- Volume 70, Issue 12, 1989

Using antisera made against peptides corresponding to different regions of the large subunit of herpes simplex virus type 1 ribonucleotide reductase we have probed proteolytic fragments of this protein and found that at least a part of its unique N-terminal domain is not necessary for enzyme activity. This non-essential region encompasses the domain previously predicted to be composed of β sheets with a well buried core of hydrophobic residues. Truncated forms of the large subunit are generated in vivo and are located almost exclusively in the nucleus.

A serological survey of monkeys was conducted to determine the prevalence of antibody to human herpesvirus 6 (HHV-6). Two-hundred and fifteen sera from 10 species of monkeys were examined by an immunofluorescent antibody (IF) assay. The antibody was found in monkeys from eight of the 10 species examined, but was not detected in silvered lutongs or cotton-top tamarins. The prevalence of antibody was highest in squirrel monkeys. Sera with high antibody titres were examined further by Western blot analysis and the neutralizing antibody test and the antibody levels were compared with that from a patient with exanthem subitum. On Western blotting, monkey and human sera that were antibody-positive to HHV-6 antigen gave similar reactions with antigen components of almost the same M r. Furthermore, sera that were antibody-positive by the IF test were also positive by the neutralizing antibody test and their titres in the two tests were comparable. These results suggest the existence of HHV-6 or an HHV-6-related virus in monkeys.

Using a guinea-pig model of genital herpes simplex virus (HSV) infection we explored the protection afforded by preinfection immunization with HSV glycoproteins. Glycoprotein immunogens prepared by recombinant DNA technology were found to be as effective as immunogens purified from HSV-infected cell cultures. Immunized animals developed less severe primary disease and also experienced less frequent recurrent infections. Protection was influenced by both adjuvant and route of administration. These studies suggest that recombinant HSV glycoproteins may be effective immunogens for human clinical trials, but that the development of an effective vaccine will require identification of new potent adjuvants that are safe for human use.

Vaccinia virus particles contain a protein kinase with an M r of 62K calculated from sedimentation rate. We have sequenced the SalI G restriction fragment of the vaccinia virus genome near to the right inverted terminal repeat and have identified two genes which share 36% amino acid identity with each other and are related to the family of protein kinase genes. One gene, designated B1R, encodes a 34·2K protein which shares 27% identity with a protein kinase encoded by the herpes simplex virus type 1 US3 gene and contains conserved motifs characteristic of protein kinases of serine/ threonine specificity. The second gene, B12R, encodes a protein of 33·3K which is poorly related to known protein kinases and lacks specific amino acids at several highly conserved key positions. The deduced partial amino acid sequence of a gene in the corresponding region of the cowpox virus genome is identical to B12R except for one conservative amino acid substitution. Both of the vaccinia virus genes are transcribed towards the right-hand end of the genome early during infection. It is possible that the product of either or both of these genes associates to form a homo- or heterodimer that represents the 62K virion-associated protein kinase.

The gene encoding the major capsid protein (hexon) of human adenovirus type 40 (Ad40) has been isolated and sequenced. Comparison of the predicted amino acid sequence of the Ad40 hexon with the corresponding polypeptide of the human enteric adenovirus, Ad41, reveals an overall identity of 88%. The majority of the changes in sequence are located in two areas, amino acids 131 to 287 and 390 to 425. Regions in the hexon protein that vary between Ad40 and Ad41 (subgroup F) were the same regions that varied between Ad2 and Ad5 (subgroup C) suggesting that these areas of the protein represent type-specific antigenic determinants. Other areas were conserved within members of a subgroup but varied between subgroups. Fitting of the Ad40 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that the variable regions are located in the l1, l2 and l4 loops that form the surface of the virion. Of major significance is the absence in Ad40 of the highly acidic region present in both Ad2 and Ad5. In Ad2 this region stretches down into the d-strand of the β-barrel forming the P1 domain. Molecular modelling indicates that the amino acids in Ad40 which correspond to the acidic region of Ad2 can also be accommodated in the eight-stranded β-barrel, thereby maintaining the integrity of the barrel. Since the acidic region is also absent from the hexon of Ad41, the sequence of amino acids that replaces the acidic residues may be responsible for some of the distinctive biological properties of the subgroup F adenoviruses.

An assay system has been developed for the adenovirus endoproteinase which utilizes the synthetic peptide MSGGAFSW, derived from the cleavage site of the adenovirus type 2 (Ad2) protein pVI. MSGGAFSW was shown to be cleaved at the G–A bond when incubated with a source of Ad2 proteinase. Digestions were readily monitored by either fast protein liquid chromatography or thin layer electrophoresis, enabling the rapid production of quantitative data. Comparison of the peptide assay with a previously described [35S]methionine assay system showed it to be faster, cleaner and less prone to extreme conditions of pH and ionic strength. The effect on adenovirus proteinase activity of a number of inhibitors was assessed using both the [35S]methionine and peptide assays. Identical inhibitor profiles were obtained and these suggested that the adenovirus enzyme is a cysteine proteinase. The 23K gene product, thought to be the proteinase, contains cysteine and histidine residues at positions 122 and 54, respectively, that could constitute part of the active site of a cysteine proteinase. These amino acids and their surrounding residues are conserved in all serotypes examined and appear to bear some resemblance to those in the putative active sites of the 3C proteinases in picornaviruses.

Peptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX↓G or M(L)XGG↓X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.

Adenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA–complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn 2+ ions for Mg 2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6·4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for a least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription.

Evidence is presented here which indicates that the adenovirus DNA-binding protein (DBP) is phosphorylated at a tyrosine residue early in infection. This was suggested by the discovery that a proportion of the label in 32P-labelled DBP was resistant to alkali, and was substantiated by acid hydrolysis of DBP immunoprecipitates and by immunoblotting with a monoclonal antibody against phosphotyrosine. Treatment of [35S] methionine-labelled DBPs with chymotrypsin produced fragments of apparent M r 45K and 39K whereas digestion of 32P-labelled DBP resulted in fragments of 45K and 26K. Consideration of the distribution of 32P label and its alkali stability in these fragments suggested that chymotrypsin cleaved populations of DBP at different sites depending on their phosphorylation states. The conservation, in all of the seven adenovirus serotypes sequenced, of a tyrosine residue (at amino acid 195 in adenovirus type 2) together with its surrounding residues, suggests that phosphorylation/dephosphorylation at this tyrosine residue may be important in various functions ascribed to the DBP.

We have used enzymic amplification of specific nucleic acid sequences followed by hybridization, for the rapid detection and typing of human picornaviruses after cell culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected from the 5′ non-coding region of the genome representing highly conserved regions. Sequences specific to enteroviruses and rhinoviruses were used as probes. The assay was able to identify all the picornavirus reference strains analysed and it was also possible to discriminate between enteroviruses and rhinoviruses by the hybridization procedure. When 29 picornavirus clinical isolates were analysed, all except one were detected by gel electrophoresis and a specific hybridization signal was obtained with all except three strains using the oligonucleotide probes.

The complete nucleotide sequence of the genome of coxsackievirus A9 (CAV-9) has been determined from cDNA cloned in Escherichia coli. Excluding the 3′ poly(A) stretch, the RNA genome is 7452 nucleotides long and encodes a single polyprotein of 2201 amino acids. Comparison of the nucleotide and predicted amino acid sequences with those of the coxsackieviruses B1, B3 and B4 reveals a surprising degree of homology, with overall amino acid homologies of 86·9%, 86·2% and 87·0%, respectively. In contrast, there is much less homology to another coxsackie A virus, CAV-21, 60.4% overall amino acid homology. This demonstrates the high degree of diversity within the CAV group and indicates that the current classification does not directly correlate with molecular genetic properties. One major feature of CAV-9 is an insertion, relative to all other enteroviruses sequenced to date, which is located at the C terminus of VP1, and includes an arginine-glycine-aspartic acid tripeptide. Such sequences in a number of other proteins are known to have activity in promoting attachment to cell receptors and the implications for CAV-9 receptor binding are discussed.

Antigenic variants of foot-and-mouth disease virus (FMDV) of serotype C (isolate C-S8c1) were selected upon serial passage of the virus in cell culture in the absence of anti-FMDV antibodies. The variants rose from frequencies of < 10–2 in the initial plaque-purified FMDV C-S8c1 preparation, to 0·1 to 1 in three passaged populations. The proportion of antigenic variants was quantified using a new in situ plaque immunotest. A nitrocellulose filter is applied to the agar overlay of a FMDV plaque assay, and allows recovery of infectious virus from individual plaques. A second filter is placed directly on the cell monolayer and binds enough virus to permit colorimetric visualization of plaques by an enzyme-linked assay using monoclonal antibodies (MAbs). Either all or a fraction of plaques from passaged FMDV failed to react with MAb 4G3, an antibody that recognizes an epitope located within residues 144 to 150 of capsid protein VP1. Some variants rapidly dominated the viral population, and others were maintained at low levels. RNA from unreactive viruses included mutations that resulted in amino acid substitutions at the epitope recognized by MAb 4G3. We discuss models for the selection of antigenic variants of FMDV in the absence of antibodies, and implications for the antigenic diversification of RNA viruses.

Parenteral immunization of BALB/c mice at 3 months of age with inactivated influenza virus vaccine elicited a haemagglutinin (HA)-specific serum IgG antibody response. The magnitude of this response declined with advancing age at the time of vaccination. By contrast, HA-specific IgA and IgG antibody levels observed in lung lavage fluids of mice immunized at 1 and 2 years of age were comparable to those of 5 month old mice when inactivated influenza virus vaccine was administered intragastrically. The secretory immune response was not fully developed in the first 3 weeks of life. However, the HA-specific IgA and IgG responses to oral vaccination in sera were reduced in 1 or 2 year old mice when compared to 5 month old mice. These data demonstrated the preservation of the virus-specific secretory IgA response in the pulmonary fluids of aged mice after oral vaccination with inactivated influenza virus vaccine. An age-dependent difference of systemic and mucosal immunity was evident in orally immunized mice.

Influenza viruses of the H1N1 subtype have been continually circulating in pigs in the U.S.A. for at least 50 years. To examine the level of antigenic variation in these swine viruses, a panel of 60 monoclonal antibodies (MAbs) to the haemagglutinin (HA) of recent swine isolates was prepared. Evaluation of neutralization escape mutants selected with these MAbs defined four antigenic sites on the HA, two of which overlap. Swine viruses isolated over 24 years in an enzootic area in Wisconsin were examined by ELISA and haemagglutination inhibition (HI) with these MAbs and the results indicated that the antigenic sites defined by these MAbs were highly conserved in these viruses. In comparing recent H1N1 viruses from pigs, turkeys, ducks and humans, changes in the antigenic sites were detected on the basis of HI reactivity. However, results of ELISA with these viruses clearly showed that the antigenic sites were still present on almost all H1N1 viruses of swine origin; thus, altered reactivity of these viruses in HI tests with MAbs was not a reflection of changes in the antigenic sites defined by the MAbs. It seems likely that the variation detected in these viruses occurs by a mechanism other than immune selection.

As shown previously, after inoculation into the footpad of a mouse the lymphocytic choriomeningitis (LMC) virus multiplies locally. Beginning on day 6 or 7 after infection, the foot undergoes a delayed-type hypersensitivity (DTH) reaction which consists of two distinct phases that are mediated by CD8+ cells and CD4+ cells, respectively, and at about the same time the virus is eliminated. In general, for terminating infection of the mouse with LCM virus the CD8+ cytotoxic/suppressive T lymphocyte (CTL) is essential; we have now determined the cells that mediate control of the virus in a tissue undergoing a specific DTH reaction. Depletion, in infected mice, of all T lymphocytes by treatment with anti-Thy-1 monoclonal antibody prevented virus elimination from the foot, and the same was true when the CD8+ CTLs were removed. Depletion of the CD4+ helper/suppressor subset only marginally impaired the ability of the mice to rid themselves of the virus. The conclusion that here too the principal antiviral element is the CD8+ CTL was confirmed by experiments in which footpad-infected mice were adoptively immunized with virus-immune splenocytes from syngeneic mice selected for subclasses of T lymphocytes, or from mice differing in defined regions of the major histocompatibility complex (MHC), and also by experiments in which monocytes were virtually absent. However, CD8+ CTL alone or cells from MHC recombinant mice with identity in class I loci were never as antivirally active as unseparated splenocytes from syngeneic donor mice. Since the CD8+ cells’ performance could be optimized by interleukin-2, we assume that the CD4+ T lymphocytes function as accessory cells; the same probably applies to monocytes.

Virus-specific cytotoxic T lymphocytes (CTL) mediate their antiviral activity either by direct lysis of infected cells, or by the release of soluble lymphokines, or by a combination of the two. We have examined the role played by interferon-gamma (IFN-γ) and tumour necrosis factor (TNFα) in virus clearance. In vitro the amount of IFN-γ synthesized by some lymphocytic choriomeningitis virus-specific H-2-restricted CTL clones was quantitatively too small to correlate with a direct antiviral activity in vivo. However, treatment of mice with a neutralizing monoclonal antibody to IFN-γ significantly inhibited the clearance of virus from the spleens of acutely infected mice given adoptive transfers of immune spleen cells. Additionally, mice treated with exogenous recombinant murine IFN-γ 24 h before or at the same time as virus inoculation showed reduced virus titres in their spleens. Hence, IFN-γ displayed a direct antiviral effect in vivo. In contrast, treatment of mice with recombinant TNFα had no effect on virus clearance and thus TNFα is unlikely to play a significant role in this acute viral infection.

A number of CD4+ T cell lines were compared for their ability to act as target cells for human immunodeficiency virus (HIV) infection in syncytium- and plaque-forming assays. MT-4 and C8166 cells were the most sensitive indicator cells for HIV- and simian immunodeficiency virus (SIV)-induced cytopathic effects, and gave rise to macroscopic (MT-4) and microscopic (C8166) plaques. The MT-4 plaque assay was evaluated for the measurement of HIV- and SIV-neutralizing antibodies.