RESUMO
BACKGROUND: Human respiratory syncytial virus (RSV) is classified into antigenic subgroups A and B. Thirteen genotypes have been defined for RSV-A and 20 for RSV-B, without any consensus on genotype definition. METHODS: We evaluated clustering of RSV sequences published in GenBank until February 2018 to define genotypes by using maximum likelihood and Bayesian phylogenetic analyses and average p-distances. RESULTS: We compared the patterns of sequence clustering of complete genomes; the three surface glycoproteins genes (SH, G, and F, single and concatenated); the ectodomain and the 2nd hypervariable region of G gene. Although complete genome analysis achieved the best resolution, the F, G, and G-ectodomain phylogenies showed similar topologies with statistical support comparable to complete genome. Based on the widespread geographic representation and large number of available G-ectodomain sequences, this region was chosen as the minimum region suitable for RSV genotyping. A genotype was defined as a monophyletic cluster of sequences with high statistical support (≥80% bootstrap and ≥0.8 posterior probability), with an intragenotype p-distance ≤0.03 for both subgroups and an intergenotype p-distance ≥0.09 for RSV-A and ≥0.05 for RSV-B. In this work, the number of genotypes was reduced from 13 to three for RSV-A (GA1-GA3) and from 20 to seven for RSV-B (GB1-GB7). Within these, two additional levels of classification were defined: subgenotypes and lineages. Signature amino acid substitutions to complement this classification were also identified. CONCLUSIONS: We propose an objective protocol for RSV genotyping suitable for adoption as an international standard to support the global expansion of RSV molecular surveillance.
Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Evolução Molecular , Genoma Viral , Genótipo , Humanos , Filogenia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/isolamento & purificação , Proteínas Virais/genéticaRESUMO
Human metapneumovirus (hMPV) is a newly identified paramixovirus, associated with respiratory illnesses in all age groups. Two genetic groups of hMPV have been described. The nucleotide sequences of the G and F genes from 11 Argentinean hMPV strains (1998-2003) were determined by RT-PCR and direct sequencing. Phylogenetic analysis showed that hMPV strains clustered into two main genetic lineages, A and B. Strains clustered into A group were split into two sublineages, A1 and A2. All strains belonging to group B clustered with representative strains from sublineage B1. No Argentinean strains belonged to sublineage B2. F sequences showed high percentage identities at nucleotide and amino acid levels. In contrast, G sequences showed high diversity between A and B groups. Most changes observed in the deduced G protein sequence were amino acid substitutions in the extracellular domain, and changes in stop codon usage leading to different lengths in the G proteins. High content of serine and threonine residues were also shown, suggesting that this protein would be highly glycosylated. The potential sites for N- and O-glycosylation seem to have a different conservation pattern between the two main groups. This is the first report on the genetic variability of the G and F protein genes of hMPV strains in South America. Two main genetic groups and at least three subgroups were revealed among Argentinean hMPV strains. The F protein seems to be highly conserved, whereas the G protein showed extensive diversity between groups A and B.
Assuntos
Genes Virais/genética , Variação Genética , Glicoproteínas/genética , Metapneumovirus/genética , Infecções por Paramyxoviridae/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Argentina , Pré-Escolar , Sequência Consenso , Humanos , Lactente , Metapneumovirus/classificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Especificidade da EspécieRESUMO
A total of 47 clinical samples were identified during an active surveillance program of respiratory infections in Buenos Aires (BA) (1999 to 2004) that contained sequences of human respiratory syncytial virus (HRSV) with a 60-nucleotide duplication in the attachment (G) protein gene. This duplication was analogous to that previously described for other three viruses also isolated in Buenos Aires in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003). Phylogenetic analysis indicated that BA sequences with that duplication shared a common ancestor (dated about 1998) with other HRSV G sequences reported worldwide after 1999. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but both copies of the duplicated segment accumulated nucleotide substitutions in more recent viruses at a rate apparently higher than in other regions of the G protein gene. The evolution of the viruses with the duplicated G segment apparently followed the overall evolutionary pattern previously described for HRSV, and this genotype has replaced other prevailing antigenic group B genotypes in Buenos Aires and other places. Thus, the duplicated segment represents a natural tag that can be used to track the dissemination and evolution of HRSV in an unprecedented setting. We have taken advantage of this situation to reexamine the molecular epidemiology of HRSV and to explore the natural history of this important human pathogen.
Assuntos
Surtos de Doenças , Glicoproteínas/genética , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sinciciais Respiratórios/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Argentina/epidemiologia , Criança , Variação Genética , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade da Espécie , População UrbanaRESUMO
The intragroup antigenic diversity of the G glycoprotein of 226 human respiratory syncytial virus (HRSV) strains isolated in Buenos Aires (Argentina) and Santiago (Chile) between 1995 and 2002 was evaluated by ELISA with a panel of 14 anti-G monoclonal antibodies (MAbs). Out of 226 strains characterized, 172 (76%) belonged to group A and 54 (24%) to group B. Strains from both groups cocirculated throughout the study period in both countries, except in 1996, 2000, and 2002 when only group A strains were isolated. Within group A 23 different antigenic patterns were found as defined by the combination of reactivities with eight strain-specific anti-G MAbs. These antigenic patterns showed different behavior regarding their circulation. Some major patterns were observed in most years with variable proportions; other minor patterns were present in low proportions during 1 or 2 years and then were apparently replaced by new patterns. Some antigenic patterns occurred both in Argentina and Chile during the same epidemics. Since no strain-specific MAbs were available for group B, we could not evidence the antigenic variability within group B. These are the first data on antigenic characterization of HRSV strains isolated in Argentina and Chile. It is shown that the ELISA with MAbs directed against the G protein of RSV is a valuable tool. These results will also provide useful information for further studies to evaluate the antigenic variability of HRSV strains in relation with genetic characteristics.
Assuntos
Antígenos Virais/genética , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Variação Antigênica , Argentina/epidemiologia , Pré-Escolar , Chile/epidemiologia , Humanos , LactenteRESUMO
The genetic and antigenic variability of human respiratory syncytial virus (HRSV) strains isolated in Buenos Aires from 1995 to 2001 was evaluated by partial nucleotide sequencing of the G gene and enzyme-linked immunosorbent assay analysis with anti-G monoclonal antibodies. Phylogenetic analyses showed that 37 group A strains clustered into five genotypes, whereas 20 group B strains clustered into three genotypes. Group A showed more genetic variability than group B. A close correlation between genotypes and antigenic patterns was observed. Changes detected in the G protein of viruses from both groups included (i) amino acid substitutions and(ii) differences in protein length due to either changes in stop codon usage or sequence duplications. Three B strains from 1999 exhibited a duplication of 20 amino acids, while one B strain from 2001 had 2 amino acids duplicated. The comparison among Argentinean HRSV strains and viruses isolated in other geographical areas during different epidemics is discussed.
Assuntos
Antígenos Virais/sangue , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Sequência de Aminoácidos , Argentina , Sequência de Bases , Linhagem Celular Tumoral , Pré-Escolar , Primers do DNA , Humanos , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Human metapneumovirus (hMPV) is a virus, which was first associated with acute lower respiratory infection in children but is detected currently in all age groups. Clinical symptoms are similar to those described for respiratory syncytial virus (RSV) infections, ranging from mild respiratory illness to severe bronchiolitis and pneumonia in children. To date, no cases of hMPV have been reported in Argentina. In this study, 440 respiratory samples obtained during the period 1998-2002 from children under 5 years old with acute respiratory infection were evaluated. Routine detection for RSV, adenovirus, influenza, and parainfluenza was undertaken by immunofluorescent assay. Of the samples negative for these viruses, only 100 were available. All these samples were tested for hMPV by RT-PCR using primers for the L gene. Eleven out of 100 (11%) respiratory samples were positive for hMPV by RT-PCR. A higher frequency of detection was observed in spring. hMPV was detected in all the years studied, except in 2001. Ten out of 11 children positive for hMPV were hospitalized. Median age was 5 months. Of seven patients, five (71%) required oxygen supplementation. The most frequent diagnosis was bronchiolitis (86%), sometimes accompanied by conjunctivitis and otitis media. The present study showed that hMPV was associated with acute lower respiratory infections in children in Buenos Aires, Argentina. This evidence strongly suggests that hMPV is a common pathogen with a wide geographical distribution, which should be included in the routine diagnosis of respiratory viruses in young children.
Assuntos
Metapneumovirus/isolamento & purificação , Infecções por Paramyxoviridae/epidemiologia , Infecções por Paramyxoviridae/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Argentina/epidemiologia , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Metapneumovirus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do Ano , Proteínas Virais/genéticaRESUMO
The entire nucleotide sequence of the G gene of three human respiratory syncytial virus (HRSV) isolates (antigenic group B) has been determined. These three viruses (named BA viruses) were isolated in Buenos Aires in 1999 from specimens collected in different hospitals and at different dates. BA viruses have an exact duplication of 60 nucleotides in the G gene, starting after residue 791. This duplication is flanked by a repeat of four nucleotides (GUGU) and can fold into a relatively stable secondary structure. These features suggest a possible mechanism for the generation of a duplicated G segment. The predicted polypeptide is lengthened by 20 amino acids (residues 260-279) and this is reflected in the slower electrophoretic mobility of the G protein precursor of BA viruses compared with related viruses. The changes reported here expand the examples of drastic genetic alterations that can be introduced into the G protein sequence of HRSV while it replicates in its natural host.
Assuntos
Duplicação Gênica , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Proteínas Virais/química , Proteínas Virais/imunologia , Replicação ViralRESUMO
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.
Assuntos
Variação Antigênica , Surtos de Doenças , Variação Genética , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Sequência de Aminoácidos , Antígenos Virais , Argentina/epidemiologia , Pré-Escolar , Humanos , Dados de Sequência Molecular , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Análise de Sequência de DNA , Uruguai/epidemiologia , Proteínas Virais/genéticaRESUMO
In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4 per cent of the samples, 11.8 per cent of which were positive by SV and 7.7 per cent by CC. Out of 84 positive samples, concordance between both methods was observed in 36 per cent of the cases. We found that 46 per cent of the samples were positive only by SV, while 18 per cent were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43 per cent were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18 per cent of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity.
Assuntos
Humanos , Citomegalovirus , Infecções por Citomegalovirus , Sensibilidade e Especificidade , Cultura de Vírus , Replicação ViralRESUMO
In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4 per cent of the samples, 11.8 per cent of which were positive by SV and 7.7 per cent by CC. Out of 84 positive samples, concordance between both methods was observed in 36 per cent of the cases. We found that 46 per cent of the samples were positive only by SV, while 18 per cent were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43 per cent were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18 per cent of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity. (Au)