- Volume 68, Issue 10, 1987

A gene coding for insulin-like growth factor II (IGF II) was constructed from 16 oligodeoxynucleotides synthesized chemically and cloned into EcoRI-SalI sites of pBR322. In this gene an ATG codon for methionine was introduced for cleavage by CNBr at the beginning of mature IGF II. For expressing foreign genes, a new host- vector system, with Bombyx mori silkworm larvae as the host and B. mori nuclear polyhedrosis virus (BmNPV) as the vector, has been developed. BmNPV genomic DNA codes polyhedrin which is a major protein of inclusion bodies and is mass- produced in infected silkworm larvae. We employed this polyhedrin production system to obtain a large yield of a foreign gene product. The coding region of the carboxy- terminal half of polyhedrin was removed and the remainder was ligated with the IGF II gene in phase to create a fusion protein gene consisting of the coding region of the amino-terminal half of polyhedrin and the IGF II gene. This fusion protein gene was combined in a plasmid with the promoter and 5′ and 3′ flanking regions of the polyhedrin gene. The resulting plasmid and the wild-type BmNPV genomic DNA were cotransfected into BM-N cells, and a recombinant virus was isolated by the limiting dilution method. The silkworm larvae infected with the recombinant virus produced 3·6 mg of the fusion protein per larva and the infected BM-N cells produced 0·3 mg per ml of culture. IGF II was released from the fusion protein produced by BM-N cells infected with the recombinant virus by CNBr treatment, purified by extraction with guanidine-HCl, column chromatography and HPLC and the correct amino-terminal amino acid sequence confirmed.

The coding sequences of the hepatitis B virus surface antigen were inserted into a baculovirus transfer vector produced from Autographa califomica nuclear polyhedrosis virus (AcNPV) so that the foreign gene was under the control of the AcNPV polyhedrin promoter. Spodoptera frugiperda cells infected with the derived recombinant baculovirus produced and secreted 22 nm particles containing the hepatitis B surface antigen. The particles had morphological and antigenic properties identical to those of 22 nm particles isolated from the plasma of chronic active hepatitis patients.

Protein A-gold labelling was used to detect and locate particles and two major late antigens, polyhedrin and pl0, of Autographa californica multiply-enveloped nuclear polyhedrosis virus in thin sections of Spodoptera frugiperda cells. With polyhedrin antiserum, gold label was found over viral occlusion bodies (polyhedra), but not over the fibrous structures associated with polyhedron morphogenesis and previously thought to consist of precondensed polyhedrin. In contrast, with pl0 antiserum, gold label was found only over these fibrous structures in the nucleus and similar structures in the cytoplasm. This suggests that pl0 is a major constituent of the fibrous structures and may, therefore, play an important role in polyhedron morphogenesis.

Sigma virus, the hereditary agent of a CO2-induced paralysis of Drosophila, is classified as a rhabdovirus on a molecular basis. We have purified its genome which after 32P-labelling was used as a probe to detect mRNAs in infected cells. A cDNA copy of the entire coding region of the glycoprotein mRN A was cloned. Nucleotide and deduced amino acid sequences were determined and compared to previously known sequences of other rhabdovirus glycoproteins to determine the relatedness of Sigma virus to other viruses of this group.

The peplomer gene of feline infectious peritonitis virus (FIPV) strain 79-1146 was isolated from a genomic cDNA library by differential hybridization with RNA 2 and 3 as probes. From the nucleotide sequence a primary translation product of 1452 residues (Mr 160472) was predicted, containing an N-terminal signal sequence, a C-terminal transmembrane segment and 35 potential N-linked glycosylation sites. By SI nuclease analysis the 5′ end of the presumptive RNA 2 body was located at about 30 nucleotides upstream from the initiating AUG codon. At approximately the same position a nine nucleotide sequence ACUAAACUU was found, which was also present 37 nucleotides downstream from the open reading frame. Comparison of the sequences of the FIPV, murine hepatitis virus and infectious bronchitis virus peplomer proteins showed about 27 % overall homology, with most conservation in the C-terminal half.

We previously showed that treatment of different cell lines with interferon-α (IFN-α) concurrently inhibited both cell growth and the rise observed in 125I-labelled transferrin binding when cells are exposed to culture conditions that stimulate proliferation. To gain insight into the relationship between these two IFN-induced inhibitory processes, we investigated the effect of IFN-α on the binding of125I-labelled transferrin to Daudi cells sensitive or resistant to its antiproliferative action. We found a close correlation between the ability of IFN-α to inhibit cell growth and to inhibit transferrin receptor expression. Since growth inhibition induced by other agents is not always accompanied by an inhibition of transferrin receptor expression, the previous and present observations suggest that the inhibitory effect of IFN on this expression is at least one of the mechanisms by which IFN inhibits cell proliferation. We also observed that IFN-α did not modify transferrin receptor biosynthesis in IFN-sensitive Daudi cells, suggesting that IFN-α may change the processing of the transferrin receptor molecules, making them unable to bind transferrin.

Murine T cell clones specific for the Kunjin (KUN), West Nile (WN) and Murray Valley encephalitis (MVE) flaviviruses were generated in vitro following priming in vivo. Clones were isolated by limiting dilution and maintained in culture with antigen stimulation and interleukin-2 (IL-2). The cells were characterized as having the Thy 1+, L3T4+ and Lyt 2− phenotype by immunofluorescence. All clones proliferated strongly and secreted high levels of IL-2 and IL-3 in response to homologous antigen. Both KUN- and WN-specific clones showed extensive cross-reactivity to KUN and WN antigen, but recognized MVE to a lesser extent. In contrast, MVE-specific clones cross-reacted strongly with both KUN and WN. These data show that antigen-specific, major histocompatibility complex-restricted L3T4+ T cells are generated during flavivirus infection and are cross-reactive for viruses of the same subgroup.

Treatment of adult mice with gold sodium thiomalate made the normally non-lethal Semliki Forest virus and Sindbis virus infections lethal and increased the virulence of Langat and West Nile viruses. These changes were associated with an enhanced virus invasion of the brain. Depression of the humoral antibody response was not observed.

Ectromelia virus-specific cytotoxic T (Tc) cell precursors were present in the draining popliteal lymph node of all strains of mice tested at 2 to 3 days after footpad inoculation of a high dose (105 p.f.u.) of the virulent Moscow strain of ectromelia virus. To detect this response it was necessary to culture lymph node cells from infected mice in the presence of T cell growth factors and to use the more sensitive neutral red assay for measuring cytotoxicity. Cells with lytic activity were virus-specific, major histocompatibility complex-restricted Tc cells. C57BL/6J resistant mice, which express a single dominant gene conferring innate resistance had virus-specific Tc cell precursors 1 to 2 days sooner than did susceptible BALB/b mice. This Tc cell-mediated immune response early after infection could account for the barrier to virus dissemination known to operate 1 to 2 days after infection to slow virus passage into the lymphoreticular system.

We have recently reported that adenovirus replication is inhibited by human recombinant interferon-γ, but not by recombinant interferon-α, in a dose-dependent manner. The aim of this study was to determine whether the antiviral effect of recombinant interferon-γ could be linked to interferon-induced alteration at the membrane level, inhibiting either adenovirus penetration of or release from WISH cells. Adsorption and penetration were investigated with an 125I-labelled adenovirus binding assay. To test defective virus release, the presence of newly synthesized virus proteins in the cytoplasmic and nuclear compartments was investigated. Binding studies showed that interferons-γ and -α did not modify adenovirus attachment and penetration. Interferon-γ but not interferon-α inhibited hexon protein synthesis in the cytosol as well as its accumulation in the nuclear compartment. The synthesis of polypeptides III, IV and VI was also inhibited. In cells infected before interferon-γ treatment, its addition could be delayed up to 2 h after the infection to produce an inhibition of virus yield greater than 1 log10 unit (90 % inhibition). We conclude that interferon-γ acts on an intracellular step before or at adenovirus protein synthesis, probably through a mechanism not shared with interferon-α.

Neonatal ferrets are protected against infection with influenza virus by colostral and milk-derived anti-influenza virus IgG after suckling on an immune mother. The levels of IgG elicited and then transmitted to neonates were similar when mothers were immunized by either live infection or killed vaccines. Maternal anti-influenza virus IgA and IgM appears not to cross the neonatal gut epithelium although both are present in maternal serum and milk.

Synthetic peptides corresponding to six predicted immunogenic sites on human rhinovirus type 2 (HRV2) have been tested for their reactivity with an anti-virion antibody and for their ability to elicit neutralizing antibody. Four of the peptides reacted with HRV2 antiserum in an indirect ELISA. Rabbit antisera produced to three of these four peptides, one each from VP1, VP2 and VP3, reacted with the virus in an indirect ELISA and with the corresponding proteins by Western blotting. Furthermore, antiserum to one of the peptides, designed to cover the neutralization epitope NIm-II on VP2, not only reacted well in a sandwich ELISA and in an immunoprecipitation test but also neutralized virus infectivity.

A murine model is described in which recrudescence of herpes simplex virus (HSV) type 1 was achieved. C3H mice were shaved and irradiated with u.v. B light 3 days before being infected epidermally with a clinical isolate of HSV. Seven weeks or longer following the primary infection, the survivors were again shaved, irradiated with u.v. and mildly tape-stripped. Recrudescent lesions occurred in up to 80 % of mice at the site of the original lesion in most cases, but also occasionally at other sites. Skin painting with u.v.-irradiated urocanic acid (a substance suggested to be a photomediator of the immunosuppressive effects of u.v.) in place of u.v.-irradiation induced some recrudescence but was not as efficient as u.v.-irradiation. Antibody titres to HSV had no value in predicting whether recrudescence would occur but lymphoproliferative responses in draining lymph nodes may provide some indication of viral activity at the epidermal site. A hypothesis is developed that u.v.-irradiation before primary infection with HSV induces a suppressive immune response to the virus which affects the virus-host interaction and accounts for a high incidence of recrudescent lesions on subsequent stimulus.

Previous work has shown that a novel protein kinase is induced after infection of cultured cells with herpes simplex virus type 1 (HSV-1). Separately, it has been reported that the protein encoded by HSV-1 gene US3 shows similarity in its amino acid sequence to members of the protein kinase family of eukaryotes. We have investigated the possibility that these two observations are connected by preparing an antiserum to a synthetic oligopeptide corresponding to the carboxy-terminal eight amino acids of the US3 protein. This antiserum reacted on immunoblots with a polypeptide of apparent molecular weight 68000 from extracts of cells which had been infected with HSV-1. The antiserum also reacted strongly with a 68000 molecular weight species from a preparation of the novel HSV-1 protein kinase which had been extensively purified and resolved from other protein kinases. In addition, the purified preparation phosphory-lated a protein species, also of 68000 apparent molecular weight, when incubated with [γ-32p]ATP. These data are consistent with gene US3 encoding the novel protein kinase induced after infection of cells with HSV-1.

We prepared vaccinia virus variants by introducing part of the HindIII D fragment of the DNA of the parental Lister (LO) strain (temperature-resistant and forming large plaques and pocks) into the attenuated LC16m8 strain (temperature-sensitive and forming small plaques and pocks) by the use of a homologous recombination technique in vivo. Special attention was paid to the HindIII D fragment, since this fragment has an extra XhoI site in LC16m8 which is absent from LO. After HindIII D of LO was introduced as a calcium phosphate precipitate into rabbit kidney (RK13) cells which had been infected with LC16m8, five virus variants (LOTC-1 to LOTC-5) forming much larger plaques than LC16m8 were cloned. In LOTC-2, LOTC-4 and LOTC-5, the introduction of at least part of HindIII D of LO into the corresponding HindIII D region of the LC16m8 genome was apparent as judged by the disappearance of the XhoI site, whereas variants LOTC-1 and LOTC-3 retained the site. The biological characteristics of all the LOTC variants were similar to each other. Their plaque size and pock size were similar to those of LO, whereas they were rather akin to LC16m8 with regard to temperature sensitivity and neurovirulence. The present results strongly suggested that part of the HindIII D fragment was involved in determining biological characteristics affecting plaque size and pock size, but had little influence on temperature sensitivity and neurovirulence.

Sinc, with two alleles p7 and s7, is the major gene determining the incubation period of all strains of scrapie in mice. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and we have used a cDN A copy of the hamster PrP mRNA to carry out restriction fragment length polymorphism (RFLP) analysis of different inbred mouse strains including VM(Sinc p7) and VM(Sinc s7) congenic mice. In VM(Sinc p7) mice, a 5·5 kb XbaI fragment hybridized to the PrP cDNA sequence whereas VM(Sinc s7) congenic mice had a 3·8 kb XbaI fragment. The VM × VM (Sinc s7) congenic F1 mice had both the 5·5 kb and the 3·8 kb fragments. The Sinc s7 donor mouse strain, C57BL, had the 3·8 kb fragment suggesting that the Sinc gene and the gene coding for PrP are linked, and could even be the same gene. Other Sinc p7 inbred mice (IM and MB) had the 5·5 kb fragment but so too did some Sinc s7 strains (RIII and VL), implying that the XbaI site polymorphism is not functionally involved in the difference between the two Sinc alleles. We have mapped the polymorphic XbaI site to the 3′ flanking region of the PrP gene. TaqI and HhaI were also found to show polymorphisms in the inbred mouse strains studied. The apparent RFLP with HhaI may be a result of differences in methylation rather than in sequence.

The physical map of the genome (209 kbp) of insect iridescent virus type 6, also known as Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, was constructed for the restriction enzymes ApaI, Asp718, PvuII and SphI using a gene library of viral DN A containing the complete viral genome. Although the CIV genome is linear, it was found that the restriction maps were circular, because of the unique structure of the genome of this virus.