The immune response to vaccinia virus is significantly reduced after scarification with TK- recombinants as compared to wild-type virus.

Although it is unlikely that large-scale vaccination against smallpox will ever be required again, it is conceivable that the need may arise to vaccinate against a human orthopoxvirus infection. A possible example could be the emergence of monkey poxvirus (MPV) as a significant human disease in Africa. Vaccinia virus (VV) recombinants, genetically modified to carry the immunogenic proteins of other pathogenic organisms, have potential use as vaccines against other diseases present in this region. The immune response to parental wild-type (wt) or recombinant VV was examined by binding and functional assays, relevant to protection: total IgG, IgG subclass profile, B5R gene product (gp42)-specific IgG, neutralizing antibodies and class 1-mediated cytotoxic lymphocyte activity. There was a substantial reduction in the immune response to VV after scarification with about 10(8) PFU of recombinant as compared to wt virus. These data suggest that to achieve the levels of immunity associated with protection against human orthopoxvirus infection, and to control a possible future outbreak of orthopoxvirus disease, the use of wt VV would be necessary.

Vaccinia virus recombinants encoding the truncated structural gene region of Venezuelan equine encephalitis virus (VEEV) give solid protection against peripheral challenge but only partial protection against airborne challenge with virulent VEEV.

Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 10(8) PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines.

Recombinant vaccinia viruses protect against Clostridium perfringens alpha-toxin.

Recombinant vaccinia viruses that expressed the nontoxic C-domain of Clostridium perfringens alpha-toxin were constructed. The J2R (thymidine kinase [TK] gene) and B13R (serpin 2 [SPI-2] gene) loci were used as insertion sites for the clostridial DNA, and expression of the foreign protein was measured in each case. A double recombinant that encoded the alpha-toxin truncate at the B13R locus and the protective antigen of Bacillus anthracis at the J2R locus was also constructed. Although differences in expression of the alpha-toxin C-domain were recorded, all of the vaccinia recombinants protected mice against a lethal challenge with alpha-toxin demonstrating that a recombinant vaccinia virus can be used to provide protection against a toxin challenge that is known to be solely antibody mediated.

Improved protection against Venezuelan equine encephalitis by genetic engineering of a recombinant vaccinia virus.

An improved vaccine is needed against Venezuelan equine encephalitis (VEE) virus because the existing live attenuated vaccine, TC-83, causes a high incidence of adverse effects, and the Formalin-inactivated vaccine, C-84, does not protect against airborne infection. A recombinant vaccine had previously been constructed in which the VEE structural proteins were expressed by vaccinia virus. Although protection against subcutaneous challenge with VEE was achieved, the vaccine had limited efficacy against aerosolized virus. We made a similar construct (WR100) and compared its performance with that of a recombinant vaccinia virus which had been altered in two ways (WR103) in order to improve its performance as a vaccine: a synthetic promoter was inserted upstream of the VEE coding sequence to increase the amount of VEE proteins produced, and a single nucleotide in the E2 glycoprotein gene was altered to enhance immunogenicity. The WR103 virus expressed greater amounts of VEE proteins on the surface of infected cells than did WR100, and this difference was found to correspond to a 3.5-fold increase in VEE protein production. Sera from mice immunized with WR103 contained elevated levels of antibody to VEE, and enhanced protection against subcutaneous challenge with the pathogenic Trinidad donkey strain was achieved. This altered construct could form the basis for a better vaccine against VEE.

Improved protection against Venezuelan equine encephalitis by genetic engineering of a recombinant vaccinia virus

An improved vaccine is needed against Venezuelan equine encephalitis (VEE) virus because the existing live attenuated vaccine, TC-83, causes a high incidence of adverse effects, and the Formalin-inactivated vaccine, C-84, does not protect against airborne infection. A recombinant vaccine had previously been constructed in which the VEE structural proteins were expressed by vaccinia virus. Although protection against subcutaneous challenge with VEE was achieved, the vaccine had limited efficacy against aerosolized virus. We made a similar construct (WR100) and compared its performance as a vaccine: a synthetic promoter was inserted upstream of the VEE coding sequence to increase the amount of VEE proteins produced, and a single nucleotide in the E2 glycoprotein gene was altered to enhance immunogenicity. The WR103 virus expressed greater amounts of VEE proteins on the surface of infected cells than did WR100, and this difference was production. Sera from mice immunized with WR103 contained elevated levels of antibody to VEE, and enhanced protection against subcutaneous challenge with the pathogenic Trinidad donkey strain was achieved. This altered construct could form the basis for a better vaccine against VEE.(Au)

Baculovirus replication: phosphorylation of polypeptides synthesized in Trichoplusia ni nuclear polyhedrosis virus-infected cells.

A number of polypeptides synthesized specifically in Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus (T. ni MNPV)-infected Spodoptera frugiperda cells are phosphorylated both early and late in infection. Certain non-structural proteins and the major basic internal protein are the main phosphoproteins detected in infected cells. The polyhedron protein was not phosphorylated. Many cell proteins continue to be phosphorylated throughout infection. Pulse-chase experiments have shown that some polypeptides are stably phosphorylated whereas other polypeptides (including the major basic protein) have phosphates which cycle on and off. One polypeptide was substantially labelled only after a chase with unlabelled orthophosphate. Fractionation of cells into nucleus and cytoplasm showed that polypeptides located in both the cytoplasm and nucleus were phosphorylated.

Baculovirus replication: inhibition of Trichoplusia ni multiple nuclear polyhedrosis virus by [E]-5-(2-bromovinyl)-2'-deoxyuridine.

[E]-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) inhibits the replication of the baculovirus Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus in Spodoptera frugiperda cells. Virus-specific DNA synthesis and late protein synthesis are suppressed by the drug. BVdU is phosphorylated by deoxythymidine (deoxycytidine) kinase present in both uninfected and virus-infected cells, and in its 5'-triphosphate form it inhibits DNA polymerase activity in virus-infected cells. The effect of the BVdU is not completely reversible. Phosphonoacetic acid, phosphonoformic acid and Acyclovir have no effect on baculovirus replication. Acyclovir fails to compete with deoxycytidine and thymidine as substrates for pyrimidine deoxynucleoside kinase in virus-infected and uninfected cells.

Isolation of a bisegmented double-stranded RNA virus from Thirlmere reservoir.

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 X 10(6) and 2.09 X 10(6). The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.

Induction of a nonoccluded baculovirus persistently infecting Heliothis zea cells by Heliothis armigera and Trichoplusia ni nuclear polyhedrosis viruses.

A nonoccluded singly enveloped baculovirus (baculovirus X) persistently infects Heliothis zea (IMC-HZ-1) cells in culture. Singly enveloped nuclear polyhedrosis viruses from H. zea and Heliothis armigera, and multiply enveloped nuclear polyhedrosis viruses from Trichoplusia ni, Spodoptera frugiperda, and Spodoptera littoralis were all found to induce baculovirus X. Experiments are reported which use metabolic inhibitors and inactivated inducing virus to show that it is probable that a structural component of the virus, most likely a protein, is responsible for inducing baculovirus X. The persistent virus is induced to replicate by uv-inactivated virus but not by heat-inactivated inducing virus. The virus is not induced to replicate by a number of metabolic inhibitors in the absence of an inducing virus. Inhibition of transcription and translation prevents the induction of the persistent virus by an inducing virus. Inhibition of DNA replication has no effect on the induction of the virus. This suggests that the persistent virus genome is present in abundance in all cells.

The replication and titration of iridescent virus type 22 in Spodoptera frugiperda cells.

A plaque assay for iridescent virus type 22 (from Simulium sp.) using Spodoptera frugiperda cells has been devised, and the kinetics of growth of the virus in this cell line have been determined. The virus particle/p.f.u. ratio was 75 +/- 8, and the p.f.u./TCID50 ratio was 0.56 +/- 0.11.

The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus in cultured Spodoptera frugiperda cells.

The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus have been determined. Two inhibitors of protein synthesis-cycloheximide and puromycin-were irreversible inhibitors of virus multiplication. Three inhibitors of nucleic acid synthesis-actinomycin D, cytosine arabinoside and camptothecin- prevented virus multiplication; only camptothecin was reversible. Rifampicin had no effect on virus multiplication.