- Volume 81, Issue 11, 2000

The continual threat posed by newly emerging influenza virus strains is demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong. Currently, immunization against influenza virus infection is fairly adequate, but it is imperative that improved vaccines are developed that can protect against a variety of strains and be generated rapidly. Since humoral immunity is ineffective against serologically distinct viruses, one strategy would be to develop vaccines that emphasize cellular immunity. Here we report the successful protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours internal genes 98% homologous to HK156. This strategy also protected mice that are deficient in antibody production, indicating that the immunity is T-cell-mediated. In the course of these studies, we generated a highly pathogenic H5N1 reassortant which implicated NP and PB2 as having an important contribution to pathogenesis when present with a highly cleavable H5. These results provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus and have important implications for the development of novel strategies for the prevention and treatment of HK156 infection and the design of future influenza vaccines.

Following binding to cell surface sialic acid, entry of influenza viruses into cells is mediated by endocytosis. Productive entry of influenza virus requires the low-pH environment of the late endosome for fusion and release of the virus into the cytoplasm and transport of the virus genome into the nucleus. We investigated novel mechanisms to inhibit influenza virus infection using highly specific inhibitors of protein kinase C. We found that one inhibitor, bisindolylmaleimide I, prevented replication of influenza A virus in a dose-dependent manner when added at the time of infection, but had little specific effect when added 2 h after infection had commenced. Virus yields dropped by more than 3 log units in the presence of micromolar levels of bisindolylmaleimide I. Influenza B virus replication was also inhibited by bisindolylmaleimide at micromolar concentrations. We carried out experiments to determine the point in infection that was blocked by bisindolylmaleimide I, and determined that entry of viral ribonucleoproteins (vRNPs) into the nucleus was prevented. Upon drug washout vRNP nuclear entry resumed, showing that bisindolylmaleimide I is reversible. Bisindolylmaleimide I did not affect virus binding and was apparently not acting as a weak base, because its effects were independent of the pH of the external growth medium. These experiments show that bisindolylmaleimide I blocks replication of different types of influenza virus in a dose-dependent and reversible manner, and that virus entry into the cell is inhibited.

Depending on their major histocompatibility complex (MHC) haplotype, inbred mouse strains are either resistant (H2-d, BALB/c), susceptible (H2-k, C3H) or partially resistant (H2-d×k, BaCF1) to intracerebral infection with the neurotropic rodent-adapted measles virus (MV) strain CAM/RBH. Here, mortality is demonstrated to be correlated directly with virus spread and virus replication in the CNS and to be inversely correlated with the activation of MV-specific T cells. Previously, it has been shown that primary CD4+ T cells alone are protective in the resistant background. In the susceptible background, CD4+ T cells acquire protective capacity after immunization with a newly defined CD4+ T cell epitope peptide. In the partially resistant mice, CD4+ T cells provide help for CD8+ T cells and protect in cooperation with them. It seems that the lytic capacity of CD8+ T cells is crucial in providing protection, as MV-specific Ld-restricted CD8+ T cells, which are highly lytic in vitro after transfer, protect naive animals against MV-induced encephalitis (MVE). In contrast, Kk-restricted CD8+ T cells with low lytic capacity do not protect. In the MVE model, CD4+ T cells are able to protect either alone (resistant mice), through cooperation with CD8+ T cells (intermediate susceptible) or after immunization as secondary T cells (susceptible mice). CD8+ T cells are able to protect alone after immunization if they are cytolytic. Thus, susceptibility and resistance depend upon the functional composition of CD4+ and CD8+ T cells governed by the MHC haplotype.

Binding of human respiratory syncytial virus (HRSV) to cultured cells was measured by flow cytometry. Using this assay and influenza virus as a control virus with a well-characterized receptor, a systematic search of cell surface molecules that might be implicated in HRSV binding was carried out. Treatment of cells with different enzymes or with other reagents suggested that heparin-like glycosaminoglycans (GAGs) were involved in attachment of HRSV, but not influenza virus, to host cells. This was further confirmed by a lack of binding of HRSV to CHO-K1 mutant cell lines deficient in glycosylation or GAGs biosynthesis and by an inhibition of binding after preincubation of virus with heparin and other GAGs. The degree of sulfation, more than the polysaccharide backbone of GAGs, seems to be critical for virus binding.

Sequence analysis was performed of all or part of the genes encoding the fusion (F), polymerase (L) and attachment (G) proteins of two French non-A/non-B avian pneumovirus (APV) isolates (Fr/85/1 and Fr/85/2). The two isolates shared at least 99·7% nt and 99·0% aa sequence identity. Comparison with the F genes from subgroup A, subgroup B or Colorado APVs revealed nt and aa identities of 70·0–80·5% and 77·6–97·2%, respectively, with the L gene sharing 76·1% nt and 85·3% aa identity with that of a subgroup A isolate. The Fr/85/1 and Fr/85/2 G genes comprised 1185 nt, encoding a protein of 389 aa. Common features with subgroup A and subgroup B G proteins included an amino-terminal membrane anchor, a high serine and threonine content, conservation of cysteine residues and a single extracellular region of highly conserved sequence proposed to be the functional domain involved in virus attachment to cellular receptors. However, the Fr/85/1 and Fr/85/2 G sequences shared at best 56·6% nt and 31·2% aa identity with subgroup A and B APVs, whereas these isolates share 38% aa identity. Phylogenetic analysis of the F, G and L genes of pneumoviruses suggested that isolates Fr/85/1 and Fr/85/2 belong to a previously unrecognized APV subgroup, tentatively named D. G-based oligonucleotide primers were defined for the specific molecular identification of subgroup D. These are the first G protein sequences of non-A/non-B APVs to be determined.

Efficacious lentiviral vaccines designed to induce cytotoxic T lymphocytes (CTL) in outbred populations with a diverse repertoire of MHC class I molecules should contain or express multiple viral proteins. To determine the equine infectious anaemia virus (EIAV) proteins with epitopes most frequently recognized by CTL from seven horses infected for 0·5 to 7 years, retroviral vector-transduced target cells expressing viral proteins were used in CTL assays. Gag p15 was recognized by CTL from 100% of these infected horses. p26 was recognized by CTL from 86%, SU and the middle third of Pol protein were each recognized by 43%, TM by 29%, and S2 by 14%. Based on these results, it is likely that a construct expressing the 359 amino acids constituting p15 and p26 would contain epitopes capable of stimulating CTL in most horses.

To test the in vivo anti-simian immunodeficiency virus (SIV) efficacy of interferon (IFN)-β-engineered lymphocytes, peripheral blood lymphocytes harvested from two uninfected macaques were transduced with a retroviral vector carrying a constitutively expressed IFN-β gene and reinfused, resulting in approximately 1 IFN-β-transduced cell out of 1000 circulating cells. The gene-modified cells were well tolerated and could be detected for at least 74 days without causing any apparent side effects. These two animals together with three untreated control macaques were then infected with SIVmac251. The two IFN-β-infused macaques are in good health, 478 days after infection, with a reduced plasma virus load and sustained numbers of CD4+ and CD8+ cells. Throughout the study, the proportion of IFN-β-transduced cells has been maintained. Of the three control macaques, two were characterized by a high plasma virus load and a decrease in CD4+ cells. One was moribund and was sacrificed 350 days after infection and the other now has fewer than 100 circulating CD4+ cells/ml. Unexpectedly, the third control macaque, which, like the two IFN-β-infused animals, had a low plasma virus load and a maintenance of CD4+ and CD8+ cell number, was characterized by a permanent level of serum IFN-β, of unknown origin, already present before SIV infection. Although no definite conclusion can be made in view of the limited number of animals, these data indicate that further exploration is warranted of an IFN-β-based anti-human immunodeficiency virus gene therapy.

In a cell-free system of uninfected HeLa cells, programmed with poliovirus RNA, extraneous radiolabelled 14S subunits assembled with endogenous 14S subunits and interacted with newly synthesized RNA to form virions (160S). This result suggests that 14S subunits are the key intermediates in the encapsidation of poliovirus RNA.

Although the association remains controversial, enteroviruses have been implicated in the aetiology of several chronic diseases of humans. To further understand the mechanism of enterovirus persistence and its relationship to organ pathology, virus infectivity and viral RNA kinetics in the heart and other target organs during acute and persistent phases of murine coxsackievirus B3 infection were investigated. These studies revealed a biphasic pattern of virus clearance. Thus, there was a rapid but incomplete clearance of viral RNA from the myocardium following the acute phase of virus replication, which paralleled the elimination of virus infectivity. The mean half-life of viral RNA between days 5 and 14 post-infection (p.i.) was 13·4 h. In contrast, a much slower rate of decline in viral RNA levels was observed during the post-infectious inflammatory phase of myocarditis. The mean half-life of viral RNA between days 14 and 90 p.i. was 14·1 days. Viral RNA persisted in the myocardium beyond the resolution of inflammation and was still detectable in a proportion of animals 90 days after infection. Clearance of viral RNA from other target organs occurred more rapidly, but the rate of clearance was largely independent of the level of viral RNA present during the acute phase of infection. Thus, while antiviral immune responses effectively eliminated infectious virus, clearance of residual viral RNA from the myocardium and other target organs was significantly delayed, despite a prolonged inflammatory response. These findings suggest that clearance of persistent enterovirus infection requires mechanisms different from those responsible for the elimination of virus infectivity.

The Dutch swine vesicular disease virus (SVDV) isolate NET/1/92 was one of the first isolates belonging to a new SVDV antigenic group. This strain was completely sequenced and was shown to have 93% similarity with the UKG/27/72 isolate. To enable antigenicity, replication, maturation and pathogenicity studies of NET/1/92, an infectious full-length cDNA clone, designated pSVD146, was prepared. The in vitro and in vivo biological properties of the virus derived from pSVD146 were studied by analysing antigenicity, plaque morphology, growth curves and virulence in pigs. The epitopes of newly prepared monoclonal antibodies were roughly mapped by fusion-PCR. Fine mapping of epitopes at the amino acid level was achieved by introducing single amino acid mutations in pSVD146. Two new amino acids important in epitope formation were located in VP1; one was mapped in the C-terminal end and the second is thought to be located in the H–I loop. Growth curve and plaque sizes in vitro were similar between virus derived from pSVD146 and the parent wild-type virus. In virulence studies in pigs, the lesions score, neutralization titres and the seroconversion rates were comparable between virus derived from pSVD146 and the parent strain. Since virus derived from pSVD146 had the same biological properties as the parent strain NET/1/92, the full-length infectious cDNA clone pSVD146 will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.

High doses (>1·56×107 p.f.u.) of purified preparations of human adenovirus types 3, 5 and 8 exhibited definite pyrogenic activity when injected intravenously into rabbits. Complete pyrogenic tolerance was obtained not only with homologous types but also with heterologous types of adenovirus. No pyrogenic cross-tolerance was observed between each of these three adenovirus types and paramyxovirus pyrogen or bacterial lipopolysaccharide. Adenovirus pyrogenicity was retained after UV-inactivation, whereas it was inactivated by heating at 56 °C for 30 min. Adenovirus pyrogenicity was not neutralized by mixing with homologous type-specific antiserum but non-pyrogenic doses (107 p.f.u.) of adenovirus types 3, 5 and 8 became highly pyrogenic in the presence of type-specific antibodies at the optimal virus:antibody ratio. This enhanced pyrogenicity depended upon the virus–antibody complex. From these results, it is probable that the pyrogenic activity of the virus–antibody complex, rather than the pyrogenic activity of the virions, is the main contributor to fever in adenovirus infection under actual physiological conditions.

The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) can transform cells independently of p53 degradation. The precise mechanisms underlying this transformation are not yet completely understood. Here it is shown that BPV-1 E6 interacts with CBP/p300 in the same way as described for the E6 proteins of oncogenic human papillomaviruses. This interaction results in an inhibition of the transcriptional coactivator function of CBP/p300 required by p53 and probably by other transcription factors. The comparison of the CBP/p300-binding properties of BPV-1 E6 mutants previously characterized in transcription and transformation studies suggests (i) that the E6–CBP/p300 interaction may be necessary, but not sufficient, for cell transformation, and (ii) that the transcriptional activator function, inherent to the E6 protein, is not derived from forming a complex with CBP/p300.

The ubiquitous human polyomaviruses BK (BKV) and JC (JCV) persist with no adverse effects in immunocompetent individuals. Virus-induced pathogenesis has been linked to virus reactivation during impaired immune conditions. Previous studies have shown a significant difference between the VP1 DNA sequences of JCV obtained from control urine samples and those in progressive multifocal leukoencephalopathy brain samples. This difference could not be detected when comparing normal control urinary JCV DNA with DNA sequences from chronic progressive multiple sclerosis patients. Since BKV and JCV are readily activated in systemic lupus erythematosus (SLE) patients, the presence of specific strains, related to VP1 DNA sequences, was investigated in these patients. VP1 DNA sequences in 100 urine samples from 21 SLE patients and 75 urine samples from 75 healthy pregnant women were analysed and compared to previously reported sequences. The results show that the VP1 sequence profiles of JCV and BKV excreted by SLE patients do not differ significantly from those excreted by immunocompetent individuals. The European JCV subtypes 1A or 1B were represented among all JCV-positive urine specimens, while BKV VP1 sequences showed complete, or almost complete, identity with the MM or JL strains. Different urine samples from the same patient collected over a 1 year period were predominantly stable. BKV VP1 DNA in urine specimens from healthy pregnant women was only detected during the third trimester of their pregnancy. These results argue against SLE-specific JCV and BKV strains and suggest reactivation of the viruses rather than recurrent re-infections of patients with SLE.

Murine gammaherpesvirus-68 (MHV-68) was originally isolated from a bank vole by passage through mouse brain. Given its ability to replicate in mouse brain and its subsequent reisolation from trigeminal ganglia, it was originally considered to be an alphaherpesvirus. Molecular studies have now firmly established MHV-68 to be a gammaherpesvirus. Other gammaherpesviruses have been suggested to cause and in some cases shown to cause neurological disease. Given the isolation history of MHV-68, we have studied the ability of this virus to gain access to, to replicate in and to persist in the mouse CNS. Following intranasal inoculation the virus was not generally neuroinvasive. However, in mice with a deletion of the type-I interferon receptor gene, peripheral virus titres are higher and perivascular CNS infection was observed. There was no evidence of virus spread via olfactory routes. Direct intracerebral inoculation of virus was fatal with widespread infection and destruction predominantly of meningeal and ependymal cells. Hippocampal pyramidal neurons, oligodendrocytes, Bergmann glia cells in the cerebellar cortex and neural progenitor cells in the rostral migratory stream were also infected. A similar infection was observed in younger mice. CNS infection following virus reactivation was investigated by implantation of infected glial cells. Implantation into a brain ventricle led to widespread fatal infection, principally involving ependymal and meningeal cells. Implantation into the striatum resulted in a predominantly neuronal infection. Implantation of cells into mice transiently treated with the antiviral thionucleoside analogue 2′-deoxy-5-ethyl-β-4′-thiouridine resulted in survival with detection of virus-infected cells in the brain 1 year later.

Latency-associated nuclear antigen (LANA), encoded by ORF 73 of Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus-8), may play an important role in the persistence of the viral episome by tethering it to host chromosomes during mitosis. It also has been suggested from its amino acid sequence features that LANA may have transcription-regulatory activity. Here, it is reported that LANA interacts with activating transcription factor (ATF) 4/cAMP response element-binding protein (CREB) 2, a member of the ATF/CREB family of transcription factors, and represses the transcriptional activation activity of ATF4/CREB2. Repression by LANA is independent of the DNA-binding ability of ATF4/CREB2, since LANA also represses transactivation of ATF4/CREB2 fused to the GAL4 DNA-binding domain and does not affect the DNA-binding ability of ATF4/CREB2 in an electrophoretic mobility shift assay. The putative leucine zipper domain of LANA is required for binding to the relatively conserved basic region/leucine zipper domain (bZIP) of ATF4/CREB2, suggesting that the interaction may involve leucine zipper dimerization.

The herpesvirus saimiri (HVS) gene product encoded by ORF73 shares a limited homology with the ORF73 encoded protein of Kaposi’s sarcoma-associated herpesvirus (KSHV). It has recently been shown that the KSHV ORF73 protein is expressed during a latent infection and co-localizes with host cell chromosomes, suggesting that it plays a role in episomal maintenance by tethering viral genomes to host cell chromosomes. At present the role of the HVS ORF73 gene product is unknown. However, the expression of HVS ORF73 in a stably transduced human carcinoma cell line, where the HVS genome persists as a non-integrated circular episome, has recently been shown. In this report, the characterization of the HVS ORF73 protein and the mapping of its functional domains are described. The results suggest that the HVS ORF73 gene encodes a 64 kDa nuclear protein. Moreover, the amino terminus contains two functional nuclear localization signals, whereas the carboxy terminus is required for the distinctive speckled nuclear distribution pattern as observed with both the HVS and KSHV ORF73 proteins.

Glycoprotein B (gB, gpUL55) is the major antigen recognized by the neutralizing humoral immune response against human cytomegalovirus (HCMV). The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids (aa 552–635) for antibody binding. In this study, the structural requirements for antibody binding to AD-1 have been determined. The domain was expressed in prokaryotic and eukaryotic systems and analysed in immunoblots under reducing and non-reducing conditions. In addition, AD-1 was purified in an immunologically active form and the concentration of sulphydryl groups was determined. The data clearly show that the only form that is recognized by antibodies is a disulphide-linked monomer of AD-1. The disulphide bond is formed between cysteines at amino acid positions 573 and 610 of gB.