Mariana Viegas

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

ABSTRACT 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.

Wild-type Measles Virus in Brain Tissue of Children with Subacute Sclerosing Panencephalitis, Argentina

We studied eight children who had measles at 6 to 10 months of age during the 1998 Argentine measles outbreak and in whom subacute sclerosing panencephalitis developed 4 years later. We report the genetic characterization of brain tissue–associated measles virus samples from three patients. Phylogenetic relationships clustered these viruses with the wild-type D6 genotype isolated during the 1998 outbreak. The children received measles vaccine; however, vaccinal strains were not found.

Sixteen years of evolution of human respiratory syncytial virus subgroup A in Buenos Aires, Argentina: GA2 the prevalent genotype through the years

Human respiratory syncytial virus (HRSV) is the main viral cause of acute lower respiratory tract infections (LRTI) in children worldwide. In recent years, several preclinical trials with vaccine candidates have been reported. It is in this sense that molecular epidemiological studies become important. Understanding viral dispersion patterns before and after the implementation of a vaccine can provide insight into the effectiveness of the control strategies. In this work we analyzed the molecular epidemiology of HRSV-A over a period of sixteen years (1999-2014) in Buenos Aires. By bioinformatic tools we analyzed 169 sequences of the G glycoprotein gene from hospitalized pediatric patients with LRTI. We found that GA2 was the most prevalent genotype (73.35%). GA5 genotype co-circulated in our region until 2009 when it was no longer detected, except in 2011. The recently globally emerging ON1 lineage with a 72-nt duplication increased its frequency to become the only lineage detected in Buenos Aires in 2014. By discrete phylogeographic analysis of global ON1 strains we could determine that Panama could be the location of the MRCA dated June 20, 2010; and this lineage could be introduced in Argentina from Spain in April 2011. This analysis also showed temporary and geographical clustering of ON1 strains observed as phylogenetic clades with strains exclusively associated from a single country, nevertheless among our 44 ON1 strains from three outbreaks (2012-2014) we could also detect posterior reintroductions and circulation from United States, Cuba, South Korea, and Spain. The continuous phylogeographic analysis of one sublineage of Argentine ON1 strains allowed us to establish that there could be a local clustering of some strains even in neighborhoods. This work shows the potential of this type of bioinformatic tools in the context of a future vaccine surveillance network to trace the spread of new genetic lineages in human populations.

Phylogenetic and molecular analyses of human parainfluenza type 3 virus in Buenos Aires, Argentina, between 2009 and 2013: The emergence of new genetic lineages

Despite that human parainfluenza type 3 viruses (HPIV3) are one of the leading causes of acute lower respiratory tract infections in children under five, there is no licensed vaccine and there is limited current information on the molecular characteristics of regional and global circulating strains. The aim of this study was to describe the molecular characterization of HPIV3 circulating in Buenos Aires. We performed a genetic and phylogenetic analysis of the HN glycoprotein gene. Between 2009 and 2013, 124 HPIV3-positive samples taken from hospitalized pediatric patients were analyzed. Four new genetic lineages were described. Among them, C1c and C3d lineages showed local circulation patterns, whereas C3e and C3f comprised sequences from very distant countries. Despite the diversity of the described genotypes, C3a and C3d predominated over the others, the latter was present during the first years of the study and it was progressively replaced by C3a. Molecular analyses showed 28 non-synonymous substitutions; of these, 13 were located in potentially predicted B-cell epitopes. Taken together, the emergence of genetic lineages and the information of the molecular characteristics of HN protein may contribute to the general knowledge of HPIV3 molecular epidemiology for future vaccine development and antiviral therapies.

Phylogenetic analysis of rubella viruses isolated in 2008 outbreak in Argentina

BACKGROUND A rubella outbreak was recorded in Buenos Aires during 2008. OBJECTIVES The objective of this communication is to present the genetic and phylogenetic analyses of wild-type RUBV circulating in Buenos Aires during the 2008 outbreak. STUDY DESIGN Throat swab samples collected from patients diagnosed with rubella between June 2008 and December 2008 were inoculated in cell culture and 23 isolates were sequenced. RESULTS Phylogenetic analysis of the WHO-recommended window (nt 8731-9469) of the E1 envelope glycoprotein was performed and all isolates clustered with the 2B genotype. CONCLUSIONS Genotype 2B seems to be endemically circulating in the Southern cone of Latin America, thus causing recent outbreaks.

Unravelling respiratory syncytial virus outbreaks in Buenos Aires, Argentina: Molecular basis of the spatio-temporal transmission

Respiratory syncytial virus (RSV) is the main viral cause of hospitalization due to acute lower respiratory tract infections in infants worldwide. Several vaccines against RSV are under research and development, which are about to be approved. We evaluated transmission patterns in different settings to determine age-specific vaccination targets from a viral perspective. We sequenced the G glycoproteins ectodomain of a constant clinical sampling between two epidemic outbreaks in a limited geographical region and performed phylogeographic analyses. We described a spatio-temporal transmission between local strains, which were originated in the center of the analyzed area and then spread to others. Interestingly, that central area reported the highest population density of the region and also showed overcrowding. This information should be considered by public health systems to evaluate vaccination at all ages in those areas to decrease viral transmission and in lower density populations only susceptible children should be vaccinated.

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.

Influenza virus: 16 years’ experience of clinical epidemiologic patterns and associated infection factors in hospitalized children in Argentina

Background Influenza is an important cause of acute lower respiratory tract infection (aLRTI), hospitalization, and mortality in children. This study aimed to describe the clinical and epidemiologic patterns and infection factors associated with influenza, and compare case features of influenza A and B. Methods In a prospective, cross-sectional study, patients admitted for aLRTI, between 2000 and 2015, were tested for respiratory syncytial virus, adenovirus, influenza, or parainfluenza, and confirmed by fluorescent antibody (FA) or real-time polymerase chain reaction (RT-PCR) assay of nasopharyngeal aspirates. Results Of 14,044 patients, 37.7% (5290) had FA- or RT-PCR-confirmed samples that identified influenza in 2.8% (394/14,044; 91.4% [360] influenza A, 8.6% [34] influenza B) of cases. Influenza frequency followed a seasonal epidemic pattern (May–July, the lowest average temperature months). The median age of cases was 12 months (interquartile range: 6–21 months); 56.1% (221/394) of cases were male. Consolidated pneumonia was the most frequent clinical presentation (56.9%; 224/394). Roughly half (49.7%; 196/394) of all cases had previous respiratory admissions; 9.4% (37/394) were re-admissions; 61.5% (241/392) had comorbidities; 26.2% (102/389) had complications; 7.8% (30/384) had nosocomial infections. The average case fatality rate was 2.1% (8/389). Chronic neurologic disease was significantly higher in influenza B cases compared to influenza A cases (p = 0.030). The independent predictors for influenza were: age ≥6 months, odds ratio (OR): 1.88 (95% confidence interval [CI]: 1.44–2.45); p<0.001; presence of chronic neurologic disease, OR: 1.48 (95% CI: 1.01–2.17); p = 0.041; previous respiratory admissions, OR: 1.71 (95% CI: 1.36–2.14); p<0.001; re-admissions, OR: 1.71 (95% CI: 1.17–2.51); p = 0.006; clinical pneumonia, OR: 1.50 (95% CI: 1.21–1.87); p<0.001; immunodeficiency, OR: 1.87 (95% CI: 1.15–3.05); p = 0.011; cystic fibrosis, OR: 4.42 (95% CI: 1.29–15.14); p = 0.018. Conclusion Influenza showed an epidemic seasonal pattern (May–July), with higher risk in children ≥6 months, or with pneumonia, previous respiratory admissions, or certain comorbidities.

Dengue Virus 1 Outbreak in Buenos Aires, Argentina, 2016

The largest outbreak of dengue in Buenos Aires, Argentina, occurred during 2016. Phylogenetic, phylodynamic, and phylogeographic analyses of 82 samples from dengue patients revealed co-circulation of 2 genotype V dengue virus lineages, suggesting that this virus has become endemic to the Buenos Aires metropolitan area.

Respiratory Syncytial Virus. Clinical Epidemiological Pattern and lethality associated factors in Children Admitted in a Pediatric Hospital: 15 years’ experience

Background: Respiratory Syncytial Virus (RSV) is the main agent that causes Acute Lower Respiratory Tract Infection (ALRI) in children. Objective: to describe the clinical and epidemiological pattern and the lethality factors associated to RSV infection. Methods: Prospective, cross sectional study of patients admitted for ALRI at “R.Gutierrez” Children Hospital, 2000-2014. Virological diagnosis of respiratory virus: RSV, adenovirus (AV), influenza (IF) and parainfluenza (PIV) was made by fluorescent antibody assay of nasopharyngeal aspirates or real time-PCR (IF). Results: from a total of 13.309 patients included, 38.6%(5118) had positive samples; RSV was predominant all through the study period 81% (4146/5118) without significant annual variations (71-88). It shows a seasonal epidemic pattern (median of epidemiological weeks of viral activity onset and offset:1935) and agrees with the lowest average temperature months. RSV was followed by IF: 7.4%, PIF:6% and AV:4.5%. From 5118 RSV cases the median of age was 7 months (0-216 months), 74%<1 year, 43%<6 months, 20%<3 months; 56.5% males; most frequent clinical feature was bronchiolitis 60.6%; 27% recorded previous admissions for respiratory causes. Comorbidity was found in 41.5% (1717/4146) being the most frequents: recurrent obstructive bronchitis (74%) and congenital heart disease (14%). Complications were detected in 25% of cases, 6.5% had nosocomial infections. Lethality was 1.9 %(78/4108). From 78 RSV fatal cases the median of age of was 5 months (0-180 months), 27%<3months; most frequent clinical feature was pneumonia 53%. Comorbidity was found in 65.4% (51/78) being the most frequents: recurrent obstructive bronchitis 50.9% (26/51) and congenital heart disease 33%(17/51).Moderate to severe malnourishment OR 2.28 (1.19-4.36) p<0.01, congenital cardiopathy 3.53 (1.95-6.39) p<0.01 and the presence of chronic neurological disease OR 3.25 (1.65-6.39) p<0.01 were the independent predictors for VSR lethality. Conclusion: RSV showed an epidemic pattern (May-July) and affected mostly young children. RSV lethality was more associated with malnourishment, congenital cardiopathy and the presence of chronic neurological disease. BACKGROUND AND AIMS • In Argentina, respiratory infections are the third cause of death in children under five years old. • Respiratory Syncytial Virus (RSV) is the main agent that causes Acute Lower Respiratory Tract Infection (ALRI) in children. • The aim of this study was to describe the clinical and epidemiological pattern and the lethality factors associated to RSV infection. MATERIAL AND METHODS • Study design: Prospective, cross sectional study including patients admitted for ALRI at “R.Gutierrez” Children Hospital, 2000-2014. • Active epidemiological surveillance with a specific case report form. • Inclusion criteria: all patients admitted for ALRI. • Virological diagnosis of respiratory viruses: RSV, adenovirus (AV), influenza (IF) and parainfluenza (PIV) was made by fluorescent antibody assay of nasopharyngeal aspirates or real time-PCR (IF). • Genotypes: G-glycoprotein gene ectodomain was amplified by RT-PCR and Sanger sequenced in an ABI3500 (Applied Biosystems). • Statistical Methods: Epiinfo 7 was used for data analysis. Risk factors were calculated using Odds Ratio (OR) as a measure of association with 95% confidence interval (CI); for multivaried analysis we used SPSS v.15.0 and a probability of less than 0.05 was considered significant. CONCLUSIONS • RSV showed an epidemic pattern (May-July) and affected mostly young children. • RSV lethality was more associated with malnourishment, congenital heart disease and the presence of chronic neurological disease. 546 angelagentile21@gmail.com Negative samples Positive samples RSV 81% (4146) Influenza 7.4% (378) 61,4% (8191) 38.6% (5118) n=13.309 PIV 6% (307) AV 4.5% (230) RESULTS • RSV was predominant all through the study period 81% (4146/5118) without significant annual variations (71-88). • It showed a seasonal epidemic pattern (median of epidemiological weeks of viral activity onset and offset:19-35) RSV Seasonal Pattern. 2000-2014 Population • RSV incidence remained high throughout the winter until spring. • The peaks were observed between epidemiological weeks (EW) 25-30 (June) except the years 2010 and 2012 that showed earlier peaks from 18 EW (May), coinciding with the lowest average temperature months every year. Annual Distribution of respiratory viruses (RSV, Adenovirus, Influenza, Parainfluenza) 2000-2014 (n=4146) n % Gender (male) 2342 56,5 Median of age= 7 months old (0-216 months) < 3 months 837 20.2 < 6 months 1774 42,9 < 12 months 3057 73,9 Prematurity 569 13,7 Neonatal respiratory pathology 472 11,4 Immunosuppressed 79 1,9 Malnourished 205 5 Previous admissions for respiratory causes 1101 26,6 Comorbidities (41,5%) n= 1717 Recurrent obstructive bronchitis 1270 74 Congenital heart disease 258 15 Chronic neurological disease 174 10,1 Complications (24%) n=981 Media acute otitis 307 31.3 Respiratory distress 323 32,9 Atelectasis 177 18 Sepsis 146 14,8 RSV cases Population Features RSV fatal cases Population Features (n=78) n % Gender (male) 39 50 Median of age= 5 months (1-180 months) < 3 months 27 34,6 < 6 months 40 51,3 < 12 months 60 76,9 Prematurity 19 24,4 Neonatal respiratory pathology 20 25,6 Immunosuppressed 2 2,6 Malnourished 16 20,5 Previous admissions for respiratory causes 34 43,6 Comorbidities (65,4%) n= 51 Recurrent obstructive bronchitis 26 50,9 Congenital heart disease 17 33,3 Chronic neurological disease 10 19,6 Complications (94,9%) n=74 Respiratory distress 64 82,1 Sepsis 26 33.3 Atelectasis 11 14,1 The most frequent clinical presentation was bronchiolitis (60.6%) Nosocomial infections rate: 6.5% (258/3986) Lethality rate 1.9% (78/4108). The most frequent clinical presentation was Pneumonia (53%) Nosocomial infections rate: 48,1% (37/77) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 200