Genetic variability of group A and B human respiratory syncytial viruses isolated from 3 provinces in China.

The genetic variability of HRSV in China was studied using nucleotide sequencing of the hypervariable C-terminal region of the G protein gene and phylogenetic analysis on 80 isolates obtained from three children's hospitals over a period of three epidemic seasons, 1990/1991, 2000/2001, and 2003/2004. The results showed that 76/80 of these isolates belonged to group A and 4/80 belonged to group B. Phylogenetic analysis revealed that most of the group A isolates were genotype GA2 (74/76 isolates), and the other two isolates were GA3 and GA5. All group B isolates clustered into genotype GB3. There was substantial variation among the GA2 isolates, with nucleotide sequence and amino acid homologies ranging from 88.1-100% and 78.4-100%, respectively, in the hypervariable C-terminal region of the G protein gene. One group B virus, HRSV/Beijing/B/04/11, contained a 60-nucleotide duplication in the C-terminal region of the G protein, which was similar to what has been reported previously for isolates in several countries. This is the first report on the genetic diversity of human respiratory syncytial virus isolated during epidemic periods from children in China. These data provided a preliminary evaluation of patterns of circulation and the genetic diversity of isolates associated with HRSV epidemics within China.

Stevens-Johnson syndrome and toxic epidermal necrolysis after vaccination: reports to the vaccine adverse event reporting system.

We conducted a telephone survey of reports of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) to the Vaccine Adverse Event Reporting System. We identified six cases of SJS or TEN after vaccination without other obvious triggers, suggesting that SJS and TEN might very rarely be caused by vaccination. Confirmation of this hypothesis will likely require controlled studies.

The regulation of type 7 adenylyl cyclase by its C1b region and Escherichia coli peptidylprolyl isomerase, SlyD.

Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2. The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit alpha (Galpha(s)). In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD protein can inhibit the Galpha(s)- and/or forskolin-activated activity of both soluble and membrane-bound AC7. Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli slyD-deficient strain. This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6. This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.

Human respiratory syncytial virus surface glycoproteins F, G and SH form an oligomeric complex.

In order to study structural associations, RSV surface glycoproteins were evaluated using heparin agarose affinity chromatography (HAAC). When RSV-infected cell lysate was analyzed by HAAC, all three surface glycoproteins, (F, G and SH), were eluted. Similarly, when separate lysates from Vero cells infected with vaccinia recombinants expressing F (vvF), G (vvG) and SH (vvSH) proteins were subjected to HAAC, only vvF and vvG expressed proteins bound to heparin, whereas vvSH expressed protein did not bind. When lysates from vvF, vvG and vvSH-infected Vero cells were mixed prior to HAAC, only F and G bound heparin. In contrast, following co-infection of Vero cells with vvF, vvG and vvSH, all three proteins were detected subsequent to HAAC. Following HAAC of A2-infected cell lysate and lysate from vvF, vvG and vvSH co-infected Vero cells, two high molecular weight complexes of 175 Kd and 210 Kd, respectively, were identified that reacted with anti-F, anti-G and anti-SH antisera. In addition, anti-SH antiserum was able to co-precipitate RSV F, G and SH. Using HAAC and a NaCl step gradient we demonstrated that a fraction of RSV F, G and SH eluted at higher salt concentrations than either purified F or G protein. Taken together, these data suggest that RSV F, G and SH glycoproteins can form an oligomeric complex within infected cells and this complex has a higher affinity for heparin than either G or F protein alone.

Group II, but not group I, metabotropic glutamate receptors in the rat nucleus accumbens contribute to amphetamine-induced locomotion.

Recently, it was reported that blocking metabotropic glutamate receptors (mGluRs) in the rat nucleus accumbens (NAcc) prevents the generation of locomotion by amphetamine (AMPH) in this site. In these studies, the non-selective group I/group II mGluR antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine [(R,S)-MCPG] was used. The present study used more selective receptor antagonists to examine the specific contribution of group I and group II mGluRs to this effect. When co-injected bilaterally with AMPH into the NAcc, the group II selective mGluR antagonist (2S)-alpha-ethylglutamic acid [EGLU; 0.5-5.0 nmole/side] dose-dependently blocked the locomotion and rearing produced by AMPH. Equimolar concentrations of the group I selective antagonist (R,S)-1-aminoindan-1,5-dicarboxylic acid [AIDA; 0.5-5.0 nmole/side] were without effect. As previously reported for (R,S)-MCPG, neither of these receptor antagonists produced locomotor effects when injected alone in these concentrations into the NAcc. These results suggest that group II, but not group I, mGluRs in the rat NAcc contribute importantly to the ability of AMPH to produce locomotor activation.

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate.

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED(50)] = 0.28 +/- 0.11 microg/ml) and cp-52 (ED(50) = 0.55 +/- 0. 14 microg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.

Phenotypic expressions of CCR5-delta32/delta32 homozygosity.

OBJECTIVE: As blockade of CC-chemokine receptor 5 (CCR5) has been proposed as therapy for HIV-1, we examined whether the CCR5-delta32/delta32 homozygous genotype has phenotypic expressions other than those related to HIV-1. DESIGN: Study subjects were white homosexual men or men with hemophilia who were not infected with HIV-1. In this study, 15 CCR5-delta32/delta32 homozygotes were compared with 201 CCR5 wild-type (+/+) subjects for a wide range of clinical conditions and laboratory assay results ascertained during prospective cohort studies and routine clinical care. CCR5-delta32 genotype was determined by polymerase chain reaction, followed by single-stranded conformational polymorphism analysis. RESULTS: Hypertension and conditions attributable to hemophilia were the only diagnoses frequently found in clinical records of CCR5-delta32/delta32 study subjects. Based on blood pressure measurement and treatment history, CCR5-delta32/delta32 homozygotes had a 2.8-fold higher prevalence of hypertension than age-matched CCR5-+/+ study subjects (95% confidence interval [CI], 1.2-6.4; p = .01); none of the homozygotes had severe hypertension. Hematologic measures were generally similar across the genotypes, but total lymphocyte counts were approximately 20% higher in CCR5-delta32/delta32 study subjects than in CCR5-+/+ study subjects (p < .05). Among patients with hemophilia who were infected with hepatitis C virus (HCV), mean alanine aminotransferase levels were 117% higher among CCR5-delta32/delta32 homozygotes (p < .05), but serum HCV levels did not differ by CCR5-delta32 genotype. CCR5-delta32/delta32 homozygous study subjects had a lower prevalence of antibodies to measles virus than those with other genotypes, but this association was not confirmed in a group of blood donors. The prevalence of antibodies to nine other common viruses, HBV, and HCV was not related to CCR5 genotype. CONCLUSIONS: CCR5-delta32/delta32 homozygotes are generally similar to wild-type persons. Confirmatory investigations are required to determine whether hypertension, increased lymphocyte counts, and higher hepatic enzyme levels in the presence of HCV infection represent true phenotypic expressions of this genotype. CCR5-delta32/delta32 homozygosity does not provide broad protection against viral infections.

The mumps virus neurovirulence safety test in Rhesus monkeys: a comparison of mumps virus strains.

Wild type mumps viruses are highly neurotropic and a frequent cause of aseptic meningitis in unvaccinated humans. To test whether attenuated mumps viruses used in the manufacture of mumps vaccines have neurovirulent properties, a monkey neurovirulence safety test (MNVT) is performed. However, results with several mumps virus MNVTs have raised questions as to whether the test can reliably discriminate neurovirulent from nonneurovirulent mumps virus strains. Here, various mumps virus strains representing a wide range of neuropathogenicity were tested in a standardized MNVT. A trend of higher neurovirulence scores was observed in monkeys inoculated with wild type mumps virus versus vaccine strains, although differences were not statistically significant. Results indicated the need for further examination and refinement of the MNVT or for development of alternative MNVTs.

Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.

Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines.

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.

Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein.

A recombinant human antibody fragment, designated RSV Fab 19, efficiently neutralizes respiratory syncytial virus (RSV). Here we report the results of our sequence analysis of antibody escape mutants that identified F glycoprotein amino acids critical for binding of human or murine RSV F-neutralizing antibodies.

Kinetics of immunologic responses after primary MMR vaccination.

To study the kinetics of humoral as well as cellular immunity to measles and to test for associated immunosuppression 124 12 month old children were studied twice, before routine MMR and either 14, 22, 30, or 38 days after vaccination. Plaque reduction neutralization (PRN) titres were determined at these time points and lymphocytes were evaluated to identify changes in proportions of phenotype, their capacity to generate cytokines and to respond to blast transformation (BT) to measles hemagglutinin (HA), tetanus toxoid and Candida antigen. The PRN titre and BT to HA plateaued at 30 days and CD8+ and NK cells increased after immunization. Interleukin 2, 4, and 10 showed no significant changes. There was mild suppression of BT at 14 and 22 days post-immunization Interferon-gamma was the principal cytokine produced after primary measles immunization, suggesting primary measles immunization induces predominantly a TH1 type response.

Differential modulation of the immune response by breast- or formula-feeding of infants.

Spontaneous integrin expression on CD4+, CD8+ and CD19+ lymphocytes at 6 months was significantly lower in breastfed than formula-fed infants (p < 0.05). In another study of 59 formula-fed and 64 breastfed 12-month-old children blast transformation and cytokine production by lymphocytes, and T cell changes were measured before and after measles-mumps-rubella vaccination (MMR). Before vaccination, lymphocytes of breastfed children had lower levels of blast transformation without antigen (p < 0.001), with tetanus toxoid (p < 0.02) or Candida (p < 0.04), and lower interferon-gamma production (p < 0.03). Fourteen days after the live viral vaccination, only the breastfed children had increased production of interferon-gamma (p < 0.02) and increased percentages of CD56+ (p < 0.022) and CD8+ cells (p < 0.004). These findings are consistent with a Th1 type response by breastfed children, not evident in formula-fed children. Feeding mode has an important long-term immunomodulating effect on infants beyond weaning.

Measles vaccination of infants in a well-vaccinated population.

During outbreaks of measles, measles vaccine is recommended for infants considered to be at risk who are 6 months of age and older. In a prospective trial the serologic response to early measles immunization has been evaluated in 125 infants given monovalent measles vaccine at 6 to 8.5 months of age and measles-mumps-rubella at 15 months. The response to vaccination was measured by plaque reduction neutralization (PRN) assay and enzyme immunoassay. Infants were grouped by the mother's immunization history: natural immunity (n = 60, Group 1); killed followed by live, further attenuated vaccine (n = 22, Group 2); and live, further attenuated vaccine only (n = 43, Group 3). The prevaccination geometric mean titer (GMT) by PRN for Group 1 (GMT = 69) was significantly higher than that of Group 2 (GMT = 18) or 3 (GMT = 13). Seroconversion (4-fold increase in PRN titer) rates after monovalent vaccine were 31, 71 and 76% for Groups 1, 2 and 3, respectively. Seroconversion percentages were higher when measured 6 to 8 weeks after vaccination compared with 4 to 5 weeks. After measles-mumps-rubella > or = 97% of all infants had PRN titers > 120 and were measles IgG-positive by enzyme immunoassay. These data show that as demographics shift to a well-vaccinated maternal population and susceptibility in younger infants, measles vaccination before the currently recommended age will be effective.

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections.

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.

Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease.

A formalin-inactivated respiratory syncytial virus (RSV) vaccine tested 22 years ago failed to protect infant vaccinees against RSV infection or disease. Instead, lower respiratory tract disease was enhanced during subsequent infection by RSV. Enhancement of pulmonary pathology is also observed when cotton rats are immunized with formalin-inactivated RSV and subsequently infected with this virus. A major question that must be addressed for each new paramyxovirus vaccine is whether the immunogen possesses the capacity to potentiate disease. In the present study, we evaluated a newly developed purified F and G glycoprotein vaccine over a wide dosage range for immunogenicity, efficacy and capacity to potentiate pulmonary pathology in cotton rats. In addition, a formalin-inactivated RSV vaccine, which served as a positive control for enhancement of pulmonary pathology, was evaluated simultaneously. The results of these comparisons indicate that the purified F and G glycoprotein vaccine was highly immunogenic and was efficacious even in animals that developed low levels of serum-neutralizing antibodies. Furthermore, the F and G vaccine did not induce potentiation of pulmonary pathology. In contrast, formalin-inactivated RSV potentiated RSV pulmonary histopathology, but there was a sparing of potentiation at high and low doses. Both the formalin-inactivated RSV and purified F and G preparations induced a high level of serum antibodies capable of binding to purified F and G glycoproteins but both sets of antibodies had significantly reduced neutralizing activity. These results are encouraging because they suggest that purified paramyxovirus glycoproteins might be used safely as a vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function.

Eighteen neutralizing monoclonal antibodies (MAbs) specific for the fusion glycoprotein of the A2 strain of respiratory syncytial virus (RSV) were used to construct a detailed topological and operational map of epitopes involved in neutralization and fusion. Competitive binding assays identified three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB). Thirteen MAb-resistant mutants (MARMs) were selected, and the neutralization patterns of the MAbs with either MARMs or RSV clinical strains identified a minimum of 16 epitopes. MARMs selected with antibodies to six of the site A and AB epitopes displayed a small-plaque phenotype, which is consistent with an alteration in a biologically active region of the F molecule. Analysis of MARMs also indicated that these neutralization epitopes occupy topographically distinct but conformationally interdependent regions with unique biological and immunological properties. Antigenic variation in F epitopes was examined by using 23 clinical isolates (18 subgroup A and 5 subgroup B) in cross-neutralization assays with the 18 anti-F MAbs. This analysis identified constant, variable, and hypervariable regions on the molecule and indicated that antigenic variation in the neutralization epitopes of the RSV F glycoprotein is the result of a noncumulative genetic heterogeneity. Of the 16 eptiopes, 8 were conserved on all or all but 1 of 23 subgroup A or subgroup B clinical isolates.

Evaluation in non-human primates of the safety, immunogenicity and efficacy of recombinant vaccinia viruses expressing the F or G glycoprotein of respiratory syncytial virus.

It has been shown previously that immunization with recombinant vaccinia viruses expressing the F or G envelope glycoprotein of human respiratory syncytial virus (RSV) strain A2 induced a protective immune response in the lower respiratory tract of cotton rats against live RSV challenge. As a continuation of these studies, the safety, immunogenicity and efficacy of these recombinant vaccinia viruses was evaluated in non-human primates. Rhesus and patas monkeys were each inoculated intradermally at separate sites with the vaccinia-A2-F or vaccinia-A2-G recombinant or the parental vaccinia virus WR strain and the dermal lesion sizes were compared. Vaccinia-A2-F and vaccinia-A2-G recombinants produced lesions that were 5- to 15-fold smaller in area than vaccinia-WR. These studies indicated that insertion of either RSV gene into the thymidine kinase (TK) gene of vaccinia-WR significantly attenuated the virus for rhesus and patas monkeys. The immunogenicity of vaccinia-A2-F and vaccinia-A2-G was evaluated in squirrel, rhesus, African green, owl and patas monkeys. In four of the five species tested, the vaccinia-RSV recombinants stimulated levels of RSV serum-neutralizing antibodies considered to be protective for the lower respiratory tract of human infants and cotton rats. Interestingly, the level of RSV serum-neutralizing antibodies correlated with the size of the lesion. A boost in RSV serum-neutralizing antibody titres was not observed following a second inoculation. Owl monkeys inoculated with a single intradermal dose of vaccinia-A2-F and vaccinia-A2-G were completely resistant to infection of the lower respiratory tract with live RSV.(ABSTRACT TRUNCATED AT 250 WORDS)