Evolutionary Dynamics and Temporal/Geographical Correlates of Recombination in the Human Enterovirus Echovirus Types 9, 11, and 30

The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.Human enteroviruses (HEV) are single-stranded, positive-sense RNA viruses in the virus family Picornaviridae. Infections with these viruses are enterically transmitted and normally cause subclinical or mild symptoms, but they are also capable of causing a wide array of often severe disease presentations, including aseptic meningitis, encephalitis, and acute flaccid paralysis. Echovirus type 9 (E9) and E11 are serotypes of species B enteroviruses and are among the most common etiological agents of aseptic meningitis (14). The first epidemiological study of E9 investigated isolates obtained from sick children in 1995 (24). E9 is chiefly associated with mild infections affecting children over the age of 5 years and teenagers, although it can cause more severe syndromes in neonates and immunosuppressed patients (14). E11 infections, which predominantly affect infants less than 1 year old, have been associated with large outbreaks of uveitis in infants (15, 21), sepsis, and other neonatal systemic illnesses with high mortality rates (14, 23).Circulating strains and genotypes of E9 (2, 8, 11, 14) and E11 (4, 7, 21) have been characterized in many different countries through analysis of the structural genome regions, principally VP1. In common with other mammalian RNA viruses, both E9 and E11 show rapid accumulation of nucleotide substitutions over time, but their epidemiologies are distinct. E9 is associated with widespread, large-scale seasonal outbreaks and displays a regular epidemic pattern of outbreaks occurring approximately every 3 years (8, 14). Outbreaks of E11 are less common, occur irregularly, and often last for several years (14, 25).In contrast to time-related diversification observed in the capsid-encoding regions of the HEV genome, nonstructural region genes are subject to frequent recombination (7, 18, 19, 26, 30), leading to the concept of separate, modular evolution of the structural and nonstructural regions of the HEV genome (20). Recombination in the nonstructural gene region of HEV is limited to members of the same species (27). In a recent large-scale investigation of echovirus type 30 (E30) molecular epidemiology, phylogenetic analysis of the 3Dpol region revealed the existence of a number of discrete, bootstrap-supported clades, allowing circulating E30 variants to be classified into a series of distinct recombinant forms (RFs) (22). Individual RFs became very rapidly disseminated over large geographical distances through repeated cycles of emergence, dominance, and disappearance in a 3- to 5-year cycle.In the current study, we carried out parallel investigations of the molecular epidemiology and evolution of clinically presenting isolates of E9 and E11 collected in 16 countries in Europe, central Asia, Southeast Asia, and West Africa over a 14-year period. By comprehensive sequencing of these isolates in the structural (VP1) and nonstructural (3Dpol) genome regions, it was possible to explore the dynamics of sequence diversification and recombination. The turnover of individual recombinant groups and the phylogeographical correlates of recombination were determined and compared to those of E30, providing new insights into the nature of the global spread of these important viral pathogens and the role of recombination in enteroviral evolution.