Full-Genome Sequences of Seven Fatal Enterovirus 71 Strains Isolated in Shenzhen, China, in 2014

The whole-genome sequences of seven fatal enterovirus 71 (EV71) strains, isolated in southern China, in 2014, were determined. The complete genome sequences of these strains displayed close relationships to native EV71 strains and showed 94.2% to 99.8% identity to each other. All of these strains were assigned to subgenotype C4a based on phylogenetic analysis of the VP1 gene.

From March 2014 to September 2014, the Shenzhen Center for Disease Control and Prevention (CDC) received death reports of eight children with severe HFMD infections. Fecal specimens or anal swabs from these cases were archived at the Department of Microbiology, Shenzhen CDC. Seven of 8 specimens (87.5%) were detected as positive for EV71 by real-time reverse transcription-PCR (RT-PCR), and the other specimen (1/8 [12.5%]) was detected as positive for CV-A16 (12). Next, these strains were isolated by culturing clinical samples in rhabdomyosarcoma (RD) cell lines. Detailed epidemiological data for seven fatal EV71 strains are listed in Table 1.
A pair of universal primers, EVA-F30 (5=-TTAAAACAGCCT GTGGGTTGTACCCACCCA-3=) and EVA-R36 (5=-GCTATTCT GGTTATAACAAATTTACCCCCACCAGTC-3=), were used to amplify full-length genomes of these strains by a one-step RT-PCR method, as described previously (12). Amplified DNA products were sequenced by TaKaRa (Japan) using a primer-walking method. Contigs were assembled using sequencer version 4.9. The raw genome sequences were examined using BioEdit version 7.2.5 before submission to GenBank. Molecular phylogeny was investigated using the program MEGA 6.06 (13).
The complete genome sequences of seven EV71 strains were composed of 7,405 nucleotides (nt), excluding the poly(A) tail. The 5=-UTR was found to be 742 nt, followed by an open reading frame (ORF) encoding the structural protein P1 (2,586 nt), the nonstructural proteins P2 (1,734 nt) and P3 (2,259 nt), and the 3=-UTR (81 nt). The contents of A, C, G, and U of the seven EV71 genome sequences were 27.05 to 27.27%, 23.97 to 24.19%, 23.70 to 23.92%, and 24.83 to 25.02%, respectively, with GϩC contents To date, a vaccine against EV71 has not been commercially introduced (14, 15), but family-and kindergarten-based early intervention programs and timely treatment may reduce the incidence of severe and fatal cases.
Nucleotide sequence accession numbers. The full-length genome sequences of seven fatal EV71 isolates have been deposited in GenBank under the accession numbers listed in Table 1.

ACKNOWLEDGMENTS
Funding for this research was provided by the Shenzhen Center for Disease Control and Prevention, Shenzhen, China. The views and conclusions from this report are those of the authors and do not necessarily represent the official opinion of Shenzhen CDC.
We are grateful to the pediatricians who are from the sentinel surveillance system for HFMD in Shenzhen, China.