- Volume 79, Issue 8, 1998

We have proposed that hepatitis C virus should be classified into eleven genetic groups (types) which further divide into more than 80 genotypes (subtypes). However, only eight genetic groups (1–6, 10 and 11) have been defined on the basis of the full- length sequence. Hence, the entire nucleotide sequences of three HCV isolates in genetic groups 7–9 have now been determined. Phylogenetic analysis over the full-length sequences of these three isolates, along with 30 more in the other eight genetic groups, indicated that genetic groups 6–9 and 11 have bifurcated from a common branch and groups 3 and 10 from another. In the former branch groups 7 and 11, and groups 8 and 9, are closely related. Consequently, HCV can be classified into either eleven (1–11) or six groups (1; 2; 3 and 10;

4; 5; 6–9 and 11), allowing a clear separation of group and genotype similarity within the NS5b region or a subregion of 1093 nt. When pairwise comparison of 1093 nt in the NS5b sequence was performed on 106 HCV isolates of 36 genotypes in eleven genetic groups, they were classified into either eleven (1–11) or six (1; 2; 3 and 10; 4; 5; 6–9 and 11) genetic groups. However, group and genotype similarities were not clearly separable in either classification. The overlapping range was smaller using the classification into eleven genetic groups as compared to six genetic groups (2·7 vs 4·7%). These results indicate that HCV might not have evolved in the two-tiered fashion, at least in a strict sense.

We previously found two cell lines (MT-2 and PH5CH) that were susceptible to hepatitis C virus (HCV) infection. Analysis of the infectivity of sera from HCV-positive blood donors for MT-2 and PH5CH cells suggested the cell tropism of HCV. To investigate further the cell tropism of HCV, the dynamics of HCV populations during culture were examined using three MT-2 clones and three PH5CH clones, infected with inoculum 1B-2. To type HCV populations in these infected cells, the HCV hypervariable region 1 (HVR1) in these cloned cells was characterized by sequence analysis and HpaII digestion analysis, which could distinguish three major HVR1 types (I, II and III) derived from the inoculum 1B-2. It was found that genomes containing HVR1 type I became predominant in MT-2 clones, and genomes containing HVR1 type II became predominant in PH5CH clones during culture after inoculation. These results suggest that inoculum 1B-2 contains both lymphotropic and hepatotropic HCV species, which can be distinguished by HVR1 type. To search for cell type- specific sequences in regions other than HVR1, three HCV cDNA clones (3·4 kb of the 5′ noncoding region to the nonstructural 2 region) containing HVR1 type I obtained from HCV-infected MT-2C cells, and three HCV cDNA clones containing HVR1 type II obtained from HCV-infected PH5CH7 cells were sequenced. Following a comparison of the sequences, 11 amino acids were identified as candidates for determinants of the cell tropism of HCV.

Infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is widely distributed in human populations. Viruses related to GBV-C/HGV have been recovered from several New World primate species, including tamarins, owl monkeys and marmosets. To understand more about the relationship between GB viruses and their hosts, we used primers from the 5′ non-coding (5NC), non-structural 3 (NS3) and NS5 regions in nested polymerase chain reactions to screen for related viruses infecting non-captive chimpanzees (Pan troglodytes, troglodytes and verus subspecies). Sequences from the 5NCR and NS5 regions were amplified from samples taken from 3 of 39 chimpanzees, and from one chimpanzee in the NS3 region. Sequence comparisons of each region revealed that the GB virus infecting chimpanzees was distinct from both GBV-C/HGV and from any of the known GBV-A sequences, but was more closely related to human viruses. GB viruses recovered from different chimpanzees were more diverse than variants of GBV-C/HGV found in humans, with 25% sequence divergence in the 5′NCR and 20% (9·5% amino acid) sequence divergence in NS5 between variants recovered from the troglodytes and verus subspecies, compared with 7·4% and 10·4% (1·9% amino acid) divergence amongst GBV-C/HGV variants infecting humans. Finding GBV-C/HGV-related viruses in an Old World monkey species suggests that GB-like viruses may be widely distributed in simians, and suggests a close evolutionary relationship with their natural hosts.

An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5′ untranslated region (UTR), a luciferase gene and the 3′ UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase underthe control of a mammalian promoter. Only a low level of luciferase expression was ob tained in cells coinfected with AcT7HCVLuc and either AcCAT7 or AdexCAT7. In contrast, high-level luciferase expression was detected when the same cells were coinfected with FPVT7HCVLuc and either AcCAT7 or AdexCAT7. We further constructed a recombinant fowlpox virus with its HCV minigene extended to contain the whole HCV core protein region. Significantly high levels of expression of HCV core protein were detected in MT-2, COS7 and Vero cells by coinfection with the recombinant fowlpox virus and AdexCAT7. A coinfection system consisting of recombinant fowlpox virus and Adex- CAT7 was established for high level of expression of a target gene in various cells.

The mechanisms were investigated of haematopoietic progenitor growth inhibition, observed after in vitro infection of cord blood mononuclear cells (CBMNC) by a clinical isolate of dengue 3 (29–56DSS). The level of virus replication was not different when CBMNC were inoculated with 29–56DSS compared with a prototype strain of dengue 3 (H-87) which had no inhibitory effect. An inhibitory effect was also observed when cell-free and heat-inactivated supernatants from 29–56DSS cultures, but not from H-87 cultures, were added to cultures of normal CBMNC, suggesting an indirect mechanism via the release of soluble suppressive factor(s). Macrophage inflammatory protein-1a (MIP-1α ) was detected at a significantly higher level in 29–56DSS cultures than in controls. Blocking experiments with anti-MIP-1α antibody demonstrated that the inhibitory effect was related at least partly to high MIP-1α levels. To our knowledge, this is the first report suggesting an indirect effect of dengue infection on haematopoiesis mediated by a suppressive cytokine.

The 17D-204 vaccine manufactured in South Africa (17D-204-SA) and a large plaque variant (17D-LP) derived from it were highly virulent in adult mice. The LD50 of 17D-LP virus was 0 2 p.f.u. for mice following intracerebral inoculation. In comparison, a medium plaque variant derived from 17D-LP, termed 17D-MP virus, was found to be attenuated in adult mice following the same route of inoculation (> 104 p.f.u./LD50). Replication of 17D-MP virus was decreased in infected mouse brains compared to 17D-LP virus. Also, 17D-MP virus was slightly temperature sensitive at 39·5 °C. Compared to its parent viruses, 17D-204-SAand 17D-LP, 17D-MP virus had one unique mutation at nt 8045 in the genome which resulted in a single amino acid substitution (Pro → Ser) at residue 137 of the NS5 protein and appeared to be the mutation responsible for the attenuation of 17D-MP virus. This is the first time that altered virulence of a flavivirus caused by mutation in a non-structural protein gene, other than NS1, has been reported.

Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal antibodies (MAbs) and 12 overlapping fragments of the protein expressed in E. coli. Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid. This domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appears to be the most immunodominant region.

A mutational and genetic analysis of the poliovirus protein 3A has led to the identification of a single amino acid mutant virus with a restrictive phenotype to form plaques in Vero cells. This mutant (I46T 3A) can be grown and amplified in HeLa cells, where virus replication takes place at wild-type levels. However, Vero cells infected with this virus cannot complete the growth cycle. I46T 3A virus has a defect in the ability to kill Vero cells, as estimated by FACS analysis of propidium iodide uptake by dead cells. Since these defects are observed under conditions where no abnormalities in the rate of synthesis and processing of the mutant polyprotein occur, the inability to induce the cytopathic effect in infected Vero cells denotes the existence of a defect in the activity of 3A, but not the level of expression of the viral genome. As a consequence of this impaired capability to generate the cytopathic effect, I46T 3A mutant viruses cannot be titrated by plaque assay in Vero cells. Only revertant viruses with the wild-type sequence arise and form lysis plaques in Vero cells. Our results suggest a role for the 3A protein (or a precursor thereof) in the virus- induced cytopathic effect. The mutant virus characterized in this work may be a useful tool to understand how poliovirus kills infected cells and carries out the final step of its life-cycle, the release of virus progeny.

Within the sequence motif conserved at the extreme ends of the influenza virus vRNAs, a unique natural variation, U or C, is observed at position 4 of the 3′ end. To test the role of this nucleotide, two isogenic A/WSN/33 viruses, carrying either C4 or U4 nucleotide at the ′ end of the neuraminidase (NA) gene, were generated. Compared with the C4 virus, the U4 virus exhibited delayed synthesis of vRNA and stimulation of mRNA synthesis with prolonged accumulation in influenza virus-infected cells. The mRNA/ vRNA ratio was increased up to 20-fold by the C4 → U4 substitution suggesting that the U4nucleo- tide greatly stimulated transcription of the vRNA template. In isolated virion, the U4 virus had higher NA activity than the C4 virus. In MDBK cells, the U4 virus grew to lower haemagglutination (HA) titres but with higher infectivity than the C4 virus, with a corresponding increase in the ratio of p.f.u./HA units of about 10- to 40-fold. Western blot analysis of isolated virion showed that the ratio of two surface proteins, HA/NA, was greatly decreased in the U4 virus. This suggests that the position 4 nucleotide is a genetic determinant for the repertoire of surface antigens and their ratio could be changed without detrimental effects on virus growth. Results could be used to design genetically engineered influenza virus for vaccination. The observed down-regulation of transcription by C4 nucleotide is consistent with its potential role in segment-specific regulation of influenza virus gene expression, especially PB1, PB2 and PA proteins, during virus infection.

Infection of macaques with attenuated simian immunodeficiency virus (SIV) induces potent superinfection resistance that may be applicable to the development of an AIDS vaccine but little information exists concerning the conditions necessary for the induction of this vaccine effect. We report that only a high dose of attenuated SIVmac protected macaques against intravenous challenge with more virulent virus 15 weeks after primary infection. Three of four animals given 2000–20000 TCID50 of SIVmacC8, a molecular clone of SIVmac251(32H) with a 12 bp deletion in the nef gene, essentially resisted superinfection with uncloned SIVmac. In two animals challenge virus was never detected by PCR and in one animal challenge virus was detected on one occasion only. Although animals given 2– 200 TCID50 of attenuated virus were superinfected they were spared from the loss of CD4 cells seen in infected naive controls. Protection from superinfection did not correlate with immune responses, including the levels of virus-specific antibodies or virus-neutralizing activity measured on the day of challenge; although, after superinfection challenge, Nef-specific CTL responses were detected only in animals infected with high doses of attenuated SIV. Unexpectedly, cell-associated virus loads 2 weeks after inoculation were significantly lower in animals infected with a high dose of attenuated SIV compared to those in animals infected with a low dose. Our results suggest that the early dynamics of infection with attenuated virus influence superinfection resistance.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for the infectivity of virions produced by non-permissive cells. The primary replicative defect of Vif particles involves either synthesis or stability of viral DNA, but the mechanism of this defect is unknown. Here, we report the results of a detailed analysis of HIV-1 DNA synthesis by isogenic VifNmutants produced by different chronically infected H9 clones, which exhibit different degrees of impairment in their replicative capacity. We found that the degree of impairment of DNA synthesis by the mutant particles always correlated with the degree of their loss of infectivity. This impairment appears to be global, with a defect increasing along with synthesis of longer viral DNA species. We conclude that the primary replicative defect of Vif virus involves the capacity of the reverse transcription complex of HIV-1 to efficiently elongate viral DNA, resulting in an inability to produce full-length viral DNA genomes.

Based on available sequence data, a phylogeny of small ruminant lentiviruses (SRLV) was established for env, pol, gag and LTR fragments using maximum likelihood, neighbour-joining and minimum evolution reconstruction techniques. To reconstruct topologies as accurately as possible, phylogenetic parameters like base composition, percentage divergences, kappa, the gamma shape parameter alpha and codon position-specific rates were estimated prior to the reconstruction of trees. Divergences between fragments of SRLV ranged from 16% in gag and pol to 22% in env and 35% in LTR. The codon position bias found and the ratios of synonymous to nonsynonymous substitutions were inversely related to overall divergence, indicating the existence of both negative and positive Dar winian selection in SRLV genes. The phylogenetic trees reconstructed with relative substitution rates assigned to the codon positions revealed an interesting relationship between lentiviruses from sheep and goats. Overall, at least six different clades could be differentiated, with no clear separation of SRLV strains derived from goats (caprine arthritis- encephalitis virus) or sheep (maedi-visna virus). Trees generated with fragments from different coding regions were in good agreement with each other as well as with trees generated with different phylogeny reconstruction methods. In this work, clear indications of the existence and epidemiological importance of cross-species transmission were found.

An important characteristic of the E6 proteins derived from oncogenic associated human papillomaviruses (HPVs) is their ability to target the cellular tumour suppressor protein, p53, for ubiquitin mediated degradation. Several studies have attempted to address the important characteristics of both E6 and p53 for this activity in vitro, but the equivalent determinants have not been extensively assessed in vivo. Indeed, recent studies indicate differences between the in vitro and the in vivo degradation assays. We have performed an extensive analysis of the ability of a range of HPV-18 E6 mutants to direct p53 degradation in vivo. In addition, we have also compared the ability of HPV-18 E6 to direct the degradation of different oligomeric forms of p53 both in human and in murine cells. The results of these studies show that mutants of E6 exhibit very similar phenotypes both in vitro and in vivo. In contrast, mutants of p53 show markedly different susceptibilities in vitro and in vivo to E6-induced degradation, and this is further affected by the nature of the cell type in which the assays are performed. Finally, using a cell line temperature sensitive for the E1 ubiquitin-activating enzyme we have been able to show directly that this enzyme is involved in the process of E6-mediated degradation of p53 in vivo.

Proliferative responses to human papillomavirus type 16 (HPV-16) E5 peptides were determined for short-term cell lines derived from peripheral blood mononuclear cells of 75 women. Cell lines from 16 of the 75 women proliferated in response to stimulation with pooled E5 peptides; this was most common for patients with low-grade squamous cervical intraepithelial lesions (LSIL; 6 of 15 patients, 40%) and less frequent for asymptomatic women with no cervical lesions (4 of 20, 20%), those with high-grade squamous intraepithelial lesions (HSIL; 5 of 33,15%) and others with cervical cancer (1 of 7, 14%, P= 0·027). Amongst these patients, proliferative responses were exclusive to those that were positive for HPV-16 DNA(12 of 41, 29%; c.f. none of 13 HPV-16 DNA-negative subjects exhibited a proliferative response; P=0·023) and were again most prevalent amongst HPV-16 DNA-positive LSIL (6 of 14, 43%), as compared with HPV-16 DNA-positive HSIL (5 of 23, 22%) or HPV-16 DNA-positive cervical cancer patients (1 of 4, 25%, P > 0·05). In contrast, for asymptomatic women, responsiveness was statistically independent of HPV-16 DNA status, i.e. responsiveness in HPV-16 DNA-positive and DNAnegative subjects was observed in 3 of 15 (20%) and 1 of 5 (20%) cases, respectively (P > 0·05). There were no associations between detection of HPV-16 mRNA and proliferative responses (P > 0·05). These data suggest that HPV-16 E5-specific T-helper activity is depressed amongst women with HSIL lesions.

Human papillomavirus type 60 (HPV-60) is the only virus type that has been identified in epidermoid cysts. In this study, HPV-57 DNA was found in three out of 18 plantar epidermoid cysts with different histological features from HPV-60-associated cysts, using PCRand Southern hybridization. The HPV-57- associated cysts had features resembling an HPV-2- specific cytopathic effect. The sequences of two HPV-57 DNA clones isolated from two patients were identical, but differed at some positions from those of HPV-57a and HPV-57b. This putative new subtype was tentatively designated as HPV-57c, and may be associated with plantar epidermoid cysts showing histological features resembling the HPV-2 cytopathic effect.

Bovine herpesvirus type 1 (BHV-1) glycoprotein H (gH) is a structural component of the virion which forms a complex with glycoprotein gL. To study the role of BHV-1 gH in the virus infectious cycle, a gH null mutant was constructed in which the gH coding sequences were deleted and replaced by the Escherichia coli lacZ cassette. The BHV-1 gH null mutant was propagated in frans-complementing MDBK cells, stably transfected with plasmid pMEP4 containing the BHV-1 gH gene under the control of the inducible mouse metallothionein promoter. Experiments with the BHV-1 gH null mutant showed that gH is essential in the infectious cycle of the virus and is specifically involved in virus entry and cell-to-cell spread. The lack of infectivity of virions devoid of gH is not due to a defect in attachment. Moreover, PEG-induced fusion of virions to target cells provides evidence that BHV-1 gH is required for virion penetration.

We have identified the herpes simplex virus type 2 (HSV-2) UL55 gene product using a rabbit polyclonal antiserum raised against a recombinant 6 × His-UL55 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 23 kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor phosphonoaceticacid. Indirect immunofluorescence studies localized the UL55 protein within and at the periphery of the nucleus as discrete granules at late times post-infection, and nuclear fractionation studies showed that the protein was associated with the nuclear matrix of infected cells. Moreover, these discrete regions containing the UL55 protein were found to be adjacent to compartments, designated assemblons, containing the capsid protein ICP35. However, the UL55 protein was not detected in purified virions. These results suggest that the UL55 protein of HSV-2 may play an accessory role in virion assembly or maturation.

Studies on Marek’s disease virus serotype 2 (MDV2) are important for understanding the natural nonpathogenic phenotypes of MDV. We determined the 16770 bp nucleotide sequence of the MDV2 genome located in the right part the of unique long region. The analysis revealed 12 complete open reading frames (ORFs) with high amino acid sequence identities to the gene products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1 ranging from the UL41 to UL51 genes. Except for the MDV2 UL41 gene, all of the identified genes were confirmed to be transcribed with 3 ′-coterminal mRNAs and/or a unique transcript in the virus-infected cells. Transcriptional patterns for the regions of the MDV2 UL48 to UL49.5 genes were notably different from the similar area of MDV serotype 1.

The location of the BcLFI promoter of Epstein-Barr virus (EBV) has been identified by primer extension, which indicates that the 1 site of the BcLFI mRNA is located at nucleotide 137676 of the EBV genome. According to deletion analysis, the region upstream from nucleotide 38 is not essential for transcription of BcLFI. A 23 bp region in the promoter, from nucleotide 38 to 16, was identified as necessary for regulating the expression of BcLFI, i.e. the promoter activity is activated by 12-O-tetradecanoylphorbol 13-acetate but is repressed by phosphonoacetic acid. The results presented also demonstrate that the oriLyt sequence in cis is essential for enhancing the expression of BcLF1.