Differentiation of influenza B lineages circulating in different regions of Brazil, 2014-2016, using molecular assay.

Background: Two antigenically and genetically distinct lineages of influenza B viruses (B/Victoria and B/Yamagata) have been co-circulating worldwide since 2002. Virological surveillance is essential to differentiate between both lineages with a view to the annual updating of the B component for the trivalent or quadrivalent influenza vaccine composition. Methods: The samples analyzed in the present study were collected by influenza sentinel units located in the Southeast, Midwest, North, and Northeast regions of Brazil, part of the National Influenza Virus Surveillance Network, coordinated by the Ministry of Health of Brazil. A total of 870 influenza B positive samples by reverse transcription real - time polymerase chain reaction (RT-qPCR), collected during 2014, 2015, and 2016 influenza seasons, were submitted to the influenza B lineage genotyping panel for characterization as B/Yamagata or Victoria lineages using RT-qPCR. Results: Of the 197 samples analyzed in 2014, a total of 160 (81 %) corresponded to the B/Yamagata lineage, 19 (10 %) to the B/Victoria lineage, and 18 (9 %) to indeterminate lineages. Of the 190 samples analyzed in 2015, a total of 124 (65 %) corresponded to the B/Yamagata lineage; 55 (29 %) to the B/Victoria lineage, whereas 11 (6 %) were of indeterminate lineages. Of the 483 samples analyzed in 2016, a total of 297 (62 %) corresponded to the B /Victoria lineage; 174 (36 %) to the B/Yamagata lineage and 12 (2 %) to indeterminate lineages. This cross-sectional study revealed influenza B virus (IBV) infection in all age groups, and among them, the highest prevalence was observed in individuals between 11 and 49 years of age Our findings demonstrate the match between influenza B virus lineages recommended by the World Health Organization (WHO) for the trivalent vaccine composition to be used in the Southern Hemisphere (SH) and the predominant circulating viruses during the 2014, 2015, and 2016 seasons.

Use of Sanger protocols to identify variants of concern, key mutations and track evolution of SARS-CoV-2.

Vaccination and the emergence of SARS-CoV-2 variants mark the second year of the pandemic. Variants have amino acid mutations at the spike region, a viral protein central in the understanding of COVID-19 pathogenesis and vaccine response. Variants may dominate local epidemics, as Gamma (P.1) in Brazil, emerging in 2020 and prevailing until mid-2021. Different obstacles hinder a wider use of Next-Generation Sequencing for genomic surveillance. We describe Sanger based sequencing protocols: i) Semi-nested RT-PCR covering up to 3.684 kb (>96 %) spike gene; ii) One-Step RT-PCR for key Receptor Binding Domain (RBD) mutations (codons 417-501); iii) One-Step RT-PCR of partial N region to improve genomic capability. Protocols use leftovers of RNA extracted from nasopharyngeal swabs for quantitative RT-PCR diagnosis; with retro-transcribed DNA sequenced at ABI 3500 using dye termination chemistry. Analyses of sequences from 95 individuals (late 2020/early 2021) identified extensive amino acid variation, 57 % with at least one key mutation at the Receptor Binding Domain, with B.1.1.28 lineage most prevalent, followed by Gamma and Zeta variants, with no Delta variant observed. The relatively low cost and simplicity may provide an accessible tool to improve surveillance of SARS-CoV-2 evolution, monitor new variants and vaccinated breakthroughs.

Human bocavirus in hospitalized children under 5 years with acute respiratory infection, São Paulo, Brazil, 2010.

The aims of this study were to investigate the human bocavirus (HBoV) frequency and genotypes in hospitalized children <5 years presenting acute respiratory infections (ARI) within the São Paulo metropolitan area. Nasopharyngeal samples from 300 patients, previously screened for common respiratory viruses, were tested by qPCR for the NSP1 and NP-1 genes. The VP1/2 gene in positive samples was then amplified by PCR and sequenced. A total of 49 positive HBoV cases (16.3%; mean Ct value of 34.41) were detected with the mean age being 18.1 months (range 1 month to 5 years) and the median age being 1 year of age. Children aged between 0 and 12 months had higher detection rates of HBoV (69.4%; 34/49; mean Ct = 34.45) than children from other age groups (30.6%; 15/49; mean Ct = 34.34). No significant differences were observed between HBoV Ct levels and clinical illness. The occurrence was more frequently associated with fall (38.8%; 19/49) and spring (36.7%; 18/49). All 12 sequenced isolates were identified as HBoV-1, displaying minor genetic variation compared to the Swedish reference strains ST1 and ST2 (99.1-99.7% nt). The sole identification of HBoV-1 supports the hypothesis that this particular genotype is strongly related to ARI, and contributes to the role of this virus in the aetiology of respiratory diseases.