Molecular epidemiology of enteroviruses associated with hand, foot, and mouth disease in South India from 2015 to 2017

Abstract

Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by human enteroviruses and is clinically characterised by fever with vesicular rash on the hands, feet, and mouth. While enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) were the major etiological agents of HFMD in India earlier, the data on recently circulating enteroviruses associated with HFMD are sparse. Here, we describe the molecular epidemiology of enteroviruses associated with HFMD in South India from 2015 to 2017. We used archived enterovirus real-time reverse transcription (RT) PCR-positive vesicle swab and/or throat swab specimens from clinically suspected HFMD cases collected from four secondary-care hospitals in South India between July 2015 and December 2017. PCR amplification and sequencing were done based on the 5'VP1, 3'VP1, VP2, or 5´NCR regions to identify enterovirus types. Genetic diversity among enteroviruses was inferred by phylogenetic analysis. Of the 107 enterovirus RNA real-time RT-PCR-positive HFMD cases, 69 (64%) were typed as CVA6, 16 (15%) were CVA16, and one (1%) was CVA10, whereas in 21 (20%) cases, the virus was not typeable by any of the methods used in the study. The majority of HFMD cases (89, 83%) were in children less than five years old, while 11 (10.3%) were in adults. 5'VP1 yielded the maximum number of enteroviruses genotyped, and phylogenetic analysis showed that the CVA6 strains belonged to subclade D3, while the subclades of CVA16 and CVA10 were B1c and D, respectively. The predominant etiological agent of HFMD in South India during 2015-2017 was CVA6, followed by CVA16 and CVA10.

Fig. 1

The algorithm of enterovirus detection and typing in clinical specimens

Fig. 2

Rash in children with hand, foot, and mouth disease. (A) erythematous macules on the palms, (B) crusted lesions on the knee, (C) papules on the foot, (D) erythematous macules on the soles

Fig. 3

Phylogenetic trees of CVA6 based on partial 5’VP1 (A), 3’VP1 (B), VP2 (C), and 5’NCR (D) sequences of the study strains and other representative global strains. Phylogenetic relationships were inferred using the neighbour-joining method with the Kimura 2-parameter (K2P) model with 1000 bootstrap replicates for each data set. The GenBank accession number and the place and year of collection are shown for each strain. The clades and subclades are colour coded from node to tip (clade A in dark green, clade B in yellow, clade C in blue, subclades D1 in pink, D2 in light green, and D3 in red). The study strains are indicated by blue text. The scale bar represents nucleotide substitutions per site.

Fig. 4

Phylogenetic analysis of CVA16 based on partial 5’VP1 (A), VP2 (B), and 5’NCR (C) sequences of the study strains and reference strains. Phylogenetic relationships were inferred using the neighbour-joining method with the Kimura 2-parameter (K2P) model. The statistical reliability was estimated for each node using 1000 bootstrap replicates. The branches are colour coded as clade A in dark green, subclade B2 in yellow, subclade B3 in blue, subclades B1a in pink, B1b in violet, and B1c in red. The study strains are indicated by blue text. The scale bar represents nucleotide substitutions per site.

Fig 5

Phylogenetic tree of the CVA10 strain from this study and global representative strains, based on partial 5’VP1 sequences. Phylogenetic relationships were inferred using the neighbour-joining method with the Kimura 2-parameter (K2P) model with 1000 bootstrap replicates. The clades and subclades are colour coded from node to tip (clade A in dark green, clade B in yellow, clade C in blue, clade D in red, clade E in violet, F in pink, and G in light green). The strain from this study is indicated by blue text. The scale bar at the bottom indicates the relative phylogenetic distance.