Screening and detection of human enterovirus 71 infection by a real-time RT-PCR assay in Marseille, France, 2009–2011 C.Y.Q. Tan, G. Gonfrier, L. Ninove,

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Screening and detection of human enterovirus 71 infection by a real-time RT-PCR assay in Marseille, France, 2009–2011 C.Y.Q. Tan, G. Gonfrier, L. Ninove, C. Zandotti, A. Dubot-Pérès, X. de Lamballerie, R.N. Charrel Clinical Microbiology and Infection Volume 18, Issue 4, Pages E77-E80 (April 2012) DOI: 10.1111/j.1469-0691.2012.03769.x Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1 Specimen processing and EV71 detection. (a) Flowchart for clinical specimen processing. CSF, cerebrospinal fluid; BGM, Buffalo Green Monkey renal cells; MRC5, human foetal lung fibroblasts; IFA, immunofluorescence assay. (b) Nucleotide sequences of the hybridization probes for the specific amplification of EV71. The positions of all probes are those relative to EV71 prototype strain BrCr (accession No. U22521). †As described in Tan et al. [5]. Clinical Microbiology and Infection 2012 18, E77-E80DOI: (10.1111/j.1469-0691.2012.03769.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 2 Phylogenetic and genetic analyses of the detected EV71 strains with all complete genome and genogroup C2 VP1 sequences available in GenBank. Aligned nucleotide sequences were analysed phylogenetically in MEGA v3.1 (neighbour-joining method, Jukes-Cantor model, 1000 pseudoreplicates). CVA16-G-10 (accession No. U05876) was added as an outlier for both trees. (a) In the VP1 coding region, MRS/09/3663, MRS/11/8134 (both 891 nt) and MRS/10/8229 (354 nt) clustered reliably (bootstrap 91%) with sequences of the C2 subgenogroup, which included FJ824734, a fatal strain isolated in France in 2007. (b) Full-length MRS/09/3663 grouped reliably (bootstrap 100%) with FJ172159, isolated in 2008 in Singapore. (c) MRS/09/3663 was used as the query sequence with a 500-nt sliding window and steps of 20 nt (neighbour-joining method, Kimura 2-parameter model, 100 pseudoreplicates) in SimPlot v3.5.1. Signals between 75% and 100% were observed across the entire genome with FJ172159 exclusively. No other evidence of possible recombination events was observed. Clinical Microbiology and Infection 2012 18, E77-E80DOI: (10.1111/j.1469-0691.2012.03769.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions