Human Respiratory Syncytial Virus Subgroup Research Articles

Structure-antigenicity relationship studies of the central conserved region of human respiratory syncytial virus protein G.

BBG2Na is a recombinant protein, composed in part of carrier protein BB and of the central conserved domain of the attachment glycoprotein G of human respiratory syncytial virus (HRSV) subgroup A. This protein is a potent vaccine candidate against HRSV. G2Na contains several contiguous B-cell epitopes, occupying sequential positions in the linear sequence of the protein. One of the epitopes contains four cysteines that are completely conserved in known strains of HRSV and form a 'cysteine noose' motif. In this study, we analysed circular dichroism (CD) spectra of BBG2Na and its B-cell epitopes. We also used NMR and molecular dynamics simulations to determine the three-dimensional structure of the cysteine noose domain. We observed significant structural differences related to the length of peptides containing the cysteine noose. These differences show good correlation with the immunogenic activity of the peptides. It is shown that a single Val(171) addition induces a pronounced structure stabilization of the cysteine noose peptide G4a (1-4/2-3) (residues 172-187), which is associated with a 100-fold increase in its antigenicity vis-à-vis a G-protein specific monoclonal antibody.

Antigenic and Genetic Characterization of Twenty-six Strains of Human Respiratory Syncytial Virus (Subgroup A) Isolated During Three Consecutive Outbreaks in Havana City, Cuba

Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100%) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus.

A bovine respiratory syncytial virus strain with mutations in subgroup-specific antigenic domains of the G protein induces partial heterologous protection in cattle

Bovine respiratory syncytial virus (BRSV) strains are tentatively divided in subgroups A, AB and B, based on antigenic differences of the G protein. A Dutch BRSV strain (Waiboerhoeve: WBH), could not be assigned to one of the subgroups, because the strain did not react with any monoclonal antibody against the G protein. We describe here that the WBH strain has accumulated critical mutations in subgroup-specific domains of the G protein gene, which also occur but then independently in G protein genes of BRSV subgroup A or B strains. Although the comparison of nucleotide residues 256–792 of the G gene of the WBH strain with those of subgroup A and B strains showed that the G gene of the WBH strain is different from that of BRSV subgroup A and B strains, the sequence divergence was not more than observed within the G genes of human respiratory syncytial virus subgroup A or B strains. The WBH strain did not induce severe disease after experimental infection of calves, and induced partial protection against a heterologous challenge. Despite the dissimilarity of the conserved central regions of the G protein of the WBH strain and that of the challenge strain, a secondary antibody response against this region was induced in WBH-infected calves after challenge. We conclude that complete BRSV virus can partially protect against a BRSV infection with a strain that contains an antigenic dissimilar G protein.

Linear antigenic and immunogenic regions of human respiratory syncytial virus N protein.

Three linear antigenic regions on the N protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were identified by using peptides which reacted in ELISA with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the protein: Thr11 to Gly30 (N3 peptide), Ser231 to Ala250 (N25 peptide) and Thr371 to Leu391 (N39 peptide). The site represented by the N39 peptide reacted with four subgroup A-specific MAbs. There were minor variations in the amino acid epitope dependencies of each of these MAbs. Two additional antigenic regions Ser131 to Arg150 and Ala181 to Leu200, were represented by peptides that reacted with human convalescent sera, but these peptides did not differentiate between acute and convalescent sera from RSV-infected humans. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the N protein from a nucleocapsid-containing homogenate derived from extracts of RSV-(subgroup A and/or B)-infected 35S-labelled cells in a radioimmuneprecipitation assay (RIPA); the sera were also used to demonstrate RSV infection in cells by immunofluorescent assay (IFA). Anti-N3 peptide sera precipitated N41, the full-length (M(r) 41,000) form of N protein, in a RIPA done on a soluble protein pool. Anti-N39 (C-terminal region) peptide sera precipitated both forms, suggesting that N38 (M(r) 38,000) is an N-terminally truncated (probably at position Tyr23 located inside the N3 peptide linear antigenic region) form of N41 protein.

Nucleotide sequence analysis of the non-structural NS1 (1C) and NS2 (1B) protein genes of bovine respiratory syncytial virus

The nucleotide and deduced amino acid sequences of the non-structural (NS) protein genes of bovine respiratory syncytial virus (BRSV) strain A51908 were determined. The NS1 and NS2 genes were 524 and 489 nucleotides long with single open reading frames, encoding polypeptides of 136 and 124 amino acids, respectively. Comparison of the NS1 gene of BRSV with the corresponding sequences of ovine respiratory syncytial virus (ORSV) and human respiratory syncytial virus (HRSV) subgroups A and B revealed 82, 67 and 65% identity at the nucleotide level, and 89, 69 and 68% identity at the amino acid level, respectively. The nucleotide identity of the NS2 gene of BRSV to those of ORSV and HRSV subgroups A and B was 80, 69 and 67% and the predicted amino acid identity was 87, 84 and 83%, respectively. The intergenic sequences of the NS1-NS2 and NS2-N gene junctions of BRSV were determined and found to be 13 nucleotides shorter and 29 nucleotides longer, respectively, than the corresponding regions of HRSV.

Linear antigenic and immunogenic regions of the respiratory syncytial virus P protein.

Three linear antigenic regions on the P protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were represented by peptides that reacted with monoclonal antibodies and with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the P protein: Pro91 to Asp110, Ser161 to Lys180 and Glu221 to Phe241. The role of individual amino acids in the epitopes defined by monoclonal antibodies was determined. Two monoclonal antibodies reacting with the same antigenic site were found to detect epitopes that had different amino acid dependencies. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the P protein from RSV-infected 35S-labelled cell extracts in a radioimmune precipitation assay. These findings have implications for forthcoming structural-functional studies of RSV capsid component interactions and also for serological diagnosis of RSV infection.

Identification of subgroups of bovine respiratory syncytial virus.

The occurrence of antigenic variation among nine isolates of bovine respiratory syncytial virus (BRSV) was determined by examining their reaction patterns to human respiratory syncytial virus (HRSV) subgroup A and B monoclonal antibodies (MAbs) by enzyme immunoassay and radioimmunoprecipitation with fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using MAbs and polyclonal antisera to HRSV and BRSV. Shared epitopes were demonstrated on four of five structural proteins between BRSV and both subgroups A and B of HRSV. The nine isolates of BRSV showed different patterns of reactivity in enzyme immunoassays with panels of MAbs to HRSV subgroups A and B. Major variations in the molecular weights of the P (phosphoprotein) and F (fusion protein) proteins were demonstrated among the BRSV isolates tested. These results suggest that BRSV belongs to a different antigenic grouping than HRSV and that BRSV is composed of two distinct subgroups.

Genes 1 and 2 of pneumonia virus of mice encode proteins which have little homology with the 1C and 1B proteins of human respiratory syncytial virus

Genes 1 and 2 of pneumonia virus of mice (PVM) consist of 410 and 571 nucleotides and encode proteins of 113 and 156 amino acids respectively. The proteins show no extensive (gene 1 analogous to 1C) or low (gene 2 analogous to 1B) homology to their presumed counterparts in human respiratory syncytial virus (HRSV). The strongest homology is between regions of approximately 35 amino acids located near the carboxy termini of the gene 2 product and the 1B protein with 29% identity, although a lower level of homology can be detected throughout much of these proteins (18% identity overall). These observations contrast with the conservation of 1C and 1B proteins between subgroups of HRSV and with the conservation of nucleocapsid proteins between HRSV and PVM.

Evolution of the G and P genes of human respiratory syncytial virus (subgroup A) studied by the RNase A mismatch cleavage method

The G and P genes of human respiratory syncytial viruses (subgroup A), isolated between 1961 and 1989, were analyzed by RNase A one-dimensional fingerprinting, using the Long strain as the reference. Total RNA extracted from cells infected with the different isolates was hybridized to radiolabeled antisense G or P RNA probes of the Long virus. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products analyzed by gel electrophoresis. Comparative analysis of the cleavage patterns revealed extensive genetic heterogeneity in both genes among viruses isolated in different epidemics. In contrast, 13 viruses isolated in Montevideo during a 3-month period showed much more restricted heterogeneity; thus, 11 viruses represented the predominant type of this outbreak and only 2 other viruses generated different RNA cleavage patterns distantly related to the major type. Statistical analysis of the results obtained indicated progressive accumulation of genetic changes with time along cocirculating evolutionary lineages within the same antigenic subgroup of RS virus. The results are discussed in terms of a model for RS virus evolution.

Preparation and Characterization of Monoclonal Antibodies Directed against Five Structural Components of Human Respiratory Syncytial Virus Subgroup B

Mouse hybridomas producing antibodies against the structural proteins of strain WV4843, a subgroup B strain of respiratory syncytial (RS) virus, were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the virus. After immunoprecipitation test with [35S]methionine-labelled extracellular virions, 35 clones found to produce antibodies against the fusion (F) protein, six against the member (M) protein, 21 against the nucleocapsid (NP) and eight against the phospho- (P) protein were further characterized. Immunoprecipitation with [3H]glucosamine-labelled intracellular virus polypeptides detected nine hybridoma cell lines producing antibodies against the large glyco- (G) protein of the virus. By competitive binding ELISA tests with monoclonal antibodies against each of the structural components, a minimum of two, 24, four, 15 and three epitopes were detected on the G, F, M, NP and P proteins, respectively. Eleven monoclonal antibodies directed against nine epitopes of the F protein could neutralize the infectivity of the virus. In contrast, none of the nine monoclonal antibodies against G could neutralize the infectivity of the virus. In order to find out more about the antigenic relationship between human and bovine RS virus strains all monoclonal antibodies were reacted with subgroup A RS virus and also with three different strains of bovine RS virus and one strain of caprine RS virus in immunofluorescence, ELISA and immunoprecipitation tests. In addition, 31 previously developed monoclonal antibodies against subgroup A virus were reacted with the bovine and caprine strains. The numbers of monoclonal antibodies of subgroup B specific for the B type of the two human subgroups were 9/9, 3/35, 0/6, 0/21, 0/8, for the G, F, M, NP and P proteins, respectively. No antigenic variations were found between the three bovine strains and the caprine strain. They did not react with the nine monoclonal antibodies against the G protein of subgroup B, nor did they react with nine monoclonal antibodies against subgroup A. Most but not all of the monoclonal antibodies against the other structural proteins of the two human RS virus subgroups reacted with the four strains. All 11 monoclonal antibodies against the F protein of subgroup B that could neutralize the infectivity of subgroup B also reacted with the bovine strains and neutralized their infectivity. It is concluded that although the bovine strains share many epitopes with the two human subgroups, they are antigenically distinct from the human viruses.

Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity.

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus.