- Volume 80, Issue 3, 1999

In order to map antigenically important regions of glycoprotein B (gB) of pseudorabies virus (PrV), a panel of recombinant fragments of gB expressed in E. coli and truncated fragments of gB generated by cleavage of purified native gB with trypsin and cyanogen bromide was analysed by using 26 monoclonal antibodies directed against gB. Three continuous epitopes were localized in the vicinity of the N terminus of gB, between amino acids (aa) 59 and 126. One continuous epitope mapped between residues 214 and 279. The residues involved in the assembly of eight discontinuous epitopes were located between aa 540 and 734. The constituents of two discontinuous epitopes were harboured in a segment encompassing aa 540-646. The clustering of continuous epitopes at the extreme N terminus of PrV gB and the locations of residues involved in the assembly of discontinuous epitopes of PrV gB are in good agreement with data on epitope locations in gB homologues from other herpesviruses.

In permissive cells, human cytomegalovirus encodes the protein US28, a functional CC chemokine receptor. US28 polyadenylated mRNA could be detected by RT-PCR as early as 2 h post-infection. US28 mRNA appeared after major IE1 transcripts (UL123), but before transcripts of the early genes pp65 (UL83) and gB (UL55), and the late gene pp150 (UL32). This temporal appearance indicates that US28 is transcribed earlier than previously reported. Furthermore, US28 mRNA could be detected in semi- and non-permissive cells.

Human herpesvirus-8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus, encodes a protein, referred to as HHV8-Vcyc, with sequence similarity to human G1 cyclins, in particular of the D type. HHV8-Vcyc is expressed in Kaposi’s sarcoma and functional analysis suggests that it can activate cyclin-dependent kinases (cdk) and thereby trigger inactivation of the retinoblastoma protein (pRb), indicating that HHV8-Vcyc may contribute to the oncogenic potential of HHV-8. We show here that HHV8-Vcyc can activate transcription of the human cyclin A gene in quiescent cells, a property shared with known transforming oncogenes. Transcriptional activation by HHV8-Vcyc depends on an E2F-binding site in the cyclin A promoter, and cdk6 kinase activity is required. The ability of HHV8-Vcyc to activate cyclin A gene expression is shared by D-type cyclins and cyclin E. Unlike D-type cyclins, HHV8-Vcyc is unable to trigger phosphorylation of the pRb-related protein p107 and fails to induce dissociation of p107 from E2F. Unlike cyclin E, HHV8-Vcyc fails to interact physically with E2F complexes on the cyclin A promoter. These results provide additional evidence for the notion that the HHV-8-encoded cyclin differs in several properties from cellular G1 cyclins.

Analysis of the recently completed genomic sequence of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8) revealed that ORF 50 encodes a protein with homology to the Epstein-Barr virus (EBV) transcription factor R. In this report, we show that ORF K8, contiguous to ORF 50, is interrupted by two introns and that the spliced RNA is translated into a bZip protein that has homology to the EBV transcription factor EB1. The newly characterized K8 protein forms homodimers but does not heterodimerize with other members of the bZip protein family.

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

Previous studies have shown that the human polyomavirus BK (BKV) genome contains an oestrogen response element (ERE). This isolated element binds its cognate receptor in vitro and can mediate 17beta-oestradiol-induced gene expression when linked to a heterologous promoter. The roles of the ERE- and the AP-1-binding sites in oestrogen receptor-directed transcription from the complete BKV promoter/enhancer (Dunlop strain) have been examined and the effects of the general co-activator CBP and large T- and small t-antigens on oestrogen receptor-mediated transcription have been investigated. A constitutive activated oestrogen receptor stimulated BKV promoter activity in HeLa cells. Mutations in either the ERE- or the AP-1-binding sites did not impair oestrogen receptor-induced activation of the BKV Dunlop promoter, while mutations in both binding motifs almost completely abolished oestrogen receptor-induced transcription. Simultaneous expression of large T- and small t-antigens strongly activated oestrogen receptor-mediated transcription. When expressed separately, only large T-antigen moderately stimulated oestrogen receptor-mediated transcription. The stimulatory effect of large T-antigen on the activity of the oestrogen receptor is probably indirect because no physical interaction between the two proteins was detected in a two-hybrid assay. Large T-antigen abrogated the synergistic effect on transcription between this nuclear receptor and the general co-activator CBP. The findings that the BKV early proteins amplify oestrogen receptor-mediated transcription may have important biological implications in individuals with raised oestrogen concentrations.

We have previously examined 29 cervical cell isolates for human papillomavirus type 16 (HPV-16) sequence variations in the E6, L2 and L1 coding regions, and the long control region (LCR). Twenty-five of these isolates as well as 23 additional isolates are characterized here as we present the complete E5 coding segment and the E2 hinge region. Eight amino acid variations were observed in the E5 coding segment, 13 were identified in the E2 hinge region and 5 were observed in the overlapping E4 coding segment. These amino acid variations may be relevant to differences in biological functions and may result in altered humoral or cell-mediated immune responses to HPV-16 variants. The characterization of sequence variation within high-risk HPV types might be important in the search for epidemiological correlates of cervical cancer risk. This work complements and extends HPV-16 genome sequence information from specific isolates previously reported by our group.

Two patterns of mutation are commonly observed in the polymerase gene of lamivudine [(-)2′-deoxy-3′-thiacytidine]-resistant hepatitis B virus (HBV). The M539I substitution in the conserved YMDD motif occurs independently of other changes, whereas the M539V substitution is associated with an additional upstream change (L515M). These mutations were introduced into a common background and their effects on HBV DNA replication and lamivudine resistance studied. The L515M and M539V mutations provided only partial resistance while the M539I mutation conferred a high degree of lamivudine resistance. The combination of the L515M and M539V mutations gave an intermediate level of replication competence, compared with either mutation alone, and increased resistance to lamivudine. This probably accounts for these two mutations always being observed together. The M539I mutation reduced replication competence.

The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner.

In order to elucidate the mechanisms of hepadnavirus evolution in vivo and to trace the fate of known quasispecies in a single animal during the acute phase of infection, a woodchuck (Marmota monax) was infected with the hepadnavirus woodchuck hepatitis B virus (WHV). Woodchuck 197 (W197) was injected intravenously with pooled sera collected from a chronic carrier that had been infected originally with a molecular clone of known genome sequence (WHV7). Viral genome variants from both the inoculum and the follow-up sera from W197 were characterized for the presence of quasispecies related to the WHV7 sequence. Interestingly, WHV7-related genomes were predominant 6 weeks post-infection (p.i.), whereas a highly heterogeneous virus population was present in the first viraemic serum (4 weeks p.i.). Using WHV7 as the prototype, the variability of the Pol and PreS/S regions in the first 11 weeks p.i. has been calculated. The sequence population in serum collected 6 weeks p.i. was highly homogeneous, with a mean variability of 0.36% in the region analysed. Mean variability values ranging from 0.82% to 1.61% were found in quasispecies from the other sera. The presence of possible selective pressure was analysed by means of the non-synonymous versus synonymous variation ratio (dn/d5). We found that the dn/d5 values were stable for the S ORF (ranging from 2.6 to 3.0), whereas a wider range was observed for the Pol ORF (from 1.4 to 3.0). Furthermore, from the analysis of the variability of the codon positions for the two overlapping ORFs it was found that, in most cases, non-synonymous mutations at position 1 of the Pol ORF (position 3 of the S ORF) corresponded to synonymous variation in the S (Pol) ORF, indicating independent evolution of the encoded proteins.

Parvovirus B19 is the only member of the Parvoviridae family known to cause disease in humans. Owing to the high level of cell tropism the virus can only replicate in proliferating and differentiating erythroid precursor cells, which are present in human bone marrow and foetal liver. As human bone marrow is very difficult to obtain, an alternative in vitro system for the propagation of B19 virus has been developed, based on the application of mobilized haemapoietic progenitor (apheresis) cells. These cells are routinely harvested from cancer patients after treatment with recombinant human granulocyte/macrophage colony-stimulating factor. Replication of parvovirus B19 in vitro is possible in these cells after stimulation with erythropoietin. Therefore, this system is an easily, accessible alternative to the use of human bone marrow in parvovirus B19 infection assays.

TT virus (TTV) is a newly identified single-stranded DNA virus. We retrospectively analysed serum samples from sixteen patients, infected with both hepatitis C virus (HCV) and TTV, and who had been treated with interferon. An elevated serum alanine aminotransferase level after interferon was associated with persistence of HCV (abnormal in five of seven patients with persistence of HCV compared with normal in all nine patients who showed eradication of HCV) irrespective of persistence of TTV. Comparison of partial viral DNA nucleotide sequences and phylogenetic analysis showed that viral strains that had a high identity to the prototype virus were more resistant to interferon than those showing low nucleotide sequence identity. Although we observed no liver cell injury caused by persistent TTV infection, the mechanism(s) of TTV resistance to interferon should be further investigated for a better understanding of viral diseases and establishment of therapy.

Partial sequences from two genomic regions of simian enteroviruses were analysed and their relatedness to other picornaviruses was compared. Of the 18 simian viruses included in the analysis, sequences were obtained from eleven strains for at least one genomic region. In the 5′ non-coding region, SV6, SV19, SV26, SV35, SV43 and SV46 (simian viruses) and BA13 (baboon virus) clearly grouped together with human enteroviruses, whereas SV4, SV28 and SA4 (South African isolate) were more distantly related. In the 3D RNA polymerase-coding region, SV26, SV35, SV43 and SV46 could be clearly identified as enteroviruses and fell into the previously defined cluster A, which contains such human viruses as coxsackievirus A16 and enterovirus 71. However, although SV6 and BA13 were also enterovirus-like, they did not belong to any known genetic cluster of human enteroviruses. Moreover, while SV18 could be recognized as a picornavirus, it did not directly group with members of the genus Enterovirus.

Phylogenetic analysis was used to examine the evolutionary relationships within a group of coxsackie B viruses that contained representatives of the major serotypes of this group and 45 isolates of swine vesicular disease virus (SVDV) from Asia and Europe. Separate analyses of sequence data from two regions of the viral genomes encoding the VP1 and 3BC genes both revealed that the SVDV belonged to a single monophyletic group which could be clearly distinguished from all other sampled coxsackieviruses. Regression analysis revealed that within the SVDV clade at least 80% of the synonymous variation in evolutionary divergence between isolates was explained by time, indicating the existence of an approximate molecular clock. Calibration of this clock according to synonymous substitutions per year indicated the date of occurrence of a common ancestor for the SVDV clade to be between 1945 and 1965.

The complete RNA genome of avian encephalomyelitis virus (AEV) has been molecularly cloned and sequenced. This revealed AEV to be a member of the Picornaviridae and consequently it is the first avian picornavirus for which the genome has been sequenced. Excluding the poly(A) tail the genome comprises 7032 nucleotides, which is shorter than that of any mammalian picornavirus sequenced to date. An open reading frame commencing at nucleotide 495 and terminating at position 6896 (6402 nucleotides) potentially encodes a polyprotein of 2134 amino acids. The polyprotein sequence has 39% overall amino acid identity with hepatitis A virus (HAV; genus Hepatovirus), compared to 19 to 21% for viruses from the other five picornavirus genera. Eleven cleavage products were predicted. The highest identity (49%) with HAV was in the P1 region, encoding the capsid proteins. The 5′ and 3′ untranslated regions (UTRs) comprise 494 and 136 nucleotides, respectively. The 5′ UTR is the shortest of any picornavirus sequenced to date and, unlike HAV, it does not contain a long polypyrimidine tract.

The aim of this study was to investigate the importance of cellular immunity in foot-and-mouth disease in cattle, in particular to determine whether a CD8+ T-cell response could be detected, as these cells may play a role in both immunity and virus persistence. As attempts to characterize classical cytotoxic T cells had yielded non-reproducible results, largely due to high backgrounds in control cultures, a proliferation assay was developed that was demonstrated to detect antigen-specific, MHC class I-restricted bovine CD8+ cells responding to foot-and-mouth disease virus (FMDV). Proliferative CD8+ T-cell responses were detected consistently from 10 to 14 days following infection with FMDV and typically lasted 3-4 weeks. The role of CD8+ T cells in control of the disease, in particular their relevance for the establishment of persistence, may now be investigated.

A recombinant live vector vaccine was produced by insertion of cDNA encoding the structural proteins (P1) of foot-and-mouth disease virus (FMDV) into a replication-competent human adenovirus type 5 vaccine strain (Ad5 wt). Groups of cattle (n = 3) were immunized twice, by the subcutaneous and/or intranasal routes, with either the Ad5 wt vaccine or with the recombinant FMDV Ad5-P1 vaccine. All animals were challenged by intranasal instillation of FMDV 4 weeks after the second immunizations. In the absence of a detectable antibody response to FMDV, significant protection against viral challenge was seen in all of the animals immunized twice by the subcutaneous route with the recombinant vaccine. The observed partial protection against clinical disease was not associated with a reduction in titre of persistent FMDV infections in the oropharynx of challenged cattle.

The partial sequence of a hepatitis E virus (HEV-US1) isolated from a patient in the United States (US), suffering from acute viral hepatitis with no known risk factors for acquiring HEV, has been reported. These sequences were significantly different from previously characterized HEV isolates, alluding to the existence of a distinct human variant. In this paper, we report the near full-length sequences of HEV-US1 and a second US isolate (HEV-US2). HEV-US2 was identified in a US patient suffering from acute viral hepatitis. These sequences verify the presence of a new HEV strain in North America and provide information as to the degree of variability between variants. The HEV-US nucleotide sequences are 92% identical to each other and only 74% identical to the Burmese and Mexican strains. Amino acid and phylogenetic analyses also demonstrate that the US isolates are genetically distinct, suggesting the presence of three genotypes of HEV. Serum from the second US patient induced hepatitis following inoculation into a cynomolgus macaque. Within 2-4 weeks, HEV-US2 RNA was detectable in both the serum and faecal material coinciding with elevated serum alanine transaminase levels. Infection resolved as antibody titres increased 8 weeks post-inoculation.