- Volume 67, Issue 4, 1986

The American Society for Virology Fifth Annual Scientific Meeting. University of California, Santa Barbara, CA 93102, U.S.A., June 22–26, 1986. The American Society for Virology will hold its 5th Annual Scientific Meeting on 22–26 June 1986 at the University of California, Santa Barbara, CA 93102.

Symposia and Workshops are scheduled to cover current research on animal, bacterial, insect and plant viruses, including molecular, genetic, immunological and medical aspects.

The American Society for Virology seeks to advance and promulgate knowledge relevant to all fields of virology. Membership is open to virologists worldwide.

For further information, address inquiries to: Sidney E. Grossberg, M.D.; Secretary-Treasurer; American Society for Virology; Department of Microbiology; Medical College of Wisconsin; 8701 Watertown Plank Road; Milwaukee, Wisconsin 53226, U.S.A.

International Symposium on Medical Virology. Disneyland Hotel, Anaheim, California, U.S.A., November 12–14, 1986. Sponsor: Medical Microbiology Division, Department of Pathology, University of California, Irvine, Medical Center

For information contact: Dr Luis M. de la Maza; Department of Pathology, Route 84; University of California, Irvine, Medical Center; 101 City Drive South Orange, California 92668; Tel: (714) 634-6868

Double-Stranded Rna Virus Symposium. Mansfield College, Oxford, U.K., September 9–13, 1986. The Symposium will include sessions covering the molecular biology and genetics as well as the biology, epidemiology and pathology of all double-stranded RNA-containing viruses of animals, plants, fungi/yeasts and bacteria.

For further information, please contact:

Symposium Organizer. D. H. L. Bishop; Natural Environment Research Council; Institute of Virology; Mansfield Road; Oxford OX1 3SR United Kingdom

Programme Committee. Bernard N. Fields; Dept. of Microbiology and Molecular Genetics Harvard Medical School; Boston, MA 02115, U.S.A.

W. K. Joklik Dept. of Microbiology and Immunology Duke University Medical Center Durham, NC 27710, U.S.A.

Aaron J. Shatkin Dept. of Cell Biology Roche Institute of Molecular Biology Nutley, NJ 07110, U.S.A.

The genetics of natural resistance to the development of latent infection in the trigeminal ganglia of mice inoculated in the lip with herpes simplex virus type 1 (HSV-1) was examined. Based on coefficients of a logistic regression relating latency to strain and HSV-1 concentration, inbred strains of mice formed a continuum of resistance ranging from most resistant (C57BL/6J) to most susceptible (PL/J). When these results were analysed along with latency data derived from studies employing a non-fatal concentration of HSV-1, three subpopulations were identified among these strains: resistant (C57BL/10J, BALB/cByJ, C57BL/6J), moderately resistant (DBA/2J, SWR/J, A/J, AKR/J, DBA/1J) and susceptible (PL/J, LP/J, CBA/J). Results from F1 hybrids between resistant and moderately resistant strains (B6D2F1/J, B6AF1/J) and between resistant and susceptible strains [(C57BL/6J × CBA/J)F1, (C57BL/6J × LP/J)F1)] indicated that resistance is dominant. Data from both inbred and congenic strains failed to show an association between H-2 and resistance to the development of a latent infection. Studies of mortality also indicated that a continuum was present, with C57BL/10J, C57BL/6J and DBA/1J being most resistant and PL/J mice most susceptible. When inbred strains were categorized on the basis of resistance to the development of latent infection and mortality, five groups could be identified. Group A are strains resistant to both mortality and latency (C57BL/6J, C57BL/10J, DBA/1J) while group B consists of one strain (BALB/cByJ) intermediate in resistance to mortality but resistant to latency. Group C are strains intermediate in resistance to mortality and susceptible to latency (LP/J, CBA/J) while Group D are strains susceptible to mortality and intermediate in susceptibility to latency (AKR/J, SWR/J, DBA/2J). Group E consists of one strain (PL/J) susceptible to both mortality and latency. These results indicate that host factors play an important role in the establishment of latent infection in vivo.

The aim of this study was to establish whether cytotoxic T cells (Tc), raised against respiratory syncytial virus (RSV) in the mouse, are specific to the strain of immunizing virus, or cross-reactive between virus strains. Several recent studies using monoclonal antibodies have begun to define the antigenic variation among strains of RSV. It is likely that a successful RSV vaccine will need to contain antigenic determinants from more than one subtype, but since only the highest levels of neutralizing antibody are able to give complete protection against RSV infection, a vaccine may also need to elicit a cellular immune response. We have recently described H-2-restricted, RSV-specific Tc following RSV infection in the mouse; we present here evidence that polyclonal RSV-specific Tc in the mouse recognize syngeneic target cells infected with every human strain of RSV tested, whatever the subtype. The only RSV strain that appeared not to be recognized was bovine RSV, which seems unable to infect mouse cells; however, bovine cells, infected with bovine RSV and fixed with glutaraldehyde, primed mice for Tc which recognized human strains of RSV.

The virulence of different vaccine and wild-type yellow fever (YF) viruses for young adult mice was compared using both intracerebral and intranasal routes of administration. Thirty-five different YF viruses killed mice within 12 days following intracerebral inoculation. In contrast, only seven of those examined killed mice following intranasal administration; these were Asibi virus (YF-AS), the French neurotropic vaccine, two out of three 17DD vaccine substrain viruses (Brazil, Colombia but not Dakar) and three out of six wild-type isolates (YF-B7, YF-B12 and YF-B15). None of eight distinct preparations from the 17D-204 vaccine substrain was virulent by the intranasal route. Thus, strains of YF virus can be distinguished on the basis of their virulence for mice if the intranasal route is used. Evidence of heterogeneity in mouse virulence within the populations was obtained using large and small plaques selected from wild-type stocks of virus. Following intranasal inoculation, a YF-AS small plaque variant was more virulent than either its parent virus or a large plaque variant. On the other hand, a large and a small plaque variant from a non-virulent wild-type strain could not be distinguished in these tests.

A monoclonal radioimmunoassay (RIA) was developed for detection of dengue virus in infected cell culture fluids and blood samples from dengue patients. Antibodies used to construct the RIA were selected on the basis of high binding avidity, the demonstration of synergism in competitive binding assays and empirical trials with different antibody combinations. Optimal binding of all four dengue virus serotypes was achieved by use of a flavivirus group-reactive and a dengue virus complex-reactive antibody attached to a solid-phase support and a single flavivirus subgroup-reactive antibody as radiolabelled probe. A ‘simultaneous sandwich’ format and prolonged (18 h) incubation at 37 °C yielded optimal results. The limit of sensitivity of the RIA for detection of dengue type 2 virus was 2.7 log10 mosquito 50% infectious doses (MID50). The assay was tenfold more sensitive for dengue type 2 than for dengue types 1 and 3 viruses and 100-fold more sensitive than for dengue type 4 virus. Specificity, assessed using over 500 disease control human sera, was increased by addition of monoclonal anti-tetanus blocking antibodies, resulting in a false positive rate of only 0.2%. Heterologous dengue virus antibodies were shown to inhibit the RIA in assays performed with artificial immune complexes. Acute phase human sera containing 104.2 to 107.6 MID50 but no detectable antigen by RIA, were also shown to inhibit binding of the homologous dengue virus serotype; this effect was attributed to heterologous antibody from a prior infection. Among 116 viraemic sera from dengue patients, the RIA was positive in 43 to 47% of patients with dengue type 1, 2 or 3 infections but in only 10% of the dengue type 4 cases. Virus was more frequently detected in cases of primary infection (54%) than in cases of superinfection (16%). Despite the limitations imposed by immunological interference, the antigen capture RIA appears useful as a rapid diagnostic technique for dengue surveillance.

Interferon (α, β and γ) susceptibility was tested in a human cell line, UVr-1, a u.v. light-resistant variant of RSa cells; the latter have high sensitivity to both u.v. lethality and the cell profileration inhibition (anticellular) effect of human interferon (HuIFN) preparations. UVr-1 cells were less sensitive than the parental RSa cells to the inhibitory effects of HuIFN preparations, as measured by cell proliferation and the incorporation of [3H]deoxythymidine and [3H]deoxyadenosine into acid-insoluble cellular material. Nevertheless, UVr-1 cells exposed to HuIFN showed almost the same enhanced levels of antiviral activity and pppA(2′p5′A)n synthetase activity as similarly treated RSa cells. Further, UVr-1 cells had much the same binding capacity for 125I-labelled HuIFN-αA. Thus, it seems likely that the variant has an increased resistance to the anticellular effect but not to the antiviral effect of HuIFN preparations. UVr-1 cells showed no significant difference from RSa cells in u.v.-induced DNA repair synthesis. However, when a comparison was made between the susceptibility of normal fibroblasts and fibroblasts from patients with Cockayne's syndrome, characterized by an altered u.v. sensitivity but no alteration of DNA repair replication synthesis, the Cockayne's syndrome fibroblasts, CCK-3 and CCK-4, were more susceptible to HuIFN-β as judged by cell proliferation and deoxythymidine incorporation tests.

Three clones of Daudi cells, DIF3, DIF8 and DIF9, have been isolated which possess specific interferon (IFN) receptors even though these cells are resistant to both the antiviral and antiproliferative actions of human IFN-α. Studies with 125I-labelled cloned human IFN-α subspecies showed that clone DIF3 bound IFN in a manner identical to that of the IFN-sensitive parental cells. The initial peak of binding observed 30 min after treatment of either Daudi or DIF3 cells at 37 °C with IFN-α2 or IFN-α8 was, however, either absent or much reduced when using DIF8 cells which are resistant to the antiproliferative action of IFN-α2 and -α8. Similarly, no initial peak of binding was observed after treatment of DIF9 cells with IFN-α2 at 37 °C. In accord with its reduced biological activity, the binding of 125I-IFN-α1 to either Daudi or DIF8 cells was considerably lower than that of either of the other two IFN-α subspecies and followed the form of a hyperbola without an initial transitory peak of binding. It is suggested that the phenotype of IFN resistance of DIF8 and DIF9 cells may be related to a defect in receptor activation whereas the same phenotype of DIF3 cells would appear to be unrelated to IFN binding.

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.

Cognate genes of members of the Palyam serogroup of orbiviruses have been identified previously, and their relatedness to the prototype virus was determined by blot hybridization of the genome segments of members of the serogroup using Palyam genomic RNA and isolated Palyam RNA segments as probes. In this study, the genetic relatedness of nine Palyam serogroup isolates was determined by reciprocal blot hybridizations of genomic RNA from each virus to the segments of all members of the group. The number and identity of highly related genes varied between isolates. For example, CSIRO Village and Palyam were related in genes 2 and 6, while Bunyip Creek and Vellore were related in genes 2 and 6. However, CSIRO Village and Bunyip Creek were highly related to D'Aguilar in all genes except 2 and 6, suggesting that there may have been genetic reassortment of Palyam serogroup dsRNA segments. Genes 2 and 6 were correlated consistently with serotype specificity. Genes 5, 7 and 9 were highly related among all members of the group. The Indian strains, Palyam and Vellore, were highly related in genes 1, 3 and 8, and they exhibited weak homology to genes 1, 3 and 8 of the Australian and African strains. However, one Indian isolate, Kasba, was more closely related to strains from Africa and Australia than it was to other Indian strains. There was little evidence which indicated that geography was predictive of the genetic relationships of the strains. Thus, immunological pressure may be the most important factor affecting the Palyam serogroup gene pool.

A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine strains of all three serotypes of poliovirus. The antigenic and molecular evolution of the Sabin strain of poliovirus type 3 was then followed throughout the entire period of virus excretion. Novel strains appeared at 8, 42 and 52 days post-vaccination and were the products of both intertypic recombination between type 2 and type 3 poliovirus in regions of the genome coding for non-structural proteins and of point mutations in the region coding for the structural proteins. Excretion of virus continued for 73 days. All strains examined reacted with all monoclonal antibodies specific for the main immunodominant antigenic site of type 3 poliovirus, but variation was observed at other, immunorecessive sites. These findings have possible implications for the evolution of the virus in vaccinees or in epidemics and are consistent with the known antigenic stability of the virus.

The monovalent ionophore monensin, which interferes with cellular transport pathways, and the antibiotic tunicamycin, which prevents glycosylation of newly synthesized proteins, were used to examine Hantaan virus particle formation and polypeptide synthesis. Viral replication in the presence of either drug resulted in reduced antigen production as well as reduced yields of both intracellular and extracellular infectious virus. Analysis of viral polypeptides synthesized in the presence of the drugs suggested differential effects of monensin and tunicamycin on Hantaan virus. Although reduced levels of the three major structural proteins were detected with increasing concentrations of monensin, the electrophoretic migrations of the polypeptides synthesized were unaltered. In contrast, after tunicamycin treatment, G1 was no longer detectable and G2 displayed both a quantitative reduction and an apparent molecular weight reduction of approximately 3000. Both G1 and G2 were sensitive to endoglycosidases H and F with resultant electrophoretic mobility shifts corresponding to molecular weights of approximately 7000 for G1 and 3000 for G2. Oligosaccharides appeared to be mostly, but not entirely, of the high-mannose type.

A recombinant vaccinia virus (NP-VAC) containing cDNA corresponding to segment 5, the nucleoprotein (NP) gene of influenza A/PR/8/34 virus was used to examine the specificity of human influenza virus immune cytotoxic T lymphocytes (CTL). Effector cell preparations from two donors recognized autologous lymphocytes that had been infected with NP-VAC. Lysis was specific because cells infected with vaccinia virus were not killed and recognition was HLA-restricted. In one donor, the influenza virus-specific CTL response changed with time so that his effector cells no longer recognized autologous lymphocytes infected with NP-VAC. However, a component that was NP-specific remained because these CTL lysed the more sensitive autologous B lymphoblastoid cells that had been infected with NP-VAC. In four other donors, no NP-specific CTL response could be detected using autologous lymphocyte targets. Thus NP, an internal virus protein, is one antigen that is recognized by human influenza A virus-specific CTL, but it is likely that other individual virus components contribute to the total CTL response.

Extracts from herpes simplex virus type 2 (HSV-2)-infected cells were subjected to affinity chromatography with gel-bound Helix pomatia lectin (HPA). Only one HSV-2-specified glycoprotein was isolated by this procedure and the glycoprotein had an apparent molecular weight of 130000 (130K). The HPA-binding glycoprotein was genetically mapped, using HSV-1 × HSV-2 intertypic recombinants into the short component of the HSV-2 genome. The mapping position, electrophoretic mobility and the antigenic properties of the HPA-binding protein indicated that it was unrelated to glycoprotein C (gC), which is the HPA-binding glycoprotein in HSV-1-infected cells, and distinct from gE and gD which map in the S component. The glycoprotein was almost quantitatively precipitated by monoclonal antibody AP1, specific for glycoprotein g92K and it also reacted with monoclonal antibody 1206-3, specific for the HSV-2 glycoprotein G previously described. It is concluded that the isolated glycoprotein is identical to g92K and consequently also to the HSV-2-specific glycoprotein G.

Gene US4 of herpes simplex virus type 1 (HSV-1) has been predicted, from DNA sequence analysis, to encode a protein of molecular weight 25237 and its properties suggest it to be a membrane-associated protein. We have investigated this protein by raising antiserum to a synthetic oligopeptide corresponding to a stretch of amino acids from an internal hydrophilic region of the predicted sequence. This antiserum immunoprecipitates three glycoprotein species of apparent mol. wt. 37000, 48000 and 56000 from extracts of cells infected with HSV-1. These species are also specifically immunoprecipitated from purified virions. The in vitro translation product of gene US4 has an apparent mol. wt. of 35000. Sequence comparisons of the short unique regions of the HSV-1 and HSV-2 genomes, in combination with published mapping data for glycoprotein G (gG) of HSV-2, has led to the conclusion that the product of gene US4 of HSV-1 is the equivalent of gG.

The nucleotide sequence of the coding region of the thymidine kinase gene from each of three mutant strains of herpes simplex virus type 1 and from the parental strain, SC16, has been determined. The mutants were known to express thymidine kinase enzymes with distinct substrate binding properties. Consideration of the lesions in the genes responsible for these altered biochemical properties has led us to postulate a preliminary model for the active centre of the enzyme, involving the cooperation of three distinct regions of the polypeptide.