- Volume 67, Issue 5, 1986

For some time, the Editors of The Journal of General Virology have sought a format for presenting accounts of advances and notable events in virology, in a broader and more flexible manner than is afforded by the traditional review of a topic in depth. This article represents an experiment in such presentation. Its aim is to look at advances in virus research, and to present brief accounts of the most important and interesting work published within a calendar year. Subject to evaluation of the success of this first essay, an annual feature is envisaged.

Implementation of these fine notions, however, has demanded some compromises. Virology comprises such an awesome range of techniques and objectives, with such a disparate assembly of virus types, that rigorous limits were necessary. Thus, we have confined our attention to animal viruses, and explicitly avoided any pretension to constructing a systematic review of all virology; the idea of selectivity was pre-eminent.

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

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

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.

Four bacteriophages (A16, CK235, φ1.2 and K31) which specifically attack different encapsulated strains of Escherichia coli have been shown to be related to bacteriophage T7 (which is unable to grow on encapsulated hosts). The conclusion that phages A16 and CK235 are related to T7 is based on (i) similarities in the pattern of expression of intracellular phage proteins, (ii) early appearance, in infected host cells, of a phage DNA-specific RNA polymerase and (iii) hybridization (albeit to a low extent) of A16 DNA and of CK235 DNA to T7 DNA. The first two criteria also apply to phages φ1.2 and K31 but hybridization of their DNAs with T7 DNA could not be detected. The RNA polymerases of CK235 and A16 have similar template specificities and the same applies to the RNA polymerases of φ1.2 and K31. None of the new RNA polymerases can use T7 DNA as template.

Intranasal administration of defective interfering (DI) influenza virus (A/WSN) ensured the survival of 80% of C3H mice otherwise lethally infected with WSN by the intranasal route, whereas a control group which received β-propiolactone-inactivated DI virus in place of DI virus died at 7.4 days post-infection. DI virus-treated mice developed significantly less lung consolidation than controls although qualitatively the cellular pathology in the two groups was indistinguishable. Surprisingly, in view of the accepted mode of action of DI virus interference, multiplication of infectious virus in the lung, production of viral haemagglutinin (HA) antigen and neuraminidase, and the distribution and amount of viral antigen in cells as shown by immune labelling were unaffected by the presence of active DI virus. Furthermore, assays of lung extracts showed that DI virus was not stimulating significantly greater amounts of interferon than the control inactivated DI virus. An alternative explanation arises from the fact that the pathology of influenza in inbred mice is immune (T lymphocyte)-mediated. Thus, since there is no evidence that DI virus affects virus multiplication we suggest that DI virus is responsible for ameliorating the damaging host responses. Another aspect of the immune response modulated by DI virus was the enhancement of local haemagglutination-inhibiting (HI) antibody in the lung, with peak increases of up to 10-fold over the relevant controls being demonstrated at 5 days after infection. This antibody was presumably complexed to HA antigen in the lung as activity was only demonstrated after elution at low pH. It had no detectable neutralizing activity (< 10% HI: neutralization ratio of convalescent serum) which accounts for the coexistence of local antibody and virus infectivity. Mice infected with virus alone or which received β-propiolactone-inactivated DI virus in addition to a lethal dose of WSN did not develop significant amounts of lung antibody. No differences were seen in serum HI titres. The increased level of antibody could not be attributed to the presence of greater amounts of HA antigen in lungs of mice treated with DI virus, as ELISA showed no significant difference from control preparations. The possibility that the two modulated immune responses are linked through HI antibody blocking access of T cells to cell membrane-borne HA antigen is discussed.

Ten of 12 Chandipura virus tdCE mutants, which exhibit temperature-dependent restriction of growth in chick embryo (CE) cells but not in BS-C-1 cells, showed deficient transcriptase activity in vitro at 39 °C relative to wild-type virus. A gradation in transcriptional activity at 39 °C in vitro was observed. Reversion of the tdCE phenotype to unrestricted growth in CE cells at 39 °C was accompanied by partial restoration of normal transcriptase activity at 39 °C, suggesting that reversion was mediated by either extragenic or intragenic suppression. Viral protein synthesis was reduced or absent in CE cells at 39 °C indicating that transcription was also defective in vivo under these conditions. Induction of heat-shock proteins in CE cells at 39 °C occurred normally in tdCE mutant-infected cells and RNA methylation in vitro was unaffected.

Three hybridoma antibodies, prepared against the RSN-2 strain of human respiratory syncytial (RS) virus, have been used to identify antigenic variation between 41 isolates of RS virus collected from widely separated geographical regions over a period of 29 years. One antibody was directed against an antigenic site on the virus fusion protein, VP70. This site was shared by 21 virus isolates tested and its recognition by the antibody was sensitive to the presence of 2-mercaptoethanol. The remaining two antibodies used react against the virus phosphoprotein, VPP32. Two independent sites were recognized on VPP32 by these antibodies. One antibody reacted with all of the virus isolates screened while the second reacted with only 21 out of the 41 virus isolates. On the basis of the variable epitope, two antigenic types of human RS virus were identified. The distribution of each antigenic group among 28 RS virus isolates from the Grampian Region, north-east Scotland, collected between 1982 and 1984 was determined. The reactivity of these antibodies was examined using immunofluorescence staining and by immunoblotting; the latter technique also revealed that the electrophoretic mobility of VPP32 varied in parallel with the variable antigenic site.

The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression.

Description of the interactions between bovine herpesvirus type 1 (BHV-1) and cattle was performed by the method known as kinetic logic. This logical formalization uses variables with two possible values, 1 and 0, which tell whether an element is present or not at a significant level. To each variable is associated a function which tells if the element is being produced at a significant rate. The temporal relation between a variable and its associated function is given by specific time delays. The BHV-1 infection system is described by a set of five logical equations which tell in what conditions each function is on or off. The five functions are: V, development of viral multiplication; R, development of reactivation of the latent virus; A, development of an immune response; G, establishment of the viral genome; M, development of a memory of a first immune response. Several examples are detailed in a dynamic analysis, in connection with known experimental data.

Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing a limited outbreak of poliomyelitis in a vaccinated population.

Morphological and immunocytological studies have demonstrated the presence of paramyxovirus antigens in Paget's bone disease tissue and in particular antigens related to measles virus and respiratory syncytial virus. To examine the relationship between measles virus and Paget's bone disease we used in situ hybridization and a cloned measles virus DNA probe specific for the nucleocapsid protein to detect and locate measles virus RNA sequences in Paget's bone tissue. In five patients with the disease, measles virus RNA sequences were detected not only in 80 to 90% of the multinucleated osteoclasts where there is morphological and immunocytological evidence of measles virus activity but also in 30 to 40% of mononucleated bone cells, mainly osteoblasts, osteocytes, fibroblasts and lympho-monocytes. In contrast, no hybridization was observed in bone tissue from three control patients without signs of Paget's bone disease. These results indicate that the host cell range for measles virus in Paget's disease is more widespread than has been supposed. They also demonstrate the usefulness of the in situ hybridization method to detect viral genetic information in cells where viral antigenic activity is not detectable. These observations further support the hypothesis that measles virus is involved in the pathogenesis of Paget's bone disease.

Frog virus 3 (FV3) assembles in morphologically distinct assembly sites in the cytoplasm of infected cells. As the assembly sites form, the intermediate filaments (IF) aggregate, delimit the assembly sites, and remain so throughout infection. To determine the molecular basis of reorganization of IF, we analysed the vimentin of uninfected and FV3-infected cells by two-dimensional gel electrophoresis. The results showed that (i) the vimentin was more acidic in FV3-infected cells than in uninfected cells, (ii) the acidification of vimentin in FV3-infected cells was possibly due to a fourfold increase in phosphorylation, and (iii) the phosphorylation of vimentin preceded the reorganization of IF around virus assembly sites. A temperature-sensitive mutant of FV3 (ts9467), which at the non-permissive temperature neither reorganized IF nor formed assembly sites, failed to increase the phosphorylation of vimentin. Together, the above results suggest that changes in phosphorylation may modulate IF organization and that changes in IF organization are required for FV3 assembly site formation.

High multiplicity infection of mouse fibroblast L-2 cells with mouse hepatitis virus (MHV) resulted, within 6 h, in a decline in total protein synthesis to about 7% of that observed in uninfected cells. The amount of intracellular total translatable RNA, however, increased approximately threefold, as a result of the accumulation of virusencoded mRNAs. MHV-infected cells could be superinfected with vesicular stomatitis virus, demonstrating that MHV infection did not irreversibly alter the cellular translational machinery to the exclusion of non-MHV mRNAs. Comparative polysome analysis from MHV-infected and uninfected L-2 cells showed that MHV infection resulted in an increase in single 80S ribosomes and in a shift from longer to shorter polysomes. These observations suggest first, that MHV infection inhibits total protein synthesis at a very early stage, as evidenced by the increase in 80S ribosomes, and, second, that the increased number of viral mRNAs produced after infection compete with cellular mRNAs for cellular ribosomes. In vitro translation of RNA extracted from MHV-infected and mock-infected cells suggested that levels of cellular mRNAs were decreased after infection. This suggestion was confirmed by demonstrating the loss of cellular actin mRNA, using a radiolabelled cDNA probe, as a consequence of MHV infection.

Secretory IgA (sIgA) and IgG from porcine milk and serum, respectively, [3H]uridine-labelled virus, swine testis and pig kidney cell lines were used to examine the neutralized virus-cell interaction. Transmissible gastroenteritis virus (TGEV), 99.99% neutralized by immunoglobulin, was able to attach to the cells. Moreover, sIgA enhanced virus attachment. However, the neutralized virus was unable to enter cells, as demonstrated by the action of proteinase K which removed it from the cell surface. It was also found that pre-attached virus was still neutralizable and that IgG and sIgA had similar TGEV-neutralizing capacities.

Latent Japanese encephalitis virus (JEV) infection was shown in inapparently congenitally infected Swiss albino mice after their mothers had been given JEV intraperitoneally during pregnancy. Only one of 37 (2.7%) of the baby mice showed persistence of infectious virus at 5 weeks of age. Reactivation of JEV in Swiss albino mice was demonstrated by stimulation with allogeneic spleen cells from Parks strain mice at 21 weeks of age; reactivation was demonstrated in 41% of the inapparently infected mice. The spleen cells of congenitally infected mice had depressed [3H]thymidine uptake following stimulation with concanavalin A, and depressed ability to induce a graft-versus-host response.

Twelve geographical isolates of Oryctes baculovirus were cloned in DSIR-HA-1179 cells. The DNA of these isolates was analysed by restriction endonuclease digestion with the enzymes BamHI, EcoRI, HindIII and PstI. Each isolate showed slightly different restriction fragment electrophoresis profiles. Most of the changes were due to small insertions or deletions of DNA, although two isolates lacked a single restriction site. The sites of genotypic change were not randomly distributed but were mainly associated with regions that have been shown to contain reiterated sequences.

We have developed an enzyme-linked immunosorbent assay for murine leukaemia virus using methanol-fixed cells as antigen. Specific antisera clearly recognized viral antigens within cells adherent to the microtitre plates. This test was used to quantify infection, to monitor virus multiplication and to demonstrate virus neutralization by antisera. The ELISA was as sensitive as, and faster and easier to perform than, the XC plaque assay and could even quantify large amounts of virus. It can easily be adapted for assaying other viruses.

The complete sequence of the RNA segment that codes for a major outer capsid protein (VP5) of bluetongue virus serotype 10 has been determined from overlapping cDNA clones inserted into pBR322. The segment 5 RNA of the virus (M5 RNA) is deduced to be 1638 base pairs long (1.05 × 106 daltons) and has an open reading frame in one strand capable of coding for a protein with a calculated size of 59163 daltons (526 amino acids) and a net charge of -4.5 at neutral pH.