Etiology of Multiple Non-EV71 and Non-CVA16 Enteroviruses Associated with Hand, Foot and Mouth Disease in Jinan, China, 2009-June 2013

Abstract

Hand, foot, and mouth disease (HFMD) is an infectious disease caused by human enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enteroviruses. It is of interest that other enteroviruses associated with HFMD in Jinan have been rarely reported. The aim of the present study is to detect and characterize the circulating serotypes of non-EV71 and non-CVA16 enteroviruses associated with HFMD in Jinan city, Shandong province, China. A total of 400 specimens were collected from clinically diagnosed HFMD cases in Jinan from January 2009 to June 2013. All specimens were infected with non-EV71 and non-CVA16 enteroviruses previously confirmed by RT-PCR or real-time PCR according to the protocols at that time. The GeXP-based multiplex RT-PCR assay (GeXP assay) was performed to investigate the pathogen spectrum of 15 enteroviruses (coxsackieviruses A4, A5, A6, A9, A10, A16; coxsackieviruses B1, B3, B5; Echoviruses 6, 7, 11, 13, 19 and EV71) infections associated with HMFD. For GeXP assay negative samples, reverse transcription nested PCR (nested RT-PCR) based on the 5' -untranslated region (5'- UTR) sequence and phylogenetic analysis were conducted to further explore the etiology of multiple enteroviruses. The results showed that a total of twenty serotypes of enteroviruses (including EV71 and CVA16) were identified by GeXP assay and nested RT-PCR. The most circulating twelve serotypes of enteroviruses with HFMD in Jinan from 2009 to June 2013 were EV71, CVA16, CVA10, CVA6, CVA12, CVA2, Echo3, CVA4, CVA9, CVB1, CVB3 and Echo6. CVA10 and CVA6 were the most prevalent pathogens other than EV71 and CVA16 in Jinan and their most prevalent seasons were spring and summer, and a slight increase was observed in autumn and early winter. It should be noted that mixed-infections were identified by GeXP assay and the phylogenetic tree clearly discriminated the multiple pathogens associated with HFMD. Our results thus demonstrate that there was a clear lack of a reliable testing method for EV71 and CVA16 and multiple non-EV71 and non-CVA16 enteroviruses associated with HFMD were present in Jinan. The GeXP assay combined with nested RT-PCR based on 5'-UTR region could meet the need for the national surveillance of multiple enteroviruses or the investigation of epidemic outbreaks triggered by enteroviruses in the future.

Fig 1. The enterovirus serotypes identified in hand-foot-mouth disease during 2009 to June 2013, in Jinan, China.

Fig 2. Monthly distribution of non-EV71 and non-CVA16 enterovirus serotypes during 2009 to June 2013 in Jinan, China.

Fig 3. Monthly distribution of CVA6 associated with HFMD during 2009 to June 2013 in Jinan, China.

Fig 4. Monthly distribution of CVA10 associated with HFMD during 2009 to June 2013 in Jinan, China.

Fig 5. Phylogenetic analysis based on the 5’-UTR region of HFMD cases during 2009 to June 2013 in Jinan, China.

A total of 29 representative sequences (representing 12 serotypes) from this study and 12 reference strains worldwide were used to build the tree. Jinan sequences were labeled by black solid dot. Abbreviations: JN, Jinan; CHN, China.