- Volume 76, Issue 3, 1995

Human parvovirus B19 is the aetiological agent of the common childhood disease erythema infectiosum (fifth disease). The infection is usually benign and self-limiting, but in adults cases of severe arthritis which may persist for years have been reported. Neutralizing antibodies directed against the structural proteins are usually produced shortly after the infection. The immune response against the third major protein, the nonstructural protein NS-1, of parvovirus B19 has not been characterized so far. We cloned and expressed the full-length NS-1 protein and fragments thereof in Escherichia coli. The purified recombinant proteins were used to investigate the presence of antibodies to the NS-1 protein in sera from patients with parvovirus B19 infection. Specific antibodies could be detected in sera from patients suffering from severe parvovirus B19-associated arthritis using Western blot analysis and an ELISA. Sera from patients with acute or past infection without complications did not contain detectable levels of immunoglobulin to NS-1. The use of subfragments of the NS-1 protein allowed localization of the antigenic domains in the carboxy-terminal region of the protein.

The temporal relationship between primary genital human papillomavirus (HPV) infections and the induction of antibodies against viral antigens has not been established. In order to address this question we studied a cohort of 110 women and 48 men with multiple heterosexual partners, who were followed for 220 person-years during which they made 583 visits to the sexually transmitted diseases clinic of the Amsterdam Public Health Service. At each visit spatula or brush samples from multiple anogenital and oral sites were collected for HPV DNA analysis by PCR. Serum samples were also collected and analysed for serological reactivity to peptides derived from the L1, L2 and E2 regions of HPV types 6, 16 and 18 as well as to bovine papillomavirus and HPV-16 virus-like particles. Seroconversions for at least three antigens were found among 16/158 patients. Of these, 10/16 were HPV-positive and in 5/16 cases seroconversions occurred concomitantly with the detection of HPV DNA. Analysis of participants who were HPV-negative at entry, but became HPV DNA-positive during follow-up revealed that antibodies against several HPV antigens were regularly induced at the time of a new HPV infection, in particular the IgG responses against HPV-16 virus-like particles and against the HPV-16 E2-derived peptide 245. Whereas the responses induced among the women with new HPV-16 infection tended to continuously increase, the responses among the men with any type of new HPV infection were mostly transient and disappeared during follow-up. In conclusion, we find that antibody responses to multiple viral antigens are often induced following the detection of genital HPV infection, that the type-specificity of the response is limited and that transient responses are common.

Equine herpesvirus type 1 (EHV-1) strain Ab4 gene 67 has no counterpart in any herpesvirus sequenced to date. To identify and characterize the product of EHV-1 gene 67, we have expressed the putative amino acids 11 to 260 encoded by gene 67 as a β-galactosidase fusion protein in Escherichia coli. The expressed fusion protein has been used to generate an antiserum raised against the gene 67 product. Immunoblotting and immunoprecipitation experiments have revealed that the anti-67 serum specifically recognizes a polypeptide with an M r of 36000 (the 36K polypeptide) in infected cell extracts. The gene 67 protein is regulated as an early polypeptide in EHV-1 strain Ab4 infected cells and post-translational modification experiments have revealed that the protein is phosphorylated, but not glycosylated. The gene 67 protein has been transiently expressed in BHK-21/C13 cells using plasmid pCMV67, which contains the putative gene 67 ORF under the control of the cytomegalovirus immediate early promoter. Immunoblotting experiments with anti-67 have shown that the 36K protein is expressed at high levels in transfected cells. From both immunofluorescence and cellular fractionation experiments it is concluded that the gene 67 protein is associated with intracellular membranes and produces novel ribbon or filament-like structures within the cytoplasm of infected cells. We have demonstrated that the gene 67 product is a component of the virion nucleocapsid/tegument.

A collection of neutralizing monoclonal antibodies (MAbs) produced against the LWVRT 60.1, Jasper and N1 strains of infectious pancreatic necrosis virus (IPNV) were selected for the analysis of VP2 epitopes. Previous characterization of the LW and JA MAbs allowed the identification of continuous and discontinuous epitopes but the topological localization of these sites remained obscure. The ability of these MAbs to differentiate individual epitopes was evaluated by additivity and competition assays using antigen-coated plates and by surface plasmon resonance (SPR), an automated biosensor system that is able to retain the conformation integrity of proteins. IPNV-neutralizing MAbs defined a major, conformational-dependent and immunodominant area where continuous epitopes represent portions of a larger discontinuous epitope. Moreover, weakly neutralizing MAbs could interact with internal sites, located within the foldings of the polypeptide chain. Anti-VP2 MAbs prepared against the European serotype N1 recognized and competed for epitopes present on the North American strains LWVRT 60.1 and Jasper. Attempts to establish the proximity of VP2 and VP3 epitopes have been made by SPR. Results indicate that these major structural proteins do not overlap in the virion.

The nucleotide sequences of the small (S) genomic RNAs of six California (CAL) serogroup bunyaviruses (Bunyaviridae: genus Bunyavirus) were determined. The S RNAs of two California encephalitis virus strains, two Jamestown Canyon virus strains, Jerry Slough virus, Melao virus, Keystone virus and Trivittatus virus contained the overlapping nucleocapsid (N) and nonstructural (NSs) protein open reading frames (ORFs) as described previously for the S RNAs of other CAL serogroup viruses. All N protein ORFs were 708 nucleotides in length and encoded a putative 235 amino acid gene product. The NSs ORFs were found to be of two lengths, 279 and 294 nucleotides, which potentially encode 92 and 97 amino acid proteins, respectively. The complementary termini and a purine-rich sequence in the 3′ non-coding region (genome-complementary sense) were highly conserved amongst CAL serogroup bunya-virus S RNAs. Phylogenetic analyses of N ORF sequences indicate that the CAL serogroup bunyaviruses can be divided into three monophyletic lineages corresponding to three of the complexes previously derived by serological classification. The truncated version of the NSs protein, which is found in five CAL serogroup bunyaviruses, appears to have arisen twice during virus evolution.

Processing of Japanese encephalitis (JE) virus nonstructural (NS) proteins expressed by recombinant vaccinia viruses was analysed to characterize the responsible viral protease. Analysis of the processing of polyprotein NS2A-2B-3′ containing the N-terminal 322 amino acids of NS3 revealed products consistent with cleavages at the predicted intergenic junctions as well as at one or possibly two sites within NS2A. Cleavage at the alternate site(s) containing the cleavage sequence motif within NS2A could possibly explain the production of the NS1′ protein in JE virus-infected cells. Polyprotein NS2A-d2B-3′ containing a large deletion within NS2B was cleavage-defective, despite the presence of the proposed NS3 protease domain. Cleavage of NS2A-d2B-3′ was restored if NS2B or NS2A-2B was supplied in trans, providing evidence that NS2B is strictly required for NS3 proteolytic activity. NS2B- or NS3-specific sera raised against the bacterial TrpE fusion protein co-precipitated NS2B and NS3 or NS3′ from the lysate of JE virus or recombinant virus-infected cells. Thus both protease components are associated as a complex, presumably representing the active JE virus protease. JE virus and the analogous dengue 4 (DEN-4) protease components were employed to examine the activity of heterologous proteases. The defective cleavage of JE virus NS2A-d2B-3′ was complemented by heterologous DEN-4 NS2B, whereas the defective cleavage of DEN-4 NS2A-d2B-3′ was not corrected by heterologous JE virus NS2B. This suggests that the heterologous JE virus NS2B-DEN-4 NS3 protease is not active, despite the considerable sequence conservation of NS2B and NS3 between the two viruses. The cleavage activity was restored by replacement of the C-terminal 80 amino acids of JE virus NS2B with the corresponding DEN-4 sequence, consistent with the notion that the C-terminal region contains amino acid residues for interaction with DEN-4 NS3.

The initial replication of lactate dehydrogenase-elevating virus (LDV) in mice, its invasion of the central nervous system (CNS) and infection of anterior horn neurons in C58 and AKXD-16 mice were investigated by Northern and in situ hybridization analyses. Upon intraperitoneal injection, LDV replication in cells in the peritoneum was maximal at 8 h post-infection (p.i.). Next, LDV infection was detected in bone marrow cells and then in macrophage-rich regions of all tissues investigated (12 to 24 h p.i.). By 2 to 3 days p.i., LDV RNA-containing cells had largely disappeared from all non-neuronal tissues due to the cytocidal nature of the LDV infection of macrophages. In the CNS at 24 h p.i. LDV replication was very limited and confined to cells in the leptomeninges. LDV replication in the cells of the leptomeninges should result in the release of progeny LDV into the cerebrospinal fluid and thus its dissemination throughout the CNS. However, in C58 and AKXD-16 mice, which are susceptible to paralytic LDV infection, only little LDV RNA and few LDV-infected cells were detectable in the spinal cord until at least 10 days p.i. Extensive cytocidal infection of anterior horn neurons occurred only shortly before the development of paralytic symptoms between 2 and 3 weeks p.i. The reason for the relatively long delay in LDV infection of anterior horn neurons is not known. No LDV RNA or LDV RNA-containing cells were detected in the brain, except in the leptomeninges at early times after infection.

The nucleocapsid (N) and polymerase (L) genes of the vaccine strain of rinderpest, and the 5′ and 3′ terminal domains of the genome have been sequenced. Together with previously published data, this completes the sequence of the entire genome of rinderpest virus. The viral genome is 15 881 bases in length, similar to that of measles virus and slightly longer than that of canine distemper virus. The L gene is identical in length to that of measles virus, encoding a 2183 amino acid protein with a calculated M r of 248 100. The L protein sequence of morbilliviruses is highly conserved, more than 75% of residues being identical or conserved in all three sequences currently available. The N protein was, as for the other sequenced genes where comparison is possible, essentially identical to that of the virulent parent. In addition, we have determined the terminal sequences of two virulent strains of rinderpest and compared the sequences of virulent and non-virulent strains.

An in vitro cleavage/initiation assay was used to analyse cleavage site choice and transcription initiation by the influenza virus polymerase. A synthetic mRNA which is cleaved by the polymerase to produce a single 11 base primer fragment was altered around this cleavage site. Depending upon the mutations made, alternative cleavage sites were used. This system was then used in extracts from recombinant vaccinia virus infected cells which express the polymerase. These extracts require the addition of a synthetic vRNA in order to induce cleavage and initiation activity. The data show that the choice of cleavage site is wholely controlled by the mRNA and does not depend upon interactions with the vRNA template. However, the site of initiation of the cleaved primer on the template is influenced by template-primer interactions.

From immunological and phylogenetic analyses of H3 influenza viruses isolated from pigs and ducks in the People’s Republic of China (China), Hong Kong, Taiwan and Japan, between 1968 and 1982, we arrived at the following conclusions. The H3 haemagglutinin and N2 neuraminidase genes from swine isolates can be segregated into four mammalian lineages, including: (i) the earliest human strains; (ii) early swine strains including Hong Kong isolates from 1976–1977; (iii) an intermediate strain between the early swine and recent human strains; and (iv) recent human strains. In this study we found an unusual swine strain (sw/Hong Kong/127/82) belonging to the third lineage which behaved like those of the early swine-like lineage in the haemagglutination inhibition test; but neuraminidase inhibition profiles with monoclonal antibodies indicated that this virus is related to late human strains. On the basis of pairwise comparisons of complete or partial nucleotide sequences the genes encoding the three polymerase proteins (PB2, PB1, PA), the nucleoprotein, the membrane protein and possibly the nonstructural proteins of sw/Hong Kong/127/82 are of the swine H1N1 lineage, whereas genes encoding the two surface glycoproteins belong to the human H3N2 lineage. In contrast, all RNA segments of one swine isolate (sw/Hong Kong/81/78) are similar to those of recent human H3N2 viruses. This study indicated that frequent interspecies infections between human and swine hosts appeared to occur during 1976–82. Although the evolutionary rates of human (0.0122/site/year), swine (0.0127/site/year) and avian (0.0193/site/year) virus genes are similar when based upon synonymous substitutions, nonsynonymous substitutions indicated that viral genes derived from human and swine viruses evolved about three times faster (0.0026–0.0027/site/year) than those of avian viruses (0.0008/site/year). Furthermore, the evolutionary mechanism by which human and swine H3 haemagglutinin genes evolve at a similar rate, based on nonsynonymous substitutions, appeared to be quite different from previous evidence which showed that human H1 haemagglutinin genes evolved three times faster than those of swine viruses. However, comparison of the number of nonsynonymous substitutions in the antigenic sites (A–E) of haemagglutinin molecules demonstrated that swine viruses evolve at a rate that is about one fifth to one tenth that of human viruses, reflecting the conservative nature of the antigenic structure in the former.

New classes of mutants of influenza virus A/seal/Mass/1/80 are described in which the haemagglutinins (HA) have lost their protease cleavability by trypsin, but can be activated by elastase, chymotrypsin or thermolysin in different cell types. The same proteases that were required for activation of infectivity of the mutants also activated haemolysis and cell-fusing properties. The protease activation (pa)-mutants were nonpathogenic for chickens, but induced a protective immune response against a highly pathogenic challenge virus. The failure of the mutants to be activated by trypsin, but instead to be activated by the other proteases employed, was related to amino acid exchanges around the HA cleavage site. The cleavability of the chymotrypsin and elastase pa-mutants is most likely determined by replacement of Arg-1 by neutral amino acids such as Ile, Thr, Met or Leu, depending on the substrate specificity of the activating proteases. Cleavage activation of the thermolysin pa-mutants, on the other hand, became possible by insertion of a single Leu residue at position 4 of the HA2 polypeptide, which compensates for the loss of the Gly residue at the N terminus of the fusion peptide due to thermolysin cleavage. The correction of the mutations in revertants confirmed the conclusions drawn from sequence analyses of the pa-mutants.

AKR/Gross virus-specific cytotoxic T lymphocytes (CTL) from C57BL/6 (B6) mice are H-2Kb-restricted and recognize epitopes encoded by the prototype endogenous ecotropic murine leukaemia virus (Emv) AKR623. Four CTL epitopes have been identified by the use of synthetic peptides corresponding to AKR623-encoded amino acid sequences. Here we present both functional and nucleotide sequence data indicating that three closely related Emv share all of these CTL epitopes. We also found that one other murine leukaemia virus (MuLV) was not susceptible to lysis by these CTL. This is the ecotropic component of the LP-BM5 virus complex that causes murine AIDS. Nucleotide sequencing revealed that three of the four epitopes, including the immunodominant peptide, are altered in this virus. The other epitope was unchanged. These data implied that the inability of anti-AKR/Gross virus CTL to lyse cells infected with the LP-BM5 ecotropic (BM5eco) MuLV was due to the functional loss of three of the four CTL epitopes. Using recombinant vaccinia and Sindbis virus vectors, we have shown that the BM5eco-encoded form of the immunodominant epitope, which differs only by an arginine for lysine substitution at the N-terminal residue, fails to induce a CTL response in B6 mice. Immunization with BM5eco-infected cells also failed to induce MuLV-specific CTL. In light of the long in vivo passage history of the LP-BM5 complex in B6 mice, our results are consistent with a contribution of CTL-mediated immune selection to the evolution of the BM5eco MuLV.

We examined the effect of human foamy virus (HFV) infection on the expression of human major histocompatibility complex molecules. Our data show that in vitro HFV infection of U373-MG glioblastoma cells results in increased expression of class I human leukocyte antigen (HLA) and transcripts. Transient transfection assays of plasmids containing the reporter gene chloramphenicol acetyl transferase driven by different 5′ deletions of the HLA-A11 class I promoter allowed identification of cis-acting elements involved in this regulation. HFV infection has two opposite effects on the HLA class I promoter: transactivation of the HLA-A11 promoter through a positive regulatory element located in the -525 to -335 region upstream of exon 1 and down-regulation of transcriptional activity driven by the -335 to -205 class I promoter region. Additional experimental data indicate that the effect of HFV on HLA class I expression is not mediated by the interferon pathway.

A murine monoclonal antibody (MAb F-91-55) raised against the complex of soluble CD4 and human immunodeficiency virus type 1 (HIV-1) gp120 had previously been found to inhibit syncytium formation without inhibiting the interaction of CD4 with gp120, and its binding site was localized within the first two domains (D1/D2) of CD4. We investigated whether this antibody inhibited the infectivity of HIV-1 in the CD4+ T cell lines A3.01, Sup-T1 and H9. We also examined the effect of the antibody on syncytium formation between these cells and chronically infected H9 cells. Syncytium formation was found to depend critically on the incubation medium used. The effect of the MAb on HIV-1 infectivity was very limited with A3.01 and Sup-T1 cells, although it inhibited syncytium formation between A3.01 or Sup-T1 and chronically infected H9 cells. In contrast, the MAb inhibited significantly the infectivity of HIV-1 in H9 cells, but it also inhibited syncytium formation between H9 and chronically infected H9 cells to a greater extent than in the case of the other cell lines. Our results indicate that cellular systems used for syncytium assays differ in their susceptibility to inhibitory antibodies. In the A3.01 and Sup-T1 cell systems, the differences in the ability of the MAb to block viral entry or syncytium formation raise the possibility that the mechanisms of interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion.

One of the hallmarks of human immunodeficiency virus type 1 (HIV-1) infection is the decline in CD4+ T lymphocytes which precedes the progression from an asymptomatic state to AIDS. Apoptosis (programmed cell death) is one of the mechanisms proposed to mediate this depletion. Infectious and inactivated preparations of HIV-1LAI were compared for their potential to induce apoptosis. Analysis with fluorescence-activated cell sorting using the DNA intercalative compound propidium iodide demonstrated that apoptosis occurred only with infectious HIV-1, implying that cell surface binding and signalling by the virus alone were insufficient to trigger apoptosis. Apoptosis was further confirmed by the presence of characteristic digestion of host cell DNA and morphologically by nuclear condensation observed by transmission electron microscopy. HIV infection of CD4+ T cell lines generated an accumulation of the cells in G2/M phase of the cell cycle and cells undergoing apoptosis appeared to originate from the pool of cells in the G1 phase. Inhibitors of HIV replication were used to identify the point in the virus replicative cycle at which apoptosis is induced. The reverse transcriptase inhibitor, ddI, or the HIV protease inhibitor, RO31-8959 (Saquinavir), were added either 2 h before or 6 h after HIV inoculation. Only ddI inhibited HIV-induced apoptosis when added before inoculation; however, neither treatment was effective in preventing HIV-induced apoptosis when applied 6 h after inoculation. These data indicate that apoptosis requires a single round of reverse transcription and the expression of virion proteins, but not the maturation of progeny virions. Two agents which compete with HIV for binding to CD4+ T cells, dextran sulphate and the anti-CD4 MAb Leu3a, were effective at preventing apoptosis when added 6 h after infection, implying that a subsequent gp120-CD4 interaction at the surface of an infected cell was required to complete the apoptotic process.