Molecular epidemiology of human enterovirus 71 at the origin of an epidemic of fatal hand, foot and mouth disease cases in Cambodia

Abstract

Human enterovirus 71 (EV-A71) causes hand, foot and mouth disease (HFMD). EV-A71 circulates in many countries and has caused large epidemics, especially in the Asia-Pacific region, since 1997. In April 2012, an undiagnosed fatal disease with neurological involvement and respiratory distress occurred in young children admitted to the Kantha Bopha Children's Hospital in Phnom Penh, Cambodia. Most died within a day of hospital admission, causing public panic and international concern. In this study, we describe the enterovirus (EV) genotypes that were isolated during the outbreak in 2012 and the following year. From June 2012 to November 2013, 312 specimens were collected from hospitalized and ambulatory patients and tested by generic EV and specific EV-A71 reverse transcription PCR. EV-A71 was detected in 208 clinical specimens while other EVs were found in 32 patients. The VP1 gene and/or the complete genome were generated. Our phylogenetic sequencing analysis demonstrated that 80 EV-A71 strains belonged to the C4a subgenotype and 3 EV-A71 strains belonged to the B5 genotype. Furthermore, some lineages of EV-A71 were found to have appeared in Cambodia following separate introductions from neighboring countries. Nineteen EV A (CV-A6 and CV-A16), 9 EV B (EV-B83, CV-B3, CV-B2, CV-A9, E-31, E-2 and EV-B80) and 4 EV C (EV-C116, EV-C96, CV-A20 and Vaccine-related PV-3) strains were also detected. We found no molecular markers of disease severity. We report here that EV-A71 genotype C4 was the main etiological agent of a large outbreak of HFMD and particularly of severe forms associated with central nervous system infections. The role played by other EVs in the epidemic could not be clearly established.

Figure 1

Maximum likelihood phylogenetic tree of 269 EV-A71 VP1 sequences. The branches are color-coded according to the location of sample collection (Cambodia=red, Vietnam=blue, Thailand=purple and China=green). For better clarity, taxon names are not shown, and sequences from other locations beside the four countries are not color-coded. The sequences isolated from Cambodian patients with different disease categories of severity are marked by white triangles for hand, foot and mouth disease, white rectangles for central nervous system involvement and black circles for cardiopulmonary failure. The tree was built using the maximum likelihood method based on the GTR+G4 model. The robustness of nodes was assessed with 1000 bootstrap replicates. Bootstrap values <70 are not shown.

Figure 2

Maximum likelihood (ML) phylogenetic tree of 140 VP1 sequences from all four enterovirus species (A, B, C and D). The tree was built using the ML method based on the GTR+G4 model. The robustness of nodes was assessed with 1000 bootstrap replicates. Bootstrap values <70 are not shown. The sequences isolated from Cambodian patients with different disease categories of severity are marked by white triangles for hand, foot and mouth disease, white rectangles for central nervous system involvement and black circles for cardiopulmonary failure.