| Abstract: | Globally, echovirus 30 (E30) is one of the most frequently identified enteroviruses and a major cause of meningitis. Despite its wide distribution, little is known about its transmission networks or the dynamics of its recombination and geographical spread. To address this, we have conducted an extensive molecular epidemiology and evolutionary study of E30 isolates collected over 8 years from a geographically wide sample base (11 European countries, Asia, and Australia). 3Dpol sequences fell into several distinct phylogenetic groups, interspersed with other species B serotypes, enabling E30 isolates to be classified into 38 recombinant forms (RFs). Substitutions in VP1 and 3Dpol regions occurred predominantly at synonymous sites (ratio of nonsynonymous to synonymous substitutions, 0.05) with VP1 showing a rapid substitution rate of 8.3 × 10−3 substitutions per site per year. Recombination frequency was tightly correlated with VP1 divergence; viruses differing by evolutionary distances of >0.1 (or 6 years divergent evolution) almost invariably (>97%) had different 3Dpol groups. Frequencies of shared 3Dpol groups additionally correlated with geographical distances, with Europe and South Asia showing turnover of entirely distinct virus populations. Population turnover of E30 was characterized by repeated cycles of emergence, dominance, and disappearance of individual RFs over periods of 3 to 5 years, although the existence and nature of evolutionary selection underlying these population replacements remain unclear. The occurrence of frequent “sporadic” recombinants embedded within VP1 groupings of other RFs and the much greater number of 3Dpol groups than separately identifiable VP1 lineages suggest frequent recombination with an external diverse reservoir of non-E30 viruses.The genus Enterovirus in the family Picornaviridae is a group of nonenveloped RNA viruses that cause a wide range of diseases in humans and other mammals. Enteroviruses contain a positive-sense RNA genome of approximately 7,500 nucleotides encoding a polyprotein that after cleavage yields structural (capsid proteins VP1 to VP4) and nonstructural (2A to 3D) proteins. Primary infection with an enterovirus leads to viral replication in the tissue around the gastrointestinal tract, followed by a transient viremia and sometimes migration into other tissues (6, 44). Although infection in immunocompetent individuals is often asymptomatic or causes mild febrile illness, enteroviruses are a common etiological agent in aseptic meningitis, encephalitis, and paralysis in individuals of all ages, with persistent and/or widely disseminated systemic infection in immunosuppressed individuals and neonates (12, 19, 23).Enteroviruses were originally classified as polioviruses, coxsackie virus type A or B viruses, or echoviruses (enteric cytopathic human orphan viruses), depending upon the infectious properties of the virus such as pathogenicity in mice (reviewed in reference 22). From the 1960s onwards, enteroviruses within these groups were further differentiated into serotypes originally by using panels of specific neutralizing antisera and, more recently, by sequence comparisons of structural gene regions such as VP1 (9, 34, 38, 43). There are currently over 100 recognized human enterovirus serotypes that fall into four main species (designated A to D) using phylogenetic analysis (54). The Enterovirus genus additionally contains several other species infecting primates, cattle, and pigs and has recently been expanded to include the genetically related human rhinovirus A and B (54).The species B serotype, echovirus 30 (E30), is a major cause of meningitis in both children and adults. Among the many serotypes associated with this disease presentation, E30 is generally the most commonly isolated in Europe (8, 31, 49), the United States (10, 37), Asia (1, 60), and South America (33). E30 infections typically occur as a series of outbreaks every 3 to 5 years, frequently over large geographical areas. For example, high frequencies of E30 detection in meningitis cases and surveillance programs were reported for 2000 to 2001 throughout Europe, including Denmark (58), Belgium (57), Cyprus (45), Germany (46), and France (3, 5), and again in 2005 to 2006 (8). Similarly, in the United States, long-term surveillance by the Centers for Disease Control and Prevention revealed peaks of E30 isolation in 1981, 1991 to 1993, 1997, and 2003 (10, 37). The underlying basis for this periodicity in E30 infections and the possible association of different genetic variants of E30 with outbreaks are currently poorly understood.At any one time point, a range of different species B enterovirus serotypes circulate in human populations. The evolution of enteroviruses occurs through genetic drift and, over much longer periods, antigenic diversification in the structural gene region encoding the virus capsid (7, 14, 25, 30, 51, 55); it may also occur by recombination between the capsid and nonstructural coding parts of the genome and the 5′ untranslated region (2, 13, 16, 20, 26, 28, 29, 35, 39, 41, 47, 48, 53). To date, almost all documented examples of recombination have been limited to members of the same species (e.g., between species B serotypes), with the exception of the 5′ untranslated region, where only a single genetic group can be identified within human species A and B and a second with species C and D (48).In this study, we have carried out an extensive investigation of VP1 sequence divergence and recombination through sequencing the 3Dpol region of E30 isolates and samples collected from several European countries, Southeast Asia, and Australia over a combined 8-year observation period. Using this geographically diverse sample collection, our aims were to document the time span and geographical extent of different E30 variants as they emerged and spread during the observation period. The identification of individual recombinants of E30 provides the means to document in detail the dynamics of E30 population turnover, geographical ranges of enterovirus transmission networks, and, ultimately, the relationship between the emergence of new variants of E30 and longer-term changes in disease associations and pathogenicity. |
