- Short report
- Open Access
Isolation of an imported subgenotype B5 strain of human enterovirus A71 in Chongqing City, China, 2014
© The Author(s). 2016
- Received: 29 March 2016
- Accepted: 22 June 2016
- Published: 29 June 2016
Enterovirus A71 (EV-A71) is the main pathogen responsible for large outbreaks of hand, foot, and mouth disease (HFMD) in mainland China, and the dominant EV-A71 strains belong to subgenotype C4. To date, only one imported subgenotype B5 of EV-A71 has been reported in Xiamen City Fujian Province, 2009.
Here, we report on another imported subgenotype B5 of EV-A71 isolated from a HFMD patient in Chongqing City in 2014 (strain CQ2014-86/CQ/CHN/2014, hereafter refer as CQ2014-86). The VP1 coding sequence and the whole genome sequence revealed that strain CQ2014-86 shares the high nucleotide identity with Vietnamese strains isolated in 2011–2013, suggesting that strain CQ2014-86 may have been imported from Vietnam. In the 5’UTR, P2 and P3 regions, recombination events were found between strain CQ2014-86 and other EV-A, such as coxsackievirus A4 (CV-A4), CV-A5, CV-A14 and CV-A16.
This is the second report on importation of subgenotype B5 of EV-A71 in China, implying that we need to pay more attention to the importation of different subgenotypes of EV-A71.
- Human enterovirus A71
- Subgenotype B5
Human enterovirus A71 (EV-A71) is a neurotropic pathogen that can cause severe hand, foot, and mouth disease (HFMD) in children under 5 years old. It belongs to species EV-A (genus enterovirus, family picornaviridae, order Picornavirales). Following large scale outbreaks of HFMD in 2008 in mainland China, it has been categorized “C” group notifiable infectious diseases by the Ministry of Health of China [1–3]. Since then, China has built a HFMD laboratories network across the whole country, and this network is crucial for monitoring the prevalence of EV serotypes associated with HFMD patients, especially EV-A71. The molecular epidemiology of EV-A71 in mainland China reflects the pattern of circulation of subgenotype C4 viruses. In this study, we report on an imported subgenotype B5 of EV-A71 isolated from a HFMD patient in Chongqing City, China, in 2014.
In total, 210 clinical samples were collected in Chongqing City in 2014. Viruses were isolated from the original clinical specimens by propagation in human rhabdomyosarcoma (RD) and human larynxarcinoma (HEp-2) cells by conventional methods. Viral RNA was extracted using QIAamp Viral RNA mini kit (QIAGEN, Germany). Real-time reverse transcription-polymerase chain reaction , molecular typing and phylogenetic analysis was performed as described previously . And strain CQ2014-86 was identified as subgenotype B5 of EV-A71.
The viral RNA was converted to cDNA by a random priming strategy. The cDNA was amplified using primers according to the previous report , and a primer-walking strategy was used to close the gaps as necessary. PCR products were purified for sequencing using a QIAquick Gel Extraction Kit (QIAGEN), after which the amplicons were sequenced bidirectionally using fluorescent dideoxy-chain terminators and an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The 5′-segment sequence was determined using a 5′-Rapid Amplification of cDNA Ends Core Set (Takara Biomedicals, Dalian, China), according to the manufacturer’s instructions.
The nucleotide sequences of EV-A71 strains was aligned using Bioedit sequence alignment editor software (version 5.0). Maximum likelihood (ML) trees were constructed using the best-fit Kimura 2-parameter + I model of nucleotide substitution in Mega software (version5.03) . Similarity plot and bootscanning analyses were performed using the Simplot program (version 3.5.1; Stuart Ray, Johns Hopkins University, Baltimore, MD, USA).
Not surprisingly, all EV-A71 strains belonged to subgenotype C4a except one strain, strain CQ2014-86/CQ/CHN/2014 (hereafter refer as strain CQ2014-86), which was identified as subgenotype B5. Until now, only one whole genome sequence of subgenotype B5 from mainland China has been published (JN964686-Xiamen/FJ/CHN/2009) . Here, we report the full-length genome sequence of a subgenotype B5 of EV-A71 (strain CQ2014-86) isolated in Chongqing City, China, during HFMD surveillance. The sequence of strain CQ2014-86 has been deposited in the GenBank database under the accession number KU647000. Strain CQ2014-86 was isolated from a throat swab specimen from a 4-year-old girl. The genome length of CQ2014-86 is 7412 nucleotides. Similarity between the only two strains that have full-length genomic sequences (Chongqing and Xiamen strains) isolated in mainland China is 95.75 % at the nucleotide level and 98.49 % at the amino acid level.
CQ2014-86 shares the highest nucleotide identity with the Vietnamese strains. The nucleotide identities among them ranged from 97.5 to 99.9 %. The high similarity indicated that the strains had the same origin. Therefore, it is assumed that the virus was imported from neighboring countries and regions, possibly from Vietnam. Furthermore, the virus isolated from Xiamen City clustered with the strains from Taiwan, and the nucleotide identities between them ranged from 95.9 to 99.6 %, hence, the Xiamen strain may originate from Taiwan.
The earliest subgenotype B5 of EV-A71 was isolated in Sarawak, Malaysia in 2000, and then in Yamagata, Japan in 2003 [7, 8]. The subgenotype B5, which is spread widely throughout the world, especially in Southeast Asia, has caused several disease outbreaks in Japan , Vietnam , Thailand , Singapore , Malaysia , and Taiwan (2008, 2012) [12, 13].
In China, most of EV-A71 belonged to subgenotype C4 [1, 2]. Frequent international travel, however, may result in some other subgenotypes being imported. This is the second report on importation of the subgenotype B5 of EV-A71 following the first, which occurred 5 years previously in Xiamen City in mainland China. Therefore, monitoring of imported subgenotypes EV-A71 should be strengthened.
We would like to acknowledge the staffs of the national HFMD surveillance program in the Chongqing Center for Disease Control and Prevention for collecting specimens from patients in this study. This study was supported by the National Basic Research Program of China (973 Program): (Grant No. 2011CB504902) and Key Technologies Research and Development Program of National Ministry of Science (Grant No. 2013ZX10004-202).
YZ and WX conceived and designed the experiments. QY, YZ, DY, SZ, DW, TJ, WH and HA performed the experiments. QY and YZ analyzed the data. QY wrote the main manuscript text. All authors reviewed the manuscript.
The authors declare that they have no competing interests.
This study did not involve human participants or human experiments. The only human materials used were throat swabs from national HFMD surveillance at the instigation of the Ministry of Health P. R. of China for public health purpose. Written informed consent for the use of the clinical samples was obtained from the parents of the patient involved in this study. This study was approved by the second session of the Ethics Review Committee of the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, and the methods were carried out in accordance with the approved guidelines.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Tan X, Huang X, Zhu S, Chen H, Yu Q, Wang H, Huo X, Zhou J, Wu Y, Yan D et al. The persistent circulation of enterovirus 71 in People’s Republic of China: causing emerging nationwide epidemics since 2008. PLoS One. 2011;6:e25662.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Y, Tan X, Wang H, Yan D, Zhu S, Wang D, Ji F, Wang X, Gao Y, Chen L et al. An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong. China J Clin Virol. 2009;44:262–7.View ArticlePubMedGoogle Scholar
- Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, Mao N, Xu S, Zhu S, Cui A et al. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol J. 2010;7:94. doi:10.1186/1743-422X-7-94.View ArticlePubMedPubMed CentralGoogle Scholar
- Cui A, Xu C, Tan X, Zhang Y, Zhu Z, Mao N, Lu Y, Xu W. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD. PLoS One. 2013;8:e61451.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Y, Tan X, Cui A, Mao N, Xu S, Zhu Z, Zhou J, Shi J, Zhao Y, Wang X et al. Complete genome analysis of the C4 subgenotype strains of enterovirus 71: predominant recombination C4 viruses persistently circulating in China for 14 years. PLoS One. 2013;8:e56341.View ArticlePubMedPubMed CentralGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Mizuta K, Abiko C, Murata T, Matsuzaki Y, Itagaki T, Sanjoh K, Sakamoto M, Hongo S, Murayama S, Hayasaka K. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol. 2005;43:6171–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Tee KK, Lam TTY, Chan YF, Bible JM, Kamarulzaman A, Tong CYW, Takebe Y, Pybus OG. Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene. J Virol. 2010;84:3339–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Geoghegan JL, Tan LV, Kuhnert D, Halpin RA, Lin X, Simenauer A, Akopov A, Das SR, Stockwell TB, Shrivastava S et al. Phylodynamics of Enterovirus A71-Associated Hand, Foot, and Mouth Disease in Viet Nam. J Virol. 2015;89:8871–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Puenpa J, Theamboonlers A, Korkong S, Linsuwanon P, Thongmee C, Chatproedprai S, Poovorawan Y. Molecular characterization and complete genome analysis of human enterovirus 71 and coxsackievirus A16 from children with hand, foot and mouth disease in Thailand during 2008–2011. Arch Virol. 2011;156:2007–13.View ArticlePubMedGoogle Scholar
- Wu Y, Yeo A, Phoon MC, Tan EL, Poh CL, Quak SH, Chow VT. The largest outbreak of hand; foot and mouth disease in Singapore in 2008: the role of enterovirus 71 and coxsackievirus A strains. Int J Infect Dis. 2010;14:1076–81.View ArticleGoogle Scholar
- Huang YP, Lin TL, Kuo CY, Lin MW, Yao CY, Liao HW, Hsu LC, Yang CF, Yang JY, Chen PJ, Wu HS. The circulation of subgenogroups B5 and C5 of enterovirus 71 in Taiwan from 2006 to 2007. Virus Res. 2008;137:206–12.View ArticlePubMedGoogle Scholar
- Huang YP, Lin TL, Lin TH, Wu HS. Antigenic and genetic diversity of human enterovirus 71 from 2009 to 2012. Taiwan PLoS One. 2013;8:e80942.View ArticlePubMedGoogle Scholar