An Inexpensive and Accurate Reverse Transcription-PCR-Melting Temperature Analysis Assay for Real-Time Influenza Virus B Lineage Discrimination.

In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm ) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010-2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.

Whole-Genome Characterization of a Novel Human Influenza A(H1N2) Virus Variant, Brazil.

We report the characterization of a novel reassortant influenza A(H1N2) virus not previously reported in humans. Recovered from a a pig farm worker in southeast Brazil who had influenza-like illness, this virus is a triple reassortant containing gene segments from subtypes H1N2 (hemagglutinin), H3N2 (neuraminidase), and pandemic H1N1 (remaining genes).

Phylodynamics of influenza A(H3N2) in South America, 1999-2012.

The limited influenza A(H3N2) genetic data available from the Southern Hemisphere (particularly from Africa and Latin America), constrains the accurate reconstruction of viral dissemination dynamics within those regions. Our objective was to describe the spatial dissemination dynamics of influenza A(H3N2) within South America. A total of 469 sequences of the HA1 portion of the hemagglutinin gene (HA) from influenza A(H3N2) viruses sampled in temperate and tropical South American countries between 1999 and 2012 were combined with available contemporary sequences from Australia, Hong Kong, United Kingdom and the United States. Phylogenetic analyses revealed that influenza A(H3N2) sequences from South America were highly intermixed with sequences from other geographical regions, although a clear geographic virus population structure was detected globally. We identified 14 clades mostly (≥80%) composed of influenza sequences from South American countries. Bayesian phylogeographic analyses of those clades support a significant role of both temperate and tropical regions in the introduction and dissemination of new influenza A(H3N2) strains within South America and identify an intensive bidirectional viral exchange between different geographical areas. These findings indicate that seasonal influenza A(H3N2) epidemics in South America are seeded by both the continuous importation of viral variants from other geographic regions and the short-term persistence of local lineages. This study also supports a complex metapopulation model of influenza A(H3N2) dissemination in South America, with no preferential direction in viral movement between temperate and tropical regions.

Phylogenetic analyses of influenza A (H1N1)pdm09 hemagglutinin gene during and after the pandemic event in Brazil.

Pandemic influenza A H1N1 [A(H1N1)pdm09] was first detected in Brazil in May 2009, and spread extensively throughout the country causing a peak of infection during June to August 2009. Since then, it has continued to circulate with a seasonal pattern, causing high rates of morbidity and mortality. Over this period, the virus has continually evolved with the accumulation of new mutations. In this study we analyze the phylogenetic relationship in a collection of 220 A(H1N1)pdm09 hemagglutinin (HA) gene sequences collected during and after the pandemic period (2009 to 2014) in Brazil. In addition, we have looked for evidence of viral polymorphisms associated with severe disease and compared the range of viral variants with the vaccine strain (A/California/7/2009) used throughout this period. The phylogenetic analyses in this study revealed the circulation of at least eight genetic groups in Brazil. Two (G6-pdm and G7-pdm) co-circulated during the pandemic period, showing an early pattern of viral diversification with a low genetic distance from vaccine strain. Other phylogenetic groups, G5, G6 (including 6B, 6C and 6D subgroups), and G7 were found in the subsequent epidemic seasons from 2011 to 2014. These viruses exhibited more amino acid differences from the vaccine strain with several substitutions at the antigenic sites. This is associated with a theoretical decrease in the vaccine efficacy. Furthermore, we observed that the presence of any polymorphism at residue 222 of the HA gene was significantly associated with severe/fatal cases, reinforcing previous reports that described this residue as a potential virulence marker. This study provides new information about the circulation of some viral variants in Brazil, follows up potential genetic markers associated with virulence and allows infer if the efficacy of the current vaccine against more recent A(H1N1)pdm09 strains may be reduced.

Whole-genome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season.

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.

Whole-genome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.