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 (DENV) is a public health problem representing the most important arthropod-borne viral disease in humans. In Argentina, Northern provinces have reported autochthonous cases since 1997, though these outbreaks have originated in bordering countries, where co-circulation of more than one serotype has been reported. In the last decade, imported dengue cases have been reported in Buenos Aires, the urban area of Argentina with the highest population density. In 2009, a dengue outbreak affected Buenos Aires and, for the first time, local transmission was detected. All cases of this outbreak were caused by DENV-1. In this report, we present the full-length sequences of 27 DENV-1 isolates, corresponding to imported cases of 1999–2000, as well as local and imported cases of the 2009 and 2010 outbreaks. We analyzed their phylogenetic and phylodynamic relationships and their global and local spread. Additionally, we characterized their genomic and phenotypic features. All cases belonged to DENV-1 genotype V. The most recent ancestor for this genotype was dated ∼1934, whereas that for the 2009 outbreak was dated ∼2007. The mean rates of nucleotide substitution were 4.98E-4 and 8.53E-4 subs./site/yr, respectively. We inferred an introduction from Paraguay in 1999–2000 and mainly from Venezuela during 2009–2010. Overall, the number of synonymous substitutions per synonymous site significantly exceeded the number of non-synonymous substitutions per site and 12 positively selected sites were detected. These analyses could contribute to a better understanding regarding spread and evolution of this pathogen in the Southern Cone of South America.
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