Within-Host Rhinovirus Evolution in Upper and Lower Respiratory Tract Highlights Capsid Variability and Mutation-Independent Compartmentalization.

BACKGROUND: Rhinovirus (RV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how RV evolves within hosts during infection. METHODS: We sequenced RV complete genomes from 12 hematopoietic cell transplant patients with infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL). Metagenomic and amplicon next-generation sequencing were used to track the emergence and evolution of intrahost single nucleotide variants (iSNVs). RESULTS: Identical RV intrahost populations in matched NW and BAL specimens indicated no genetic adaptation is required for RV to progress from URT to LRT. Coding iSNVs were 2.3-fold more prevalent in capsid over nonstructural genes. iSNVs modeled were significantly more likely to be found in capsid surface residues, but were not preferentially located in known RV-neutralizing antibody epitopes. Newly emergent, genotype-matched iSNV haplotypes from immunocompromised individuals in 2008-2010 could be detected in Seattle-area community RV sequences in 2020-2021. CONCLUSIONS: RV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, RV sequences.

The Lambda Variant in Argentina: Analyzing the Evolution and Spread of SARS-CoV-2 Lineage C.37.

The second wave of COVID-19 occurred in South America in early 2021 and was mainly driven by Gamma and Lambda variants. In this study, we aimed to describe the emergence and local genomic diversity of the SARS-CoV-2 Lambda variant in Argentina, from its initial entry into the country until its detection ceased. Molecular surveillance was conducted on 9356 samples from Argentina between October 2020 and April 2022, and sequencing, phylogenetic, and phylogeographic analyses were performed. Our findings revealed that the Lambda variant was first detected in Argentina in January 2021 and steadily increased in frequency until it peaked in April 2021, with continued detection throughout the year. Phylodynamic analyses showed that at least 18 introductions of the Lambda variant into the country occurred, with nine of them having evidence of onward local transmission. The spatial--temporal reconstruction showed that Argentine clades were associated with Lambda sequences from Latin America and suggested an initial diversification in the Metropolitan Area of Buenos Aires before spreading to other regions in Argentina. Genetic analyses of genome sequences allowed us to describe the mutational patterns of the Argentine Lambda sequences and detect the emergence of rare mutations in an immunocompromised patient. Our study highlights the importance of genomic surveillance in identifying the introduction and geographical distribution of the SARS-CoV-2 Lambda variant, as well as in monitoring the emergence of mutations that could be involved in the evolutionary leaps that characterize variants of concern.

Within-host rhinovirus evolution in upper and lower respiratory tract highlights capsid variability and mutation-independent compartmentalization.

Background: Human rhinovirus (HRV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how HRV evolves within hosts during infection. Methods: We sequenced HRV complete genomes from 12 hematopoietic cell transplant patients with prolonged infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL) specimens. Metagenomic (mNGS) and amplicon-based NGS were used to study the emergence and evolution of intra-host single nucleotide variants (iSNVs). Results: Identical HRV intra-host populations in matched NW and BAL specimens indicated no genetic adaptation is required for HRV to progress from URT to LRT. Microbial composition between matched NW and BAL confirmed no cross-contamination during sampling procedure. Coding iSNVs were 2.3-fold more prevalent in capsid over non-structural genes, adjusted for length. iSNVs modeled onto HRV capsid structures were significantly more likely to be found in surface residues, but were not preferentially located in known HRV neutralizing antibody epitopes. Newly emergent, serotype-matched iSNV haplotypes from immunocompromised individuals from 2008-2010 could be detected in Seattle-area community HRV sequences from 2020-2021. Conclusion: HRV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, HRV sequences.

Genomic Characterization of Respiratory Syncytial Virus during 2022-23 Outbreak, Washington, USA.

We sequenced 54 respiratory syncytial virus (RSV) genomes collected during 2021-22 and 2022-23 outbreaks in Washington, USA, to determine the origin of increased RSV cases. Detected RSV strains have been spreading for >10 years, suggesting a role for diminished population immunity from low RSV exposure during the COVID-19 pandemic.

Evolutionary dynamics of respiratory syncytial virus in Buenos Aires: Viral diversity, migration, and subgroup replacement.

Globally, the human respiratory syncytial virus (RSV) is one of the major causes of lower respiratory tract infections (LRTIs) in children. The scarcity of complete genome data limits our understanding of RSV spatiotemporal distribution, evolution, and viral variant emergence. Nasopharyngeal samples collected from hospitalized pediatric patients from Buenos Aires tested positive for RSV LRTI during four consecutive outbreaks (2014-2017) were randomly subsampled for RSV complete genome sequencing. Phylodynamic studies and viral population characterization of genomic variability, diversity, and migration of viruses to and from Argentina during the study period were performed. Our sequencing effort resulted in one of the largest collections of RSV genomes from a given location (141 RSV-A and 135 RSV-B) published so far. RSV-B was dominant during the 2014-2016 outbreaks (60 per cent of cases) but was abruptly replaced by RSV-A in 2017, with RSV-A accounting for 90 per cent of sequenced samples. A significant decrease in RSV genomic diversity-represented by both a reduction in genetic lineages detected and the predominance of viral variants defined by signature amino acids-was observed in Buenos Aires in 2016, the year prior to the RSV subgroup predominance replacement. Multiple introductions to Buenos Aires were detected, some with persistent detection over seasons, and also, RSV was observed to migrate from Buenos Aires to other countries. Our results suggest that the decrease in viral diversity may have allowed the dramatic predominance switch from RSV-B to RSV-A in 2017. The immune pressure generated against circulating viruses with limited diversity during a given outbreak may have created a fertile ground for an antigenically divergent RSV variant to be introduced and successfully spread in the subsequent outbreak. Overall, our RSV genomic analysis of intra- and inter-outbreak diversity provides an opportunity to better understand the epochal evolutionary dynamics of RSV.

Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

Assessing the hidden diversity underlying consensus sequences of SARS-CoV-2 using VICOS, a novel bioinformatic pipeline for identification of mixed viral populations.

INTRODUCTION: Coinfection with two SARS-CoV-2 viruses is still a very understudied phenomenon. Although next generation sequencing methods are very sensitive to detect heterogeneous viral populations in a sample, there is no standardized method for their characterization, so their clinical and epidemiological importance is unknown. MATERIAL AND METHODS: We developed VICOS (Viral COinfection Surveillance), a new bioinformatic algorithm for variant calling, filtering and statistical analysis to identify samples suspected of being mixed SARS-CoV-2 populations from a large dataset in the framework of a community genomic surveillance. VICOS was used to detect SARS-CoV-2 coinfections in a dataset of 1,097 complete genomes collected between March 2020 and August 2021 in Argentina. RESULTS: We detected 23 cases (2%) of SARS-CoV-2 coinfections. Detailed study of VICOS's results together with additional phylogenetic analysis revealed 3 cases of coinfections by two viruses of the same lineage, 2 cases by viruses of different genetic lineages, 13 were compatible with both coinfection and intra-host evolution, and 5 cases were likely a product of laboratory contamination. DISCUSSION: Intra-sample viral diversity provides important information to understand the transmission dynamics of SARS-CoV-2. Advanced bioinformatics tools, such as VICOS, are a necessary resource to help unveil the hidden diversity of SARS-CoV-2.

Cost-Effective Method to Perform SARS-CoV-2 Variant Surveillance: Detection of Alpha, Gamma, Lambda, Delta, Epsilon, and Zeta in Argentina.

SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.

Virus sincicial respiratorio en niños nacidos prematuros: 19 años de vigilancia epidemiológica activa en un hospital pediátrico / Respiratory syncytial virus in preterm infants: 19 years of active epidemiological surveillance in a children's hospital

Introducción. El virus sincicial respiratorio (VSR) es el principal agente causal de la infección respiratoria aguda baja (IRAB) en pediatría. Los niños prematuros tienen mayor riesgo de complicaciones asociadas con esta infección. Los objetivos fueron describir y comparar las características clínicas y epidemiológicas asociadas a IRAB por VSR en niños/as nacidos pretérmino y a término, y establecer predictores de letalidad en los prematuros.Métodos. Estudio prospectivo, transversal, de pacientes ingresados por IRAB, en el período 2000-2018. El diagnóstico virológico se realizó mediante inmunofluorescencia indirecta o reacción en cadena de la polimerasa con transcriptasa inversa de aspirados nasofaríngeos. Se registraron las características clínico-epidemiológicas. Se desarrolló un modelo de regresión logística múltiple para establecer los predictores de letalidad en prematuros.Resultados. Se incluyeron 16 018 casos de IRAB; 13 545 (el 84,6 %) fueron estudiados; 6047 (el 45 %) positivos; VSR predominó en el 81,1 % (4907); mostró un patrón epidémico estacional; el 14 % (686) fueron prematuros.Los prematuros mostraron mayor frecuencia de comorbilidades, antecedentes respiratorios perinatales, cardiopatía congénita, desnutrición, enfermedad respiratoria crónica, displasia broncopulmonar, hospitalización previa por IRAB y enfermedad neurológica crónica (p < 0,001); requirieron más cuidados intensivos, mayor tiempo de internación y mayor tasa de letalidad (p < 0,01). La cardiopatía congénita fue predictor independiente de letalidad por VSR en prematuros [OR 3,67 (1,25-10,8), p = 0,01].Conclusión. VSR mostró un patrón epidémico, afectó a prematuros con ciertas comorbilidades con mayor morbimortalidad que los de término. La letalidad por VSR en prematuros se asoció con la cardiopatía congénita.

Introduction. Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection (ALRTI) in pediatrics. Preterm infants are at a higher risk for complications. We aimed to describe and compare the clinical and epidemiological characteristics associated with ALRTI due to RSV in preterm and term infants and to establish the predictors of fatality among preterm infants.Methods. Prospective, cross-sectional study of patients admitted due to ALRTI in the 2000-2018 period. Viral diagnosis was done by indirect immunofluorescence or reverse transcription polymerase chain reaction in nasopharyngeal aspirates. Clinical and epidemiological characteristics were recorded. A multiple logistic regression model established the predictors of fatality among preterm infants.Results. A total of 16 018 ALRTI cases were included; 13 545 (84.6 %) were tested; 6047 (45 %) were positive; RSV was prevalent in 81.1 % (4907), with a seasonal epidemic pattern; 14 % (686) were preterm infants.Comorbidities, perinatal respiratory history, congenital heart disease, malnutrition, chronic respiratory disease, bronchopulmonary dysplasia, prior hospitalization due to ALRTI, and chronic neurological disease (p < 0.001) were more common among preterm infants; they required more intensive care and a longer length of stay, and had a higher fatality rate (p < 0.01). Congenital heart disease was an independent predictor of fatality due to RSV among preterm infants (OR: 3.67 [1.25-10.8], p = 0.01).Conclusion. RSV showed an epidemic pattern and affected more preterm infants with certain comorbidities, with a higher morbidity and mortality, compared to term infants. RSV fatality among preterm infants was associated with congenital heart disease.

Respiratory syncytial virus in preterm infants: 19 years of active epidemiological surveillance in a children's hospital. / Virus sincicial respiratorio en niños nacidos prematuros: 19 años de vigilancia epidemiológica activa en un hospital pediátrico.

INTRODUCTION: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection (ALRTI) in pediatrics. Preterm infants are at a higher risk for complications. We aimed to describe and compare the clinical and epidemiological characteristics associated with ALRTI due to RSV in preterm and term infants and to establish the predictors of fatality among preterm infants. METHODS: Prospective, cross-sectional study of patients admitted due to ALRTI in the 2000-2018 period. Viral diagnosis was done by indirect immunofluorescence or reverse transcription polymerase chain reaction in nasopharyngeal aspirates. Clinical and epidemiological characteristics were recorded. A multiple logistic regression model established the predictors of fatality among preterm infants. RESULTS: A total of 16 018 ALRTI cases were included; 13 545 (84.6 %) were tested; 6047 (45 %) were positive; RSV was prevalent in 81.1 % (4907), with a seasonal epidemic pattern; 14 % (686) were preterm infants. Comorbidities, perinatal respiratory history, congenital heart disease, malnutrition, chronic respiratory disease, bronchopulmonary dysplasia, prior hospitalization due to ALRTI, and chronic neurological disease (p < 0.001) were more common among preterm infants; they required more intensive care and a longer length of stay, and had a higher fatality rate (p < 0.01). Congenital heart disease was an independent predictor of fatality due to RSV among preterm infants (OR: 3.67 [1.25-10.8], p = 0.01). CONCLUSION: RSV showed an epidemic pattern and affected more preterm infants with certain comorbidities, with a higher morbidity and mortality, compared to term infants. RSV fatality among preterm infants was associated with congenital heart disease.

Introducción. El virus sincicial respiratorio (VSR) es el principal agente causal de la infección respiratoria aguda baja (IRAB) en pediatría. Los niños prematuros tienen mayor riesgo de complicaciones asociadas con esta infección. Los objetivos fueron describir y comparar las características clínicas y epidemiológicas asociadas a IRAB por VSR en niños/as nacidos pretérmino y a término, y establecer predictores de letalidad en los prematuros. Métodos. Estudio prospectivo, transversal, de pacientes ingresados por IRAB, en el período 2000-2018. El diagnóstico virológico se realizó mediante inmunofluorescencia indirecta o reacción en cadena de la polimerasa con transcriptasa inversa de aspirados nasofaríngeos. Se registraron las características clínicoepidemiológicas. Se desarrolló un modelo de regresión logística múltiple para establecer los predictores de letalidad en prematuros. Resultados. Se incluyeron 16 018 casos de IRAB; 13 545 (el 84,6 %) fueron estudiados; 6047 (el 45 %) positivos; VSR predominó en el 81,1 % (4907); mostró un patrón epidémico estacional; el 14 % (686) fueron prematuros. Los prematuros mostraron mayor frecuencia de comorbilidades, antecedentes respiratorios perinatales, cardiopatía congénita, desnutrición, enfermedad respiratoria crónica, displasia broncopulmonar, hospitalización previa por IRAB y enfermedad neurológica crónica (p < 0,001); requirieron más cuidados intensivos, mayor tiempo de internación y mayor tasa de letalidad (p < 0,01). La cardiopatía congénita fue predictor independiente de letalidad por VSR en prematuros [OR 3,67 (1,25-10,8), p = 0,01]. Conclusión. VSR mostró un patrón epidémico, afectó a prematuros con ciertas comorbilidades con mayor morbimortalidad que los de término. La letalidad por VSR en prematuros se asoció con la cardiopatía congénita.

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.

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.

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.

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 glycoprotein's 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.

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.