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Measles virus infection of microvascular endothelium in vivo and ensuing endothelial cell activation may be important in the pathogenesis of subsequent inflammation in target organs. This study investigated the capacity of measles virus to induce procoagulant activity, in vitro, in endothelial cells isolated from human umbilical cord veins. Endothelial cells were infected with a clinical isolate of measles virus propagated in Vero cells. Cells were also incubated with bacterial lipopolysaccharide (10 micrograms/ml), herpes simplex virus type 1, cytomegalovirus or culture medium alone as positive and negative controls, respectively. Endothelial cell procoagulant activity was measured in a one-stage clotting assay. Measles virus stimulated both a time and dose-dependent endothelial cell procoagulant response by the induction of tissue factor synthesis, confirmed by both immunocytochemistry and its dependence on factor VII for activity. This activity was reduced by u.v.-irradiation of the virus. Infected cells were analysed by double immunofluorescent staining for both tissue factor and measles virus N-protein, and examined using confocal scanning laser microscopy. Cells expressing tissue factor were also positive for the measles virus N-protein. Low levels of interleukin-1 were detected in some viral inocula derived from measles virus-infected Vero cells, however neutralising antibody to interleukin-1 failed to inhibit the endothelial cell procoagulant response to measles virus, whereas it significantly reduced procoagulant activity induced in endothelial cells by recombinant interleukin-1. The capacity of measles virus to induce endothelial tissue factor in vitro, may be relevant to the thrombotic vasculopathy associated with measles virus infection in vivo.

The envelope glycoprotein B of human cytomegalovirus (CMV) is a major target of the neutralizing antibody response against this virus, and hence has importance as a potential subunit vaccine. PCR was utilized to amplify DNA encoding the dominant antigenic determinant on this molecule, AD-1 (codons 552 to 635), and DNA sequencing was carried out in order to compare nucleotide variation in AD-1 between clinical isolates of CMV and the laboratory strain AD169. Wild-type CMV strains isolated from AIDS patients were not only more likely to possess nucleotide substitutions (19/24 compared to 5/25, P < 0.0001) than those from renal transplant recipients, but they also exhibited a greater degree of nucleotide sequence divergence (6.94 versus 0.82 substitutions/1000 bp, P < 0.0001; 96.0 to 100% versus 99.4 to 100% similarity). Increased sequence variation in the AIDS patients did not correlate with absolute peripheral blood CD4+ T cell level (r = 0.33, P > 0.1). Only two strains from AIDS patients and one strain from the renal transplant recipients possessed nucleic acid substitutions that resulted in codon changes, indicating that AD-1 is relatively well conserved amongst clinical isolates of CMV. The demonstration of strains with codon changes within neutralizing epitopes, however, highlights the importance of taking into consideration the presence of these strains within the wild-type virus population when preparing subunit vaccines.

Cytotoxic T cell recognition of virus-infected cells requires the presentation of viral peptides by class I HLA molecules on the cell surface. We report here that cytomegalovirus (CMV) infection of human fibroblasts results in a progressive decrease in the cell surface expression of class I HLA and beta 2-microglobulin (beta 2m) such that in the late stages of infection the majority of infected cells have no detectable surface class I HLA. Coincident with decreased surface expression of class I HLA was an increase in his cytoplasmic expression. Confocal scanning laser microscopic analysis demonstrated that class I HLA and beta 2m accumulate in a perinuclear compartment inside the CMV-infected cell. Our data thus support the concept that CMV infection induces altered transport of class I HLA to the cell surface. We suggest that the virus has evolved this mechanism as a strategy to avoid T cell recognition of infected cells.

Previous studies have shown that beta 2 microglobulin (beta 2m) is associated with glycoproteins present on the envelope of urinary human cytomegalovirus (CMV). beta 2m is non-covalently associated with the alpha chain of human leukocyte antigen (HLA) class I antigens and therefore it was of interest to determine whether the class I alpha chain is also associated with the beta 2m-CMV complex. Using a panel of monoclonal antibodies recognizing different conformational determinants on the HLA class I heterodimer or free beta 2m, we have shown that beta 2m but not the alpha chain of HLA class I could be immunoprecipitated from 125I-surface-labelled virions purified directly from urine. We therefore conclude that host class I HLA alpha chains are not associated with beta 2m on the envelope of urinary CMV.

The heterogeneity of low passage human cytomegalovirus (HCMV) strains was determined by HindIII typing of 28 clinical isolates from transplant patients. These data have shown that, in general, each patient's strain has a unique restriction profile, usually comprising combinations of HindIII sites present in one or more of the tissue culture-adapted strains AD169, Towne and Davis. To map sequence changes in a more refined manner we performed detailed analyses of 33 low passage clinical isolates, including those aforementioned, analysing a sequence within glycoprotein B containing a major neutralizing epitope. A 149 bp sequence containing the epitope (amino acids 608 to 625) was amplified using the polymerase chain reaction, the products were cloned and their DNA sequence was determined. Comparison of the DNA and deduced amino acid sequences with those of HCMV strain AD169 revealed that there was a high degree of conservation of the epitope between the 33 clinical isolates. However 10 of the isolates possessed silent mutations and three isolates contained mutations producing amino acid changes within the neutralizing epitope. The possible functional significance of these changes is discussed.

We have previously reported that human cytomegalovirus (CMV) from urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies that can capture CMV grown in vitro. We report here that CMV exists in vivo in body fluids such as urine as beta 2 microglobulin (beta 2 m)-coated particles. We have demonstrated the presence of beta 2m on CMV purified directly from urine by Western blotting and have shown that the beta 2m was associated with the viral envelope. Urinary CMV could be specifically bound by an affinity column comprising a monoclonal antibody specific for beta 2m bound to Sepharose. The beta 2m-coated urinary CMV could not be neutralized by hyperimmune globulin, human immune sera or murine monoclonal antibodies that could neutralize CMV grown in cell culture. We conclude that the binding of beta 2m by CMV masks the important antigenic sites necessary for neutralization which are recognized by man's immune response. We propose that CMV has evolved this mechanism of coating itself in a host protein as a mechanism of evading the host immune response and facilitating transmission between individuals.

Adult athymic Nu/Nu mice showed increased susceptibility to lethal infection with murine cytomegalovirus (MCMV) when compared to their heterozygous T cell-competent Nu/+ littermates. However, the extent of this increase in susceptibility varied dramatically depending on the genetic background of the mice carrying the Nu/Nu gene. Genetically susceptible Balb/c (H-2d) mice showed a greater than 316-fold difference between the LD50 of Nu/Nu and Nu/+ littermates. In marked contrast, the genetically resistant CBA (H-2K) strain was characterized by only a 16-fold difference in resistance between Nu/Nu and Nu/+ mice, and furthermore, the athymic CBA Nu/Nu mice were no susceptible than the T cell-competent Balb/c Nu/+ strain. These results together with previous observations strongly suggest that the (H-2k)-associated resistance of the CBA strain is mediated by non-T cell-dependent early defence mechanisms.

We have previously demonstrated that human cytomegalovirus (CMV) binds the host protein beta 2 microglobulin (beta 2m) from body fluids or from cell culture media. In this report we have examined the effect of the beta 2m on viral infectivity. We have shown that the addition of human purified beta 2m, or a fraction of foetal calf serum corresponding to bovine beta 2m, to culture medium increased the amount of infectious extracellular CMV, compared to that from cells grown in serum-free medium. Metabolic labelling experiments demonstrated that this effect was not due to an increase in the amount of extracellular virus but to an increase in the infectivity of the virus present in extracellular fluids. We concluded that the binding of beta 2m by CMV increased its infectivity. We have shown that CMV and beta 2m compete for binding sites on fibroblasts. As the main binding site on cells for beta 2m is the class I HLA heavy chain we compared the binding of CMV to the Raji and Daudi cell lines which express or lack expression of class I HLA molecules. The binding of radiolabelled beta 2m-coated CMV was significantly higher to Raji cells than to Daudi cells. Furthermore, CMV could compete with beta 2m for binding to Raji cells, although the reverse was not true. These results demonstrate that CMV can use class I HLA molecules as a virus receptor. We propose that when coated with beta 2m, CMV has the capacity to displace beta 2m from the class I HLA heavy chain-beta 2m dimer on the cell surface and bind to cells. The fact that beta 2m enhances infectivity suggests that such binding leads to productive infection of cells.

We previously reported that a host protein, beta 2 microglobulin (beta 2m) inhibited the detection of human cytomegalovirus (CMV) in urine specimens by enzyme immunoassay and postulated that beta 2m bound to the virus particle and masked the viral antigenic determinants. We report here that CMV strain AD169 grown in cell culture bound human beta 2m when this protein was added to cell culture fluids or when the virus was added to urine. Such binding was not seen with herpes simplex virus. CMV could also bind bovine beta 2m from foetal calf serum in cell culture fluids. The use of radiolabelled beta 2m in other experiments showed that CMV bound beta 2m after release from cells and that the bound beta 2m did not represent acquisition of class I HLA molecules during budding from host cell membranes. Immunoprecipitation studies showed that beta 2m was bound by two viral envelope proteins beta 2m BP1 (beta 2m-binding protein 1) and beta 2m BP2 of molecular masses 36,000 and 65,000 daltons respectively. beta 2m could not bind to separated viral proteins under reducing or non-reducing conditions. We propose that interaction of these two proteins on the viral surface is required to enable CMV to bind beta 2m.

The delayed-type hypersensitivity (DTH) response in mice infected with murine cytomegalovirus (MCMV) was measured by ear swelling following a challenge with heat-treated MCMV. DTH was dose-dependent and could be detected as early as 3 days post-infection with peak responses occurring between days 15 and 21 post-infection. The DTH response was found to be specific for MCMV since it could not be elicited by either herpes simplex virus type 1 or influenza A virus in MCMV-primed mice. The elicited DTH response was greater in mice primed with attenuated compared with virulent MCMV. The DTH response was shown to correlate positively with the genetically determined resistance status of mouse strains to this virus. Previous research has shown that resistance to lethal infection with MCMV is controlled by H-2-linked genes since mice having the k haplotype are more resistant than mice having the b or d haplotype of the H-2-complex. Also, non-H-2-linked genes in CBA, C3H, C57BL/10 and probably other strains confer resistance. Resistant strains (C3H [H-2k], CBA [H-2k]) developed greater DTH responses than those of susceptible strains (BALB/c [H-2d], C57BL/10 [H-2b]) inoculated with the same dose of virus. In addition, the genetically resistant mouse strains B10.BR [H-2k] and BALB.K [H-2k] gave a significantly greater DTH response than that of the corresponding congenic strains C57BL/10 [H-2b], BALB/c [H-2d] and BALB.B [H-2b] which are genetically susceptible to the virus. Also, the DTH response of C57BL/10 [H-2b] was significantly higher than that of BALB.B [H-2b] which correlates with their relative genetic resistance to MCMV, indicating the importance of non-H-2-linked genes. Furthermore, in addition to the response of greater magnitude, resistant strains (CBA, C3H, B10.BR) produced DTH responses to MCMV by day 3 compared with day 5 post-infection for susceptible BALB/c mice. These findings indicate that the magnitude and the time of appearance of the DTH response correlates positively with the genetically determined resistance status, although the role of DTH responses in controlling MCMV infections remains to be determined.

The role of antibodies as mediators of genetically determined resistance to murine cytomegalovirus (MCMV) in mice has not been elucidated. The ability of mice with different MCMV resistance phenotypes to produce an antibody response to MCMV was investigated in order to assess whether the host genotypes that control resistance also influence antibody production. Antibodies to MCMV in the sera of resistant (BALB.K, CBA/CaH, B10.BR) and susceptible [BALB/c, BALB.B, C57BL/10ScSn (B10), B10.D2, B10.A, A/J] mice were determined by ELISA and/or a complement-requiring neutralization assay. IgM antibodies were produced by all strains of mice as early as 3 to 5 days post-infection (p.i.) with maximum titres observed after 10 days p.i. for some strains, whilst IgG antibodies were produced by 5 to 7 days p.i. with maximum titres at 20 days p.i. IgA antibodies were not detected in the sera of MCMV-infected mice. Virulent MCMV induced higher antibody titres than either attenuated or u.v.-inactivated forms of the virus. Although high doses of virulent virus delayed the early production of IgM antibody they did not adversely affect the kinetics of IgG antibody production. High titres of neutralizing antibodies were detected as early as day 3 post-inoculation of virulent virus; when attenuated virus was used in the neutralization assay, this was found to be more easily neutralized than salivary gland-derived virus. Interestingly, although guinea-pig complement greatly enhanced antibody-mediated neutralization of MCMV, mouse complement was also effective at enhancing neutralization. Although genetically determined resistance to MCMV is an early event with the resistant phenotype being demonstrable during the first few days of the infection, there was no evidence that antibodies were responsible for this resistance since neither antibody titres nor the time of first appearance of antibody correlated with resistance status. However, these results do not exclude a more general role for antibody in limiting MCMV infection, especially in immunity to re-infection since passively transferred antibodies from resistant or susceptible mouse strains lowered virus titres in MCMV-infected animals.