Use of baculoviruses as biological insecticides.

Naturally occurring baculoviruses can be used to control a wide range of insect pests. Most baculoviruses are used as biopesticides, that is, they are sprayed onto high-density pest populations in a manner akin to the use of synthetic chemical pesticides. However, other strategies that use the biological features of the viruses are also possible and should increase as we expand our knowledge of baculovirus ecology. In order to develop a baculovirus control program, several areas need to be studied before progressing to large scale field studies and commercialization. These range from laboratory efficacy testing and the development of production systems to detailed study of pest behavior and the development of appropriate application strategies.

Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles.

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 A in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryo-electron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 microg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV.

Recombinant baculovirus-synthesized African horsesickness virus (AHSV) outer-capsid protein VP2 provides protection against virulent AHSV challenge.

African horsesickness virus serotype 4 (AHSV-4) outer-capsid proteins VP2 or VP2 and VP5, prepared from single or dual recombinant baculovirus expression vectors grown in Sf9 insect cells, were administered in different amounts to horses and the neutralizing antibody responses were measured. Control and vaccinated horses were challenged with virulent AHSV-4 6 months later and monitored post challenge. The results indicated that two inoculations of extracts containing VP2 and VP5, or VP2 alone, in doses of 5 micrograms VP2 or more per horse, were sufficient to elicit protection against African horsesickness (AHS) disease. The recombinant VP2 protein is a potential candidate vaccine for AHS in horses.

Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes.

Plasmodium sporozoites collected from oocysts, haemocoel and salivary glands of the mosquito show profound differences in their biological properties such as motility, ability to induce protective immune response and infectivity for vertebrate host cells. Sporozoites from salivary glands are much more infectious than those from oocysts and haemocoel. Differential expression of proteins, such as the circumsporozoite (CS) protein and the thrombospondin-related adhesive protein (TRAP), implicated in sporozoite recognition and entry into hepatocytes may account for the development of infectivity during ontogeny. We have carried out a series of experiments to: (i) analyse the expression and localization of TRAP in P.falciparum sporozoites during development in the mosquito; and (ii) elucidate the biochemical and adhesive properties of recombinant TRAP. Our data indicate that TRAP is not expressed in oocysts, whereas variable amounts of CS protein are found in this parasite developmental stage. Hemocoel sporozoites display the distinct phenotypes TRAP- CS protein+ and TRAP+ CS protein+ at a frequency of 98.5 and 1.5% respectively. Salivary gland sporozoites are all TRAP+ CS protein+. We also provide experimental evidence showing that recombinant TRAP binds to the basolateral cell membrane of hepatocytes in the Disse's space and that sulfated glycoconjugates function as TRAP ligands on human hepatocytes.

Development of a competitive ELISA for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein.

A competitive ELISA based on the reaction between a monoclonal antibody (mAb) and a recombinant nucleoprotein of the peste des petits ruminants virus (PPRV) was developed. This protein was obtained in large quantities from insect cells infected with a PPR nucleoprotein recombinant baculovirus (N-B). The competitive ELISA was compared with the virus neutralisation test (VNT) for detecting specific antibodies to PPRV in sheep and goats. The time consuming VNT is the only prescribed test that is capable of distinguishing between PPRV and the cross-reactive rinderpest virus (RPV). The competitive ELISA involves the simultaneous addition of the mAb and antibodies present in a positive serum, leading to competition for a specific epitope on the N-B. Optimum conditions were obtained by using serum samples which had positive or negative neutralising activity against PPRV or RPV. A negative cut-off point was determined on PPRV-negative sera from RPV-vaccinated cattle. A threshold value of 48 per cent inhibition, calculated from the mean for this population plus 2.7 standard deviations, was used in routine testing. A total of 683 sera were analysed by the competitive ELISA and the VNT. A good correlation (r = 0.94) was observed between the titres obtained in the two tests, with 80 sera that were from laboratory sources. The agreement between the two tests was determined on 271 field sera (kappa = 0.825). Their relative sensitivity (94.5 per cent) and specificity (99.4 per cent) were assessed on the 148 laboratory sera plus the 271 sera used for the determination of kappa.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, crystallization and preliminary X-ray diffraction studies of alpha-toxin of Clostridium perfringens.

Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C. The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

Induction of protective cytotoxic T cells with viral proteins.

Induction of CD8+, class I-restricted T cells by non-infectious, exogenous antigens has been documented for model protein antigens such as ovalbumin and for major histocompatibility complex restricted short peptides in viral and tumor systems. However, the protective capacity of cytotoxic T cells induced by conventional proteins has not been tested in vivo so far. We, therefore, evaluated the induction of protective cytotoxic T cells against three different full-length recombinant viral proteins derived from a baculovirus expression system, i.e. the glycoprotein and nucleoprotein of lymphocytic choriomeningitis virus (LCMV) and the nucleoprotein of vesicular stomatitis virus (VSV). These viral proteins induced cytotoxic T cells in a T helper cell-independent fashion which lysed infected target cells in vitro and protected mice from viral replication, immunopathological disease and growth of a tumor expressing the same antigen as a tumor antigen. These results are surprising, since it had been shown earlier for completely inactivated nonreplicating viral vaccines and again here for beta-propiolactone-inactivated VSV or UV-light inactivated LCMV that nonreplicating viral vaccines were incapable of inducing protective cytotoxic T cells. Our data show that immunization of mice with as little as 10 micrograms of non-infectious viral proteins triggered long-lasting CD8+ T cell-mediated antiviral immunity. It was found that the protein alone was only weakly able to induce cytotoxic T cells, and that association with cellular debris functioned as an adjuvant. These findings may be relevant for our understanding of the phenomenon of cross-priming and have obvious implications for vaccine strategies.

Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection.

Insect cells co-infected with appropriate recombinant baculoviruses synthesize double-shelled, virus-like particles (VLPs) with bluetongue virus (BTV) VP2 proteins representing serotype 1 (BTV-1), 2 (BTV-2), 10 (BTV-10), 13 (BTV-13) or 17 (BTV-17) as previously reported for BTV-10 (French, T.J., Marshall, J.J.A. and Roy, P. J. Virol. 1990, 64, 5696-5700). The derived particles were purified and used to vaccine sheep, either as single VLP types (BTV-10, BTV-17) or as a combination of all five serotypes. Control sheep received saline. The virus-neutralizing antibody responses were measured. Depending on the experiment, the sheep were challenged with homologous (BTV-10, -13, -17) or selected heterologous (BTV-4, -11, -16) viruses either after 4 months or 14 months, and the disease, viraemias and clinical reactions monitored. The results indicated that two doses of 10 micrograms of VLPs elicited a long-lasting immune response which protected the sheep against challenge with the homologous virulent virus. In certain cases, partial protection was afforded against challenge by heterologous BTV serotypes.

Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases.

The sequence of Rift Valley fever virus L segment that we published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA molecule. Here, we have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 negative-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called 'polymerase module', we identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented negative-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the 'polymerase module', we have determined the position of the conserved residues in these polymerases and discuss their possible functions in light of the available structural information.

Localization of T helper cell epitopes in the vesicular stomatitis virus: the nucleoprotein is responsible for serotype cross-reactive T help.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is known to contain the biologically relevant sites for virus-neutralizing antibodies as well as T helper (Th) cell epitopes. The capacity of other VSV proteins to elicit potent Th cell responses has not yet been investigated. Additionally, a short-lived cross-reactivity between the two serologically distinct VSV serotypes Indiana (IND) and New Jersey (NJ) on the T helper cell level has been reported. Here we address the question of whether the VSV nucleoprotein (N) or matrix protein (M) can elicit T help to VSV-G-specific B cells and which of the VSV proteins contains the elements responsible for the IND/NJ cross-reactivity. The N, G, and M of the VSV Indiana serotype produced in a recombinant baculovirus system were assayed for the ability to activate VSV-specific Th cells to induce immunoglobulin class switch of neutralizing antibody responses in vivo in C57BL/6 (H-2b) mice. All three VSV-IND proteins helped in the production of neutralizing IgG antibodies to the homologous VSV-Indiana serotype but only VSV-IND N was able to trigger an IgG response to the heterologous VSV-New Jersey serotype. This data suggest that Th epitope(s) in the VSV-IND N are responsible for the observed cross-reactivity of T helper cells.

Structures associated with the expression of rabies virus structural genes in insect cells.

When rabies virus structural genes were expressed in insect cells, major observed alterations were a high level of cytoplasmic vacuolization caused by the matrix protein M2 and the glycoprotein G. Ring-like structures, 16 nm in diameter, were observed in cell-free extracts from insect cells that expressed the N protein alone. Hexagonally shaped structures, 16-20 nm in diameter, and regular lattice aggregates of the same structures appeared on co-expression of N and M1 proteins. Co-expression of the four structural proteins led to the formation of cell surface blebs containing the structures corresponding to N and M1 proteins.

Analyses of alternative poly(A) signals for use in baculovirus expression vectors.

Synthetic, duplicated, or heterologous sequences encompassing putative 3' mRNA processing (polyadenylation, poly(A)) sites have been analyzed with regard to their involvement in processing mRNA species synthesized from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The data indicate that a synthetic sequence based on the 3' processing signal of the rabbit beta-globin gene is used more efficiently than an SV40-based, early gene poly(A) signal, or the AcNPV 1629 ORF poly(A) signal. Evidence is presented that indicates that the hexanucleotide AAUAAA alone is inadequate to effect mRNA processing. This supports the view that like other eukaryotic mRNAs additional signals are involved.

Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VP0, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein.

The cDNA sequences encoding the VP0, VP3, and VP1 structural proteins of poliovirus type 3 (strain P3/Leon/37) have been individually cloned and used to prepare recombinant baculoviruses based on Autographa californica nuclear polyhedrosis viruses (AcNPV). Expression of the proteins was monitored in virus-infected Spodoptera frugiperda cells. Both the individually expressed VP0 protein and VP0 derived from the complete poliovirus coding region were shown to incorporate myristic acid. A significant improvement in VP0 protein yield was obtained when the amino terminal glycine of VP0 was changed to alanine (VP0/ala), suggesting that the presence of glycine or its myristylation is unfavorable for VP0 expression. Even so, the expression levels of the poliovirus capsid proteins were low by comparison with those obtained for other foreign genes (e.g., lacZ). The reason does not appear to be due to protein instability or, from the studies undertaken with VP0, the sequences that flank the AUG codon. Using recombinant baculoviruses that express VP0, VP1, and VP3 (or VP0/ala, VP1, and VP3), poliovirus-like particles (VLPs) were isolated from infected S. frugiperda cells. Examination by electron microscopy of the VLPs purified by CsCl gradient centrifugation revealed structures corresponding in size, appearance, and antigenicity to those expected for poliovirus procapsids; however, the yields of particles were low when compared to those derived from a construct (AcLeon) that expresses the complete coding region of poliovirus type 3, indicating that procapsid synthesis from the P1 precursor is a more favored route.

Incidence of feline immunodeficiency virus reactive antibodies in free-ranging lions of the Kruger National Park and the Etosha National Park in southern Africa detected by recombinant FIV p24 antigen.

Lion sera from the Kruger National Park (KNP) dating back to 1977 and from the Etosha National Park (ENP), obtained from 1989 to 1991, have been analysed by ELISA and Western blot analyses using a genetically engineered antigen representing the p24 structural protein of feline immunodeficiency virus (FIV). It was concluded that some 83% of 98 KNP lion sera reacted with the p24 antigen, while none of 28 ENP lion sera reacted. A few other KNP felids (cheetahs and genets) gave samples that did not react with the FIV p24 antigen. For the KNP lions, apart from a lower prevalence in cubs (50%), no particular trends were demonstrated in terms of age, sex, date or origins of the samples. In Western blot and radio-immunoprecipitation analyses the lion sera reacted with the engineered p24 antigen, as well as with the p15 and p24 gag proteins and the p50 gag precursor protein from FIV, indicating that the agent is probably a lentivirus related to FIV. The ELISA with the engineered p24 antigen required less serum and appears to be more sensitive at detecting FIV-reactive antibodies than assays with available commercial kits.