- Volume 76, Issue 5, 1995

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains (RQP[T/S]LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish. The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe. The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units). The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids. Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization. Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues. Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV.

DNA of Yaba virus, a member of the poxviruses, was mapped by cross-hybridization between fragments of various restriction enzymes. The genome was approximately 135 kb in length and possessed two characteristic features of poxviruses: cross-links and inverted terminal repeats at both termini. Hybridization of fragments of Yaba virus DNA to known vaccinia virus DNA fragments indicated that the thymidine kinase (TK) gene mapped within the 0.9 kb XhoI-HincII fragment between 52.5 and 53.5 kb from the left end of the genome. The fragment could rescue the TK+ phenotype in TK− cells preinfected with a TK vaccinia virus mutant. Nucleotide sequencing of the fragment revealed an ORF capable of encoding 181 amino acids. The sequence TAAAAATGAAAAATTA upstream of the ORF was considered to be the promoter and the downstream sequence TTTTTAT to be the early transcription termination signal. These sequences are in good accord with the consensus regulatory sequences for the expression of early genes of other known poxviruses. The amino acid sequence similarity among the poxvirus TK genes suggests that Yaba virus is most closely related to swinepox virus and less similar to fowlpox virus.

The African swine fever virus (ASFV) j13L gene encodes a 177 amino acid protein (19.0 kDa) with a putative transmembrane domain between residues 32 and 52. There is a potential signal peptide cleavage site at residue 54 and several possible motifs for phosphorylation and myristylation. Rabbit antisera raised against a synthetic peptide from the C terminus of the j13L ORF identified proteins of 25–27 kDa in cells infected with a recombinant vaccinia virus expressing the j13L ORF, in ASFV-infected cells and in purified extracellular ASF virions. In ASFV-infected cells the j13L protein was expressed late during infection and exhibited size variation (25–27 kDa) between the different ASFV strains. Nucleotide sequence analysis of the gene in these strains showed that these size differences were due to variation in the number and sequence of tandemly repeated amino acid repeats. Although ASFV-infected animals made antibodies to the j13L protein, no protection was observed when pigs were vaccinated with a recombinant vaccinia virus expressing the j13L ORF.

Replication of bovine papillomavirus type 1 (BPV-1) DNA has been shown to require two viral proteins known to interact in a molecular complex: E2, a transcription activator, and E1, another nuclear phosphoprotein, which binds to the replication origin and for which helicase/ATPase activities have previously been reported. Here we characterize the BPV-1 E1 ATPase activity. In contrast to Seo et al., (Proceedings of the National Academy of Sciences, USA, 90, 702–706, 1993), we were able to detect this activity in the absence of nucleic acid in partially purified preparations of either E1 protein or of E1–E2 protein complex. Measurements of specific activity and kinetic parameters gave similar values for preparations of various kinds. ATPase activity was quantitatively retained by immunoprecipitates obtained by using anti-E1 or, in the case of E1–E2 complex, anti-E2 antibodies. Significantly, preparations of bacterially expressed glutathione S-transferase-E1 fusion protein exhibited levels of DNA-independent ATPase activity comparable to those of baculovirus-expressed E1. The presence of nucleic acids of various types, including stoichiometric amounts of a BPV-1 ori DNA fragment containing E1 and E2 binding sites, did not grossly affect E1 ATPase activity, the most notable effect being a 2-fold stimulation by unspecific ssDNA. Altogether, our results indicate that BPV-1 E1 possesses an intrinsic ATPase activity which does not depend on the presence of nucleic acid; moreover, they render unlikely any modulation of E1 ATPase activity due to binding either E2 protein or target DNA sequences, or as a result of protein phosphorylation.

The study of viral infectivity and detection of viral capsid antigens of the major cervical cancer-associated human papillomavirus (HPV) type, HPV-16, requires knowledge of which epitopes are exposed in clinical specimens of infected tissue or on intact capsids. To define the antigenic epitopes of HPV-16, antisera to 66 overlapping synthetic peptides corresponding to the HPV-16 capsid proteins L1 and L2 and to seven peptide analogues were tested in immunoperoxidase stainings of consecutive sections from formalin-fixed, paraffin-embedded HPV infected tissue. Antisera against eleven different peptides from L1 and against seven different peptides from L2 recognized the HPV capsid antigen. Most epitopes were only found on the capsid antigen of certain genital HPV types, but four antigenic epitopes in L1 were detectable also in cutaneous wart specimens. All antigenic epitopes in L2 were restricted to genital HPV types and four L2 epitopes were only detectable in HPV-16 or HPV-33 positive specimens. The surface exposure of the antigenic epitopes was investigated by comparing the reactivity of the antipeptide antisera with intact or disrupted virions or capsids of HPV-11, HPV-16 and bovine papillomavirus (BPV). Twenty antipeptide sera from L1 and seven antipeptide sera from L2 were reactive with intact HPV-16 capsids at titres up to 1:146000. Sixteen of these antisera were also reactive with disrupted HPV-16 capsids. Cross-reactivity with disrupted HPV-11 and BPV was detected for eleven and six antisera, respectively, whereas intact HPV-11 or BPV virions showed only weak cross-reactivity. In conclusion, the HPV-16 L1 and L2 capsid proteins contained multiple antigenic epitopes, most of which were shared with one or several additional HPV types.

Infection of dendritic cells (DC) by human immunodeficiency virus (HIV) has been disputed. Employing a fluorescence-activated cell sorter, DC, identified by the absence of membrane markers for T, B, natural killer (NK) and monocytic cells and by high levels of MHC class II DR antigen, were shown to express low levels of CD4. Immunomagnetic beads were used to separate blood low density cells, which are enriched for DC, into CD4-positive and -negative populations. Examination of these cells by electron microscopy showed an increase in the percentage of cells with DC morphology in the CD4-positive fraction and a reduction in the CD4-negative fraction. Electron microscopy of semi-purified DC preparations infected in vitro for 5 days with HIV-1 revealed morphologically distinct veiled DC with mature virions on the cell surface and virus budding through the cell membrane. Further evidence for the growth of HIV in DC was provided by experiments in which DC were extensively depleted of contaminating lymphocytes and monocytes prior to infection. Estimation of provirus load by a nested PCR indicated that after 5 days an infection level of one provirus copy per five cells could be achieved. After 7 days the provirus copy number could exceed the cellular genome copy number, suggesting that some cells had more than one provirus. Infectious virus could not be demonstrated in these cultures after 24 h but was detected after 5 or 7 days. Infection of DC in the presence of antibodies against CD4 was inhibited and suggests infection occurs via a CD4-dependent pathway. These results confirm that DC are susceptible to HIV infection in vitro. The immunological consequences of DC infection in vivo may be significant in the pathogenesis of AIDS.

We have characterized and mapped variable and conserved neutralization epitopes of serogroup A strains of aquatic birnaviruses. Epitope mapping using monoclonal antibodies (MAbs) and Escherichia coli-expressed deletion fragments of VP2 of the N1 strain of infectious pancreatic necrosis virus (IPNV) demonstrated that two variable epitopes, H8 and B9, depend on the variable region between amino acid 204–330. A conserved neutralization epitope, F2, was shown to depend on the same region as epitopes H8 and B9 but was additionally dependent on amino acids between 153–203. The neutralization epitopes H8, B9 and F2 were also shown to overlap by a competitive binding assay. One conserved neutralization epitope, AS-1, was not exposed on any of the recombinant VP2 deletion fragments and was therefore not possible to map. However, the MAbs AS-1 and F2 were partly competitive indicating that these epitopes are overlapping. All neutralization epitopes were independent of a conserved non-neutralization epitope, E4. Our results demonstrate that the central third of VP2 contains several partly overlapping neutralization epitopes, both variable and conserved among serogroup A strains of IPNV.

The nucleotide sequence encoding the C terminus of the nucleocapsid protein of measles virus (MV) is the most variable in the genome. The sequence of this region is reported for 21 new MV strains and for virus RNA obtained from cases of subacute panencephalitis (SSPE) tissue. The nucleotide sequence of a total of 65 MV strains has been analysed using the CLUSTAL program to determine the relationships between the strains. An unrooted tree shows that eight different genotypes can be discerned amongst the sequences analysed so far. The data show that the C-terminal coding sequence of the nucleocapsid gene, although highly variable between strains, is stable in a given strain and does not appear to diverge in tissue culture. It therefore provides a good ‘signature’ sequence for specific genotypes. The sequence of this region can be used to discriminate new imported viruses from old ‘endemic’ strains of MV in a geographical area. The different genotypes are not geographically restricted although some appear to be the mainly ‘endemic’ types in large areas of the world. In global terms there appears to be at least four cocirculating genotypes of MV. The low level of divergence in the Edmonston lineage group isolated before 1970 indicates that some isolates are probably laboratory contaminants. This applies to some SSPE isolates such as the Hallé, Mantooth and Horta-Barbosa strains as well as some wild-type isolates from that period.

The induction of macrophage procoagulant activity (PCA) has been shown to correlate with the development of fulminant hepatic necrosis after infection with murine hepatitis virus strain 3 (MHV-3). However, comparatively little is known about the early events in cells after viral infection leading to PCA expression. Accordingly, we investigated the early cellular events in the induction of macrophage PCA by MHV-3. MHV-3 stimulation of macrophages did not result in a detectable increase in intracellular calcium levels nor did stimulation of macrophages by calcium ionophores result in induction of PCA, suggesting that calcium transients were neither necessary nor sufficient for induction of PCA by MHV-3. Treatment of cells with phorbol myristate acetate had no effect on PCA induction; however, inhibition of protein kinase C (PKC) by staurosporine or H7 resulted in attenuation of macrophage PCA following MHV-3 stimulation (P < 0.05 compared with untreated macrophages), suggesting that although activation of PKC alone is insufficient for PCA induction, PKC may be an integral component of PCA induction by MHV-3. We have previously demonstrated that dimethyl prostaglandin E2 inhibited induction of PCA by MHV-3. In this study, treatment of cells by agents that increase intracellular cAMP (forskolin, isobutylmethyl xanthine) significantly inhibited PCA induction (P < 0.02). These results demonstrate that induction of macrophage PCA by MHV-3 involves PKC, but proceeds independently of changes in intracellular calcium, and that PCA expression is down-regulated by increases in intracellular cAMP.

The 5′-terminal untranslated region (5′ UTR) of the uncapped hepatitis A virus (HAV) RNA contains two pyrimidine-rich sequences; one about 20 nucleotides (nt) in length in the vicinity of the AUG initiation codon (nt 706–726), and a longer one (about 40 nt) encompassing nt 100 to 140. The latter includes a 13 nt ‘core’ sequence (positions 126–138 in the HM175 strain) which is 80% identical to the pyrimidine-rich tract of poliovirus type 1 RNA (Mahoney strain). Representative cDNAs of the entire 5′ UTR of HAV RNA were inserted in the intercistronic region of the bi-cistronic plasmid pSV-GH/CAT between the genes coding for the human growth hormone (GH) and bacterial chloramphenicol acetyltransferase (CAT). When COS-7 cells were transfected with these constructs they transiently expressed CAT indicating that the 5′ UTR of HAV was efficiently directing internal initiation of translation of the reporter gene. Under similar conditions the 5′ UTR of poliovirus type 2 (Lansing strain) was 30% more efficient in directing the expression of the CAT gene. Removal of the ‘core’ sequence from the 5′-distal pyrimidine-rich stretch extending between nt 117 and 131 in the HAV 5′ UTR reduced the CAT activity in the lysates of transfected cells by 40%, whereas point mutations engineered in this segment strongly decreased (80% inhibition) the HAV-driven expression of the reporter gene. Limited mutations systematically introduced in the reiterated (U)UUUCCC motifs of the 5′-distal pyrimidine-rich tract identified two major functional domains extending between nt 100–106 and 113–119. Substitutions in these hexanucleotides abrogated internal initiation of translation, whereas similar changes in the neighbouring domains (nt 107–112 and 120–126) had no effect on the expression of the reporter gene, suggesting that the 5′-most pyrimidine-rich tract is indeed part of the structure(s) recognized by ribosomes and associated factors at initiation of translation and that the hexanucleotides 100–106 and 113–119 constitute and important part of it. Although HAV replicates better at 33°C than at 37°C, incubation of transfected cultures at 33 °C delayed the expression and slightly reduced the level of CAT activity in the cell lysates, but the overall effect of the mutations remained unchanged.

A method is described for identifying different genotypes of hepatitis C virus (HCV) by restriction endonuclease cleavage of sequences amplified by PCR from the 5′ non-coding region. Using the enzymes HaeIII-RsaI and HinfI-MvaI, followed by cleavage with BstU1 or ScrFI, it was possible to identify and distinguish HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5 and 6. The method was used to investigate the prevalence of these genotypes in 723 blood donors in 15 countries, the largest survey to date, and one which covered a wide range of geographical regions (Europe, America, Africa and Asia). These results, combined with a review of the existing literature, indicate the existence of several distinct regional patterns of HCV genotype distribution, and provide the framework for future detailed epidemiological investigations of HCV transmission.

In this study, we characterized the B cell and T cell responses to the hydrophilic portion of hepatitis C virus (HCV) core protein in two strains of mice and identified the respective antigen determinants. BALB/c (H-2d) and C57BL/6 (B6:H-2b) mice were immunized by a subcutaneous injection of recombinant HCV core protein together with Freund’s complete adjuvant. The level of antibody production, as determined by ELISA, was consistently higher in BALB/c than in B6 mice. However, antibodies in sera from each strain bound to the N-terminal region of the core protein within amino acids 1 to 28 (MSTNPKPQRKIKRNTNRRPQDVKFPGGG), according to an experiment using non-overlapping peptides that covered the hydrophilic portion of HCV core protein. The T cell responses were also higher in BALB/c than in B6 mice with respect to the proliferative responses of the draining lymph node cells in vitro. By limiting dilution cultures of the draining lymph node cells in vitro repetitively stimulated with recombinant core protein, T cell clones were established from both strains of mice and characterized. The surface markers of these clones were Thy-1.2+, CD3+, TCRαβ+, CD4+ and CD8−. The proliferative responses were inhibited in the presence of anti-CD4 or anti-MHC class II monoclonal antibodies. The T cell lines in BALB/c mice recognized an epitope in HCV core at amino acids 72 to 91 (EGRAWAQPGYPWPLYGNEGL). The T cell lines in B6 mice recognized an epitope at amino acids 55 to 74 (RPQPRGRRQPIPKARQPEGR). Thus, mice with different MHC haplotypes recognized different non-overlapping T cell antigenic determinants of HCV core proteins.

A human hepatoma cell line constitutively expressing proteins of hepatitis C virus (HCV) was established by transfection with cDNA encoding part of the virus non-structural (NS) genome region. Proteins consistent with authentic processing at NS3/NS4A, NS4A/NS4B and NS4B/NS5A were identified. Pulse-chase experiments indicated that the cleavage between NS3 and NS4A occurred first and cleavage at other sites followed. Expression of specific surface antigens in response to the presence of HCV proteins was analysed by flow cytometry. A significant increase in CD26 expression was observed in cells expressing the HCV proteins. CD26 plays an important role in cellular signal transduction. Its upregulation in response to the presence of HCV proteins may play a role in viral pathology.

A Chinese hamster ovary cell line was established which abundantly expresses the second envelope protein (E2) of hepatitis C virus under the control of an exogenous promoter. The expressed E2 protein was found to be a glycoprotein of 58 kDa by immunoprecipitation with sera from patients that had chronic hepatitis C. Using this cell line as antigen in immunofluorescence tests, as high as 93% of patients with non-A non-B hepatitis had antibodies against E2 protein. In Western blots using SDS-denatured E2 protein, however, the detectability of the antibody was drastically reduced to 30%. Immunoprecipitation assays and ELISA, using both native and denatured E2 protein, revealed that antibodies to E2 protein were present in most of the chronic hepatitis C patients and that they reacted only to the native forms.

COS-7 cells transfected with parvovirus B19-simian virus 40 (SV40) hybrid vectors have previously been shown to express B19 structural proteins. In this study the morphology and antigenicity of B19 proteins expressed in these cells were investigated. At 84 h after transfection, approximately 10% of the COS-7 cells expressed B19 antigen, and the yield was equivalent to 2 × 103 to 2 × 105 B19 particles/transfected cell. The B19 proteins self-assembled into capsids that were morphologically and antigenically similar to native B19 virions, and could substitute for native antigen in a B19 IgM assay. Recombinant capsids lacking the recently described 11 kDa protein also resembled native virions.

E5 is the smallest transforming protein encoded by the human papillomaviruses (HPVs). It has been shown to promote anchorage-independent growth in established NIH 3T3 cells, an activity that is enhanced in the presence of epidermal growth factor (EGF). It is thought that this activity of E5 is brought about by an increase in the half-life of stimulated EGF receptors, possibly through the perturbation of receptor processing. Recent studies have also shown that E5 can co-operate with HPV-16 E7 to stimulate proliferation of primary rodent cells. Using haemagglutinin I epitope-tagged E5 proteins, we have compared the mitogenic activity of HPV-6 and HPV-16 E5. Both tagged proteins retain the ability to bind to the cellular 16 kDa H+-ATPase protein. In addition, both HPV-6 and HPV-16 E5 retain the ability to co-operate with E7 in primary rodent cells, although HPV-16 E5 is considerably more active than HPV-6 E5 in these mitogenic assays. Interestingly, transfection of a plasmid over-expressing c-Raf appears to be capable of functionally substituting for E5 in the co-mitogen assays. Polyclonal cell lines derived from baby rat kidney cells co-transfected with E7 and E5 genes continue to express both the E5 and E7 mRNA, although the level of E5 expression is very low and protein cannot be detected. These polyclonal lines appear to be immortal and in some cases demonstrate anchorage-independent growth, an activity which is enhanced by the addition of EGF.

Two strains of influenza A virus were isolated from pigs in northern Japan in 1992. Serological tests showed that the haemagglutinin (HA) and neuraminidase (NA) antigens were more closely related to those of recent human H1N1 viruses than to those of swine H1N1 viruses. The HA and NA genes of isolate A/sw/Obihiro/5/92 were shown to be closely related to those of current human H1N1 viruses. Evolutionary trees constructed from nucleotide sequences showed that the HA and NA genes of A/sw/Obihiro/5/92 were apparently on a branch cluster containing human strains isolated between 1990 and 1992.

A mouse model of Sendai virus infection was adopted to examine the in vivo neurovirulence of parainfluenza viruses. A nested polymerase chain reaction detected the Sendai virus nucleoprotein gene in the olfactory bulbs of intranasally infected mice for at least 168 days post-infection (p.i.) and virus-specific messenger RNAs for 28 days p.i. Viral proteins were histochemically detected in some olfactory neurons for 7 days p.i. They were also found in glomeruli of the olfactory bulbs but not in the mitral cells and the tufted cells. No virus was detected in the whole brain not including the olfactory bulbs. When mice were inoculated with UV-inactivated virus, the viral RNA was present in the olfactory bulbs for a short period of 14 days, with no demonstrable viraemia. These results demonstrate that the parainfluenza virus directly accesses the central nervous system via olfactory neurons and establishes long-term persistence in the nerve tissue.