- Volume 80, Issue 2, 1999

Echoviruses induce a wide spectrum of diseases in man, the most severe being meningitis. In neonates, however, a severe systemic infection can be observed, leading to death. Serum albumin is the most abundant protein in plasma and most interstitial fluids, and its functions include osmoregulation and transport and delivery of hydrophobic molecules such as fatty acids and steroids. The results of cold-synchronized one-step growth analysis of echovirus 7 infection and sucrose-gradient analysis of A-particles suggest that physiological concentrations of albumin block echovirus 7 infection by inhibiting uncoating. The blockage was reversible and was still effective when albumin was added 30 min after virus adsorption. Inhibition of uncoating was confirmed by using rhodanine, a known specific inhibitor of echovirus uncoating. After removal of the albumin blockage, addition of rhodanine perpetuated the inhibition. Serum and interstitial albumin concentrations may limit echovirus infection in vivo and thereby act as an extracellular determinant for echovirus tropism.

Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3′-distal to the putative helicase-encoding region. Expression from these vectors in E. coli resulted in discrete protein products with smaller than expected molecular sizes. This confirmed that an active viral protease was produced in E. coli and that the protease was capable of cleaving the expressed protein at defined sites. Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG). The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.

Porcine alveolar macrophages (AMphi) are the dominant cell type that supports the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro. In order to determine the characteristics of the virus-receptor interaction, the attachment of PRRSV to cells was examined by using biotinylated virus in a series of flow cytometric assays. PRRSV bound specifically to AMphi in a dose-dependent manner. Binding of PRRSV to AMphi increased gradually and reached a maximum within 60 min at 4 degrees C. By confocal microscopy, it was shown that different degrees of PRRSV binding exist and that entry is by endocytosis. Virus uptake in vesicles is a clathrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH4Cl and chloroquine, it was demonstrated that PRRSV uses a low pH-dependent entry pathway. In the presence of these reagents, input virions accumulated in large vacuoles, indicating that uncoating was prevented. These results indicate that PRRSV entry into AMphi involves attachment to a specific virus receptor(s) followed by a process of endocytosis, by which virions are taken into the cell within vesicles by a clathrin-dependent pathway. A subsequent drop in pH is required for proper virus replication.

Although North American and European serotypes of porcine reproductive and respiratory syndrome virus (PRRSV) are recognized, only the genome of the European Lelystad strain (LV) has been sequenced completely. Here, the genome of the pathogenic North American PRRSV isolate 16244B has been sequenced and compared with LV. The genomic organization of 16244B was the same as LV but with only 63.4% nucleotide identity. The 189 nucleotide 5′ non-coding region (NCR) of 16244B was distinct from the LV NCR, with good conservation (83%) only over a 43 base region immediately upstream of open reading frame (ORF) 1a. Major differences were found in the region encoding the non-structural part of the ORF1a polyprotein, which shared only 47% amino acid identity over 2503 residues of the six non-structural proteins (Nsps) encoded. Nsp2, thought to have a species-specific function, showed the greatest divergence, sharing only 32% amino acid identity with LV and containing 120 additional amino acids in the central region. Nsps encoded by the 5′-proximal and central regions of ORF1b had from 66 to 75% amino acid identity; however, the carboxy-terminal protein CP4 was distinct (42% identity). The ORF 1a-1b frameshift region of 16244B had 98% nucleotide identity with LV. Consistent with previous reports for North American isolates, the six structural proteins encoded were 58 to 79% identical to LV proteins. The 3′ NCR (150 nucleotides) was 76% identical between isolates. These genomic differences confirm the presence of distinct North American and European PRRSV genotypes.

Hypervariable region 1 (HVR1) sequences of 96 clones at six time-points representing 27 variants in two major and one minor group were identified in a patient with chronic hepatitis C virus (HCV) infection over 3 years. Major and selected minor variants were used to design synthetic peptides corresponding to the HVR1 C terminus. Peptide ELISA reactivity with IgG was plotted against the corresponding clone frequency, and three patterns emerged: (1) three peptides were unreactive; (2) antibodies against two peptides followed emergence of the corresponding variant, suggesting isolate-specificity; (3) antibodies against four peptides preceded the appearance of the corresponding variant, indicating cross-reactivity or previous exposure. Cross-reactivity was investigated further: sera from six time-points were tested against 11 unrelated HVR1 peptides, seven of which (63.6%) showed cross-reactivity at all time-points. Cross-reactivity of nine patient-specific peptides tested against a panel of 45 heterologous sera from chronic HCV carriers ranged between 0 and 20%. Only three of 27 variants appeared at more than one time-point and in two cases specific and/or cross-reactive HVR1 antibodies coexisted with the corresponding variant, consistent with emergence of escape mutants. In addition, analysis of HVR1 IgG reactivity within a group of closely related patient-specific peptides revealed a loss of reactivity in one peptide attributable to a single amino acid substitution. Interferon-alpha treatment considerably reduced viral RNA but, paradoxically, heterogeneity increased.

The rubella virus (RV)-encoded protein NSP90, which contains the retinoblastoma protein (Rb)-binding motif LXCXE, interacts with Rb and RV replication is reduced in cells lacking Rb. Whether the LXCXE motif of RV NSP90 itself is essential for Rb binding and virus replication is not known. Therefore, in the present study, the functional role of this motif was investigated by site-directed mutagenesis in a plasmid from which infectious RV RNA can be produced. Three critical mutations in the motif, two substitutions at the conserved cysteine residue (C --> G and C --> R) and a deletion of the entire motif, were created. A cell-free translated NSP90 C terminus polypeptide containing the deletion did not bind to Rb and a polypeptide carrying the C --> R substitution had barely detectable binding affinity for Rb. Rb binding by the C --> G mutant was reduced significantly compared to that of wild-type protein. Correlating with the binding results, mutant viruses containing the LXRXE and LXGXE motifs had a reduction in replication to < 0.5% and 47% of the wild-type, respectively, while deletion of the motif was found to be lethal. By the first serial passage, replication of the LXRXE-carrying virus had increased from < 0.5% to 2% of the wild-type. Sequencing of the genome of this virus revealed a nucleotide change that altered the motif from LXRXE to LXSXE, which is a known Rb-binding motif in two protein phosphatase subunits. Thus, our results clearly demonstrate that the LXCXE motif is required for efficient RV replication.

In rotavirus-infected cells, the non-structural proteins NSP5 and NSP2 localize in complexes called viroplasms, where replication and assembly occur. Recently, we have demonstrated direct interaction of NSP5 with NSP2, and as a consequence of that, up-regulation of NSP5 hyperphosphorylation. To investigate a possible structural role for the NSP2-NSP5 interaction, we analysed the cytoplasmic distribution of the two proteins in transfected cells by immunofluorescence using specific antibodies. Here we report that NSP2 and NSP5 can drive the formation of viroplasm-like structures (VLS) in the absence of other rotaviral proteins and rotavirus replication. Several NSP5 deletion mutants were constructed and expressed in combination with NSP2. Both the N- and C-terminal domains of NSP5 were found to be essential for VLS formation. Only one mutant, with an internal deletion of residues 81–130, was able to interact with NSP2 to form VLS. Analysis of the phosphorylation capacity of the different mutants in vivo indicated that hyperphosphorylation of NSP5 is necessary, but not sufficient, for VLS formation. Our results suggest a role for the non-structural protein NSP5 in the structure of viroplasms mediated by its interaction with NSP2.

The deduced amino acid sequences of the outer capsid protein, VP7, of serotype G1 rotavirus clinical isolates collected over a 6 year period (1990-1995) in Melbourne, Australia, were examined. Phylogenetic analysis characterized the sequences into two discrete clusters representing two of the four global lineages of human G1 VP7 proteins. Antigenic characterization using a panel of serotype G1-specific neutralizing monoclonal antibodies classified lineage II isolates (1990-1993) as monotype G1a while lineage I isolates were classified as monotype G1b (1993-1995). Examination of the sequences of the neutralization epitope regions of VP7 revealed a particular amino acid substitution at residue 94 in region A (Asp --> Ser/Thr) that correlated with lineage and monotype designation. Our results indicated that temporal genetic variation of the VP7 of serotype G1 rotaviruses was associated with changes in the antigenicity of these isolates.

An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein. The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG. Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter-intron expression cassette expressed RG at much higher levels than vectors lacking the intron. These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells.

The nucleotide sequences of the L gene and 5′ trailer region of Ebola virus strain Mayinga (subtype Zaire) have been determined, thus completing the sequence of the Ebola virus genome. The putative transcription start signal of the L gene was identical to the determined 5′ terminus of the L mRNA (5′ GAGGAAGAUUAA) and showed a high degree of similarity to the corresponding regions of other Ebola virus genes. The 3′ end of the L mRNA terminated with 5′ AUUAUAAAAAA, a sequence which is distinct from the proposed transcription termination signals of other genes. The 5′ trailer sequence of the Ebola virus genomic RNA consisted of 676 nt and revealed a self-complementary sequence at the extreme end which may play an important role in virus replication. The L gene contained a single ORF encoding a polypeptide of 2212 aa. The deduced amino acid sequence showed identities of about 73 and 44% to the L proteins of Ebola virus strain Maleo (subtype Sudan) and Marburg virus, respectively. Sequence comparison studies of the Ebola virus L proteins with several corresponding proteins of other non-segmented, negative-strand RNA viruses, including Marburg viruses, confirmed a close relationship between filoviruses and members of the Paramyxovirinae. The presence of several conserved linear domains commonly found within L proteins of other members of the order Mononegavirales identified this protein as the RNA-dependent RNA polymerase of Ebola virus.

The surface glycoprotein, HEF, of influenza C virus (C/Johannesburg/1/66) has been shown to undergo a post-translation conformational change that is evident in a dramatic change of electrophoretic mobility. If the corresponding gene is expressed in the absence of other viral proteins, this folding process does not occur at all or only very inefficiently. A chimaeric protein, HEF-HA(Tail), in which the short cytoplasmic tail (Arg-Thr-Lys) of HEF was replaced by the cytoplasmic tail of the haemagglutinin of an influenza A virus (fowl plague virus) was constructed. In contrast to the wild-type protein, the chimaeric protein was detected on the cell surface. No further improvement of the surface expression was observed when both the transmembrane domain and the cytoplasmic tail were replaced by the corresponding domains of either the influenza A haemagglutinin or gp40, an endogenous protein of MDCK cells. For the HEF-HA(Tail) construct this study shows that a substantial amount of the protein is converted to the 100 kDa mature form that is observed in virus-infected cells. The HEF-HA expressed on the cell surface reacted positively in esterase and haemadsorption assays, indicating that it was present in a biologically active form. The results show that the short cytoplasmic tail of HEF has a negative effect on the folding and surface transport of this protein. How this effect may be prevented during a virus infection is discussed.

Dobrava hantavirus (DOB) was isolated from the striped field mouse (Apodemus agrarius) trapped on Saaremaa Island, Estonia, and its genetic and antigenic characteristics were subsequently analysed. Phylogenetic analysis showed that the Estonian DOB strain, together with several wild strains carried by Apodemus agrarius, forms a well-supported lineage within the DOB clade. The topography of the trees calculated for the S, M and L nucleotide sequences of the Estonian DOB suggests a similar evolutionary history for all three genes of this virus and, therefore, the absence of heterologous reassortment in its evolution. A cross-neutralization comparison of the Estonian virus with the prototype DOB, isolated from a yellow-necked mouse (A. flavicollis) in Slovenia, revealed 2- to 4-fold differences in the end-point titres of rabbit and human antisera. When studied with a panel of 25 monoclonal antibodies (MAbs), the Estonian and Slovenian DOB isolates showed similar antigenic patterns that could be distinguished by two MAbs. Genetic comparison showed sequence differences in all three genome segments of the two DOB isolates, including an additional N-glycosylation site in the deduced sequence of the G2 protein from the Estonian virus. Whether any of these mutations relates to the different rodent hosts rather than to the distant geographical origin of the two isolates remains to be resolved. Taken together, our observations suggest that A. agrarius, which is known to harbour Hantaan virus in Asia, carries another hantavirus, DOB, in north-east Europe.

The cdk-inhibitor p21(CIP1/WAF1) inhibits the activities of cyclin-dependent kinases and proliferating cell nuclear antigen, thereby repressing cell-cycle progression and DNA replication. Transforming oncogenes, such as E1A of human adenovirus 5 (Ad5), may interfere with such growth-inhibitory proteins. In this study, we show that in various Ad5E1-transformed cells, p21(CIP1/WAF1) is expressed and that, in general, expression is not downregulated. In addition, colony-formation assays show that in Ad5E1-transformed cells highly overexpressed p21(CIP1/WAF1) can still cause growth inhibition. FACS experiments indicate, however, that a G1 arrest induced by moderate overexpression of p21(CIP1/WAF1) can be overcome by E1A. The E1A proteins may interfere with the function of p21(CIP1/WAF1) by binding. Indeed, p21(CIP1/WAF1) binds with its cyclin/cdk-binding N terminus to the transforming N-terminal and CR1 region of the E1A proteins. Together, these results lend support to the model that E1A can interfere directly with p21(CIP1/WAF1) function and thereby stimulates cell growth.

Sexual history is an established risk determinant for cervical neoplasia. It is not clear if human papillomavirus (HPV) exposure entirely explains the sexual behaviour-related risk or if other sexually transmitted agents may act as cofactors for HPV in carcinogenesis. The aim of this study was to elucidate whether HPV exposure or HPV persistence explains the sexual history-related risk of high-grade cervical intraepithelial neoplasia (CIN) using a population-based case-control study of most of the 254 women referred to colposcopy in the Vasterbotten county in Sweden because of an abnormal cervical smear during October 1993 to December 1995 and 320 age-matched women from the general population. The women were interviewed for sexual history and tested for presence of serum antibodies to HPV-16, -18 and -33 as well as for presence of HPV DNA in cervical brush samples. HPV-16, -18 and -33 seropositivity was specific for the corresponding type of HPV DNA, dependent on the lifetime sexual history and associated with a two- to threefold increased risk of CIN 3. There was no sexual history-related risk of CIN among HPV-seropositive women and adjustment for HPV DNA presence explained the sexual history-related risk of CIN. In conclusion, HPV exposure appeared to explain the sexual history-related risk of high-grade CIN.

T-helper (Th) cell-dependent IL-2 production and plasma IgG responses to virus-like particles consisting of the human papillomavirus type 16 (HPV-16) major capsid protein L1 (L1-VLP) were determined in patients with cytological evidence of cervical intraepithelial neoplasia (CIN) participating in a non-intervention prospective cohort study. IgG responses were associated with HPV-16 persistence and high-grade CIN lesions, while high frequencies of Th responses were observed in patients with both virus clearance and virus persistence, irrespective of CIN grade. The IgG response was found in conjunction with an IL-2 response to L1-VLP in 87% of the patients. Recognition of the HPV-16 L1 Th epitope (amino acids 311-335) was found to be more closely associated than recognition of L1-VLP as a whole to HPV exposure and CIN development. Among the HPV-16+ patients included in this study, those showing a Th response to amino acids 311-335 were more likely to carry the HLA DRB1*11/DQB1*0301 haplotype, while those showing an IgG response to L1-VLP were more likely to carry DRB1*0101/DQB1*0501. However, neither cell-mediated nor humoral immune responses against HPV-16 L1 appear to be sufficient for the natural control of HPV infection and CIN development.

To investigate whether there is an association between local or systemic IgG and IgA responses against human papillomavirus (HPV) type 16 virus-like particles (VLP) containing L1 and L2 and the possible influence of these responses on clearance of HPV-16 and its associated lesions, cervical mucus samples from 125 patients and plasma samples from 100 patients, all participating in a non-intervention cohort study of women with abnormal cytology, were analysed. The results show that local IgG and IgA HPV-16 VLP-specific antibodies do not correlate with virus clearance. However, systemic IgG responses were more frequently detected in patients with a persistent infection (11/24) compared with patients with cleared HPV-16 infections (3/28, P = 0.006). Furthermore, the ultimate development of high-grade lesions was associated with systemic VLP-specific IgG reactivity (P = 0.026). By contrast, systemic IgA responses were correlated with virus clearance (7/28 clearance compared with 1/24 persistence patients, P = 0.06). This correlation was statistically significant when only those clearance patients who tested HPV-16 DNA-positive at more than one visit were included in the analysis (5/11 compared with 1/24, P = 0.007). As these systemic IgA responses were not accompanied by local IgA responses, the systemic IgA responses in HPV-16 clearance patients are suggested to be a by-product of a successful cellular immune response induced at the local lymph nodes, mediated by cytokines.

Sera from French in-patients were tested for the presence of the TT virus (TTV) genome using PCR and degenerate primers located in ORF1. Thirty-six sequences were determined and compared with those deposited in databases, revealing a high degree of genetic variability between TTV isolates (up to 47% for amino acid sequences). Phylogenetic analysis demonstrated the existence of three main groups corresponding to the previously described genotypes 1 and 2 and to a new genotype 3. Isolates could be assigned to distinct genotypes if their genetic distance was > 27%. No comparable genetic criteria were found for the definition of sub-types in the region studied. A 15-31 month follow-up of three haemodialysis patients proved the existence of chronic infection by TTV. In one patient, two strains belonging to different genotypes were detected at the same time. Sequences of both ORF1 and ORF2 remained unchanged for a given strain during the follow-up.

Vaccinia virus produces two different infectious forms, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Acquisition of the EEV envelope occurs by wrapping of IMV with vesicles of the trans-Golgi network (TGN). The most abundant protein in the envelope of EEV, P37, is a 37 kDa palmitylated protein encoded by the F13L gene. P37 is located in the inner side of the EEV envelope and accumulates in the TGN during infection. Deletion of gene F13L results in a severe defect in the wrapping process, although normal levels of IMV are produced. A cell line, derived from RK-13 cells, was obtained that stably expressed P37 (RK(P37)), and the properties of the protein were studied in the absence of other viral polypeptides. P37 produced in RK(P37) cells differed from P37 produced in vaccinia-infected cells in terms of hydrophobicity and intracellular distribution. Despite these differences, RK(P37) cells partially complemented the phenotypic defect of vaccinia virus P37-mutants. EEV production and cell-to-cell virus spread by mutant viruses were increased significantly in RK(P37) cells when compared to normal RK-13 cell cultures. Infection of RK(P37) cells with P37-virus substantially altered the hydrophobicity and the intracellular distribution of P37 in those cells. These results indicate the requirement of the infection context for determination of the normal palmitylation and intracellular localization of P37.

The molecular phylogeny of macropodid herpesviruses 1 and 2 (MaHV-1 and -2) has been investigated by cloning and sequencing the genes encoding glycoprotein B from both viruses. Phylogenetic reconstructions based on the putative amino acid sequences of glycoprotein B indicate that MaHV-1 and -2 are most closely related to the subfamily Alphaherpesvirinae. Within the Alphaherpesvirinae, MaHV-1 and -2 are closely associated with those herpesviruses that infect primates. This phylogenetic relationship does not fit the constraints of the proposed co-evolution theory described for other members of the Alphaherpesvirinae which have mammalian hosts.