Evolución de virus respiratorios sincitiales humanos que tienen una duplicación de 60 nucleótidos en el gen de la proteína G

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-07-0

Toward unified molecular surveillance of RSV: A proposal for genotype definition

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.Fil: Goya, Stephanie. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galiano, Mónica. Public Health England; Reino UnidoFil: Nauwelaers, Inne. Imperial College London; Reino UnidoFil: Trento, Alfonsina. Instituto de Investigación Hospital 12 de Octubre; EspañaFil: Openshaw, Peter J.. Imperial College London; Reino UnidoFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Zambon, Maria. Public Health England; Reino UnidoFil: Viegas, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.This work was supported in part by grants GR09/0023 (A. N.), GR09/0039 (J. A. M.) and GR09/0040 (I. C.) from Instituto de Salud Carlos III under a special research programme on pandemic flu. Additionally, the Biología Viral Unit is supported currently by grant SAF2012-31217 from Plan Nacional I+D+i.S

Human respiratory syncytial virus infects and induces activation markers in mouse B lymphocytes.

Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. Although human airway epithelial cells are the main target of HRSV, it has been reported that this virus can also infect professional antigen-presenting cells such as macrophages and dendritic cells, promoting upregulation of maturation markers. Here, we report that mouse spleen B220(+) B lymphocytes were susceptible to HRSV infection in vitro, probably involving a glycosaminoglycan-dependent mechanism. In contrast, neither CD4(+) nor CD8(+) T lymphocytes were infected. In B lymphocytes, HRSV infection upregulated major histocompatibility complex (MHC) class II but not MHC class I molecules and induced the expression of the activation marker CD86.Dr Mark E Peeples (Department of Immunology/Microbiology, Rush‐Presbyterian‐St Luke's Medical Center, Chicago, IL, USA) kindly provided the rgHRSV. Technical assistance of Carmen Mir is gratefully acknowledged. This study was supported by grants from Programa Ramón y Cajal and Fondo de Investigaciones Sanitarias de la Seguridad Social to DL; by grant SAF2006‐07805 from Ministerio de Educación y Ciencia to JAM; by grants from Comunidad de Madrid and SAF‐2004‐00534 from Ministerio de Educación y Ciencia to MDV; and by a joint grant from Instituto de Salud Carlos III to DL, JAM and MDV.S

TLR4-independent upregulation of activation markers in mouse B lymphocytes infected by HRSV.

Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. In addition, HRSV poses a serious health risk in immunocompromised individuals and the elderly. It has been reported that this virus can infect mouse antigen-presenting cells, including B lymphocytes. In these B cells, HRSV infection upregulates the expression of activation markers, including MHC class II and CD86, but not MHC class I molecules. Here, we report that HRSV infection of spleen B lymphocytes downregulated TLR4. Either blocking with anti-TLR4 antibody or genetic deletion, but not functional deficiency of TLR4, moderately reduced the infectivity of HRSV in B lymphocytes. HRSV-infected B lymphocytes with deleted TLR4 upregulated MHC class II and CD86 molecules to the same levels as TLR4(+) wild type B cells. Since the activation of monocytes and macrophages by HRSV was previously reported to depend on TLR4, the current study indicates that these cells and B lymphocytes respond to HRSV infection with different activation pathways.Dr. Mark E. Peeples (Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, USA) kindly provided the rgHRSV. Technical assistance of Carmen Mir is gratefully acknowledged. This work was supported by grants from Programa Ramón y Cajal, and Fondo de Investigaciones Sanitarias de la Seguridad Social to D. L.; by grant SAF2006-07805 from Ministerio de Educación y Ciencia to J. A. M.; by grants from Comunidad de Madrid and SAF-2004-00534 from Ministerio de Educación y Ciencia to M. D. V.; and by a joint grant from Instituto de Salud Carlos III to D. L., J. A. M. and M. D. V.S

The Complexity of Antibody Responses Elicited against the Respiratory Syncytial Virus Glycoproteins in Hospitalized Children Younger than 2 Years

The influence of age and maternal antibodies on the antibody responses to human respiratory syncytial virus (hRSV) glycoproteins in very young children has been a matter of controversy. Both, immaturity of the immune system at very early age and suppression of the host immune response by high level of maternal antibodies have been claimed to limit the host antibody response to virus infection and to jeopardize the use of hRSV vaccines under development in that age group. Hence, the antibody responses to the two major hRSV glycoproteins (F and G) were evaluated in children younger than 2 years, hospitalized with laboratory confirmed hRSV bronchiolitis. A strong negative correlation was found between the titre of circulating ELISA antibodies directed against either prefusion or postfusion F in the acute phase, but not age, and their fold change at convalescence. These changes correlated also with the level of circulating neutralizing antibodies in sera. As reported in adults, most neutralizing antibodies in a subset of tested sera could not be depleted with postfusion F, suggesting that they were mostly directed against prefusion-specific epitopes. In contrast, a weak negative association was found for group-specific anti-G antibodies in the acute phase and their fold change at convalescence only after correcting for the antigenic group of the infecting virus. In addition, large discrepancies were observed in some individuals between the antibody responses specific for F and G glycoproteins. These results illustrate the complexity of the anti-hRSV antibody responses in children experiencing a primary severe infection and the influence of preexisting maternal antibodies on the host response, factors that should influence hRSV serological studies as well as vaccine development

Natural History of Human Respiratory Syncytial Virus Inferred from Phylogenetic Analysis of the Attachment (G) Glycoprotein with a 60-Nucleotide Duplication

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

Toward unified molecular surveillance of RSV: A proposal for genotype definition

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.Fil: Goya, Stephanie. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galiano, Mónica. Public Health England; Reino UnidoFil: Nauwelaers, Inne. Imperial College London; Reino UnidoFil: Trento, Alfonsina. Instituto de Investigación Hospital 12 de Octubre; EspañaFil: Openshaw, Peter J.. Imperial College London; Reino UnidoFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Zambon, Maria. Public Health England; Reino UnidoFil: Viegas, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

PPARγ2 Pro12Ala polymorphism was associated with favorable cardiometabolic risk profile in HIV/HCV coinfected patients: a cross-sectional study

Background Peroxisome proliferator-activated receptor gamma-2 gene (PPARγ2) rs1801282 (Pro12Ala) polymorphism has been associated with lower risk of metabolic disturbance and atherosclerosis. The aim of this study was to analyze the association between the Pro12Ala polymorphism and cardiometabolic risk factors in human immunodeficiency virus (HIV)/Hepatitis C virus (HCV)-coinfected patients. Methods We carried out a cross-sectional study on 257 HIV/HCV coinfected patients. PPARγ2 polymorphism was genotyped by GoldenGate® assay. The main outcome measures were: i) serum lipids (cholesterol, triglycerides, high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), LDL-C/HDL-C, and atherogenic index (AI)); ii) homeostatic model assessment (HOMA-IR) values; iii) serum adipokines (leptin, adiponectin, resistin, plasminogen activator inhibitor-1(PAI-1), hepatic growth factor (HGF), and nerve growth factor (NGF)). Generalized Linear Models (GLM) with gamma distribution (log-link) were used to investigate the association between PPARγ2 polymorphism and continuous outcome variables. This test gives the differences between groups and the arithmetic mean ratio (AMR) in continuous outcome variables between groups. Results The rs1801282 CG/GG genotype was associated with low values of cholesterol (adjusted arithmetic mean ratio (aAMR) = 0.87 (95% of confidence interval (95% CI) = 0.79; 0.96); p = 0.004) and LDL-C (aAMR = 0.79 (95% CI = 0.68; 0.93); p = 0.004). Furthermore, rs1801282 CG/GG was associated with low values of HOMA-IR (aAMR = 0.69 (95% CI = 0.49; 0.98); p = 0.038) among patients with significant liver fibrosis (F ≥ 2). Moreover, rs1801282 CG/GG was also associated with low serum values of hepatic growth factor (HGF) (aAMR = 0.61 (95% CI = 0.39; 0.94); p = 0.028), and nerve growth factor (NGF) (aAMR = 0.47 (95% CI = 0.26; 0.84); p = 0.010). The serum levels of leptin, adiponectin, resistin, and PAI-1 did not show significant differences. Conclusions The presence of PPARγ2 rs1801282 G allele (Ala variant) was associated with a protective cardiometabolic risk profile versus CC genotype in HIV/HCV-coinfected patients. Thus, PPARγ2 rs1801282 polymorphism may play a significant role in the development of metabolic disorders in HIV/HCV coinfected patients, and might have an influence on the cardiovascular risk.Funding/Support: This work has been supported by grants given by Fondo de Investigación de Sanidad en España (FIS) [Spanish Health Founds for Research] [grant numbers PI08/0738, PI11/00245; PI08/0928, and PI11/01556], and “Fundación para la Investigación y la Prevención del Sida en España” (FIPSE) [grant number 361020/10]. This work has been (partially) funded by the RD12/0017/0024 and RD12/0017/0004 projects as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). JB is an investigator from the Programa de Intensificación de la Actividad Investigadora en el Sistema Nacional de Salud (I3SNS), Refs INT10/009 and INT12/154. PGB, AFR, DPT, MAJS and MGA are supported by “Instituto de Salud Carlos III” [grant numbers FI12/00036, UIPY-1377/08, CM12/00043, CD13/00013 and CD12/00442, respectively].S

FTOrs9939609 polymorphism is associated with metabolic disturbances and response to HCV therapy in HIV/HCV-coinfected patients

BACKGROUND: The Fat Mass and Obesity-Associated Protein (FTO) gene rs9939609 single nucleotide polymorphism (SNP) has been associated with obesity, metabolic syndrome, insulin resistance (IR), and type 2 diabetes mellitus in the general population. The aim of our study was to examine for the first time the association of the rs9939609 polymorphism with metabolic disturbances, liver disease and virologic response to hepatitis C virus (HCV) therapy with pegylated-interferon-alpha plus ribavirin (pegIFNα/RBV) in human immunodeficiency virus (HIV)/HCV coinfected patients. METHODS: We carried out a cross-sectional study in 261 patients, of whom 178 were subsequently treated with pegIFNα/RBV therapy. FTO rs9939609 and IFNL3 rs12980275 polymorphisms were genotyped by GoldenGate®. The main outcomes were: 1) metabolic disturbances: insulin resistance (homeostatic model assessment (HOMA-IR)) and overweight (body mass index (BMI)); 2) liver disease (Metavir score): significant fibrosis (F ≥2) and steatosis (>10% fatty hepatocytes); and 3) virologic response to HCV treatment: sustained virologic response (SVR). RESULTS: The rs9939609 AA genotype was associated with higher values of BMI (adjusted arithmetic mean ratio (aAMR) = 1.08; 95% confidence interval (95%CI) = 1.03 to 1.14; P = 0.002) and HOMA-IR (aAMR = 1.32; 95%CI = 1.03 to 1.69; P = 0.027). Patients with an rs9939609 AA genotype had higher likelihoods of achieving values of BMI ≥27.5 kg/m2 (adjusted odds ratio (aOR) = 3.46; 95%CI =1.17 to 10.21; P = 0.024), HOMA-IR ≥2.5 (aOR = 2.09; 95%CI = 1.02 to 4.32; P = 0.045), significant fibrosis (aOR = 2.34; 95%CI =1.02 to 5.36; P = 0.045) and steatosis (aOR = 3.65; 95%CI = 1.29 to 10.36; P = 0.015). The rs9939609 AT/AA genotype decreased the likelihood of achieving SVR (aOR = 0.58; 95%CI = 0.34 to 0.99; P = 0.044). A decision tree was performed with the genotypes of HCV, IFNL3 and FTO. The incorporation of rs9939609 significantly improves the prediction of SVR (P <0.05). The overall accuracy was 68.2%. CONCLUSIONS: Patients carrying the unfavourable AT/AA genotype of rs9939609 polymorphism had higher odds of metabolic disturbances and a lower likelihood of achieving successful virologic response to HCV therapy.This work has been supported by grants given by Fondo de Investigación de Sanidad en España (FIS) [Spanish Health Founds for Research] [grant numbers PI08/0738, PI11/00245; PI08/0928, and PI11/01556], and “Fundación para la Investigación y la Prevención del Sida en España” (FIPSE) [grant number 361020/10]. This work has been (partially) funded by the RD12/0017/0024 and RD12/0017/0004 projects as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). JB is an investigator from the Programa de Intensificación de la Actividad Investigadora en el Sistema Nacional de Salud (I3SNS), Refs INT10/009 and INT12/154. PGB, DPT, MGF, MAJS and MGA are supported by “Instituto de Salud Carlos III” [grant numbers FI12/00036, CM12/00043, RD12/0017/0024, CD13/00012and CD12/00442, respectively].S