Molecular markers of influenza B lineages and clades

Abstract

Co-circulation of two influenza B virus lineages, B/Yamagata and B/Victoria, has been recognized since the late 1980s. The assessment of the prevalent lineage and the group of viruses in circulation is of importance in order to decide on the vaccine composition and evaluate its efficacy. The molecular characterization of influenza B viruses in circulation has been the aim of this study; this was approached by identifying and locating nucleotide substitutions in the influenza B virus hemagglutinin (HA) and neuraminidase (NA), specific for the lineage and/or clade. By the alignment of 3456 sequences from the influenza GISAID EpiFlu database, a high number of lineage- and group-specific nucleotide positions have been observed in the HA gene, but not in the NA gene. Additionally, an RT-PCR method has been developed, applicable directly to clinical specimens, which amplifies a short HA region that includes a group of unique molecular signatures. Twenty eight influenza B virus-positive respiratory specimens, collected in Tuscany in the seasons 2012-2013 and 2013-2014, were analyzed. The results revealed two clearly distinguishable patterns: one, more frequent, was characterized by all of the nucleotide changes associated with the B/Yamagata lineage (in most cases of Group 2), whereas the other exhibited all of the changes associated with the B/Victoria lineage. It can be concluded that the analysis of this short HA sequence can permit a rapid, highly sensitive determination of influenza B virus lineages and clades.

Figure 1

Nucleotide changes distinguishing the influenza B viruses of the B/Victoria lineage (here, represented by B/Brisbane/60/2008) from the viruses of the B/Yamagata lineage (here, represented by B/Wisconsin/1/2010, Group 3, and B/Massachusetts/2/2012, Group 2). The figure shows the changes present in a short region, about 60 nts long, of the HA, selected as described in the text. The lineage-specific markers (nts 522, 538–540, 548, 555, 558, 568) are highlighted in yellow, those that are clade specific (nt 541) in green. In blue is indicated another nucleotide substitution (nt 589), which characterizes the influenza B/Yamagata viruses of clade 2 from those of clade 3 analyzed in this study.

Figure 2

Phylogenetic tree of the HA1 portion from the HA gene of 18 sequenced samples analyzed with the neighbor joining algorithm. (The number of sequences in the tree corresponds to the patient’s number reported in Table 1).