Recent Human Influenza A/H3N2 Virus Evolution Driven by Novel Selection Factors in Addition to Antigenic Drift

Influenza A viruses are negative-strand segmented RNA viruses of the family Orthomyxoviridae that infect humans and a wide variety of animal species. Although there remains concern about the emergence of a pandemic influenza virus [1, 2], seasonal influenza epidemics consistently cause morbidity and mortality globally. Influenza viruses infect 5%–15% of the global population annually, resulting in ~500,000 deaths [3, 4]. In the United States, influenza is estimated to kill 36,000 people in an average year [5], which is significantly higher than the 15,000 deaths annually associated with AIDS [6]. Every few years, influenza epidemics boost annual mortality above average levels, causing 10,000–15,000 additional deaths [3, 4]. Influenza pandemics occur sporadically, characterized by a change in the viral hemagglutinin (HA) subtype through reassortment or introduction of a new virus (antigenic shift). Interpandemic influenza, in contrast, occurs yearly and has been characterized by acquisition of point mutations in the viral HA1 domain encoding the major antigenic sites of the HA protein, leading to serial antigenic change (antigenic drift) [7–9]. Recent studies have addressed the genomic evolutionary biology of interpandemic influenza [4, 10–13], suggesting that other viral features may play significant roles in strain selection and evolution. Co-circulation of multiple, genetically distinct strains leading to mixed infection and frequent intrasubtypic reassortment events [10, 12–14], as well as periodic “genome-wide selective sweeps’” characterized by the selection of a single clonal strain and the extinction of co-circulating strains [13], represent newly demonstrated evolutionary mechanisms, but biological correlations that lead to these phenomena are not well established. One important evolutionary event involved the emergence of the A/Fujian/411/2002-like (Fujian-like) viruses associated with the moderately severe epidemic of the 2003–2004 influenza season [16–18]. With diminished protection afforded by the influenza vaccine strains, pneumonia and influenza deaths in the United States during the 2003–2004 season exceeded the epidemic threshold for 9 consecutive weeks [19]. During the 2004–2005 A/California/7/2004-like (California-like) influenza season, the pneumonia and influenza deaths exceeded the epidemic threshold for 10 consecutive weeks [20]. During these seasons, the Centers for Disease Control and Prevention (CDC) reported an increase in deaths among children that was above the epidemic threshold [19–21]. Studies of Fujian-like viruses demonstrated the emergence of a major antigenic variant during the 2003–2004 influenza season that was derived from reassortment of 2 distinct clades of co-circulating H3N2 viruses, rather than by antigenic drift [10, 22]. These reassortant viruses contained the Fujian-like HA gene from a minor co-circulating H3N2 lineage (clade B) and the other 7 gene segments from the major circulating lineage (clade A) [10]. During the next influenza season, 2004–2005, another unexpected evolutionary event occurred. The clade A lineage that made up the majority of the H3N2 viruses during the 2003–2004 season was largely supplanted in 2004–2005 by the unreassorted clade B viruses, which acquired several HA antigenic changes to be become California-like [23, 24], but which also acquired a small number of amino acid mutations in the genes encoding the viral ribonucleoprotein complex (RNP). This derivative clade, termed here clade B′, caused a genome-wide selective sweep; that is, the replacement of the clade A viruses by the clade B′ viruses was so complete that the overall diversity of the circulating H3N2 viruses was dramatically reduced [12–14] (Figure 1). Figure 1 Genomic analyses of Fujian-like H3N2 viruses. A, Evolutionary relationships of concatenated major coding regions of influenza A viruses sampled in New York State during the period 1999–2004. The complete genomes of 35 human H3N2 influenza A virus ... In this study, we examined the hypothesis that Fujian-like viruses from the 2003–2004 influenza season (clades A and B) and the derivative California-like strain associated with the dominant selective sweep in 2004–2005 (clade B′) would exhibit different phenotypic properties that correlated with the selection of clade B′ viruses in 2004–2005 [13, 14]. The phenotypic properties of representative viruses from each clade were characterized in both cell culture and ferrets, and the role of the small number of amino acid mutations in the internal segments encoding the viral RNP of the clade B′ viruses was examined using an in vitro reporter assay.