Original Studies

Prevalence of Coxsackievirus A6 and Enterovirus 71 in Hand, Foot and Mouth Disease in Nanjing, China in 2013 Ya-Qian Hu, MS,*† Guang-Cheng Xie, MS,† Dan-Di Li, PhD,† Li-Li Pang, PhD,† Jing Xie, MS,† Peng Wang, MS,* Ying Chen, MS,* Jing Yang, MS,* Wei-Xia Cheng, PhD,* Qing Zhang, MS,† Yu Jin, MD,* and Zhao-Jun Duan, PhD† Background: Although hand, foot and mouth disease (HFMD) has been strongly associated with enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enteroviruses, studies regarding coxsackievirus A6 (CVA6) infection in HFMD are limited. The aim of this study was to identify the major etiological agents causing HFMD in Nanjing in 2013 and explore the clinical and genetic characteristics of the prevalent enterovirus (EV) types in HFMD. Methods: A total of 394 throat swabs were collected from hospitalized children diagnosed with HFMD from April to July 2013. EVs were detected by reverse transcription polymerase chain reaction of 5′ UTR sequences. Genotyping and phylogenetic analysis were based on VP4 sequences. Demographic and clinical data were obtained. Results: Of the specimens, 68.5% (270/394) were positive for EVs. The genotypes and detection rates were CVA6, 30.00% (81/270); EV71, 17.41% (47/270); HRV, 11.11% (30/270); CVA10, 3.33% (9/270); CVA2, 1.11% (3/270); CVA16, 0.74% (2/270); EV68, 0.37% (1/270); echovirus 6, 0.37% (1/270); echovirus 9, 0.37% (1/270), respectively. Patients infected with CVA6 displayed symptoms atypical of HFMD, including larger vesicles on their limbs and buttocks. Phylogenetic analysis revealed 2 genetically distinct CVA6 strains that circulated independently within the region. Patients infected with CVA6 were more likely to have abnormal periphery blood white blood cell and C-reactive protein levels, while EV71 was more likely to infect the central nervous system, as indicated by clinical manifestations and white blood cell analysis of cerebrospinal fluid. Conclusions: Multiple EV genotypes contributed to HFMD in Nanjing in 2013, and CVA6 was the dominant genotype. The clinical presentation of CVA6 infection differs from that of EV71 infection in HFMD. Key Words: hand foot and mouth disease, human enterovirus, coxsackievirus A6, EV71

H

and, foot and mouth disease (HFMD) is caused by acute enterovirus infection in humans, particularly those less than 5 years of age. The disease is characterized by a brief fever and vesicular exanthema on the hands and feet and in the mouth1,2 and imposes a heavy economic and public health burden in most Asian countries, especially in China.3 The most common etiologic agents of HFMD are EV71 and CVA16.4 Large outbreaks of EV71-associated HFMD with high morbidity and mortality have occurred in several Asian countries and regions, including Taiwan,5 Singapore,6 Malaysia,7 Japan,8 Vietnam,9 Thailand10 and China.11,12 In recent years, other EV types—including CVA6, CVA10, CVB1-3, echovirus 4 and echovirus 19—in addition to EV71 and CVA16, have been associated with increasingly frequent cases of HFMD and outbreaks around the world.13–15 Among these HFMDassociated EV-A viruses, CVA6 has attracted considerable attention; its association with an HFMD outbreak was first reported in Finland in 2008,13,16 and later in Europe and Asia.17–20 CVA6 is now spreading in Mainland China, including Shenzhen,21,22 Changchun23 and Beijing.24 There is no specific vaccine or antiviral drug available against EV71 and CVA16, although the first EV71 vaccine was tested in Bulgaria in 1976.25 To date, 5 EV71 candidate vaccines have been assessed in clinical trials in Asian countries. Of these, 3 were in Mainland China and recently completed phase III trials.26 While these appear to be effective and highly immunogenic,27,28 they were raised only against the C4 genotype of EV71 and crossimmunity for other EV71 genotypes and enteroviruses has not been assessed. Thus, information regarding the real-time etiology and clinical manifestations of HFMD are essential to develop appropriate interventions for prevention and control of HFMD. In this study, the prevalence and clinical and molecular characteristics of EV in HFMD in Nanjing, Jiangsu province were investigated.

(Pediatr Infect Dis J 2015;34:951–957) Accepted for publication June 1, 2015. From the *Nanjing Children’s Hospital, Nanjing Medical University, Nanjing, People’s Republic of China; and †National Institute for Viral Disease Control and Prevention, Chinese Center for Viral Disease Control and Prevention, Beijing, People’s Republic of China. Supported by a China Mega-Project for Infectious Disease (2013ZX10004-101) and the Prevention and Control of Novel Influenza Virus (2014ZX10004002) funded by the Ministry of Science and Technology of the People’s Republic of China and the Natural Science Foundation of Jiangsu Province (BK20141078). Ya-Qian Hu and Guang-Cheng Xie contributed equally to the article. The authors have no conflicts of interest. Address for correspondence: Zhao-Jun Duan, PhD, National Institute for Viral Disease Control and Prevention, Chinese Center for Viral Disease Control and Prevention, 155 Changbai Rd, Changping District, Beijing, People’s Republic of China. E-mail:[email protected] or Yu Jin, PhD, Nanjing Children’s Hospital, Nanjing Medical University, 72 Guangzhou Rd, Nanjing, People’s Republic of China. E-mail:[email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0891-3668/15/3409-0951 DOI: 10.1097/INF.0000000000000794

METHODS Ethical Approval This study was approved by the Ethics Committee of Nanjing Children’s Hospital, Nanjing Medical University. The guardians of all participating children provided written consent for the study.

Sample Collection A total of 394 throat swabs, as well as information on patient demographics, clinical symptoms and complications were obtained from hospitalized children diagnosed with HFMD from April to July 2013 at Nanjing Children’s Hospital, Jiangsu, China. All patients met the diagnostic criteria for HFMD proposed by National Health and Family Planning Commission of the People’s Republic of China. Patients with maculopapule and herpes in palm and feet, or erythra in buttock and knee, or even pulmonary edema, encephalitis and cardiopulmonary failure in severe cases were all enrolled in this study.

Human Enterovirus Detection Viral RNA was extracted from 200-μL throat swab samples using the Viral Nucleic Acid Extraction Kit II (Geneaid

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Biotech, New Taipei, Taiwan) according to the manufacturer’s instructions. RNA was reverse transcribed using random primers and superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) at 42°C for 1 hour to yield cDNA. EV was then detected by amplifying a 400-bp region of the 5′ UTR by nested polymerase chain reaction (PCR) using Taq PCR StarMix (GenStarBiosolutions, Beijing, China) and the outer primers S1 and AS1 and the inner primers S2and AS2 as described previously.20,29 Reactions were conducted under the following conditions: 94°C for 1 minute, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds, then 72°C for 10 minutes. Products were analyzed by electrophoresis in 1.5% agarose gels. Enterovirus-positive samples were further analyzed by nested PCR on the VP4 region using 2.5 μL RNA, reverse primer OL68-1, outer forward primer MD91, internal forward primer EVP4,30 and PrimerScript One Step RT-PCR Kit ver. 2 (TaKaRa, Dalian, China) with the following conditions: 50°C for 30 minutes, 95°C for 5 minutes, 40 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute, with a final extension at 72°C for 10 min. PCR products (645 bp) were visualized by electrophoresis in 1.5% agarose gels and sequenced by the Beijing TianyiHuiyuan Bioscience and Technology Inc.

Sequence Analysis Nucleotide sequences were aligned with those of known genomes using the National Center for Biotechnology Information (NCBI)’s BLAST tool. Phylogenetic trees were constructed using reference sequences with high homology to our sequences. All VP4 sequences were aligned by ClustalW. Phylogenetic trees were constructed based on VP4 sequences using the neighbor-joining method with a Kimura 2-parameter substitution model and 1000 bootstrap pseudo-replicates in the MEGA5 software. Viruses were genotyped according to known reference sequences.

Statistical Analysis Student’s t test, χ2 tests and Fisher’s exact test were used to compare continuous and categorical variables, respectively. Statistical analysis was performed using SPSS version 19; P values 70% are shown. Strains labeled with black dots belong to the C4a genotype. Coxsackievirus A16 G-10 was used as the outgroup.

Clinical Features and Laboratory Diagnosis Among these HFMD cases, the most common clinical presentations were fever and ulcers, followed by vomiting and upper airway symptoms. Fever peaks ranged from 37.6°C to 41.0°C. Complications included circulatory system symptoms, hepatic or renal function abnormality, and neurological complications, such as limb © 2015 Wolters Kluwer Health, Inc. All rights reserved.

shaking, drowsiness and convulsions. Patients infected with CVA6 were less likely to present with drowsiness and convulsions than were EV71 patients (P < 0.05). Most patients had lesions on the mouth, limbs and buttocks, the typical sites of HFMD. The atypical dermatologic presentation of patients infected with CVA6 was characterized by vesicles larger than those in other EV-infected patients (Fig. 4). www.pidj.com  |  953

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FIGURE 3.  Phylogenetic analysis of CVA6 isolates. Phylogenetic trees were constructed based on the VP4 sequence using the neighbor-joining method with a Kimura 2-parameter substitution model and 1000 bootstrap pseudo-replicates. Bootstrap values (%) for 1000 replicated trees are indicated at the nodes; only values >70% are shown. Coxsackievirus A16 G-10 and EV71 BrCr were used as outgroups. Peripheral blood white blood cell (WBC) counts in CVA6and CVA10-infected patients were significantly higher than in those infected with EV71 and other EVs, 12.518 × 109/L and 14.521 × 109/L, respectively, both of which are above the upper limit of the normal range (10 × 109/L). Similarly, mean serum C-reactive protein (CRP) and the proportion of patients with CRP above the normal limit (10 mg/L) were significantly higher in CVA6-infected patients than in those infected with EV71 and CVA16 (Table 1). Patients with EV71 infections had significantly

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higher WBC counts in cerebrospinal fluid, indicating central nervous system involvement, than patients with CVA6 and CVA16 infections (P < 0.05).

DISCUSSION This study shows that identifying human EVs based on the VP4 gene region, taking advantage of the divergence in relatively short VP4 sequences both within and between serotypes, enables © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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Hand, Foot and Mouth Disease

FIGURE 4.  Atypical dermatologic presentation of a patient infected with CVA6. Children infected with CVA6 frequently presented with erythema and vesicles larger than those infected with EV71 and CVA16. In this case, lesions were distributed on the buttocks (left) and calves (right).

TABLE 1.  Comparison of Clinical Presentations Among Enterovirus Genotypes Characteristic Age (M) Male, n (%) Fever peak (°C) Fever duration (day) Vomiting, n (%) Limb shaking, n (%) Drowsiness, n (%) Convulsions, n (%) Peripheral blood WBC count (106/L) Peripheral blood CRP abnormal cases, n (%) CSF WBC count abnormal cases, n (%) ECG abnormal cases, n (%) EEG abnormal cases, n (%)

EV71 (n = 47)

CVA6 (n = 81)

CVA16 (n = 2)

CVA10 (n = 9)

HRV (n = 30)

27.659 ± 18.552 33 (70.21) 38.909 ± 0.482 2.787 ± 1.693 15 (31.91) 6 (12.77) 15 (31.91) 33 (70.21) 10.249 ± 4.153 2 (4.26) 19 (40.43) 7 (14.89) 0 (0.00)

23.213 ± 18.778 54 (72.00) 39.203 ± 0.589* 1.780 ± 1.047* 18 (22.22) 5 (6.17) 6 (7.41)* 39 (48.15)* 12.518 ± 5.142* 43 (53.86)* 1 (1.23)* 17 (20.99) 1 (1.23)

19.000 ± 8.485 2 (100.00) 38.750 ± 0.354 2.000 ± 0.000 1 (50.00) 0 (0.00) 1 (50.00) 2 (100) 11.575 ± 0.049 1 (50.00) 1 (50.00) 0 (0.00) 0 (0.00)

25.111 ± 12.544 6 (66.67) 39.378 ± 0.377* 1.833 ± 1.000 2 (22.22) 1 (11.11) 1 (11.11) 6 (66.67)* 14.521 ± 4.448 4 (44.44) 0 (0.00)* 1 (11.11) 0 (0.00)

21.133 ± 18.778 22 (73.33) 38.757 ± 0.517 2.65 ± 2.022 7 (23.33) 4 (13.33) 7 (23.33) 17 (56.67) 10.750 ± 3.309 7 (23.33) 9 (30.00) 3 (10.00) 2 (6.67)

Other (n = 95) 22.758 ± 13.795* 64 (65.31) 38.942 ± 0.599 2.542 ± 2.022 24 (24.49) 13 (16.33) 24 (24.49) 64 (65.31) 10.860 ± 4.445 17 (17.35) 35 (35.71) 12 (12.24) 4 (4.08)

*P < 0.05 compared with EV71-infected patients. CVA2, EV68, echovirus 6 and echovirus 9 were not included in the analysis. CSF indicates cerebrospinal fluid; ECG, electrocardiogram; EEG, electroencephalogram.

rapid identification and can distinguish 89 isolates from 26 serotypes appearing >30 years apart.30 In our study, we matched VP4 sequences of our isolates to existing sequences using NCBI’s BLAST in NCBI to identify strains with high homology, allowing us to construct phylogenetic trees and thus identify EV genotypes. However, the VP4 region of 95 specimens could not be amplified, which is likely accounted for by 2 main reasons. Repeated freezing and thawing during transport may have degraded samples, or operator error, defects in other PCR materials, even the sensitivity and specificity of the primers may have prevented amplification. However, we repeated the experiment 3 times, suggesting that sample quality is likely the more important factor. This highlights the importance of careful preservation of samples in future studies. Sequencing of the VP4 region allowed us to identify numerous pathogens causing HFMD in Nanjing, including CVA6, EV71, CVA10, CVA2, CVA16, EV-68, echovirus 6 and echovirus 9. Comparison of the proportion of cases caused by each virus to prior study results reveals that infection rates have changed; CVA6 has become the dominant pathogenic agent and EV71 is the second largest contributor to HFMD while CVA16 is now less common than CVA10 and CVA2. HFMD is caused by a variety of pathogenic agents. EV71 and CVA16, previously the 2 most common EVs, caused largescale HFMD outbreaks in mainland China, including Linyi, Shandong province, Fuyang, Anhui province,11,12 Beijing, Shijiazhuang © 2015 Wolters Kluwer Health, Inc. All rights reserved.

and central China.32–34 However, infections with uncommon EVs including CVA6, CVA10, CVA4, CVB and echovirus have emerged in recent years. HFMD outbreaks caused by CVA6 were first reported in Finland,13 Taiwan35 and Singapore6 from 2007 to 2009. CVA6 and CVA10 began to co-circulate with increasing frequency in Finland,16 China36 and Thailand,10 drawing much attention. In this study, we also identified a diverse array of HFMD-causing viruses, including CVA6, EV71, CVA 10, CVA2, CVA16, EV68, ECHO6 and ECHO9. Supporting recent results in Shenzhen, China,21 our study found CVA6 to be the dominant causative pathogen. One of the most commonly known HFMD-associated EVs, CVA16, was detected in a very low proportion of cases. Together, our results and others’ indicate that the composition of viruses causing HFMD is changing worldwide. At present no effective vaccine or antiviral drug against HFMD is available. Large HFMD outbreaks with fatal neurological complications were caused mainly by the C4a genotype of EV71.11,12,37 Previous clinical trials studies showed that EV71 vaccines were efficacy and safety, but did not protect against CVA16 infections.38 Recent study showed that the bivalent VLP vaccines against EV71 and CVA16 could be a candidate vaccine for HFMD.39 Other bivalent formalin-inactivated EV71/ formalininactivated CVA16 vaccines were reported to elicit strong neutralizing antibody responses against both viruses in animal models but not to protect against CVA6 and CVA10 viral infections in www.pidj.com  |  955

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cell culture neutralization assay.40 The only candidate vaccine against this strain (the C4a genotype of EV71) was only likely to be effective in other Asian countries due to its specificity41; monovalent EV71 vaccines fail to prevent infections with other EVs. Although phylogenetic analysis showed that in this study all EV71 strains belong to the C4a genotype, the change in the distribution of viruses causing HFMD poses a novel challenge to development of multivalent vaccines against HFMD. Although the introduction of such vaccines is likely to take many years in the future, early diagnosis and treatment are key to prevent severe complications and relieve the economic and psychological burden. In China, HFMD caused by CVA6 has been reported sporadically from 2009 to 2011.17 However, CVA6 has since been identified as the predominant serotype associated with HFMD in Guangdong.42 An etiological agents study in Shenzhen from 2008 to 2012 indicated that CVA6 increased in prevalence from 0.5% to 27.4%,43 becoming the predominant causative agent in Shenzhen in 2013.21,22 Another study also found CVA6 to be the dominant causative agent of an unexpected outbreak of HFMD in Changchun, Jilin province in the summer of 2013.23 In Jiangsu province, EV71 and CVA16 were the major etiologic agents from 2009 to 2012.44 And 1 recent study showed EV71 was the main agent causing HFMD in Suzhou, Jiangsu province from April to July in 2013.31 However, this study found that the dominant agent of HFMD changed to be CVA6 in Nanjing, Jiangsu province in 2013. These results indicate that more attention should be paid to the possibility that the distribution of HFMD-causing agents may change rapidly. Our phylogenetic analysis of CVA6 indicated that 2 genetically distinct clusters of CVA6 strains could have been independently introduced into Nanjing; 1 may have come from Taiwan or Japan and the other from other cities in Mainland China. Some EVs—such as ECHO, EV68 and HRV—have rarely been associated with HFMD before this study. Among these viruses, HRV was detected at an especially high rate. Patients infected with HRV had similar clinical presentations and laboratory diagnosis to those of patients infected with CVA6 and EV71 (Table 1). No previous study has addressed whether HRVs, which are commonly seen in acute respiratory infections, pneumonia, acute wheezing and exacerbation of asthma,45–47 are associated with HFMD, although they have been reported to exist in the respiratory tract of 90% of healthy people older than 2 years.48,49 HRV-C has also been detected in fecal samples of children with gastroenteritis in the absence of respiratory symptoms,1 indicating that HRV-C may cause gastroenteritis. Our phylogenetic analysis indicated that most HRVs detected in HFMD patients belong to the C group, with homologies ranging from 62.3% to 100% with reported strains. Twenty HRV-C strains appeared to be closely related to LZ651 and CU139, whereas the other 5 clustered into 4 groups (see Fig., Supplemental Digital Content 1, http://links.lww.com/INF/C177). Although many previous studies have confirmed the pathogenic role of HRV-C in respiratory illness, the role of HRV-C in HFMD should be further investigated. CVA6 infections cause a range of clinical issues, including severe or atypical HFMD, herpangina and onychomadesis.19,50–53 The mucocutaneous manifestations of patients infected with CVA6 in our study (Fig. 4) fit the definition of atypical HFMD, including either (1) maculopapular rashes on the trunk, buttocks or face, or (2) large vesicles or bullae on any site of the body.54 The clinical manifestations of infection with EV71 or CVA6 differed (Table 1); EV71-infected patients experienced neurological symptoms—such as limb shaking, drowsiness and convulsions—more frequently than CVA6-infected patients. This fits with the current understanding of EV71 as a neurotropic enterovirus that can cause encephalitis, meningitis, brainstem encephalitis and even neurogenic

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pulmonary edema and death,6,12 whereas CVA6 presents with less severe complications. In addition to the difference in clinical manifestation, CVA6 infection is often associated with higher WBC counts and CRP levels, as confirmed by a 5-year study.35Thus, we speculate that neurological clinical manifestations, atypical dermatologic features, high peripheral blood WBC count and CRP levels and increased cerebrospinal fluid WBC counts are early indicators of potential disease progress. Whether laboratory data allow early identification of EVs requires further study. We conclude that multiple EV genotypes were associated with HFMD in Nanjing, Jiangsu province in 2013, where CVA6 was the dominant agent. In combination with other studies, the causes of HFMD in China appear to have changed in 2013 such that CVA6 is now the main etiologic agent. Differences between CVA6 and EV71 infection could allow early distinction and avoid severe consequences, especially in rural areas. REFERENCES 1. Lau SK, Yip CC, Lung DC, et al. Detection of human rhinovirus C in fecal samples of children with gastroenteritis. J Clin Virol. 2012;53:290–296. 2. Solomon T, Lewthwaite P, Perera D, et al. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778–790. 3. Xing W, Liao Q, Viboud C, et al. Hand, foot, and mouth disease in China, 2008-12: an epidemiological study. Lancet Infect Dis. 2014;14:308–318. 4. Chang LY, Lin TY, Huang YC, et al. Comparison of enterovirus 71 and coxsackie-virus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998. Pediatr Infect Dis J. 1999;18:1092–1096. 5. Ho M, Chen ER, Hsu KH, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med. 1999;341:929–935. 6. Wu Y, Yeo A, Phoon MC, et al. 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:e1076–e1081. 7. Ooi MH, Wong SC, Podin Y, et al. Human enterovirus 71 disease in Sarawak, Malaysia: a prospective clinical, virological, and molecular epidemiological study. Clin Infect Dis. 2007;44:646–656. 8. Hosoya M, Kawasaki Y, Sato M, et al. Genetic diversity of enterovirus 71 associated with hand, foot and mouth disease epidemics in Japan from 1983 to 2003. Pediatr Infect Dis J. 2006;25:691–694. 9. Tu PV, Thao NT, Perera D, et al. Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005. Emerg Infect Dis. 2007;13:1733–1741. 10. Linsuwanon P, Puenpa J, Huang SW, et al. Epidemiology and seroepidemiology of human enterovirus 71 among Thai populations. J Biomed Sci. 2014;21:16. 11. Zhang Y, Zhu Z, Yang W, 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. 12. Zhang Y, Tan XJ, Wang HY, 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;44:262–267. 13. Osterback R, Vuorinen T, Linna M, et al. Coxsackievirus A6 and hand, foot, and mouth disease, Finland. Emerg Infect Dis. 2009;15:1485–1488. 14. Russo DH, Luchs A, Machado BC, et al. Echovirus 4 associated to hand, foot and mouth disease. Rev Inst Med Trop Sao Paulo. 2006;48:197–199. 15. Zhu Z, Xu WB, Xu AQ, et al. Molecular epidemiological analysis of echovirus 19 isolated from an outbreak associated with hand, foot, and mouth disease (HFMD) in Shandong Province of China. Biomed Environ Sci. 2007;20:321–328. 16. Blomqvist S, Klemola P, Kaijalainen S, et al. Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland. J Clin Virol. 2010;48:49–54. 17. Lu QB, Zhang XA, Wo Y, et al. Circulation of Coxsackievirus A10 and A6 in hand-foot-mouth disease in China, 2009-2011. PLoS One. 2012;7:e52073. 18. Mirand A, Henquell C, Archimbaud C, et al. Outbreak of hand, foot and mouth disease/herpangina associated with coxsackievirus A6 and A10 infections in 2010, France: a large citywide, prospective observational study. Clin Microbiol Infect. 2012;18:E110–E118.

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19. Wei SH, Huang YP, Liu MC, et al. An outbreak of coxsackievirus A6 hand, foot, and mouth disease associated with onychomadesis in Taiwan, 2010. BMC Infect Dis. 2011;11:346. 20. Gopalkrishna V, Patil PR, Patil GP, et al. Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India. J Med Microbiol. 2012;61(Pt 3):420–425. 21. Li JL, Yuan J, Yang F, et al. Epidemic characteristics of hand, foot, and mouth disease in southern China, 2013: coxsackievirus A6 has emerged as the predominant causative agent. J Infect. 2014;69:299–303. 2 2. Yang F, Yuan J, Wang X, et al. Severe hand, foot, and mouth disease and coxsackievirus A6-Shenzhen, China. Clin Infect Dis. 2014;59: 1504–1505. 23. Han JF, Xu S, Zhang Y, et al. Hand, foot, and mouth disease outbreak caused by coxsackievirus A6, China, 2013. J Infect. 2014;69:303–305. 24. Hongyan G, Chengjie M, Qiaozhi Y, et al. Hand, foot and mouth disease caused by coxsackievirus A6, Beijing, 2013. Pediatr Infect Dis J. 2014;33:1302–1303. 25. Huang ML, Ho MS, Lee MS. Enterovirus 71 vaccine: when will it be available? J Formos Med Assoc. 2011;110:425–427. 26. Li JX, Mao QY, Liang ZL, et al. Development of enterovirus 71 vaccines: from the lab bench to Phase III clinical trials. Expert Rev Vaccines. 2014;13:609–618. 27. Zhu F, Xu W, Xia J, et al. Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. N Engl J Med. 2014;370:818–828. 28. Zhu FC, Meng FY, Li JX, et al. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2013;381:2024–2032. 29. Sapkal GN, Bondre VP, Fulmali PV, et al. Enteroviruses in patients with acute encephalitis, Uttar Pradesh, India. Emerg Infect Dis. 2009;15:295–298. 30. Ishiko H, Shimada Y, Yonaha M, et al. Molecular diagnosis of human enteroviruses by phylogeny-based classification by use of the VP4 sequence. J Infect Dis. 2002;185:744–754. 31. Chen Z, Sun H, Yan Y, et al. Epidemiological profiles of hand, foot, and mouth disease, including meteorological factors, in Suzhou, China. Arch Virol. 2015;160:315–321. 32. Liu W, Wu S, Xiong Y, et al. Co-circulation and genomic recombination of coxsackievirus A16 and enterovirus 71 during a large outbreak of hand, foot, and mouth disease in Central China. PLoS One. 2014;9:e96051. 33. Tian H, Zhang Y, Sun Q, et al. Prevalence of multiple enteroviruses associated with hand, foot, and mouth disease in Shijiazhuang City, Hebei province, China: outbreaks of coxsackieviruses a10 and b3. PLoS One. 2014;9:e84233. 34. Zhu J, Luo Z, Wang J, et al. Phylogenetic analysis of Enterovirus 71 circulating in Beijing, China from 2007 to 2009. PLoS One. 2013;8:e56318. 35. Lo SH, Huang YC, Huang CG, et al. Clinical and epidemiologic features of Coxsackievirus A6 infection in children in northern Taiwan between 2004 and 2009. J Microbiol Immunol Infect. 2011;44:252–257. 36. Xu M, Su L, Cao L, et al. Enterovirus genotypes causing hand foot and mouth disease in Shanghai, China: a molecular epidemiological analysis. BMC Infect Dis. 2013;13:489.

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Hand, Foot and Mouth Disease

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Prevalence of Coxsackievirus A6 and Enterovirus 71 in Hand, Foot and Mouth Disease in Nanjing, China in 2013.

Although hand, foot and mouth disease (HFMD) has been strongly associated with enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enterovirus...
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