- Volume 76, Issue 6, 1995

Langerhans cells (LC), the dendritic antigen presenting cells of the skin, mature into potent immunostimulatory cells during migration to regional lymph nodes, where they are identified as interdigitating cells (IDC). Since mature Langerhans cells (mLC) resemble IDC in phenotype and immunostimulatory capacity, we examined whether these cells were susceptible to infection with macrophagetropic and lymphotropic strains of human immunodeficiency virus type 1 (HIV-1). Highly purified cell preparations of mLC migrating from human epidermis expressed high amounts of major histocompatibility complex (MHC) class I and II antigens and of the accessory molecules CD40, CD80 and CD86, indicative of the phenotype of potent immunostimulatory cells. CD4 expression was up-regulated on mLC during cultivation, independent of the presence of tumour necrosis factor α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the culture medium. The macrophagetropic HIV-1 strain SF162 replicated to higher titres in mLC than the lymphotropic strain IIIB. Both strains induced syncytia, with SF162 showing a more rapid cytophathic effect. Addition of TNF-α enhanced virus production, due to better cell viability under TNF-α treatment, whereas GM-CSF did not significantly influence viability of cells and replication pattern of the virus. These findings suggest that in the infected individual IDC in lymph nodes may function as target cells for HIV-1.

Investigations of human immunodeficiency virus (HIV) infection of man have benefited from the study of relevant animal models of the infection and disease. However, the ultimate models use primate species which are either endangered, not generally available, or expensive to maintain. A transgenic rabbit specifically and stably expressing human CD4 protein on T lymphocytes was assessed as a new laboratory animal model for HIV-1 infection. In vitro studies demonstrate that lymphocytes derived from the transgenic rabbits are more susceptible to HIV-1IIIB infection than those from normal rabbits. In vivo infection of huCD4-transgenic rabbits using HIV-1IIIB-infected autologous lymphocytes was demonstrated by virus isolation, detection of HIV-1-specific DNA in peripheral blood lymphocytes and seroconversion to various HIV-1 proteins. Viral DNA was detected in the tissues of one rabbit sacrificed 7 weeks post-infection and virus was isolated from lymph node. Although these transgenic rabbits are less sensitive to HIV-1 infection than man, such a small and inexpensive animal model may be a useful tool.

Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.

The CD4 molecule serves as the principal cell surface receptor common to both the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Since binding to CD4 is not sufficient to permit virus entry, HIV ‘co-receptors’ have been implicated in mediating the fusion of viral and cellular membranes necessary for completing the entry process. In order to identify candidate co-receptor molecules, a panel of monoclonal antibodies (MAbs) directed against adhesion molecules was tested for the ability of the MAbs to inhibit HIV-1-induced cell fusion (syncytium formation) and HIV-1 entry. Certain antibodies directed against CD18, CD11b and CD11c inhibited HIV-1-induced syncytium formation but not entry, in agreement with previous reports. Interestingly, certain antibodies to ICAM-3 (intercellular adhesion molecule 3) (CD50) significantly inhibited HIV-1-specific entry but not syncytium formation using human SupT1 cells. Only one antibody directed against ICAM-3 significantly inhibited HIV-1-induced syncytium formation, entry and infectivity. Our results suggest that certain epitopes of ICAM-3 may be involved in mediating HIV-1-specific entry into lymphoid and monocytoid cells.

A long-term cell line (SHK-1) supporting replication of the causal virus of infectious salmon anaemia (ISA) has been established. The cell line was developed from a culture of Atlantic salmon (Salmo salar L.) head kidney cells. CPE was observed in SHK-1 cells 12–14 days after inoculation with ISA-infective tissue material. The time for CPE to develop decreased after repeated passages of medium from infected cell cultures to new cultures. Transmission trials demonstrated that Atlantic salmon parr developed ISA after intraperitoneal injection of preparations made from infected cells and growth medium. The ISA infectivity of the cell preparations increased with incubation time of inoculated cells. Cell cultures in a second passage were found to have a higher infectivity than the primary inoculated cultures. Virus particles with a diameter of approximately 100–120 nm, and which contained an external envelope and granules were seen in electron micrographs of thin sections of infected cells. Most of the virus particles were located extracellularly close to the cell surface, and in some cases, a connection between virus and plasma membrane could be observed. This indicates that virus particles were released by budding. Enveloped virus particles of 45–140 nm in diameter were seen in abundance in electron micrographs of a negatively stained purified virus preparation. Large, highly pleomorphic particles up to 700 nm in the longest dimension were occasionally observed in unpurified preparations. The evidence is therefore strong that the virus isolated in SHK-1 cells is the aetiological agent of ISA.

This report describes the preparation of six monoclonal antibodies (MAbs) raised against a British isolate of porcine reproductive and respiratory syndrome virus (PRRSV), their characterization in terms of protein specificity and their reactivity with different PRRS viruses from Europe and the USA. Radioimmunoprecipitation and Western blotting studies of MAb reactivity with proteins from cell lysates of infected cells and purified virus revealed that four of the six MAbs (WBE1 and WBE4–6) precipitated a 15 kDa viral protein. Further studies using in vitro translated products of the Lelystad virus genome showed that this protein was the product of ORF7, the putative nucleocapsid protein. The specificity of another MAb, WBE2, was found to be for a 45 kDa protein, determined to be the product of ORF3 and demonstrated to be present in purified virion preparations. The protein specificity of the sixth MAb, WBE3 could not be determined. Thirty-three PRRSV isolates from Europe and the USA were grown in alveolar macrophages and examined by immunoperoxidase staining, using the panel of six MAbs. All European isolates were recognized by the four MAbs specific for the putative nucleocapsid, but the viruses showed different patterns of reactivity with WBE2 and WBE3. Furthermore, these two MAbs stained only a small proportion of the cells infected with certain isolates, suggesting that a single isolate may be antigenically heterogeneous. No MAbs bound to US isolates, indicating a consistent antigenic difference between the putative nucleocapsid of US and European isolates. Detergent extraction of cell lysate antigen abrogated the binding of WBE1–3, suggesting that the epitopes are conformation dependent.

An efficient mucosal vaccination has a number of obvious advantages over invasive routes of immunization. The immune response to measles virus (MV) was investigated after intranasal and intragastric coimmunization of mice with cholera toxin B (CTB) as an adjuvant. High titres of virus-specific IgG antibodies and a transient IgA response were detected in the sera after intranasal but not after intragastric immunization when CTB was used. In the presence of CTB, higher titres were reached with less antigen and fewer intranasal boosts. Neutralizing antibodies were found in all animals only after co-immunization with MV and CTB. In the nasal wash and the saliva, IgG and IgA titres were significant only in the MV plus CTB groups; IgG levels were comparable to those found after intraperitoneal (i.p.) immunization with complete Freund’s adjuvant. Specific IgA was detected in the mucosal fluids only after intranasal immunization with MV plus CTB but not after i.p. or intragastric immunization. The antibody response consisted of 99% IgG1 after MV immunization. In the CTB groups 10% IgG2b and 1% IgG2a were detected in addition to the predominant IgG1 antibodies.

The Epstein—Barr virus (EBV) open reading frame BDLF3 is predicted to code for a glycoprotein on the basis that it contains sequences with signal peptide and transdomain characteristics and nine potential N-linked glycosylation sites. No sequential or positional homologues of BDLF3 have been located in other herpes-viruses. A bacterial glutathione S-transferase (GST)-BDLF3 fusion protein was used to demonstrate that over one–third of EBV-immune human sera tested recognized the fusion protein but not GST alone on Western blots. The fusion protein was used to raise polyclonal sera in rabbits. A BDLF3 recombinant baculovirus was constructed using the full-length BDLF3 sequence (AcBDLF3). Rabbit anti-fusion protein sera and some human EBV-immune sera recognized products of approximately 30 and 55 kDa from AcBDLF3-infected insect cells by Western blotting. A peptide representing the carboxy-terminal amino acids 215–234 of the BDLF3 sequence was used to raise anti-peptide sera in rabbits. Anti-peptide serum detected a product by indirect immunofluorescence in acetone-fixed EBV-infected B cells from all cell lines tested. A diffuse band with a molecular mass of 100–150 kDa was detected by Western blot in B95–8 cell lysates, partially purified B95-8 virus and B95-8-infected cell membranes after probing with anti-BDLF3 peptide serum. This product was shown to be glycosylated after enzymatic deglycosylation of a B95-8 virus preparation using neuraminidase, O-glycosidase or N-glycosidase F. The BDLF3 protein products have no known function.

The BamHI-Z-encoded Epstein—Barr virus (EBV) replication activator (ZEBRA) is a key mediator of the switch from latency to productive cycle in EBV virus. Antibodies against ZEBRA are a marker of EBV reactivation and are regularly found among patients with infectious mononucleosis (IM) or nasopharyngeal carcinoma (NPC), but are only rarely found among healthy EBV-seropositive donors. In order to define the serologically reactive epitopes in the ZEBRA protein, we synthesized a set of overlapping peptides and tested them for reactivity with serum samples from EBV-seronegative persons, patients with NPC, IM, chronic fatigue syndrome, lymphoma or from healthy donors. Three major EBV-specific epitopes were found. These epitopes were further defined and optimized using substitution or truncation analogues of the peptides. Reactivity with epitope number 22 was found in 63% of NPC patients’ sera, with < 2% of healthy donors’ sera being positive. Serological reactivity with epitope number 19 was associated with IM (57% positive, 5% healthy donors positive). Serum antibodies against epitope 1 were found among healthy donors, but were significantly elevated among patients with NPC, IM or lymphomas. In conclusion, different serologically reactive epitopes in the ZEBRA protein associate with different EBV-associated diseases.

We recently showed that BZLF1, the gene encoding the Epstein–Barr virus (EBV) Zebra protein, was expressed in all eight nasopharyngeal carcinoma (NPC) specimens studied. We present here studies on the expression of EBV lytic cycle genes in the same eight NPC biopsies to determine if production of the ZEBRA transactivator could lead to a complete productive cycle. The tumour lesions exhibit a number of different patterns of limited lytic gene expression. In three out of eight tumours neither BRLF1 nor BMLF1 expression could be detected. Otherwise BMLF1 mRNA was expressed in all the other specimens. Three specimens also expressed BRLF1. Two specimens not only exhibited BZLF1, BMLF1 and BRLF1 transcripts, but also expressed the late gene BLLF1 which encodes the membrane protein gp220. The early gene product BBLF2 could not be detected in any of the eight NPC. However, expression of the late gene encoding the lytic truncated form of LMP1 (D1LMP) was found in seven of the eight NPC biopsies. Thus, it could be suggested that the EBV abortive lytic cycle occurred in most of the NPC studied.

Valproic acid (VPA), a simple branched-chain fatty acid having anticonvulsant activity and used in the treatment of many forms of epilepsy, markedly stimulated human cytomegalovirus (HCMV) replication in human fibroblasts (MRC-5 cells). The maximum level of stimulation was reached when cells were treated for 24 h before infection. The enhancement of virus replication correlated with an increase in the number of immediate early (IE) and early (E) antigen-positive cells. VPA also induced expression of IE antigens after transfection of fibroblasts with a plasmid containing the entire IE1–2 region. Moreover, VPA stimulated the HCMV IE1–2 promoter/enhancer-mediated expression of β-galactosidase in a stably transfected Jurkat T cell line. Recently, VPA was shown to inhibit glutathione reductase in human red blood cells, but an action through the glutathione metabolic pathway can be eliminated in this case, since VPA decreased the intracellular level of glutathione in Jurkat T cells but not in MRC-5 cells. The ability of VPA to stimulate HCMV replication provides an attractive model for studying the molecular mechanism of the regulation of HCMV IE1–2 gene expression.

The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent ‘latent’ state. Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i. In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-α (IFN-α) and by the the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-α and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of β-galactosidase was not detected after infection of IFN-α-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells. A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus. The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form. A small number of remaining linear genomes resulted from incomplete uncoating of input virus.

We examined the capability of pseudorabies virus (PRV) to replicate in vitro in porcine peripheral blood mononuclear cells (PBMC) and characterized the phenotype of infected cells. In addition, we investigated whether inactivation of various PRV proteins or the expression of a foreign gene affected this replication. Finally, we studied the replication of PRV strains in concanavalin A (Con A)-stimulated lymphocytes. The replication of PRV mutants with inactivated glycoproteins gE or gG, thymidine kinase (TK), ribonucleotide reductase (RR) or US3-encoded protein kinase (PK), and the replication of PRV vector strains expressing the envelope glycoprotein E1 of hog cholera virus (HCV) were studied. By adherence of PBMC to plastic, monocytes and lymphocytes were largely separated. Infected monocytes were analysed with an immunostaining monolayer assay and infected lymphocytes were analysed with immunofluorescence staining and flow cytometry. We found that the wild-type NIA-3 virus replicated in both lymphocyte and monocyte cultures. NIA-3 infected relatively more monocytes (> 90%) than non-adherent B cells (46–65%) and T cells (17–28%); approximately equal numbers of CD4+ and CD8+ T cells were infected. Although E1 is probably involved in adsorption of HCV to host cells, the expression of E1 by PRV vector strains did not change the level of replication. Inactivation of TK and RR, but not inactivation of gE, gG or PK, severely affected the replication in both monocytes and lymphocytes. Con A stimulation of lymphocytes restored the reduced replication of the TK mutant, but not of the RR mutant. Moreover, Con A stimulation of lymphocytes reduced the replication of the wild-type NIA-3 virus. We concluded that both viral TK and RR activity are important for efficient replication of PRV in resting lymphocytes. Furthermore, Con A-stimulated lymphocytes can restore the viral TK defect and PRV replication can also be influenced by cellular metabolism.

The Spodoptera frugiperda multiple nucleocapsid nuclear polyhedrosis virus (SfMNPV) gp41 structural protein gene was located in the 1.9 kbp EcoRI-S fragment and sequenced. An open reading frame (ORF) of 999 nucleotides was detected that encoded a protein of 332 amino acids. The gp41 gene transcript was detected after 12 h post-infection (p.i.) and remained detectable at 48 h p.i. Two major mRNAs, about 1.6 and 2.8 kb in length, were determined by Northern blot analysis. Primer extension analysis demonstrated that the gp41 gene promoter region contains three transcription start sites. Two of the gp41 gene transcription start sites were located at -42 and -41 nucleotides from the ATG translation start codon within a consensus late transcription start site (TAAG) and another transcription start site was located at -140 nucleotides from the ATG translation start codon for which no consensus motif has been determined. Comparison of SfMNPV gp41 nucleotide and amino acid sequences with the gp41 genes from Autographa californica, Bombyx mori, and Helicoverpa zea NPVs showed 60% homology of nucleotide sequences and 70% similarity of amino acid sequences.

Serial passage of nuclear polyhedrosis viruses (NPVs) through cultured cell lines results in the appearance of mutants with a complex phenotype referred to as the ‘few polyhedra’ (FP) phenotype. The altered plaque morphology and reduced occlusion production associated with the FP phenotype have been observed in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) bearing mutations in the gene encoding the 25 kDa protein (25K gene). In this study, we sequenced the 25K genes of four spontaneously occurring AcMNPV FP mutants. These mutants, together with an artificially generated FP mutant (AcFPβgal, in which the gene for β-galactosidase is fused in frame with the 25K ORF), were examined at the ultrastructural level to see if they exhibited the reduced virion occlusion and intranuclear envelopment which is associated with the FP phenotype. Observations on Spodoptera frugiperda Sf9 cells infected with the FP mutants revealed that all five mutants were impaired in virion occlusion and intranuclear nucleocapsid envelopment. The 25K mutants were also found to release two- to fivefold more infectious virus (p.f.u.) into the media of infected Sf9 cells. Marker rescue of AcFPβgal restored wild-type virion occlusion, intranuclear envelopment and levels of infectious virus production.

The role of the glycans of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) in the intracellular events of Env precursor (gp160) biosynthesis has been examined by the use of a mutant gp160 in which the cluster of conserved glycosylation sites within the gp41 domain (Asn-621, -630 and -642) has been mutated. Expression of the wild-type and mutant forms of gp160 in BHK-21 cells using recombinant vaccinia viruses has shown that the kinetics of the events occurring in the endoplasmic reticulum (ER) were normal: both Env proteins had similar kinetics of disulphide bond formation, as determined by the acquisition of CD4-binding capability, and both had similar kinetics of oligomer formation. However, in contrast to the parental molecule, mutated gp160 displayed relatively slow transport from the cis to the medial Golgi where it was retained in the oligomeric state. Transport to the trans Golgi was impaired, as determined by the sensitivity of gp160 to glycosidases. Cleavage of mutated gp160 at the gp120/gp41 junction was substantially reduced but this was apparently not due to the involvement of the gp41 glycosylation in the cleavage reaction by furin inasmuch as, in the baculovirus system, mutated gp160 could be cleaved when recombinant furin was co-expressed. The reduced cleavage in mammalian cells may thus reflect the impaired routing of mutated Env to the compartment where cleavage occurs. The glycan component of gp41 is, therefore, important for the efficient intracellular transport and processing of gp160. gp160 lacking gp41 carbohydrates is an additional example, among few others, of a protein lacking glycans that is arrested in the Golgi rather than the ER following its biosynthesis.

The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced. The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature. The three ORFs from 5′ to 3′ were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively. Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation. The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots. The protein was concluded to be σ3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral σ3 polypeptide. Sequence comparison of the avian reovirus S1 gene with its mammalian counter-part did not show any significant similarity between the two. However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of α-helix structures in the amino terminal portion of σ3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus σ1 protein.

We previously reported that the A/Leningrad/134/47/57 (H2N2) cold-adapted virus (A/Len/47) used in preparing reassortant live attenuated vaccines for children acquired 14 (11 coding) mutations in genes coding for proteins other than haemagglutinin and neuraminidase during cold-adaptation. Preservation of these mutations in genomes of viruses isolated from children on the second, fifth, or eighth day after vaccination was examined by sequence analysis. The sequence data demonstrated that all nine coding mutations selected for examination were conserved in the genomes of all 11 strains investigated, indicating that the mutations accompanying cold-adaptation and attenuation of the A/Len/47 master vaccine are highly stable.