Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.
The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product.
Worldwide G-glycoprotein phylogeny of human respiratory syncytial virus (hRSV) group A sequences revealed diversification in major clades and genotypes over more than 50 years of recorded history. Multiple genotypes cocirculated during prolonged periods of time, but recent dominance of the GA2 genotype was noticed in several studies, and it is highlighted here with sequences from viruses circulating recently in Spain and Panama. Reactivity of group A viruses with monoclonal antibodies (MAbs) that recognize strain-variable epitopes of the G glycoprotein failed to correlate genotype diversification with antibody reactivity. Additionally, no clear correlation was found between changes in strain-variable epitopes and predicted sites of positive selection, despite both traits being associated with the C-terminal third of the G glycoprotein. Hence, our data do not lend support to the proposed antibody-driven selection of variants as a major determinant of hRSV evolution. Other alternative mechanisms are considered to account for the high degree of hRSV G-protein variability. H uman respiratory syncytial virus (hRSV) is recognized as the major cause of severe acute lower respiratory tract infections (ALRI) in infants and young children worldwide (1). hRSV causes annual epidemics, and reinfections are common throughout life, although they are usually less severe than the primary infections. hRSV is also an important cause of morbidity and mortality in the elderly and in adults with cardiopulmonary disease or with an impaired immune system (2). IMPORTANCE An unusual characteristic of the G glycoprotein of human respiratory syncytial virus (hRSV) is the accumulation of nonsynonymous (N) changes at higher rates than synonymous (S) changes, reaching dN/dS valueshRSV is an enveloped, nonsegmented, negative-sense RNA virus, classified in the genus Pneumovirus within the Paramyxoviridae family (for a recent review, see reference 3). The hRSV genome encodes 11 proteins, two of them being the major surface glycoproteins of the virus envelope. These are (i) the attachment (G) protein, which mediates binding of the virus to the cell surface (4), and (ii) the fusion (F) protein, which promotes fusion of the virus and cell membrane, allowing cell entry of the viral genome (5).The G protein is a type II glycoprotein synthesized as a 32-kDa polypeptide precursor of 297 to 310 amino acids (aa), depending on the strain, and modified posttranslationally by the addition of several N-linked oligosaccharides and multiple O-linked sugar chains (6). The G-protein ectodomain (from residue 67 to the C terminus) has a central conserved region (aa 163 to 189) that includes four Cys residues (residues 173, 176, 182, and 186), and it is essentially devoid of potential glycosylation sites. This conserved region is flanked by two highly variable mucin-like segments, very rich in Ser and Thr, that are potential sites of O glycosylation. The extensive glycosylation of the G protein shapes its reactivity with both murine monoclonal antibodies (M...
Partial sequences of the G protein gene of 33 isolates from antigenic group B of human respiratory syncytial virus were determined. Phylogenetic analysis indicated that the evolutionary pattern of group B viruses is similar to that previously described for isolates of antigenic group A, including worldwide distribution of related viruses and co-circulation of viruses from different lineages during the same epidemic. Dominance of AGM GA over UCM CU transitions was observed when G sequences of group B viruses were compared, as previously found in viruses from antigenic group A. Interestingly, differences in protein length, determined by the usage of alternative termination codons, were more pronounced in group B than in group A viruses. Changes in protein length correlated with the classification of viruses in different lineages. Thus, mutations that determined termination codon usage seem to have played an important role in the diversification of group B viruses.Human respiratory syncytial virus (HRSV) is the leading cause of severe lower respiratory tract infections in infants and very young children (Collins et al., 1996). The virus is distributed worldwide and in temperate climates outbreaks of HRSV infections occur yearly as sharp peaks of activity in winter months. This epidemiological pattern differs in tropical countries where HRSV infections are distributed throughout extended periods of time, mainly during the rainy seasons (Heirholzer et al., 1994). Although severe HRSV infections occur most frequently during infancy, there is growing evidence that HRSV is an important pathogen for the elderly
The genetic and antigenic variability of human respiratory syncytial virus (HRSV) strains isolated in Buenos
During 5,230 trapping nights, 672 small mammals were trapped in the areas where most hantavirus pulmonary syndrome (HPS) cases occur in Uruguay. Yellow pygmy rice rats (Oligoryzomys flavescens) were the only rodents that showed evidence of antibodies to hantavirus, with a seroprevalence of 2.6%. The rodents were trapped in all the explored environments, and most of the seropositive rodents were found in habitats frequented by humans. Nucleotide sequences were obtained from four HPS case-patients and four yellow pygmy rice rats of the M genome segment. Sequence comparison and phylogenetic analysis showed that rodent-borne viruses and viruses from three HPS case-patients form a well-supported clade and share a 96.4% identity with the previously characterized Central Plata hantavirus. These results suggest that yellow pygmy rice rat (O. flavescens) may be the host for Central Plata, a hantavirus associated with HPS in the southern area of Uruguay.[
The antigenic and genetic diversity of G glycoprotein from 25 human respiratory viruses (group A) isolated during nine consecutive epidemics (1993-2001) in Montevideo, Uruguay, and 7 strains isolated in Buenos Aires, Argentina, in the same period were analyzed. Genetic variability was evaluated by partial sequence of the G protein gene. Phylogenetic analysis indicated that most Uruguayan and Argentinean group A isolates clustered into three genotypes: GA5, GA2, and GA1. Some strains clustered into the GA3 genotype characterized previously. The antigenic analysis was carried out with a panel of anti-G monoclonal antibodies that recognized conserved and strain-specific epitopes. A close correlation between the antigenic and genetic relatedness of the strains analyzed was observed.
Mayaro (MAYV) and Una (UNAV) are emerging arboviruses belonging to the Alphavirus genus of the Togaviridae family. These viruses can produce febrile disease with symptoms such as fever, headache, myalgia, skin rash and incapacitating poly-arthralgia. Serological studies indicate that both viruses are circulating in different countries in Latin America. Viruses need the host cell machinery and resources to replicate effectively. One strategy to find new antivirals consists of identifying key cellular pathways or factors that are essential for virus replication. In this study, we analyzed the role of the ubiquitin-proteasome system (UPS) in MAYV and UNAV replication. Vero-E6 or HeLa cells were treated with the proteasome inhibitors MG132 or Lactacystin, and viral progeny production was quantified using a plaque assay method. In addition, the synthesis of viral proteins was analyzed by Western blot and confocal microscopy. Our results indicate that treatment with proteasome inhibitors decreases MAYV and UNAV protein synthesis, and also causes a significant dose-dependent decrease in MAYV and UNAV replication. Proteasome activity seems to be important at the early stages of MAYV replication. These findings suggest that the ubiquitin-proteasome system is a possible pharmacological target to inhibit these neglected alphaviruses.
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