- Volume 74, Issue 5, 1993

Wild-type human p53 and a series of p53 point mutants isolated from Burkitt's lymphoma (BL) cell lines were tested for their ability to inhibit DNA synthesis in a p53-negative BL cell line and to bind and be degraded by the human papillomavirus type 16 E6 protein. All the mutants lost the wild-type ability to inhibit DNA synthesis, demonstrating that they are all functionally altered. Binding to E6 and consequent degradation of the p53 mutants frequently correlated with changed suppressor properties in BL cells.

Human sera were tested for antibodies against the Epstein-Barr virus (EBV) latent infection terminal proteins (TPs). Anti-TP IgG and IgA antibodies were detected by an indirect immunofluorescence assay of insect cells expressing a recombinant TP1. Out of 301 human sera of patients with EBV-related and EBV-unrelated disorders, only sera from patients with nasopharyngeal carcinoma (NPC) (32/83; 38%) showed anti-TP antibodies. Studies on serial sera from German and Hong Kong NPC patients revealed a decline of anti-TP antibodies during tumour therapy, and none of these antibodies were identified in patients with early tumour stages or in remission. Comparative studies of TP1-specific polyclonal rabbit antisera and human TP-positive sera showed clear differences in the TP epitopes recognized by each. Human antisera contained antibodies only to native epitopes in exons 2 to 7 of TP1 whereas rabbit antisera reacted only with epitopes located in the first exon and, additionally, exhibited EBV strain specificities.

Both intranasal (i.n.) and intracerebral (i.c.) inoculation of mice with wild-type equine herpesvirus type 1 (wt EHV-1) caused clinical signs and mortality. Virus could be recovered from target organs (turbinates, lungs and blood) for several days. By contrast, the thymidine kinase (TK)-deficient deletion mutant PR1 produced markedly less clinical disease following both i.n. and i.c. inoculation, and, in particular, no mortality occurred. PR1 did, however, establish productive infections following either route of inoculation. High titres of virus were recovered from target organs although virus did not persist for as long as wt EHV-1 and no viraemia was detected. Primary i.n. infection of mice with either wt EHV-1 or PR1 protected against subsequent challenge with wt EHV-1 5 weeks later. I.n. inoculation of specific pathogen-free (EHV-free) foals with PR1 produced results similar to those observed after infection of mice. Clinical signs were milder than for wt EHV-1 and pyrexia was short-lived or absent. PR1 could be recovered from nasal mucus at high titres but it persisted for only 5 days post-infection compared to 11 days in the case of wt EHV-1. No viraemia was detected in foals infected with PR1. On challenge with wt EHV-1, foals given a primary infection with the mutant were partially protected; but a viraemia with a TK+ EHV-1 was observed. These results demonstrate that our TK- mutant PR1 is markedly less pathogenic than wt EHV-1, despite being able to replicate in the host. The use of TK-deficient mutants of EHV-1 as potential vaccines in the horse is discussed.

Although rabies outbreaks in most parts of the world tend to be host species-specific the rabies currently enzootic in the Canadian province of Ontario is hosted by two wildlife species, the red fox and the striped skunk. Previous studies employing monoclonal antibody panels failed to identify any host-specific differences in Ontario rabies virus street isolates, but certain observations suggested the existence of more than one viral strain in terrestrial mammals of this region. The extent of variation of the rabies virus circulating within this region has been re-examined using molecular biology techniques. The N gene of several independent isolates was amplified using PCR and the resulting products were compared by restriction enzyme analysis and, in some cases, by DNA sequencing. This analysis confirmed that there was indeed no host-specific variation in the portion of the viral genome under study but there were, however, very clear and consistent differences in the virus from distinct geographical regions.

Antibody-dependent enhancement (ADE) of dengue virus infection occurs when neutralizing antibodies at sub-neutralizing concentrations or non-neutralizing antibodies form complexes with the virus. These virus-antibody complexes can then attach to a Fcγ receptor-bearing cell, via the Fc portion of the immunoglobulin, resulting in an increased number of infected cells. ADE may be responsible in part for the most severe clinical manifestations of dengue virus infection which include haemorrhage and shock. Three classes of human Fcγ receptors exist, FcγRI, FcγRII and FcγRIII. In this study, we examined the effects of neuraminidase on ADE of dengue virus infection mediated by the low-affinity FcγRII. K562 cells, which express only FcγRII, treated with neuraminidase resulted in augmentation of ADE of dengue virus infection by human anti-dengue antibodies. This augmented ADE of infection could be blocked by anti-FcγRII monoclonal antibody IV.3. Incubation of neuraminidase-treated K562 cells with IgG-coated human red blood cells resulted in an increase in the percentage of rosette formations compared with the untreated K562 cells. A bispecific antibody directed against FcγRII and dengue virus (IV.3 × 2H2) enhanced virus infection. Neuraminidase also augmented ADE mediated by this antibody, but to a much lesser degree (by 50%) compared with that seen using conventional human anti-dengue antibody (by 200 to 300%). Fluorescence-activated cell sorting analysis of neuraminidase-treated K562 cells showed that the number of FcγRII-specific antibodies that bind to FcγRII increases by 15 to 20% after treatment with neuraminidase. These results indicate that neuraminidase augments ADE of dengue virus infection and that the augmented ADE is mediated through FcγRII.

We report the first complete nucleotide sequence of the picornavirus coxsackievirus B5 (CB5), strain 1954/UK/85, an isolate from a case of hand-foot-and-mouth disease. We have compared the sequence with those of other coxsackie B viruses, coxsackievirus A9, poliovirus and swine vesicular disease virus (SVDV). The genes encoding the three major capsid proteins are most closely related to those of SVDV but the 5′ and 3′ non-coding regions and the P3 gene are more similar to the corresponding regions in the other coxsackie B viruses than to those of SVDV. These observations are considered in the light of the antigenic and biochemical relationships between SVDV and CB5.

The paradox that group-specific neutralizing antibodies (NA) exist in the majority of human immunodeficiency virus type 1 (HIV-1)-infected patients, whereas the NA response against autologous HIV-1 virus isolates is highly type-specific, motivated us to study the type- and group-specific NA responses generated upon presentation of escape virus, and the viral epitopes involved in the escape. Patients with demonstrable escape virus all developed group-specific NA, which were detectable after a delay and disappeared prior to disease development. The sera tested inhibited the binding of recombinant soluble gp120IIIB to cell-associated CD4, but group-specific virus neutralization required binding of NA to HIV-1 prior to viral attachment to target cells. Consecutive escape virus isolates were tested for sensitivity to neutralization by heterologous sera. Only minor differences were demonstrated, suggesting that the majority of the change in neutralization sensitivity is driven by the selective pressure of type-specific NA. Furthermore, no differences were observed in sensitivity to neutralization by anti-carbohydrate neutralizing monoclonal antibodies or the lectin concanavalin A, indicating a conserved nature of certain carbohydrate neutralization epitopes during escape. Finally the V3 sequence of three sets of consecutive virus isolates were analysed revealing amino acid mutations in V3 sequences of all escape virus isolates. The biological significance of these variations was confirmed further by the demonstration of changes in sensitivity to neutralization by anti-V3 monoclonal antibodies. These results strongly suggest a participation of the NA response against the V3 loop in the immunoselection of escape virus.

Three cynomolgus macaques were immunized with recombinant envelope protein preparations derived from simian immunodeficiency virus (SIV). Although humoral and cellular responses were elicited by the immunization regime, all macaques became infected upon challenge with 10 MID50 of the 11/88 virus challenge stock of SIVmac251-32H. The polymerase chain reaction was used to amplify proviral SIV gp120 sequences present in the blood of both immunized and control macaques at 2 months post-infection. A comparison of the predominant sequences found in the region from V2 to V5 of gp120 failed to differentiate provirus recovered from either immunized or control animals. A detailed investigation of sequences obtained from the hypervariable V1 region identified a mixture of sequences in both immunized and control macaques. Some sequences were identical to those previously detected in the virus challenge stock, whereas others had not been detected previously. Phenogram analysis of the new V1 sequences found in immunized animals revealed that they were quite distinct from those from the virus challenge stock and that they included alterations to potential N-linked glycosylation sites. In contrast, new sequence variants recovered from the control animals were closely related to sequences from the virus challenge stock. The difference in diversity of new V1 sequences recovered from immunized and control macaques was highly significant (P < 0.001). Thus, the presence of preexisting immune responses to SIV envelope protein is associated with greater genetic change in the V1 region of gp120. These data are discussed in relation to the epitopes of SIV gp120 that may confer protection from in vivo challenge.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M r of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

The complete nucleotide sequence of the P gene of simian virus 41 (SV41) was determined. The gene was found to be 1406 nucleotides long and to contain a relatively small open reading frame encoding a cysteine-rich V protein with a calculated M r of 24076. We have demonstrated that RNA-editing events occur in SV41 P gene transcripts and that the ratio of edited mRNAs to faithfully copied mRNA (P-mRNA: V-mRNA) is about 1:5 at either 24 or 40 h post-infection. The mRNA with two G insertions was capable of encoding a P protein of 395 amino acids with a predicted M r of 41992. A kinetic study of P and V proteins by Western blot analysis showed that in virus-infected cells the amounts of both proteins were almost equal although the V-mRNA was considerably more abundant than the P-mRNA. Alignment of the SV41 P and V proteins with those of nine other paramyxoviruses demonstrated that irregular gaps were present around the RNA-editing sites.

Sequence analysis of the region of the mumps virus genome encoding the putative small hydrophobic protein gene confirms that it is a highly variable region. Jeryl Lynn, the mumps vaccine strain used in the U.K., is shown to be a mixture of two closely related viruses, both probably of American origin.

Turkish tick-borne encephalitis (TTE) virus causes an acute form of meningoencephalomyelitis in sheep in the north-western region of Turkey. The clinical syndrome resembles louping ill (LI) and the viruses responsible for both LI and TTE are members of the tick-borne encephalitis (TBE) complex of the Flaviviridae. The envelope protein gene of TTE virus was reverse-transcribed, amplified, cloned and sequenced. Alignment of the resultant sequence with those from other viruses of the TBE complex reveals that TTE virus is more closely related, at both nucleotide and amino acid levels (84.6% and 96% respectively), to the Central European (CEE) subtype of the TBE virus, usually associated with human disease. The relationship with LI virus is more distant (83% and 93.5% respectively). These studies support the assertion that the ovine encephalomyelitis found in Turkey is caused by a virus that is genetically distinct from known strains of both LI and CEE viruses and from a number of other known viruses of the TBE complex.

Cattle infected in utero with bovine viral diarrhoea virus (BVDV) often develop a lifelong persistent infection (PI). During this PI, BVDV infects many cell types including peripheral blood mononuclear cells (PBMNC). To define the lymphoid cell populations in which BVDV persists PBMNC subpopulations were separated using monoclonal antibodies to cell surface markers. Separated cells were analysed by a sensitive PCR assay for BVDV, in conjunction with flow cytometry to identify antigen-containing cells and with viral infectivity assays. The results indicate that BVDV establishes a productive PI in monocytes and T cells bearing the marker BoCD4, BoCD8 or gamma-delta T cell receptor. BVDV was not detected in B cells as a productive nor a latent infection.

Seven mutant viruses were derived from a Scottish strain of louping ill virus using a virus envelope-specific neutralizing monoclonal antibody. None of the mutants was neutralized and immunofluorescence microscopy confirmed that they did not bind to this antibody. Four mutants showed reduced mouse neurovirulence compared with parent virus and two mutants failed to induce protective immune responses in mice challenged with virulent tick-borne encephalitis virus. The mutants with the lowest virulence showed poor or undetectable haemagglutinating activity. The nucleotide sequence of the envelope glycoprotein gene of each of the seven mutants was determined and the deduced amino acid sequence was compared with parent virus. For each mutant, only a single amino acid codon change was detected and all the amino acid substitutions occurred within amino acid positions 308 to 311. A change from the amino acid aspartate to asparagine at amino acid position 308, which represented a potential glycosylation site, was the most effective substitution in reducing mouse neurovirulence. The results demonstrate the importance of critical sites within the envelope glycoprotein as determinants of virus virulence.

The phenotype of the rotavirus SA-11 mutant tsB carrying a thermosensitive mutation in gene 3, which encodes VP3, was characterized further from both infected cells and purified viral particles. The mutant phenotype was initially identified as negative for in vivo double- and single-stranded RNA synthesis. Our results show that the in vitro transcriptional properties of the tsB mutant at the restrictive temperature were identical to those of the wild-type strain. Similar results were obtained with respect to the VP3-associated guanylyl-transferase activity. Analysis of viral particles made by mutant-infected cells at the restrictive temperature showed that only empty single-shelled particles were assembled. This indicates that viral morphogenesis is halted after the initial viral transcription and before RNA replication, suggesting that VP3 may be required as part of the replicase system but not for subviral particle assembly. These data suggest that such a phenotype is not due to alteration of a VP3 function related to transcription.

Clones have been constructed that contain full-length cDNA copies of cowpea mosaic virus RNA1 and RNA2, downstream of the cauliflower mosaic virus 35S promoter. The clones, when linearized downstream of the viral sequences, give rise to cowpea mosaic virus-like symptoms when inoculated onto cowpea plants. Viral RNA and virions can be detected in the inoculated plants, demonstrating that the clones are directly infectious.

Multiple copies of the Johnsongrass mosaic virus coat protein synthesized in Escherichia coli can readily assemble to form potyvirus-like particles. This E. coli expression system has been used to identify some of the key amino acid residues, within the core region of the coat protein, required for assembly. The two charged residues R194 and D238 previously proposed theoretically to be involved as a pair in the construction of a salt bridge crucial for the assembly process were targeted for site-directed mutagenesis. The results from our experiments suggest that the two residues are required for the assembly process but are not necessarily involved as a pair in a common salt bridge.