Epidemiology, development of molecular diagnostic of human enterovirus and recombination and evolutionary dynamics of human coxsackievirus A6

Abstract

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by several genotypes of human enterovirus species A and represent a significant global public health concern. Enterovirus 71 (EV71) infections cause a deadly neurological disease for which no specific anti-viral drugs or vaccines has received regulatory approval. Published reports have described a dramatically increased incidence of coxsackievirus A6 (CV-A6) infections associated with HFMD since their occurrence in 2008 in Finland. The aim of this study was to evaluate the burden of human enteroviruses associated to HFMD and herpangina in patients and establish epidemiological profiles of these viruses in Thailand in 2012. Detection and genotype determination of enteroviruses were accomplished by reverse transcription-polymerase chain reaction and sequencing of the VP1 region. Enterovirus-positive samples were differentiated into 17 genotypes (CV-A 4, A5, A6, A8, A9, A10, A12, A16, A21, B1, B2, B4, B5, echovirus 7, 16, 25 and EV71). The result showed CV-A6 (33.5%), followed by CV-A 16 (9.4%) and EV71 (8.8%) as the most frequent genotypes in HFMD, CV-A 8 (19.3%) in herpangina and CAV6 (1.5%) in influenza like illness. Enterovirus infections were most prevalent during July with 34.4% in HFMD, 39.8% in herpangina and 1.6% in ILI. The higher enterovirus infection associated with HFMD and herpangina occurred in infants over one year-old. This represents the first report describing the circulation of multiple enteroviruses in Thailand. Furthermore, multiplex real-time PCR using TaqMan probes for broad detection of EVs and differentiation of EV71, CV-A6 and CV-A16 were developed and validated. The multiplex real-time PCR system which was run in two separate tubes was capable of screening and specific detection of the three selected enteroviruses, without cross-reactions with the other examined RNA viruses. The detection limit of the assays was 10 copies/µl for EV71, CV-A6, CV-A16 and panenterovirus. The overall diagnostic sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the multiplex rRT-PCR assay were greater than those of conventional RT-PCRs. The multiplex rRT-PCR assays provide the highly sensitive detection and rapid simultaneous typing of EV71, CV-A6 and CV-A16 thus rendering it feasible and attractive for large scale surveillance of EVs associated with HFMD outbreaks. Moreover, this study also investigated the relationship between these disease outbreaks with the evolutionary dynamics of CV-A6 and the appearance of novel recombinant forms (RFs) of the virus. Based on the analysis of the VP1 gene, the substitution rates of CV-A6 was estimated at 8.1 × 10-3 substitutions/site/year. There was an increasing likelihood between CV-A6 genome recombination and VP1 sequence divergence, with an estimated half-life of the RFs of 3.1 years. Bayesian phylogenetic analysis of the data showed that recently occurring recombination groups (RF-E, -F, -H, -J and -K) shared a common ancestor (RF-A). Recombination breakpoints were frequently observed between the 2A-2C gene and the 5´ untranslated region. This study revealed the potential for new CV-A6 variants to emerge and potentially modify disease outcomes of this major etiologic agent for HFMD affecting children worldwide.