- Volume 86, Issue 1, 2005

Hepatitis C virus (HCV) is a major cause of liver disease. Knowledge of HCV variability is crucial for clinical and epidemiological analysis. HCV genotype 4 (HCV-4) has become increasingly prevalent in European countries, including France, in recent years. The present study investigates the heterogeneity of HCV-4 in south-western France by phylogenetic analysis of NS5B sequences from 166 patients. The E2 region of 90 strains was also analysed. Genotype 4 accounts for 7·4 % of HCV infections in this area. Analysis of the NS5B region revealed 12 subtypes and the NS5B and E2 phylogeny data were congruent, except for one strain. The epidemiological data indicated two main groups of patients. One included intravenous drug users (IVDUs) of French origin, who were infected by homogeneous strains of subtypes 4a or 4d. The second group comprised non-IVDU patients who were infected with heterogeneous strains. This group was subdivided into patients of French origin, who were infected with eight subtypes, and patients from non-European countries (Central Africa or the Middle East), who were mainly infected with 4f, 4k, 4r and other subtypes; they showed the greatest genetic heterogeneity. This study of a large cohort of patients shows the great diversity of HCV-4 strains, and that these subtypes have spread differently.

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is an RNA molecule that is approximately 9·6 kb in length and encodes a polyprotein that is cleaved proteolytically to generate at least 10 mature viral proteins. Recently, a new HCV protein named F has been described, which is synthesized as a result of a ribosomal frameshift. Little is known about the biological properties of this protein, but the possibility that the F protein may participate in HCV morphology or replication has been raised. In this work, the presence of functional constraints in the F protein was investigated. It was found that the rate of amino acid substitutions along the F protein was significantly higher than the rate of synonymous substitutions, and comparisons involving genes that represented independent phylogenetic lineages yielded very different divergence/conservation patterns. The distribution of stop codons in the F protein across all HCV genotypes was also investigated; genotypes 2 and 3 were found to have more stop codons than genotype 1. The results of this work suggest strongly that the pattern of divergence in the F protein is not affected by functional constraints.

Novel flavivirus isolates from mosquitoes collected in northern Australia were analysed by partial genomic sequencing, monoclonal antibody-binding assays and polyclonal cross-neutralization tests. Two isolates were found to be antigenically distinct from, but related to, viruses of the Kokobera virus complex, which currently contains Kokobera (KOKV) and Stratford (STRV) viruses. Nucleotide sequence comparison of two separate regions of the genome revealed that an isolate from Saibai Island in the Torres Strait in 2000 (TS5273) was related closely to KOKV and STRV, with 74–80 and 75–76 % nucleotide similarity, respectively. An isolate from mainland Cape York in 1998 (CY1014) was found to be more divergent from KOKV and STRV, with <70 % nucleotide sequence similarity to either virus. It is proposed that isolate TS5273 represents a new subtype of KOKV and that CY1014 be classified as a novel species within the Kokobera virus complex of flaviviruses, named New Mapoon virus.

Virus genomes from the same family may exhibit a wide range in their DNA GC content, whereas viral hypermutants differ substantially in GC content from their parental genomes. As AT-rich DNA melts at lower temperatures than GC-rich DNA, use of a lower denaturation temperature during PCR should allow differential amplification of AT-rich genomes or variants within a quasispecies. The latter situation has been explored explicitly in a two-step process by using a series of well-defined viral sequences differing in their AT content. Firstly, the lowest denaturation temperature (T p) that allowed amplification of the parental sequence was determined. Secondly, differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1–3 °C lower than T p. Application of this sensitive method to two different viruses allowed us to identify human immunodeficiency virus type 1 G→A hypermutants in a situation where none were expected and to amplify AT-rich variants selectively within a spectrum of poliovirus mutants.

The C-terminal tail of the gp41 transmembrane glycoprotein of the human immunodeficiency virus type 1 (HIV-1) virion is usually thought to be inside the virion, but it has been shown recently that part of the tail is exposed on the virion exterior. Here, using a panel of antibodies, it was demonstrated that the same part of the tail is exposed on the surface of HIV-1-infected C8166 lymphoblastoid cells and HeLa cells infected with a gp41-expressing vaccinia virus recombinant. Both types of infected cell failed to react with p17 matrix protein-specific IgGs until permeabilized with saponin, confirming the integrity of the plasma membrane. Cell-surface exposure of the gp41 tail was independently demonstrated by inhibition of HIV-1-mediated cell–cell fusion by one of the gp41 tail-specific antibodies. These data also implicate the exposed region of the gp41 C-terminal tail either directly or indirectly in the viral fusion process. Its surface exposure suggests that the gp41 C-terminal tail may be a candidate for immune intervention or chemotherapy of infection.

It has been previously reported that transient corticosteroid immune suppression of ponies experimentally infected with a highly neutralization resistant envelope variant of equine infectious anemia virus (EIAV), designated EIAVΔPND, resulted in the appearance of type-specific serum antibodies to the infecting EIAVΔPND virus. The current study was designed to determine if this induction of serum neutralizing antibodies was associated with changes in the specificity of envelope determinants targeted by serum antibodies or caused by changes in the nature of the antibodies targeted to previously defined surface envelope gp90 V3 and V4 neutralization determinants. To address this question, the envelope determinants of neutralization by post-immune suppression serum were mapped. The results demonstrated that the neutralization sensitivity to post-immune suppression serum antibodies mapped specifically to the surface envelope gp90 V3 and V4 domains, individually or in combination. Thus, these data indicate that the development of serum neutralizing antibodies to the resistant EIAVΔPND was due to an enhancement of host antibody responses caused by transient immune suppression and the associated increase in virus replication.

Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous.

Pneumonia virus of mice (PVM) is a member of the subfamily Pneumovirinae and is the closest known relative of respiratory syncytial virus. Both viruses cause pneumonia in their respective hosts. Here, the genome sequences of two strains of PVM, non-pathogenic strain 15 and pathogenic strain J3666, are reported. Comparison of the genome sequences revealed 59 nucleotide differences between the two strains, 37 of which were coding. The nucleotide differences were spread throughout the genome, affecting cis-acting regulatory regions and seven of the ten genes. Development of a reverse-genetics system for PVM should allow further elucidation of the functional importance of the genetic differences between the two strains identified here.

The genomic and antigenomic 3′ ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3′ end. Under these conditions, it was found that the frequency with which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external replication promoter. It was also found that the sequences essential for replication are not required for basic mRNA synthesis as long as there is an external replication promoter at which viral RNA polymerase can enter the nucleocapsid template. The manner in which transcription and replication initiations influence each other is discussed.

The influenza A virus M2 proton channel plays a role in two stages of virus replication. The proteins of two closely related strains of the avian H7 subtype of influenza A virus, Rostock and Weybridge, were found to differ in their pH-modulating activities and activation characteristics. Of three amino acid differences at residues 27, 38 and 44 within the membrane-spanning domain, substitution at residue 44 was necessary and sufficient to account for differences in trans-Golgi pH-modulating activity, whereas changes in all three were required to switch the activation characteristics of the Weybridge M2 to those of the Rostock M2. These results not only separate the two phenomena genetically, but also indicate that the ‘unique’ activation characteristics of the Rostock M2 channel were selected specifically. In addition, they point to the importance of functional complementarity between the activation characteristics of the M2 channel and the pH of membrane fusion by haemagglutinin during virus entry.

Several NS1 mutant viruses of human influenza A/PR/8/34 (H1N1) virus were tested for their ability to induce pro-inflammatory cytokines in primary human macrophages. The findings revealed a pronounced difference in the virus-induced cytokine pattern, depending on the functionality of the NS1 protein-encoded domains. The PR8/NS1–125 mutant virus, which encodes the first 125 aa of the NS1 protein, thus lacking the C-terminal domains, induced significantly higher amounts of beta interferon, interleukin (IL) 6, tumour necrosis factor alpha and CCL3 (MIP-1α) when compared with the A/PR/8/34 wild-type virus. However, this mutant virus was as efficient as wild-type virus in the inhibition of IL1β and IL18 release from infected macrophages. Another group of viral mutants either lacking or possessing non-functional RNA-binding and dimerization domains induced 10–50 times more biologically active IL1β and five times more biologically active IL18 than the wild-type or PR8/NS1–125 viruses. The hallmark of infection with this group of mutant viruses was the induction of rapid apoptosis in infected macrophages, which correlated with the enhanced activity of caspase-1. These results indicated that the NS1 protein, through the function of its N-terminal domains, might control caspase-1 activation, thus repressing the maturation of pro-IL1β-, pro-IL18- and caspase-1-dependent apoptosis in infected primary human macrophages.

Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5′ non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.

Varicella-zoster virus (VZV) is sensitive to type I and type II interferons (IFNs), which mediate antiviral effects. In this study, it was demonstrated that IFN-β and IFN-γ inhibited the replication of VZV in vitro. Although IFN-β was more effective than IFN-γ, the level of inhibition of VZV replication achieved by the combination of both IFNs was more than additive and it was concluded that these two cytokines acted synergistically. Expression of the IFN-induced, double-stranded RNA-activated protein kinase PKR and its phosphorylation level were not modulated strongly during ongoing replication of VZV. However, in the presence of IFN-β, but not IFN-γ, PKR expression and its phosphorylation were increased, explaining in part the inhibition of virus replication by IFNs. The expression of herpes simplex virus Us11, a viral protein with several functions, including prevention of PKR activation, strongly increased the level of VZV replication.

Glycoprotein H (gH) homologues are found in all members of the herpes virus family, and gH is one of the virion envelope glycoproteins that is essential for virus entry. In this study, a recombinant soluble form of Herpes simplex virus type 1 (HSV-1) gH, in which the ectodomain is fused to the Fc-binding region of IgG, has been generated. This was expressed in mammalian cells together with gL and the resulting gHFc–gL heterodimer was purified using Protein A Sepharose. Low-affinity cell binding assays showed that gHFc–gL bound specifically to Vero cells and mutation of a potential integrin-binding motif, Arg-Gly-Asp (RGD), in gH abolished binding. CHO cells failed to bind in this assay. However, CHO cells expressing the human αvβ3 integrin bound efficiently to gHFc–gL, suggesting that HSV-1 gH can bind to cells using αvβ3 integrins and that this binding is mediated by the RGD motif in the gH ectodomain.

To analyse the function of the equid herpesvirus 4 (EHV-4) glycoprotein M homologue (gM), two different mutated viruses (E4ΔgM-GFP and E4ΔgM-w) were generated. Both gM-negative EHV-4-mutants were characterized on complementing and on non-complementing cells and compared with E4RgM, a virus where gM-expression had been repaired. It was demonstrated in virus growth kinetics that deleting gM had a more dramatic influence on EHV-4 replication than expected. Extracellular infectivity was detected 9–12 h later than in EHV-4-infected Vero cells and titres were reduced up to 2000-fold. In addition, mean maximal diameters of plaques were less than 20 % of diameters of wild-type plaques. These results are in contrast to most other alphaherpesviruses, including the closely related equid herpesvirus type 1, where deletion of gM only marginally influences the ability of viruses to replicate in cell culture. Nevertheless, analysis of infected cells by electron microscopy did not reveal a specific defect for deleting gM. It was concluded that EHV-4 gM is important for more than one step in virus replication in cell culture, influencing both efficient virus egress and cell-to-cell spread.

Human cytomegalovirus (HCMV) infection is known to modulate host gene expression and has been linked to the pathogenesis of vasculopathies; however, relevant pathomechanisms are still unclear. It was shown that HCMV infection leads to upregulation of vascular endothelial growth factor (VEGF) expression in human foreskin fibroblasts and coronary artery smooth muscle cells (SMC). Activation of VEGF transcription by HCMV infection was confirmed by transient-expression experiments, which revealed that a short promoter fragment, pLuc135 (−85 to +50), is sufficient for activation. Site-directed mutagenesis of Sp1-recognition sites within this fragment abolished the upregulation of transcription. Functional VEGF protein is released into the culture supernatant of infected SMC. Incubation of endothelial cells with supernatants from HCMV-infected SMC cultures induced upregulation of VEGF receptor-2 expression on endothelial cells, as well as a significant upregulation of DNA synthesis, implicating cell proliferation. The mean incline of DNA synthesis at 48 and 72 h post-infection was 148 and 197 %, respectively. Addition of neutralizing antibodies against VEGF completely abolished this effect. Supernatants from SMC cultures incubated with UV-inactivated virus induced a comparable effect. This virus-induced paracrine effect may represent a molecular mechanism for HCMV-induced pathogenesis, such as inflammatory vasculopathies, by inducing a proatherogenic phenotype in SMC.

The gammaherpesviruses are characteristically latent in lymphocytes and exploit lymphocyte proliferation to establish a large, persistent pool of latent genomes. Murine gammaherpesvirus 68 (MHV-68) allows the in vivo analysis of viral genes that contribute to this and other aspects of host colonization. In this study, the MHV-68 bcl-2 homologue, M11, was disrupted either in its BH1 homology domain or upstream of its membrane-localizing C-terminal domain. Each M11 mutant showed normal lytic replication in vitro and in vivo, but had a reduction in peak splenic latency. Lower infectious-centre titres correlated with lower in vivo B-cell activation, lower viral genome loads and reduced viral tRNA expression. This was therefore a true latency deficit, rather than a deficit in ex vivo reactivation. Stable, long-term levels of splenic latency were normal. M11 function therefore contributed specifically to viral latency amplification in infected lymphoid tissue.

Ostreid herpesvirus 1 (OsHV-1) is the only member of the Herpesviridae that has an invertebrate host and is associated with sporadic mortality in the Pacific oyster (Crassostrea gigas) and other bivalve species. Cryo-electron microscopy of purified capsids revealed the distinctive T=16 icosahedral structure characteristic of herpesviruses, although the preparations examined lacked pentons. The gross genome organization of OsHV-1 was similar to that of certain mammalian herpesviruses (including herpes simplex virus and human cytomegalovirus), consisting of two invertible unique regions (UL, 167·8 kbp; US, 3·4 kbp) each flanked by inverted repeats (TRL/IRL, 7·6 kbp; TRS/IRS, 9·8 kbp), with an additional unique sequence (X, 1·5 kbp) between IRL and IRS. Of the 124 unique genes predicted from the 207 439 bp genome sequence, 38 were members of 12 families of related genes and encoded products related to helicases, inhibitors of apoptosis, deoxyuridine triphosphatase and RING-finger proteins, in addition to membrane-associated proteins. Eight genes in three of the families appeared to be fragmented. Other genes that did not belong to the families were predicted to encode DNA polymerase, the two subunits of ribonucleotide reductase, a helicase, a primase, the ATPase subunit of terminase, a RecB-like protein, additional RING-like proteins, an ion channel and several other membrane-associated proteins. Sequence comparisons showed that OsHV-1 is at best tenuously related to the two classes of vertebrate herpesviruses (those associated with mammals, birds and reptiles, and those associated with bony fish and amphibians). OsHV-1 thus represents a third major class of the herpesviruses.

Cottontail rabbit papillomavirus (CRPV)-induced papillomas can progress into malignant carcinomas, remain persistent or regress. Both host immunity and virus genetic background play critical roles in these events. To test how host immunity influences CRPV-induced papilloma evolution, both EIII/JC (inbred) and New Zealand White (outbred) rabbits were treated with an immunosuppressive drug, cyclosporin A (CsA), for 80 days and the regression of three regressive constructs, H.CRPVr (a CRPV regressive strain), H.CRPVp-E6r (a progressive strain with regressive E6) and H.CRPVp-CE6rm (H.CRPVp with the carboxyl terminal of regressive E6, containing mutations at amino acid residues E252G, G258D and S259P) was checked. Papillomas induced by H.CRPVr and H.CRPVp-E6r on control inbred and outbred rabbits regressed totally around week 8, whereas papillomas on all CsA-treated rabbits grew progressively. After cessation of CsA treatment, papillomas began to regress in six outbred rabbits: 14 of 18 papillomas induced by CRPVr, 11 of 18 papillomas induced by H.CRPVp-E6r and eight of 10 papillomas induced by H.CRPVp-CE6rm regressed around week 21. In four CsA-treated inbred rabbits, two of 17 papillomas induced by H.CRPVr and one of 17 papillomas induced by H.CRPVp-E6r regressed. These data indicate that papillomas induced by a regressive CRPV strain can become persistent in the transiently immunosuppressed host. However, returning immunity can lead to regression and clearance of large papillomas (with increased antigenicity) in an outbred population, whilst these same antigenic papillomas persist in inbred rabbits.