Increased total mortality as a function of 24-h pulse pressure dipping.

Elevated pulse pressure (PP) as a difference between systolic and diastolic blood pressure is a significant risk factor of cardiovascular (CV) diseases. The goal of our study was to determine the association between PP and major adverse CV events (MACEs), and all-cause and CV mortality in the different age groups of patients with coronary artery disease (CAD) confirmed by angiography. To the PROGNOSIS study, finally there were included 891 subjects with CAD. An analysis of the receiver operating characteristic was used for predicting PP dipping among the age groups of patients. A COX proportional hazards model was used to examine the association between PP and PP dipping and risk of MACE, revascularization, CV and total mortality after adjusting for sex, diabetes, smoking and low-density lipoprotein cholesterol. The median follow-up period was 8.3 years (interquartile range: 5.3-9.0 years). There were 245 (27%) all-cause deaths including 114 (13%) CV deaths during the follow-up period. MACE occurred in 442 (50%) subjects, but coronary artery interventions (percutaneous coronary intervention or coronary artery bypass grafting) were performed in 578 subjects (65%). A Cox proportional regression analysis confirmed the relationship between PP dipping as well as PP dipping thresholds points and risk of MACE and total mortality only in the group of the oldest subjects. In contrast to younger CAD patients, PP dipping is related to MACE, CV and total mortality in very elderly CAD subjects. Nocturnal PP values tend to be higher than diurnal PP values in the oldest CAD individuals. In conclusion, in contrast to younger CAD patients, PP dipping is related to MACE, CV and total mortality in very elderly CAD subjects. Nocturnal PP values tend to be higher than diurnal PP values in the oldest CAD individuals.

Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate.

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.

The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC.

A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV-1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV-1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface-associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide-directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding

Localization of type-specific epitopes of herpes simplex virus type 2 glycoprotein G recognized by human and mouse antibodies.

Glycoprotein G is a major target for the humoral immune response against herpes simplex virus (HSV) and a prototype antigen for type-specific serodiagnosis discriminating HSV-1 and HSV-2 infections. The mature part of HSV-2 glycoprotein G-2 (gG-2) contains a unique stretch suspected to mediate type specificity, and in addition a region homologous to HSV-1 glycoprotein G-1 (gG-1). Antigenic determinants of the mature gG-2 were mapped by testing the reactivity of mouse anti-gG-2 monoclonal antibodies (MAbs) and purified human anti-gG-2 antibodies with synthetic peptides coupled to cellulose membranes. The anti-gG-2 MAbs bound to four epitopes localized in a narrow cluster within a gG-2 segment delimited by amino acids (aa) 552 and 611. This cluster was located between the predicted O-glycan-rich region and the transmembrane anchor sequence. The epitopes of the human anti-gG-2 antibodies were localized within three stretches of amino acids, two of which were overlapping with those recognized by anti-gG-2 MAbs. One of these stretches, delimited by aa 552 and 574, showed reactivity to all human HSV-2 sera tested, but not to HSV-1 sera or to purified anti-gG-1 antibodies. Neither the anti-gG-2 MAbs nor the purified human anti-gG-2 antibodies were cross-reactive to gG-1 peptides or HSV-1 antigen, although most of the epitopes were localized within the part of gG-2 which was homologous to gG-1. The findings concerning HSV-2 type-specific human antibody response to a defined stretch within gG-2 may be of importance for the further development of type-discriminating serodiagnosis.

Interaction between pseudorabies virus and heparin/heparan sulfate. Pseudorabies virus mutants differ in their interaction with heparin/heparan sulfate when altered for specific glycoprotein C heparin-binding domain.

Cell surface heparan sulfate serves as an initial receptor for a number of herpesviruses including pseudorabies virus (PrV). It has been demonstrated that the heparan sulfate-binding domain of PrV glycoprotein C is composed of three discrete clusters of basic residues corresponding to amino acids 76-RRKPPR-81, 96-HGRKR-100, and 133-RFYRRGRFR-141, respectively, and that these clusters are functionally redundant, i.e. each of them could independently support PrV attachment to cells (Flynn, S. J., and Ryan, P. (1996) J. Virol. 70, 1355-1364). To evaluate the functional significance of each of these clusters we have used PrV mutants in which, owing to specific alterations in glycoprotein C, the heparan sulfate-binding site is dominated by a single specific cluster. These mutants exhibited different patterns of susceptibility to selectively N-, 2-O-, and 6-O-desulfated heparin preparations in virus attachment/infectivity assay. Moreover PrV mutants differed as regard to efficiency of their attachment to and infection of cells pretreated with relatively low amounts of heparan sulfate-degrading enzymes. Furthermore glycoprotein C species, purified from respective mutants, bound heparin oligosaccharide fragments of different minimum size. These differences suggest that specific clusters of basic amino acids of the heparan sulfate-binding domain of glycoprotein C may support PrV binding to different structural features/stretches within the heparan sulfate chain.

Structural requirement of heparan sulfate for interaction with herpes simplex virus type 1 virions and isolated glycoprotein C.

Cell surface heparan sulfates mediate primary attachment of herpes simplex virus type 1, the first step in virus invasion of the cells. Removal of the host cell heparan sulfate results in a significantly diminished susceptibility of the cell to virus infection. On the virus envelope, glycoprotein C has been identified as the major binding site for heparan sulfate in the primary attachment of the virus to host cells. Using selectively desulfated heparins and metabolically labeled host cell heparan sulfate, we have analyzed the structural requirements of heparan sulfate to provide binding sites for glycoprotein C and the whole virus. Employing glycoprotein C affinity chromatography and a virus binding assay, we subfractionated oligosaccharides derived from heparan sulfate and partially desulfated heparin into selectively bound and unbound pools. These were chemically depolymerized and analyzed at the disaccharide level. The shortest glycoprotein C-binding fragment consisted of 10-12 monosaccharide units containing at least one 2-O- and one 6-O-sulfate group that have to be localized in a sequence-specific way, based on the finding that bound and unbound HS fragments do not differ in charge or composition. The binding sequence is found within N-sulfated blocks of heparan sulfate, although several N-acetyl groups can be tolerated within the minimal binding sequence. These minimal requirements for herpes simplex virus type 1 binding to heparan sulfate are clearly distinct from other identified protein binding sites.

Mode of interaction between pseudorabies virus and heparan sulfate/heparin.

It has been demonstrated that the efficient attachment of pseudorabies virus (PrV) is mediated by an interaction between glycoprotein C (gC) and a cellular heparin-like substance (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and t. Ben-Porat, J. Virol. 64, 278-286, 1990). According to the prevalent concept, this interaction is likely to occur between clusters of basic residues of PrV gC and the negatively charged sulfate esters and carboxylate groups of heparan sulfate/heparin. To elucidate which of the three major types of sulfate groups of heparan sulfate/heparin are involved in the interaction with PrV, we used selectively N-, 2-O-, and 6-O-desulfated samples and other modified heparins as competitors in virus-attachment assays. PrV exhibited limited preference for the specific sulfate groups of heparan sulfate/heparin in accordance with a hierarchy of 6-O- > 2-O- > N-sulfates. In addition, since selective removal of any of the specific sulfates had only a slight effect on the competition capacity of heparin, it is likely that the combination of any two of three types of sulfate groups could contribute to an interaction with PrV with an efficiency nearly equal to native, fully sulfated heparin. When tested on different cell lines the pattern of PrV requirement for the specific O-sulfate groups, i.e., 6-O-sulfates > 2-O-sulfates, remained the same. However, different minimum lengths of heparin fragments were required to inhibit PrV attachment to different cell lines, suggesting a relative virus flexibility in accommodation to different forms of heparan sulfate.

Antigenic differences between HSV-1 and HSV-2 glycoproteins and their importance for type-specific serology.

The ability to discriminate between herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) infections by serological means is of increasing importance for clinical virological diagnostics. HSV type-specific serology has a number of applications, e.g. to guide duration and dosage of antiviral therapy, to allow for stringent epidemiological analyses, to evaluate efficacy of HSV vaccine candidates, and to help the clinician during counseling of couples where one has genital herpes. The genomes of the two HSV types appear to be sufficiently intratypically stable and intertypically discrepant to permit such a discrimination. Despite recent advances in methodology, the ideal HSV type-specific serological assays still remain to be developed. The viral antigens utilized for such an assay should evoke strong antibody responses, but only against unique determinants, in order to be able to combine high sensitivity and specificity. Of all the HSV envelope proteins, the most promising candidate antigens, i.e. HSV-1 glycoprotein G (gG-1) and its HSV-2 counterpart gG-2, contain relatively long type-unique stretches of amino acids. However, whether the type-specific determinants preferentially localize to the unique or homologous stretches of gG is still unknown. Paradoxically, type-specific monoclonal antibodies to the moderately type-specific antigen, HSV-1 glycoprotein C, have hitherto been mapped to homologous rather than to type-unique stretches. A definition of human type-specific epitopes on gG-1 and gG-2, as well as a broader search for new candidate HSV antigens, might be needed to fully discriminate dual infections from high titer single HSV-1 or HSV-2 infections.

An evaluation of a hemagglutination-inhibition test for the detection of antibodies to herpes simplex virus type 1.

BACKGROUND: We have recently demonstrated the ability of herpes simplex virus type 1 (HSV-1) to agglutinate mouse red blood cells, and identified glycoprotein C (gC-1) as a major virus hemagglutinin. Based on this a classical hemagglutination-inhibition (HI) assay was developed. OBJECTIVES: Regarding significant structural differences between HSV-1 gC-1 and its herpes simplex virus type 2 (HSV-2) counterpart, gC-2, the possibility of application of a classical HI assay for the detection of HSV-1-specific antibodies was explored. STUDY DESIGN: HI antibody titers were compared with those of gC-1-specific enzyme-linked immunoassay (ELISA), and with the results of the standard gG-1- and gG-2-specific immunodot enzymatic assays for the detection of type-specific antibodies to HSV-1 and HSV-2 respectively. RESULTS: The sensitivity of HI test was 89% and 97% of that gC-1-ELISA and gG-1-immunodot respectively. Approximately 21% of serum specimens, defined as containing antibodies specific for only HSV-2, showed low HI titers. Heterotypic reactivity with purified gC-1 antigen was also observed in both ELISA and immunoblot assays. CONCLUSION: Antibodies detectable in HI assay were mainly HSV-1-specific; however, a limited degree of serologic reactivity between HSV-2-specific sera and HSV-1 hemagglutinin also occurred. Thus, our results confirmed prevalent opinion about the presence of a limited number of antigenic determinants shared by HSV-1 gC-1 and HSV-2 gC-2.

Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate.

The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations. (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-1-induced haemagglutination and virus binding to susceptible cells. (ii) A number of HSV-1 mar mutants, altered at these critical residues, were impaired in attachment to cells. (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-L-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.

Herpes simplex virus type 1-induced hemagglutination: glycoprotein C mediates virus binding to erythrocyte surface heparan sulfate.

We recently reported that herpes simplex virus type 1 (HSV-1) can cause agglutination of murine erythrocytes (E. Trybala, Z. Larski, and J. Wisniewski, Arch. Virol. 113:89-94, 1990). We now demonstrate that the mechanism of this hemagglutination is glycoprotein C-mediated binding of virus to heparan sulfate moieties at the surface of erythrocytes. Hemagglutination was found to be a common property of all gC-expressing laboratory strains and clinical isolates of HSV-1 tested. Mutants of HSV-1 deficient in glycoprotein C caused no specific hemagglutination, whereas their derivatives transfected with a functional gC-1 gene, thus reconstituting gC expression, regained full hemagglutinating activity. Hemagglutination activity was inhibited by antibodies against gC-1 but not by antibodies with specificity for glycoproteins gB, gD, or gE or by murine antiserum raised against the MP strain of HSV-1, which is gC deficient. Finally, purified gC-1 protein, like whole HSV-1 virions, showed high hemagglutinating activity which was inhibited by heparan sulfate and/or heparin and was completely prevented by pretreatment of erythrocytes with heparitinase, providing evidence that gC-1 mediates hemagglutination by binding to heparan sulfate at the cell surface. Thus, HSV-1-induced hemagglutination is gC-1 dependent and resembles the recently proposed mechanism by which HSV-1 attaches to surface heparans on susceptible cells, providing a simple model for initial events in the virus-cell interaction.

An intradermal test for the diagnosis of BHV-1 infection. The effect of repeated testing on the immune status of cattle.

The effect of the repeated delayed-type hypersensitivity (DTH) testing (an intradermal test--ID) on virus neutralizing (VN) antibody and DTH responses has been studied. Repeated intradermal injection of inactivated BHV-1 antigen elicited neither the virus neutralizing antibody nor positive skin reaction in cattle free from BHV-1 infection. On the other, hand, the same manipulation carried out in a herd infected with BHV-1 provoked seroconversion and/or positive DTH reaction in some of the animals without detectable pre-existing VN antibody. Cattle initially positive by VN and intradermal tests showed steady increase in antibody titre, and some of them developed little or no skin reaction following repeated DTH testing.

An evaluation of an intradermal test for the diagnosis of bovine herpesvirus type 1 (BHV-1) infection in cattle.

An intradermal (ID) test (a delayed-type hypersensitivity test) was used for the diagnosis of BHV-1 infection in cattle. A threshold value for the test positive results was established based on the analysis of the agreement between the results of ID test and those of virus neutralization (VN24) assay. Linear regression analysis revealed no correlation between the intensity of the skin reaction (expressed as an increase in skin-fold thickness) and the VN24 antibody titre. The sensitivity and specificity of the ID test compared to VN24 one, were 86.6% and 98.3% respectively. Of the 487 cattle with an age of over 6 months, 25 (5.1%) reacted discordantly by ID and VN24 tests, i.e. 19 seropositive animals expressed little or no skin reaction, whereas 6 seronegative individuals were positive by ID. Neither of the calves, with passively acquired antibodies in contrast to experimentally infected ones, showed distinctive skin reaction.

[Is Aujeszky's disease a zoonosis?]. / Czy choroba Aujeszkyego jest zoonoza?

Several cases of human illness with symptoms suggestive of Aujeszky's Disease (AD) at an outbreak of the disease in cattle and swine were evaluated during this study. Additionally serological studies of people in the group at high risk for illness caused by Aujeszky's Disease Virus (ADV) were carried out. In a herd of 180 bulls sudden clinical disease occurred with symptoms of muscular tremors, salivation, sweating and severe pruritus of the head. The clinical disease lasted two to eight hours and finished in death. In 7 days 10 bulls died and 60 were either killed or culled. A biological test for AD was positive. ADV was identified in the brain of a dead bull. Serological studies for the bulls were negative, however 92.9% on the premises farm were asymptomatically infected. On third to the fifth day of disease in the cattle clinical signs appeared in six of seven workers which had direct contact with diseased cattle. A pruritus of the palms, which spread onto the lower and upper arms, shoulders and back lasted several days.

The effect of some physical and serological treatments on haemofusing activity of bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 irrespective of the time of its harvesting from Madin-Darby bovine kidney cells, expressed little or no haemofusing activity. Treatment of the virus by freezing and thawing, sonication or antibody and complement enhanced this activity. Moreover haemofusing activity did not correlate with the viral capacity for fusion of susceptible cells in monolayer cultures.

Hemagglutination by herpes simplex virus type 1.

The McIntyre and HSZP strains as well as clinical isolate of herpes simplex virus type 1 were found to agglutinate C57Bl/10su and CBA mouse red blood cells. The hemagglutinating activity was inhibited by antisera that neutralized the infectivity of the virus.

[Effect of urea and uric acid on the hemolytic activity of Sendai virus]. / Wplyw mocznika i kwasu moczowego na hemolityczna aktywnosc wirusa Sendai.

It was found that haemolytic activity of Fushimi strain of Sendai virus multiplied in allantoic cavity of chicken embryos is independent on its haemagglutinating titer and also on allantoic fluid urea and uric acid content. It was shown in experiments with embryonated eggs that these two compounds have no also influence on haemolytic activity induction in Sendai virus. Moreover, the results of an experiment in which allantoic fluid was replaced by Eagle's liquid suggest that most probably the other components present in allantoic fluid do not also influence the appearance of haemolytic activity of this virus.

An evaluation of four serological tests for the detection of antibodies to bovine parainfluenza virus type 3.

Haemagglutination-inhibition (HI), virus neutralization (VN), haemolysis-inhibition (HLI-1, HLI-2) tests, and two new haemofusion-inhibition (HFI-1, HFI-2) tests, developed by us, were tested for the detection of antibodies to bovine parainfluenza virus type 3 (BPIV-3) in sera of 45 steers randomly selected from a herd naturally infected by BPIV-3. Twelve seronegative animals were then vaccinated with formalin-inactivated vaccine and 15 days later tested by the above-mentioned assays. Linear regression analysis revealed positive correlations between HI, VN, HLI-1 and HFI-1 antibody titres, which confirm the specificity of the HFI-1 test. The HLI-1 and HFI-1 assays proved to be less sensitive than the HI and VN ones, while HLI-2 and HFI-2 tests failed to detect any antibody to BPIV-3.

Attempts at the detection of antibodies to Newcastle disease virus by haemofusion-inhibition test.

Two modifications of a haemofusion-inhibition test (HFI-1 and HFI-2) were applied for the titration of antibodies to Newcastle disease virus (NDV) in chicken sera. Statistical analysis revealed a positive correlation of the HFI-1 antibody titres with those measured by the standard haemagglutination-inhibition (HI), virus neutralization (VN) and haemolysis-inhibition (HLI) tests. The same appeared true when the HFI-2 antibody titres were compared with the HI, VN, and HLI tests. Except for the several sera collected from birds immunized with formalin-inactivated vaccine, the HFI-2 antibody titres of individual serum samples were usually lower than those determined by HFI-1. The interpretation of these differences as well as some advantages and disadvantages of the proposed test are discussed.