- Volume 80, Issue 3, 1999
Volume 80, Issue 3, 1999
- Articles
-
-
-
Nuclear and nucleolar localization of an African swine fever virus protein, I14L, that is similar to the herpes simplex virus-encoded virulence factor ICP 34.5
PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.
-
-
-
-
Distribution of B-cell epitopes on the pseudorabies virus glycoprotein B
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.
-
-
-
Kinetics of transcription of human cytomegalovirus chemokine receptor US28 in different cell types
More LessIn 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.
-
-
-
Activation of cyclin A gene expression by the cyclin encoded by human herpesvirus-8
More LessHuman 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.
-
-
-
Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8) encodes a homologue of the Epstein-Barr virus bZip protein EB1
More LessAnalysis 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.
-
-
-
Development of porcine adenovirus-3 as an expression vector
More LessPorcine 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.
-
-
-
Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity
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.
-
-
-
Concerted expression of BK virus large T- and small t-antigens strongly enhances oestrogen receptor-mediated transcription
More LessPrevious 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.
-
-
-
Human papillomavirus type 16 variant lineages characterized by nucleotide sequence analysis of the E5 coding segment and the E2 hinge region
More LessWe 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.
-
-
-
Functional analysis of mutations conferring lamivudine resistance on hepatitis B virus.
More LessTwo 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.
-
-
-
A cellular protein which binds hepatitis B virus but not hepatitis B surface antigen.
More LessThe 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.
-
-
-
Hepadnavirus evolution and molecular strategy of adaptation in a new host
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.
-
-
-
Infection of apheresis cells by parvovirus B19
More LessParvovirus 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.
-
-
-
Susceptibility of TT virus to interferon therapy
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.
-
-
-
Relationships between simian and human enteroviruses
More LessPartial 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.
-
-
-
Molecular evolution of swine vesicular disease virus
More LessPhylogenetic 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.
-
-
-
Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus
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.
-
-
-
Demonstration of bovine CD8+ T-cell responses to foot-and-mouth disease virus
More LessThe 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.
-
-
-
Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins.
More LessA 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.
-
-
-
A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques
More LessThe 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.
-
-
-
Genetic diversity of equine arteritis virus
Equine arteritis viruses (EAV) from Europe and America were compared by phylogenetic analysis of 43 isolates obtained over four decades. An additional 22 virus sequences were retrieved from GenBank. Fragments of the glycoprotein G(L) and the replicase genes were amplified by RT-PCR, prior to sequencing and construction of phylogenetic trees. The trees revealed many distinctive lineages, consistent with prolonged diversification within geographically separated host populations. Two large groups and five subgroups were distinguished. Group I consisted mainly of viruses from North America, whilst group II consisted mainly of European isolates. In most instances, where the geographic origin of the viruses appeared to be at variance with the phylogenetically predicted relationships, the horses from which the viruses were recovered had been transported between Europe and America or vice versa. Analysis of the replicase gene revealed similar phylogenetic relationships although not all of the groups were as clearly defined. Virus strains CH1 (Switzerland, 1964) and S1 (Sweden, 1989) represented separate ‘outgroups’ based on analysis of both genomic regions. The results of this study confirm the value of the G(L) gene of EAV for estimating virus genetic diversity and as a useful tool for tracing routes by which EAV is spread. In addition, computer-assisted predictions of antigenic sites on the G(L) protein revealed considerable variability among the isolates, especially with respect to regions associated with neutralization domains.
-
-
-
Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein.
More LessThe non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity.
-
-
-
Differences between hepatitis C virus 5′ untranslated region quasispecies in serum and liver.
More LessIt is unclear whether the sequence populations of hepatitis C virus (HCV) quasispecies in the liver and in serum are different, as a variety of studies on this subject provide conflicting results. In the current study, the populations of HCV 5′ untranslated region (5′ UTR) sequences in paired serum and liver samples from six patients with chronic hepatitis were analysed. Liver-derived, negative-strand viral RNA was amplified with a highly strand-specific Tth-based assay, and extensive measures, including accounting for template copy number, were undertaken to lower the risk of sporadic artefactual polymorphism. Amplified sequences were compared by single-strand conformation polymorphism analysis and by direct sequencing of identified differences. In four patients, liver samples were found to contain variants within the quasispecies which were not found in serum or negative-strand viral RNA, while in the remaining two patients, low virus titre prevented a reliable quasispecies analysis. These results suggest the presence in the same individual of HCV variants differing in the 5′ UTR and possibly replicating with different kinetics.
-
-
-
Variation of hepatitis C virus following serial transmission: multiple mechanisms of diversification of the hypervariable region and evidence for convergent genome evolution.
We have studied the evolution of hepatitis C virus (HCV) from a common source following serial transmission from contaminated batches of anti-D immunoglobulin. Six secondary recipients were each infected with virus from identifiable primary recipients of HCV-contaminated anti-D immunoglobulin. Phylogenetic analysis of virus E1/E2 gene sequences [including the hypervariable region (HVR)] and part of NS5B confirmed their common origin, but failed to reproduce the known epidemiological relationships between pairs of viruses, probably because of the frequent occurrence of convergent substitutions at both synonymous and nonsynonymous sites. There was no evidence that the rate at which the HCV genome evolves is affected by transmission events. Three different mechanisms appear to have been involved in generating variation of the hypervariable region; nucleotide substitution, insertion/deletion of nucleotide triplets at the E1/E2 boundary and insertion of a duplicated segment replacing almost the entire HVR. These observations have important implications for the phylogenetic analysis of HCV sequences from epidemiologically linked isolates.
-
-
-
Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA.
The immunogenicity of variable regions of hepatitis C virus (HCV) proteins was studied by ELISA by using 543 synthetic peptides from 120 variable regions and 90 sera from HCV-infected patients. Some regions from certain genotypes were less immunogenic, or even non-immunogenic, compared with their equivalents in other genotypes. However, the mean recognition of all peptides from genotypes 1a, 1b and 3 by sera infected with genotypes 1a, 1b and 3, respectively, showed no significant differences, suggesting a similar overall immunogenicity of variable regions from these genotypes. Proteins NS4a, NS4b and NS5a were found to be the most immunogenic. Recognition of individual peptides by the sera of infected patients showed that the humoral response against HCV is patient-dependent. The work shows that 15-mer peptides may encompass several B-cell epitopes. These epitopes may lie in slightly different positions in different genotypes. Thirty-one percent of the 543 peptides were recognized by some of the 35 healthy donors. This may be a reflection of the large number of antigens to which they had been exposed, but it may also reflect a strategy of HCV to respond to immune pressure. After selection and modification, a set of 40 peptides was used to assess genotypes 1a, 1b, 1, 2 and 3 in the sera of HCV-infected patients, with sensitivities of 34.1, 48.5, 68.8, 58.3 and 48.9% and specificities of 100, 99.1, 97.1, 99.5 and 99%, respectively. The overall sensitivity and specificity for the assessment of genotypes 1, 2 and 3 were 64 and 98%, respectively.
-
-
-
Geographic distribution and evolution of Sindbis virus in Australia
More LessThe molecular epidemiology and evolution of Sindbis (SIN) virus in Australia was examined. Several SIN virus strains isolated from other countries were also included in the analysis. Two regions of the virus genome were sequenced including a 418 bp region of the E2 gene and a 484 bp region containing part of the junction region and the 5′ end of the C gene. Analysis of the nucleotide and deduced amino acid sequence data from 40 SIN virus isolates clearly separated the Paleoarctic/Ethiopian and Oriental/Australian genetic types of SIN virus. Examination of the Australian strains showed a temporal rather than geographic relationship. This is consistent with the virus having migratory birds as the major vertebrate host, as it allows for movement of virus over vast areas of the continent over a relatively short period of time. The results suggest that the virus is being periodically redistributed over the continent from an enzootic focus of evolving SIN virus. However, SIN virus strains isolated from mosquitoes collected in the south-west of Australia appear to represent a new SIN virus lineage, which is distinct from the Paleoarctic/Ethiopian and Oriental/Australian lineages. Given the widespread geographic dispersal of the Paleoarctic/Ethiopian and Oriental/Australian lineages, it is surprising that the South-west genetic type is so restricted in its area of circulation. Nucleotide sequence data from the C gene of the prototype strain of the alphavirus Whataroa were also determined. This virus was found to be genetically distinct from the SIN virus isolates included in the present study; however, it is clearly SIN-like and appears to have evolved from a SIN-like ancestral virus.
-
-
-
Amino acid substitutions in a conserved region in the stalk of the Newcastle disease virus HN glycoprotein spike impair its neuraminidase activity in the globular domain.
More LessThe ectodomain of the paramyxovirus haemagglutinin-neuraminidase (HN) glycoprotein spike can be divided into two regions: a membrane-proximal, stalk-like structure and a terminal globular domain. The latter contains all the antibody recognition sites of the protein, as well as its receptor recognition and neuraminidase (NA) active sites. These two activities of the protein can be separated by monoclonal antibody functional inhibition studies and mutations in the globular domain. Herein, we show that mutation of several conserved residues in the stalk of the Newcastle disease virus HN protein markedly decrease its NA activity without a significant effect on receptor recognition. Thus, mutations in the stalk, distant from the NA active site in the globular domain, can also separate attachment and NA. These results add to an increasing body of evidence that the NA activity of this protein is dependent on an intact stalk structure.
-
-
-
Long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism
More LessThe Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.
-
-
-
Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection
Cats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore, virus-neutralizing antibodies were detected in a Crandell feline kidney cell-based neutralization assay, but not in a neutralization assay based on primary peripheral blood mononuclear cells. Despite the induction of these FIV-specific responses, vaccinated cats were not protected. Instead, accelerated virus replication was found, an observation similar to what previous experiments using other vaccine candidates have shown. Here, the results of the present study are discussed in the light of enhancement of lentivirus infections as a complicating factor in lentivirus vaccine development.
-
-
-
Secretion of β-chemokines by bronchoalveolar lavage cells during primary infection of macaques inoculated with attenuated nef-deleted or pathogenic simian immunodeficiency virus strain mac251
More LessPrimary infection of macaques with simian immunodeficiency virus (SIV) as a model of human immunodeficiency virus (HIV) infection represents a unique opportunity to investigate early lentivirus-host interactions. In order to gain insight into immunopathogenic events taking place in the lung during lentiviral infection, we analysed lymphocyte expansion in the lung and chemokine secretion by mononuclear cells obtained by bronchoalveolar lavage (BALMCs) during primary infection by a pathogenic and a non-pathogenic SIV. Two groups of cynomolgus macaques were inoculated intravenously with a fully pathogenic isolate of SIVmac251 or with an attenuated, nef-deleted, molecular clone of SIVmac251. Spontaneous MIP-1alpha, MIP-1beta and RANTES production was assessed by ELISA in supernatants of short-term cultured BALMCs. Kinetics of haematological, virological and immunological parameters were investigated simultaneously. All 11 inoculated animals became infected. Monkeys inoculated with the nef-deleted SIV clone exhibited a significantly reduced plasma virus load and a less pronounced accumulation of lymphocytes in the lung compared to monkeys infected with the pathogenic SIVmac251 isolate. Compared to pre-infection levels, we observed an increase in the levels of RANTES, MIP1-alpha and MIP1-beta production in the two groups of monkeys, by the time of peak viraemia. Strikingly, a greater enhancement of RANTES and MIP-1alpha production was detected in monkeys infected with the attenuated virus. Given the potential influence of beta-chemokines on the immune response and virus replication, such results suggest that RANTES, MIP1-alpha and MIP1-beta could contribute to the singular features of the immune response elicited during infection of macaques with an attenuated SIV.
-
-
-
Translational effects of peptide antagonists of Tat protein of human immunodeficiency virus type 1
More LessThe Tat (trans-activator of transcription) regulatory protein of human immunodeficiency virus (HIV-1) acts by interacting with the TAR RNA domain of nascent viral transcripts and with cellular proteins to increase viral transcription. In Jurkat-derived HCLE-D36 cells, which are stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene expressed from the TAR-encoding long terminal repeat (LTR) of HIV-1, CAT protein expression is dependent on Tat. The Tat9-K-biotin peptide antagonist of Tat binds specifically to TAR RNA and competes with Tat for binding. In the cellular expression system, Tat9-K-biotin reduces Tat-dependent CAT expression. However, while the Tat antagonist greatly reduces CAT protein production and polysome association of CAT mRNA, it has little effect on CAT mRNA levels, suggesting that the antagonist works at the post-transcriptional level.
-
-
-
Characterization of the 25K FP gene of the baculovirus Bombyx mori nucleopolyhedrovirus: implications for post-mortem host degradation
More LessMutagenesis experiments on the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2′-deoxyuridine generated five mutants with a ‘few polyhedra’ (FP) phenotype. Sequence analysis of the 25K gene homologue of the BmNPV FP mutants revealed nucleotide substitutions in the coding region. Rescue experiments indicated that the FP phenotype of the BmNPV mutants resulted from mutations in the 25K coding region. Effects of infection by these FP mutants were analysed following injection of the viruses into silkworm (B. mori) larvae. Compared to infection with wild-type virus, infection with each FP mutant resulted in reduced host degradation (liquefaction). The degree to which liquefaction was blocked corresponded to the degree of functional disruption of the 25K gene product and to the extent to which polyhedron production was reduced. Electron microscopy revealed that (1) polyhedron production was reduced, (2) very few virions were occluded and those that were lacked envelopes, and (3) the basal lamina of fat-body tissue was not destroyed by infection and accumulations of virions occurred along the membrane. Typical NPV-induced liquefaction was observed following infection with a polyhedrin deletion mutant, indicating that host degradation was not related to polyhedron production. These results suggest that (1) the 25K gene product is involved in the host degradation process caused by virus infection and (2) the FP phenotype is an indirect result of disruption of the 25K gene; activation or suppression of a specific host or viral gene related to tissue degradation and polyhedron formation may be involved.
-
-
-
Nucleotide sequences and taxonomy of satsuma dwarf virus
More LessThe nucleotide sequences of genomic RNA1 (6795 nt) and RNA2 (5345 nt) of satsuma dwarf virus (SDV), a tentative member of the genus Nepovirus, were determined. The deduced genome organization of SDV showed similarities to the organization in como-, faba- and nepoviruses. There is extensive amino acid sequence similarity in the N-terminal regions of the proteins encoded by RNA1 and RNA2, as reported previously only for tomato ringspot nepovirus. However, unlike definitive nepoviruses, which have a single coat protein, SDV has two coat proteins. SDV RNA2 does not contain the long (> 1300 nt) 3′ non-coding region characteristic of some nepoviruses. Phylogenetic analysis of SDV RNA polymerase placed SDV apart from como-, faba- and nepoviruses. These unique features suggest that SDV is distinct from the Comovirus, Fabavirus and Nepovirus genera, and needs to be separated into a new genus, probably within the family Comoviridae.
-
-
-
Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase
More LessTomato ringspot nepovirus (TomRSV) RNA-1 encodes a putative NTP-binding protein (NTB), a putative viral genome-linked protein (VPg), a putative RNA-dependent RNA polymerase (Pol) and a serine-like protease (Pro), which have been suggested to be involved in viral RNA replication. Proteolytic processing of protease precursors containing these proteins was studied in Escherichia coli and in vitro. The TomRSV protease could cleave the precursor proteins and release the predicted mature proteins or intermediate precursors. Although processing was detected at all three predicted cleavage sites (NTB-VPg, VPg-Pro and Pro-Pol), processing at the VPg-Pro cleavage site was inefficient, resulting in accumulation of the VPg-Pro intermediate precursor in E. coli and in vitro. In addition, the presence of the VPg sequence in the precursor resulted in increased cleavage at the Pro-Pol cleavage site in E. coli and in vitro. Direct N-terminal sequencing of the genomic RNA-linked VPg, of the mature protease purified from E. coli extracts and of radiolabelled mature polymerase purified from in vitro translation products revealed the sequences of the NTB-VPg, VPg-Pro and Pro-Pol dipeptide cleavage sites to be Q/S, Q/G and Q/S, respectively. In vitro processing at the NTB-VPg and Pro-Pol cleavage sites was not detected upon mutation or deletion of the conserved glutamine at the -1 position of the cleavage site. These results are discussed in light of the cleavage site specificity of the TomRSV protease.
-
-
-
The complete genome sequence of the major component of a mild citrus tristeza virus isolate
The genome of the Spanish mild isolate T385 of citrus tristeza virus (CTV) was completely sequenced and compared with the genomes of the severe isolates T36 (Florida), VT (Israel) and SY568 (California). The genome of T385 was 19,259 nt in length, 37 nt shorter than the genome of T36, and 33 and 10 nt longer than those of VT and SY568, respectively, but their organization was identical. T385 had mean nucleotide identities of 81.3, 89.3 and 94% with T36, VT and SY568, respectively. The 3′ UTR had over 97% identity in all isolates, whereas the 5′ UTR of T385 had 67% identity with VT, 66.3% with SY568 and only 42.5% with T36. In the coding regions, the nucleotide differences between T385 and VT were evenly distributed along the genome (around 90% identity); this was not observed between T385 and the other isolates. T385 and T36 had nucleotide identities around 90% in the eight 3′-terminal ORFs of the genome, but only 72.3% in ORF 1a, a divergence pattern similar to that reported previously for T36 and VT. T385 and SY568 had nucleotide identities close to 90% in the 5′- and 3′-terminal regions of the genome, whereas the central region had over 99% identity. Our data suggest that the central region in the SY568 genome results from RNA recombination between two CTV genomes, one of which was almost identical to T385.
-
-
-
New defective RNAs from citrus tristeza virus: evidence for a replicase-driven template switching mechanism in their generation
Defective RNAs (D-RNAs) ranging in size from 1968 to 2759 nt were detected in four citrus tristeza virus (CTV) isolates by hybridization of electroblotted dsRNAs with two probes specific for the 5′- and 3′-terminal genomic regions. The RNAs that hybridized with both probes were eluted, cloned and sequenced. Comparison with the sequences of the corresponding genomic regions of the helper virus showed, in all cases, over 99% nucleotide identity and direct repeats of 4-5 nt flanking or in the vicinity of the junction sites. The presence of the repeats from two separate genome locations suggests a replicase-driven template switching mechanism for the generation of these CTV D-RNAs. Two of the CTV isolates that differed greatly in their pathogenicity contained an identical D-RNA, suggesting that it is unlikely that this D-RNA is involved in symptom modulation, which may be caused by another factor.
-
Volumes and issues
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)