro on the innate and adaptive immune response. We conclude with a discussion on the future considerations for using baculovirus for delivery and expression of protective genes in organ transplantation.

Baculoviruses have a unique bi-phasic life cycle and powerful promoters, which greatly facilitates their use for recombinant protein expression in insect cells. We have developed an expression system that utilizes homologous recombination in insect cells between a transfer plasmid containing a gene to be expressed and a replication-deficient virus (bacmid). Only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses using robotic liquid handlers. The bacmid has also been genetically optimized for improved protein expression and stability. We describe the application of this system for high level production of recombinant proteins.

The expression of ganglioside GD3, which plays crucial roles in normal brain development, decreases in adults but is upregulated in neoplastic cells, where it regulates tumor invasion and survival. Normally a buildup of GD3 induces apoptosis, but this does not occur in gliomas due to formation of 9-O-acetyl GD3 by the addition of an acetyl group to the terminal sialic acid of GD3; this renders GD3 unable to induce apoptosis. Using human biopsy-derived glioblastoma cell cultures, we have carried out a series of molecular manipulations targeting GD3 acetylation pathways. Using immunocytochemistry, flow cytometry, western blotting, and transwell assays, we have shown the existence of a critical ratio between GD3 and 9-O-acetyl GD3, which promotes tumor survival. Thus, we have demonstrated for the first time in primary glioblastoma that cleaving the acetyl group restores GD3, resulting in a reduction in tumor cell viability while normal astrocytes remain unaffected. Additionally, we have shown that glioblastoma viability is reduced due to the induction of mitochondrially mediated apoptosis and that this occurs after mitochondrial membrane depolarization. Three methods of cleaving the acetyl group using hemagglutinin esterase were investigated, and we have shown that the baculovirus vector transduces glioma cells as well as normal astroctyes with a relatively high efficacy. A recombinant baculovirus containing hemagglutinin esterase could be developed for the clinic as an adjuvant therapy for glioma.

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Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopox-virus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 +/- 0.1 ns to 2.1 +/- 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

Baculoviruses are lethal pathogens of insects, predominantly of the order Lepidoptera. These viruses have a bi-phasic life cycle, which greatly facilitates their use for biotechnological applications. They were exploited initially as biocontrol agents, and then engineered as protein expression vectors. The baculovirus expression vector system (BEVS) is now widely used for recombinant protein production. More recently they have become a popular choice for development as gene delivery and expression vectors in mammalian cells. This article reviews some of the major developments and patents relating to baculoviruses since their initial use as an expression tool and investigates current technologies alleviating bottlenecks in recombinant gene expression in insect cells.

The baculovirus P10 protein has always represented a mystery in the field of insect virology. Like the baculovirus polyhedrin protein it is expressed at high levels very late in infection. Homologues of the Autographa californica nucleopolyhedrovirus p10 gene are conserved in all Alphabaculoviruses and in other viruses of lepidopteran hosts yet is completely dispensable for virus replication and transmission. P10 is a microtubule interacting protein whose expression has been associated with the formation of a variety of complex and extensive cytoplasmic and nuclear structures. P10 has been associated with a number of roles during infection ranging from the formation of virus occlusion bodies, to affecting the rate of cellular and/or nuclear lysis during the final stages of the virus replication cycle. In this article we review recent work aimed at understanding the role of this enigmatic protein, putting them into context with recent advances in understanding of protein structure and function. We look back at a number of historical studies and observations, reanalysing their conclusions based on recent data and our own observations. The role of the P10 protein during baculovirus replication remains elusive, however, novel avenues of investigation have been identified that will, we are sure, eventually lead to an understanding of this protein.

An Autographa californica nucleopolyhedrovirus (AcMNPV) mutant (AcdefrT) isolated from virus-infected Trichoplusia ni (TN-368) cells produced plasma membrane blebbing and caspase-3-like activity late in infection. It also synthesized less polyhedra, but displayed enhanced budded virus formation in TN-368 cells. This phenotype resulted from dual mutations in p35 and FP-25. In this study we showed that enhanced budded virus production occurs because the hourly rate of release of virus from AcdefrT-infected cells is higher than that for AcMNPV and it continues for longer. This may be the trigger for the induction of apoptosis late in AcdefrT-infected TN-368 cells. However, laddering of host DNA was absent in TN-368 cells infected with AcdefrT, but was observed in Spodoptera frugiperda cells. Very late polyhedrin protein production and occlusion body formation was reduced in AcdefrT-infected TN-368 cells, but chitinase and capsid late gene expression remained unchanged. The AcdefrT was rescued with a copy of a baculovirus iap3, to replace the absent p35. This modification abolished most plasma membrane blebbing in AcdefrT-infected TN-368 cells, but did not affect enhanced budded virus production. These data suggest that inhibitors of apoptosis are required in T. ni cells, particularly when the production of budded virus is enhanced.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins.

The role of the microtubule-associated P10 protein of baculoviruses is not yet understood. P10 has previously been linked with the formation of a number of cytoskeletal-like or cytoskeleton-associated structures in the nucleus and cytoplasm, thought to be involved in the morphogenesis of virus polyhedral occlusion bodies. The formation of these structures was studied by immunofluorescence laser scanning confocal microscopy in TN368 cells, a model system amenable to the study of virus interaction with the host cell cytoskeleton. We show that the Autographa californica nucleopolyhedrovirus P10 protein forms two distinct cytoskeletal-like structures, microtubule-associated filaments and perinuclear tubular aggregates. P10 also associates with polyhedral occlusion bodies. Depolymerisation of the microtubule network with colchicine prevents formation of P10 filaments but not of P10 tubules. Colchicine treatment enhances the association of P10 with occlusion bodies. Transient expression of P10 showed that both filaments and tubules can form in the absence of other viral proteins. We postulate a number of possible roles of the P10 protein during virus infection and morphogenesis.

The genetic diversity of many DNA virus populations in nature is unknown, but for those that have been studied it has been found to be relatively high. This is particularly true for baculoviruses, a family of large double-stranded DNA viruses that infect the larval stages of insects. Why there should be such heterogeneity within these virus populations is puzzling and what sustains it is still unknown. It has long been recognized that some baculoviruses have a relatively wide host range, but the effect of different host species on the genotypic structure of a baculovirus population has received little attention. We provide evidence that infection of different insect species can influence the genetic diversity of a Panolis flammea nucleopolyhedrovirus (PaflNPV) population, isolated from the pine beauty moth. Variable regions of the PaflNPV genome were sequenced and novel ORFs were identified on each of the enlarged fragments. The roles of these orfs and the implications of their presence or absence within different genotypes are discussed. The variable fragments were also labelled with 32P and used as polymorphic genetic markers of genotype abundance. The proportion of polymorphic loci changed after passage in different insect species and this varied among species, suggesting a role for host selection of pathogen genotypes in the field as a mechanism for maintaining genetic diversity. These results have wide-ranging implications for understanding the ecology of insect–virus interactions in the natural environment and the evolution of baculovirus life history strategies.

Spodoptera frugiperda cells infected with Autographa californica nucleopolyhedrovirus (AcMNPV) lacking a functional anti-apoptotic p35 protein undergo apoptosis. However, such mutants replicate normally in Trichoplusia ni (TN-368) cells. An AcMNPV plaque isolate (AcdefrT) was identified during propagation of a virus deficient in p35 in TN-368 cells. This virus exhibited enhanced budded-particle formation in TN-368 cells, but was partially defective for polyhedra production in the same cells. Virus replication in AcdefrT-infected TN-368 cells was accompanied by extensive plasma-membrane blebbing and caspase activation late in infection, both features of apoptosis. Rescue of the p35 locus of AcdefrT continued to result in a reduction in polyhedra and increase in budded virus production in TN-368 cells, but no plasma-membrane blebbing was observed. The mutation was mapped to the FP-25 gene locus. This gene mutation combined with the non-functional p35 was found to be responsible for the cell-blebbing effect observed in AcdefrT-infected TN-368 cells.

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.

Infection of insect larvae with Autographa californica nucleopolyhedrovirus (AcMNPV) results in the liquefaction of the host, a process involving the action of virus-encoded chitinase and cathepsin gene products. Chitinase is localized in the endoplasmic reticulum (ER) during infection because of the presence of a C-terminal ER retrieval motif (KDEL). In this study, the KDEL coding region was removed from the chitinase gene so that expression of the modified chitinase remained under the control of its own gene promoter, at its native locus. The deletion of KDEL resulted in the redistribution of chitinase within the cell during virus infection. Chitinase lacking the KDEL motif was detectable at the plasma membrane and was also evident in the culture medium of virus-infected cells from as early as 12 h post-infection (p.i.). Secretion of chitinase from the cell continued up to 72 h p.i., until cytolysis. The biological activity of the recombinant virus in Trichoplusia ni larvae was enhanced, with a significant reduction in the lethal dose and lethal time associated with infection. Furthermore, a reduction in feeding damage caused by infected larvae was observed compared to AcMNPV-infected individuals.

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.

During infection of insect cells with Autographa californica nucleopolyhedrovirus (AcMNPV), the very late protein P10 forms large fibrillar structures in the cytoplasm and nuclei of infected cells. In this study we have used confocal microscopy in association with a novel P10 antiserum to localise and study P10 in virus-infected cells. P10 was shown to be a component of tubular-like structures that spiralled throughout the cytoplasm and nucleus of AcMNPV-infected cells. These structures were observed to colocalise partly with cortical microtubules. When microtubules were depolymerised with the drug nocodazole, P10 tubules failed to form and the protein appeared concentrated in cytoplasmic foci. For the first time, we provide direct evidence using both antibody pulldown and yeast two-hybrid experiments for the interaction of P10 with host-cell tubulin. It is suggested that this interaction may be a critical factor in AcMNPV-induced cell lysis.

During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.

A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID50 titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×106 TCID50/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×106 TCID50/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×106 TCID50/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp 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.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase gene coding region was amplified using the polymerase chain reaction, inserted into a plasmid (pROK-2) and replicated in Escherichia coli XL1–blue. The recombinant plasmid was mobilised into Agrobacterium tumefaciens LBA 4404 and inoculated into tobacco leaf discs. The presence of the expressed chitinase in foliar tissue of kanamycin-resistant plantlets of three Nicotiana tabacum cultivars (CF80, K326 and Xanthi-nc) was inferred using immunoblotting, and enzyme activity was confirmed using a fluorometric assay. Confocal laser scanning microscopy with immunofluorescent staining of foliar sections from N. tabacum Xanthi-nc expressing the viral chitinase indicated that the enzyme was restricted to the vascular tissue. Heliothis virescens larvae fed on leaf tissue expressing chitinase were not impaired either in their development to pupation or in their feeding behaviour, in comparision with their counterparts that had consumed similar amounts of untransformed tobacco leaf tissue. By contrast, when tobacco leaves were mechanically inoculated with Alternaria alternata, very few brown spots were observed at inoculation sites in chitinase-expressing tissue, whereas large and spreading lesions formed in untransformed tobacco tissue. Of all lines that were transformed, as determined by kanamycin resistance, 59% had fewer symptoms of disease (smaller disease indices) than those for untransformed controls.

The expression of heterologous genes in insect cells using baculovirus vectors is commonly used to achieve a high level of recombinant protein production. The expression system exploits the promoters from a number of hyperexpressed virus genes that are activated in the very late stages of the Autographa californica nucleopolyhedrovirus (AcMNPV) replication cycle. The most widely used of these are the polyhedrin gene (polh) and p10 (p10) gene promoters. Replacement of either the polh or p10 coding region with that of a foreign gene results in the formation of a recombinant virus with the foreign gene under control of the hyperexpressed promoter. Recombinant viruses may be propagated in insect cells cultured in vitro, for example, Spodoptera frugiperda or Trichoplusia ni cells, or may be used to infect insect larvae. In the latter case, recombinant viruses are usually prepared in which the polh and p10 remain intact, so that larvae may be infected by the natural route of feeding upon a polyhedra-containing diet. In this case, the foreign gene is expressed under control of a copy of the polh or p10 promoter, located in a nonessential region of the virus genome, for example, just upstream of the polh promoter.

Baculoviruses are used as highly efficient expression vectors for the

production of recombinant proteins in a number of insect cell lines. They

have the advantage of being safe to use and of achieving high concentrations

of the recombinant protein in the cell line of choice. The key feature of the

system is the use of a strong virus polyhedrin gene promoter to facilitate

expression of foreign genes. Substitution of the polyhedrin gene coding

region with that specifying the recombinant protein does not affect the

production of infectious virus. Further, because recombinant proteins are

made in the very late stages of infection, even cytotoxic products can be

tolerated moderately well. 

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.

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 present situation with regard to the use of baculoviruses in insect control is outlined. By virtue of their high degree of host specificity, they offer considerable advantages over chemical insecticides, but their practical use is limited by a number of factors, particularly their slow speed of action. Various approaches to the genetic modification of baculoviruses to overcome these problems are described. These have resulted in improvements in insecticidal activity in laboratory trials which are now being confirmed in the field. Thus, genetically modified baculoviruses have a promising future in pest-control programmes. Our increasing knowledge of the genetic factors which regulate their behaviour is showing how other aspects of their performance may be controlled and exploited.

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.

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 Bsu36I to improve the recovery of nonparental

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.

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.

The mouse serotonin (5-HT) receptor subtype, 5-HT7, belongs to the family of seven transmembrane G-protein-coupled receptors. To identify the structural basis for the coupling of 5-HT7 receptor to GαS we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild-type and mutant 5-HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5-HT7(E325G) and 5-HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C-terminal region of the third intracellular loop in receptor–G-protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.

Insect cells are relatively cheap to maintain and are capable of producing accurately translated and correctly processed heterologous proteins. Recent research has focused on the development of improved expression vectors for continuous, high-level production of foreign proteins, including a number of membrane-targeted receptors, in Drosophila and lepidopteran insect cells. Mosquito cells have also been employed for studies on the control of vector-borne diseases, such as malaria.

The effects of gamma-hexachlorocyclohexane (gamma-HCH) and its alpha, beta and delta isomers on the gamma-aminobutyric acid (GABA) responses of human alpha1beta3gamma2S and alpha6beta3gamma2S GABA(A) receptors expressed in Xenopus oocytes were examined by conventional two-electrode voltage-clamp techniques. Gamma-HCH induced partial inhibition of EC50 GABA responses, whereas the alpha and delta isomers produced potentiation of EC20 GABA currents. In contrast, beta-HCH had no effect on GABA currents, even at concentrations as high as 100 microM. The effects of the active HCH isomers were not influenced by alpha subunit composition because there was no significant difference in either the inhibition or potentiation of alpha1beta3gamma2S or alpha6beta3gamma2S GABA(A) receptors. Delta- and gamma-HCH antagonized picrotoxin inhibition and caused displacement of specific [35S]t-butylbicyclophosphorothionate binding. Delta-HCH potentiation was found to be additive with steroid, loreclezole and lanthanum potentiation, but nonadditive with potentiation by pentobarbital and propofol, which suggested that its activity was linked to the barbiturate site.

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.

We have synthesised the α-subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChRα, encoding the α-subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR α-subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.

Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5-HTdro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the G(alpha)s G-protein subtype. The Drosophila 5-HTdro2B receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin-insensitive G-protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G-proteins revealed that Sf9 cells lack the G-protein subtypes G(alpha i 1-3) and G(alpha)o, but express the subtype G(alpha)s and G(alpha)q.

To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and car☐y-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.

The gene encoding the major occlusion body protein,

spheroidin, of Amsacta moorei entomopoxvirus

(AmEPV) was introduced into a baculovirus

vector under control of the polyhedrin gene promoter.

A recombinant virus produced large, ovoid

occlusion body-like structures in both Spodoptera

frugiperda and Trichoplusia ni cells. These structures

resembled the spheroids found in AmEPVinfected

Lymantria dispar cells, except they were

devoid of virus particles and were not surrounded

by a membrane- or envelope-like structure. These

results were confirmed by immunofluoresence microscopy

and Western blotting using a specific antipeptide

antibody to spheroidin, and suggest that

the supramolecular assembly of spheroids is not

dependent on other EPV-encoded gene products.

Transmission electron microscopy and subcellular

fractionation experiments revealed that the spheroid-like

structures were assembled in both the

nucleus and cytoplasm of the recombinant virusinfected

cells. This contrasts with the solely cytoplasmic

localization found in AmEPV-infected cells.