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.Fil: Goya, Stephanie. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tittarelli, Estefanía. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rojo, Gabriel Lihue. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Nabaes Jodar, Mercedes Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Greninger, Alexander L.. University of Washington; Estados UnidosFil: Zaiat, Jonathan Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Viegas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentin

Emergence of intratreatment resistance to oseltamivir in pandemic influenza A H1N1 2009 virus

Background: Pandemic influenza A H1N1 2009 virus presents a new challenge to health authorities and communities worldwide. In Argentina, the outbreak was at its peak by the end of June 2009, during the southern winter. A systematic analysis of samples from patients with pandemic H1N1 2009 studied in our laboratory (Virology Laboratory, Hospital de Niños R Gutiérrez, Buenos Aires, Argentina) detected two patients presenting intratreatment emergence of the H275Y neuraminidase mutation, which confers resistance to oseltamivir. Methods: Complementary DNAs, including the 275 codon, were obtained by reverse transcriptase PCR using viral RNAs extracted from nasopharyngeal or tracheal aspirates. Conventional sequencing and pyrosequencing were performed on each sample. In order to measure the virus susceptibility to oseltamivir, 50% inhibitory concentration determinations were performed by chemiluminescence. Results: Sequential samples of two paediatric patients under oseltamivir treatment were analysed. Pretreatment samples were composed of 100% oseltamivir-sensitive variants. In case 1, the oseltamivir-resistant variant was found 8 days after the beginning of treatment. In case 2, the viral population became resistant on the second day of treatment, with 83% of the viral population bearing the mutation and this reached 100% on the seventh day. Conclusions: We describe the intratreatment emergence of oseltamivir resistance in two paediatric patients. Pyrosequencing allowed us to detect variant mixtures, showing the transition of the viral population from sensitive to resistant.Fil: Valinotto, Laura Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Diez, Roberto Alejandro. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Biosidus S. A.; ArgentinaFil: Barrero, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Farías, Julio A.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: López, Eduardo L.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin

Seroprevalence of SARS-CoV-2 among Workers in Northern Italy

Background: The spread of severe acute respiratory coronavirus 2 (SARS-CoV-2) among active workers is poor known. The aim of our study was to evaluate the seroprevalence of immunoglobulin G (IgG) among a convenience sample of workers and to identify high-risk job sectors during the first pandemic way. Methods: We conducted a cross-sectional study among workers tested for SARS-CoV-2 between 28 March and 7 August 2020, recorded by a private healthcare center located in North-West Italy. Association among seroprevalence and demographic and occupational variables was evaluated using chi square test and the seroprevalence and 95% confidence intervals (CI) were calculated. Results: We collected the results for 23568 serological tests from a sample of 22708 workers from about 1000 companies. Median age was 45 years and about 60% of subjects were male. The overall seroprevalence was 4.97% [95%CI 4.69-5.25]. No statistical difference was found among gender while seroprevalence was associated with subjects' age, geographical location, and occupational sector. Significantly higher values of positivity were observed for the logistics sector (31.3%), weaving factory (12.6%), nursing homes (9.8%), and chemical industry (6.9%) workers. However, we observed some clusters of cases in single companies independently from the sector. Then, a detailed focus on 940 food workers shown a seroprevalence of 5.21% [95%CI 3.79-6.63] and subjects who self-reported COVID-19 symptoms and who worked during lockdown had a higher probability of being infected (p < 0.001). Conclusions: Data obtained might be useful for future public health decision; more than occupation sector, it seems that failure on prevention system in single companies increase the SARS-CoV-2 transmission

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

Ichthyosis follicularis, atrichia and photophobia (IFAP) and hereditary mucoepithelial dysplasia: Two syndromes that share a common clinical spectrum

Ichthyosis follicularis, atrichia and photophobia syndrome (IFAP) is an X-linked inherited disease caused by pathogenic variants in the gene encoding the membrane-bound transcription factor peptidase, site 2 (MBTPS2). Clinical presentation includes ichthyosis follicularis, alopecia, photophobia and developmental delay. Hereditary mucoepithelial dysplasia (HMD) is a dominantly inherited disease characterized by keratitis, non-scarring alopecia, skin lesions including follicular keratosis, perineal erythema, and mucosal involvement. Recently, variants in SREBF1, a gene coding for a transcription factor related to cholesterol and fatty acid synthesis, have been associated with the disease. These two syndromes share a common clinical spectrum. Here, we describe an IFAP syndrome patient with a novel variant in the MBTPS2 gene and an HMD patient with a previously reported variant in the SREBF1 gene. In addition, we present a review of the literature describing the triad characterized by non-scarring alopecia, keratosis follicularis, and ocular symptoms common in both IFAP and HMD patients to raise awareness of these underdiagnosed diseases. We also highlight the subtle differences in clinical presentation between the two disorders to better enable differentiation.Fil: Irurzun, Inés. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Natale, Mónica Inés. Universidad de Buenos Aires; ArgentinaFil: Agostinelli, María L.. No especifíca;Fil: Lamberti, Magdalena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Montero, Maria Dolores. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Granda, Cecilia María. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Mássimo, José A.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Manzur, Graciela B.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Valinotto, Laura Elena. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Scheme of sample treatments and library preparation workflow.

<p>Different treatments are combined in the methodologies which are denoted with the letters A to I.</p

Coverage profiles of HRSV obtained per sample per methodology.

<p>A-I methodologies are indicated at the left of each profile. Genome organization with genes in green and their coding region in yellow are shown at the top of the figure. Genome regions with depth of coverage upper than 4 are underlined in orange. GenBank accession numbers are indicated in brackets. Only 6 representative samples are shown, other profiles are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199714#pone.0199714.s001" target="_blank">S1 Fig</a>.</p