Clinical features and phylogenetic analysis of severe hand-foot-and-mouth disease caused by Coxsackievirus A6

Journal Pre-proof Clinical features and phylogenetic analysis of severe hand-footand-mouth disease caused by Coxsackievirus A6

Xiaohan Yang, Yuanyuan Li, Changbin Zhang, Wenli Zhan, Jia Xie, Siqi Hu, Huiying Chai, Pan Liu, Hongyu Zhao, Bin Tang, Keyi Chen, Jian Yu, Aihua Yin, Mingyong Luo PII:

S1567-1348(19)30280-1

DOI:

https://doi.org/10.1016/j.meegid.2019.104054

Reference:

MEEGID 104054

To appear in:

Infection, Genetics and Evolution

Received date:

10 August 2019

Revised date:

24 September 2019

Accepted date:

27 September 2019

Please cite this article as: X. Yang, Y. Li, C. Zhang, et al., Clinical features and phylogenetic analysis of severe hand-foot-and-mouth disease caused by Coxsackievirus A6, Infection, Genetics and Evolution(2019), https://doi.org/10.1016/ j.meegid.2019.104054

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

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Clinical Features and Phylogenetic Analysis of Severe Hand-Foot-and-Mouth Disease Caused by Coxsackievirus A6

Xiaohan Yang a,b,1, Yuanyuan Li c,d,1, Changbin Zhang a,b,1, Wenli Zhan a,b, Jia Xie b, Siqi Hu b, Huiying Chai a,b, Pan Liu a,b, Hongyu Zhao a,b, Bin Tang a,b, Keyi Chen a,b,

a

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Jian Yu e,f, Aihua Yin a,b, Mingyong Luo a,b*

Medical Genetic Centre, Guangdong Women and Children Hospital, Guangzhou,

Medical Genetic Centre, Guangdong Women and Children Hospital, Guangzhou

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b

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P.R.China

State Key Laboratory of Respiratory Disease, National Clinical Researh Center for

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Medical University, Guangzhou, P.R.China

Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated

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Nanshan Medicine Innovation Institute of Guangdong Province, Guangzhou,

P.R.China

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Hospital of Guangzhou Medical University, Guangzhou, P.R.China

Beijing Advanced Innovation Center for Biomedical Engineering, Beihang

University, Beijing, P.R.China

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School of Biological Science and Medical Engineering, Beihang University, Beijing

P.R.China

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1 These authors contributed equally to this work.

* Corresponding author: Mingyong Luo, MD, Medical Genetic Centre, Guangdong Women and Children Hospital, Guangzhou, P.R.China, Phone: +8615920356428, E-

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mail: [email protected].

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ABSTRACT

Background: Coxsackievirus A6 (CA6) infection may lead to high hand-foot-andmouth disease (HFMD) aggregation in children. We aimed to analyze the clinical and phylogenetic features of severe CA6-associated pediatric HFMD.

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Methods: The clinical and laboratory features of 206 and 55 children with mild and severe CA6-associated HFMD, respectively, were summarized. The CA6

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regions and neighbor-joining method.

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phylogenetic tree was depicted using combinatorial analysis of the VP1-encoding

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Results: CA6 was the major pathogen both in mild and severe HFMD in 2017. Most CA6-associated severe HFMD cases showed high fever, skin rash, age younger than

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36 months, and elevated white blood cell and C-reactive protein levels, and there were

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no significant differences compared to the mild cases (p > 0.05). The severe cases

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were significantly more likely (p< 0.05) to show male sex, long fever duration, decreased oral intake, tonsil enlargement, diarrhea, vomiting, elevated levels of creatine kinase and blood glucose, and positive fecal occult-blood test results. Severe complications included aseptic meningitis (29/55, 52.7%) and pulmonary edema (6/55, 10.9%) were observed in severe cases. Furthermore, genetic analyses showed all CA6 isolates belonged to lineage E2, and two amino acid changes of V174I and T283A in VP1 may be associated with the severity of HFMD.

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Conclusions: CA6 has become a major cause of HFMD with severe systemic disorders. V174I and T283A of VP1 may be associated with the severity of CA6 infection. These findings could raise awareness of the clinical importance of CA6 infection among practitioners.

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Keywords: hand-foot-and-mouth disease; coxsackievirus A6; etiology; phylogenetic analysis

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1. INTRODUCTION

Hand-foot-and-mouth disease (HFMD) is a commonly occurring infectious disorder in children, caused by Enteroviruses (EVs), family Picornaviridae (Jubelt and Lipton, 2014). It is generally a self-limited disease that occurs in children aged younger than 5 years, and is characterized by fever, skin eruptions on the hands, feet, and other parts,

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as well as vesicles in the mouth, all of which usually recover within a week. However, some patients may rapidly develop fatal neurological and systemic complications (Ooi

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et al., 2010; Solomon et al., 2010). Of the multiple serotypes associated with HFMD,

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coxsackievirus A16 (CA16) and enterovirus A71 (EV71) are the main causative

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pathogens, especially in severe and fatal cases (Cabrerizo et al., 2014; Chen et al., 2007;

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Ooi et al., 2010; Solomon et al., 2010; Xing et al., 2014). In contrast, coxsackievirus A6 (CA6) has rarely attracted clinical attention, as the associated infections are

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typically asymptomatic or mild. However, since the CA6-associated HFMD outbreak

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in Finland in 2008, the circulation of CA6 has become more active and has been associated with several HFMD outbreaks in Asia, Europe, and the USA (Abedi et al., 2015; Centers for Disease and Prevention, 2012; Di et al., 2014; Fujimoto et al., 2012; Mirand et al., 2012; Montes et al., 2013; Osterback et al., 2009; Puenpa et al., 2013; Zeng et al., 2018).

In China, more than 1 million children contract HFMD annually, with EV71, CA16, and CA6 reported as the major causative agents (Chen et al., 2019; Di et al., 2014). The

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incidence of CA6 infection has increased dramatically in recently years; CA6 caused a large-scale outbreak of HFMD in 2017 in Guangdong, China (Zeng et al., 2018). During that outbreak, CA6 accounted for 85.5% of all such infections, and a substantial increase in the number of severe CA6-caused HFMD cases was also observed (Chen et al., 2019). CA6 has a single-stranded positive-sense RNA genome (~7 to 8 kb), which

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is similar to those of other members of the Picornaviridae. The genome is translated as a single long polyprotein that including four structural proteins, VP1 to VP4. The VP1

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protein is the major surface-accessible protein and contains many important

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neutralization epitopes and virulence determinants; it is widely used in studies focusing

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on virus evolution, serotype identification, and virulence (Acharya et al., 1989; Chang et al., 2012; Mizuta et al., 2019; Oberste et al., 1999). However, CA6 is not included in

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the routine surveillance of the Chinese Center for Disease Control and Prevention, and

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severe HFMD with CA6 infection have not been fully described. It is critical for

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guiding the development of HFMD prevention and control strategies by Elucidating the epidemiological and evolutionary characteristics of CA6. In this study, we aimed to describe the detailed clinical features of severe CA6-associated HFMD to raise awareness of the clinical importance of CA6 infection in HFMD among practitioners. Also, we performed genetic analyses of complete VP1 to investigate the molecular epidemiology of CA6 and gain a better understanding of the pathogenic mechanism behind disease severity.

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2. MATERIALS AND METHODS

2.1. Patients and Case Definition

A total of 358 hospitalized children (age range: 2 months - 9 years old) with HMFD at the Guangdong Women and Children Hospital in 2017 were included in this study. All

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CA6 infection diagnoses were confirmed with a molecular test (to be described below). The HFMD cases were defined based on the presence of oral ulcers, chiefly on the

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tongue and buccal mucosa, as well as hard and soft palate, accompanied by typical

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vesicular rashes most commonly observed on the extensor surfaces of the hands, feet, buttocks, and/or knees. By clinical manifestation, all patients were classified as having

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severe HFMD if they had any neurological complications (i.e., startle, lethargy, coma,

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limb shaking, acute flaccid paralysis, aseptic meningitis, aseptic encephalitis, or

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autonomic nervous system dysregulation) or cardiopulmonary complications (i.e.,

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cardiopulmonary failure, pulmonary oedema, rapid or slow heart rate, myocarditis or pulmonary haemorrhage), or both simultaneously; otherwise, patients were categorized as having mild HFMD (Ooi et al., 2010; Xing et al., 2014). The severe group comprised inpatients with severe HFMD, and the mild group comprised those with mild HFMD.

This study was approved by the ethics committee of the Guangdong Women and Children Hospital. Since patient information saved at the study database was delinked from individual patient identifiers, the informed consent was not needed.

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2.2. Clinical Information Collection

Data on participants’ clinical history, physical examinations, hematological, biochemical, and microbiological investigations, complications, and outcomes were collected. Information on imaging examinations including chest radiography and

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cerebrospinal magnetic resonance imaging (MRI) was also collected.

2.3. CA6 Detection and Genotyping

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Depending on the clinical symptoms of the 358 patients, we collected the appropriate

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clinical specimens (n=434), including rectal swab or fecal sample (n=341), throat swab

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(n=11), vesicular fluid (n=6), and cerebrospinal fluid (n=76). Viral RNA were extracted from these specimens using QIAamp Viral RNA Mini Kit (QIAGEN, Germany). Then,

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EV RNA was amplified using the pan-enterovirus fluorescent kit (DAAN, China) and

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CA6 was further subtyped using a CA6 RNA fluorescent PCR test kit (DAAN, China).

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All tests were performed with the 7500 Fast Real-Time PCR system (Applied Biosystems, USA).

2.4. Phylogenetic Analysis

The complete VP1-encoding regions of the CA6 isolates were amplified based on previous study (Tan et al., 2015), and the PCR products were then sequenced by ABI 3130 Genetic Analyzer (Applied Biosystems, USA). Alignments of the entire VP1 sequences were performed via ClustalW (MEGA v7.0). A phylogenetic tree was

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constructed using the neighbor-joining method (MEGA v7.0) and bootstrap analysis with 1,000 re-samplings was used to calculate confidence values.

2.5. Statistical Analysis

Data analysis was performed with SPSS 21.0 (Chicago, USA). Quantitative data were

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presented as the mean ± standard deviation and compared by an independent samples t-test, whereas those of the categorical variables were shown as frequencies and

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percentages and compared using a chi-square test. A two-sided P value＜0.05 was

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considered statistically significant in all the statistical analyses.

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3. RESULTS

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3.1. Epidemiological Features and Clinical Manifestations

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A total of 358 children hospitalized with laboratory-confirmed HFMD (261 cases with CA6 infection, and 97 cases with non-CA6 infection), from January to December 2017, were included. Of the 261 CA6-associated cases, 78.9% (206/261) showed mild HFMD and 21.1 % (55/261) showed severe HFMD (Figure 1). A total of 96.4% (53/55) of the severe HFMD and 86.4% (178/206) of the mild HFMD patients were aged younger than 36 months, and there was no significant difference between the two groups in the average age (severe HFMD: 19.3±12.6 vs. mild HFMD 20.6±13.3 months, p = 0.52) (Table 1). The mean hospitalization duration in the severe cases was significantly

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longer than that in the mild cases (9.0±4.8 vs. 6.2±2.1 days, p < 0.001). The average fever duration in the severe cases was longer than that in the mild cases (3.1±1.8 vs. 2.4±1.2 days, p = 0.009), while there was no apparent statistical difference between the groups in terms of the fever degree. As for sex ratio, 74.5% (41/55) of the severe disease patients showed male sex, which was significantly higher than the 54.9% (113/206)

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observed in the mild group (p = 0.008).

The children with CA6 infection in this study showed typical clinical symptoms

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including skin rashes/ulcer (100%), tonsil enlargement (57.1%), decreased oral intake

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(46.4%), coughing (37.9%) and diarrhea (7.3%). However, compared to the mild cases,

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the severe cases had significantly higher prevalence of decreased oral intake (61.8% vs.

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37.4%, p = 0.01), tonsil enlargement (74.6% vs. 52.4%, p = 0.003), and diarrhea (14.6% vs. 5.3%, p = 0.035) (Table 1). No significant differences were found in the prevalence

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of skin rashes/ulcer, febrile seizure, and coughing between the two groups.

All of severe cases had obvious neurologic complications (n=55, 100%), typically involved myoclonic jerk (n=38, 69.1%), febrile seizures (n=20, 36.4 %), and aseptic meningitis (n=29, 52.7%). Furthermore, two cases (3.6%) accompanied with brainstem encephalitis and presented iso-or hyper-signals on T1 and hyper-signals on T2, and demyelination changes in the white matter were evident. Additionally, six (10.9%) cases developed pulmonary edema. Limited or extensive distribution of patchy changes and decreasing radiolucency with ground-glass opacities in the lungs were observed by

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chest radiography in these children. It was noteworthy that a 60-month-old boy showed impaired consciousness during hospitalization.

3.2. Laboratory Examination

Laboratory examinations showed that the average white blood cell levels in both groups

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increased beyond the normal reference range; however, there was no difference between the two groups (14.48±6.23 vs. 14.73±4.99, p = 0.748). A majority of children

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in both groups showed elevated serum C-reactive protein levels (61.2% vs. 67.3%, p =

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0.437). All the clinical parameters were listed in Table 3. The levels of aspartate aminotransferase and creatine kinase were significantly higher in the severe group than

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the mild group (the creatine kinase level was normal in the mild HFMD group) (p =

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0.005). The severe group showed a higher percentage of hyperglycemia (52.0% vs.

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17.2%, p< 0.001) and positive fecal occult-blood test (FOBT) results (38.2% vs. 8.7%,

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p< 0.001) than the mild group.

3.3. Phylogenetic Analysis

Phylogenetic analysis of the representative CA6 strains from 16 (29.1%) severe HFMD cases

(GZ/CHN/2017/01~

GZ/CHN/2017/16)

and

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(7.8%)

mild

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(GZ/CHN/2017/17~ GZ/CHN/2017/32) showed that the sequence homologies of the 32 complete VP1 genes were displayed as 82.7%-83.6% nucleotides and 95.1%-96.4% amino acid compared to the CA6 prototype (CA6/Gdula, GenBank accession no.

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AY421764); the nucleotide and amino acid identities among the 32 isolates were 93.4%-99.9% and 97.7%-100%, respectively. Moreover, the sequence identities among the severe and mild CA6 strains were 93.9%-99.9% nt and 98.4%-100% aa, respectively.

The molecular typing results revealed that all the 32 CA6 strains detected in this study

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were clustered monophyletically into lineage E2 (Figure 2). Twenty-four (75.0%) isolates (fourteen obtained from the severe cases and ten from the mild cases) were

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closely related to virus from China between 2013 and 2016, with VP1 gene nucleotide

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identities of 95.9%-99.5%, while 7 (21.9%) isolates (one obtained from the severe cases

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and six from the mild cases) were closely related to viruses from HongKong

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(MH049747) in 2015, with identities of 97.0%-98.1% nt. The remaining isolate from the severe case group was closely related to isolates observed in Yunnan (LC412941)

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and Guangxi (MH018483) in 2015, and Shenzhen (MH716170) in China 2016, with

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nucleotide identities ranging from 97.7% to 97.9% in VP1. All CA6 isolates were observed in China over the past successive years.

3.4. Analysis of amino acid changes By alignment of the 32 isolates with 84 representative reference CA6 strains worldwide between 1992 and 2016, as well as the prototype strain Gdula and lineage E2 reference strain (KR706309/TW/2007), two main amino acid changes at the positions V174I and T283A in VP1 encoding region were found (Supplemental File 1). Sites at V174I may

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happen in 2009, accounted for 87.5% (28/32) of CA6 isolates in this study, while only 11.6% (10/86) isolates with V174I were found from 2009 to 2015 worldwide. T283A, few happened before 2017, accounted for 81.3% (13/16) of CA6 strains isolated from the severe cases, higher than 56.3% (9/16) of the mild cases. Therefore, changes of V174I and T283A may be associated with the severity of CA6-infected cases, but

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further investigation is required.

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4. DISCUSSION

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The early recognition of severe HFMD risk is critical, particularly in children with

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neurological involvement, as a small proportion of them can rapidly develop potentially

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fatal neurological injury. While most previous studies focused on EV71 and CA16 infection, CA6-associated severe HFMD remains poorly characterized. In the present

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study, CA6 was the major pathogen both in mild and severe HFMD in 2017. Most of

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the children with CA6-associated HFMD were aged younger than 36 months, and males were more often seen in severe cases. Aseptic meningitis was the main neurological complication noted in the severe cases, while pulmonary oedema was the main cardiopulmonary complication. In addition, phylogenetic analysis showed two amino acid changes, V174I and T283A, have the potential to cause changes in severity of the CA6 infection. These findings provide a better understanding of the clinical presentation and etiology of severe HFMD, which can be helpful in the formulation of appropriate treatment strategies. 13

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Consistent with previous reports (Lo et al., 2011; Montes et al., 2013), our study showed that fever than 39 °C, rashes on the palms and soles, and multiple oral ulcers were characteristic of CA6 infection. The mean age of children with CA16 infection is reportedly 43.3±6.4 months, while that of those with CA10 infection is 40.2±26 months (Yen et al., 2009). However, the target populations of CA6 infection in our study were

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aged younger than 36 months. Young children have poor immune function and a very low level of antibodies, which were potential risk factors for severe disease and death

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(Chen et al., 2007; Ooi et al., 2010; Zhang et al., 2010). Interestingly, the incidence rate

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of severe HFMD in boys was higher than that in girls, at 2.9:1 (41/14), similar to

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previous findings (Li et al., 2016; Yang et al., 2014). Boys tend to be careless about hygiene, increasing the risk of CA6 infection and transmission, and the susceptibility

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of CA6 at the host genetic level is also suggested (Chen et al., 2007; Lin et al., 2017).

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Remarkably, 52.0% of the severe cases had hyperglycemia in this study.

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Hyperglycemia is associated with fatal EV infection (Ooi et al., 2009), and is a useful clinical predictor of the presence of complicated or fatal disease. Thus, it is important for pediatricians to pay close attention to blood glucose changes in severe cases. As we know, the FOBT positivity rate was 38.2% in the severe cases was firstly reported, indicating that CA6 can cause gastrointestinal tract injury. Given the lack of data on the pathology of the children’s intestinal mucosa, further investigation is required.

Neurological injury and complications may occur in cases with EV71 infection. However, CA6-associated HFMD may also be complicated by severe neurological 14

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symptoms, with some cases displaying signs of severe neurological injury (Blomqvist et al., 2010; Logotheti et al., 2009; Richter et al., 2006). In the current study, Myoclonic jerks were the mainly neurological manifestations in the severe cases, which were seen often in EV71 infection, and could be an early indicator of neurologic involvement and sequelae (Lu et al., 2004). EVs, particularly echoviruses and coxsackievirus, accounted

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for the majority of aseptic meningitis (Nigrovic, 2013; Ooi et al., 2010). In our study, more than half of severe HFMD children accompanied with aseptic meningitis. A

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previous study in Taiwan 2011 also reported that CA6-associated HFMD children

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accompanied with aseptic meningitis (Lo et al., 2011). Through a retrograde axonal

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spread along peripheral nerves and disrupted blood–brain barrier, EVs can invade the central nervous system and resulted in aseptic meningitis or encephalitis. When

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brainstem encephalitis occurs, high signal intensities on T1 and/or T2 in the spinal cord

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and medulla oblongata can be typically identified by MRI (Li et al., 2012; Zeng et al.,

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2016). Our study showed two cases with brainstem encephalitis had high signal intensities on T2, and demyelination changes in the white matter. This suggests that CA6 could also cause brain damage. All these children recovered from the acute illnesses with no long-term sequelae.

Interestingly, six of the severe cases showed pulmonary edema-related complications and one showed impaired consciousness; such findings have been rarely reported on in previous studies (Blomqvist et al., 2010; Logotheti et al., 2009; Richter et al., 2006). Patients with pulmonary edema showed limited or an extensive distribution of patchy 15

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changes and decreasing radiolucency with ground-glass opacities in the lung fields in our study. As neurogenic pulmonary edema in EVs infection is closely associated with head injury, and it can develop very rapidly, with a mortality as high as 33% (Chang et al., 2004). The exact mechanism behind pulmonary edema development may be associated with the dysregulation of systemic and damage of brainstem nuclei, and poor

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immune function in young children (Ooi et al., 2010; Solomon et al., 2010). Although all the patients with pulmonary edema managed successfully in the pediatric intensive

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care unit (PICU), children may be left with significant debilitating morbidity (Prager et

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al., 2003). Our study adds to the enrichment of the definition of CA6 and mounting

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evidence suggesting a causal relationship between CA6 infection and pulmonary edema.

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CA6 strains are divided into six lineages (A-D, E1 and E2) based on the VP1 gene, and E2 has gradually become the predominant lineage, worldwide, since 2008 (Bian et al.,

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2015). Here, we found that all the isolates we obtained belonged to lineage E2; they

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showed high nucleic sequence and amino acid homologies with the isolates circulating in the southern and eastern provinces of China over the past successive years. The lineage E2 circulated widely across China rather than being localized to Guangdong. The Guangdong provincial Center for Disease Control and Prevention also reported that the CA6 epidemic strains in 2017 were dispersed in major clusters comprising strains from 2012-2016, and lineage E2 represented the major reason for the increase in the CA6 epidemic activity level (Zeng et al., 2018). The epidemic features of this cluster need further surveillance. 16

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As VP1 is exposed outside of the capsid of EVs and may contains critical immunologically reactive epitopes, amino acid mutations in this region could alter antigenicity and virulence. For example, site at E145Q in the receptor-binding region of VP1, is reportedly the major genetic determinant for the development of viremia and neuropathogenesis in EV71 infection and play a key role in the fitness of EV71 (Chang

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et al., 2012; Tee et al., 2010). In our study, two meaningful amino acid changes of V174I and T283A in VP1 were identified. Sites of 174 and 283 were located at or

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closed to the surface loops of VP1, which appeared to be a preferred receptor-binding

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site in many picornaviruses and characterized as necessary entry intermediates during

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infection (Belnap et al., 2000; Hewat et al., 2000; Xu et al., 2017). The two changes may affect the binding or attachment of CVA6 to host cells, and they also play

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important roles in the determinants of disease severity. The recently study revealed that

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there were no antigenic differences in change of V174I, but the antigenicities associated

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with T283A in CA6 isolates were still unclear (Mizuta et al., 2019). Additionally, six amino acid changes including V174I and T283A of CA6 VP1 circulating in Guangxi China may be associated with CA6 outbreak both in mild cases and severe cases in 2017 (Chen et al., 2019). Guangxi Province is in southern of China and adjacent to Guangdong Province, there are certain similarities in HFMD outbreak in the two regions. Thus, amino acid changes of VP1 may contribute to the outbreak of severe CA6-associated HFMD in 2017, but future investigation was need.

There were several limitations in this study. As hospitalized patients often accompany 17

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by obvious symptoms and severe complications, which may cause a slight overestimation of the severe rate. Intensive treatment administered during hospitalization might also be a potential impact on the severity of HFMD, and missing data which were not documented in records might hide potential risk factors in severe cases.

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In conclusion, CA6 has become the major pathogen associated both in mild cases and severe cases. Aseptic meningitis and pulmonary oedema were the main severe systemic

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disorders noted. In addition, the CA6 strains circulating in 2017 belonged lineage E2,

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V174I and T283A of VP1 may be associated with the severity of CA6 infection.

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Therefore, the epidemiological data in this study could raise awareness of the clinical

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importance of CA6 infection among practitioners.

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Supplementary data of this article was provided.

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Acknowledgements

This work was supported by the Guangdong Science and Technology Department (grant numbers 2014A020212246, 2016A020218011); and the Guangzhou Science, Technology and Innovation Commission (grant number 201904010452). The authors have no conflicts of interest to disclose. We would like to thank Editage (www.editage.com) for English language editing.

References 18

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Abedi, G.R., Watson, J.T., Pham, H., Nix, W.A., Oberste, M.S., Gerber, S.I., 2015. Enterovirus and Human Parechovirus Surveillance - United States, 2009-2013. MMWR Morb Mortal Wkly Rep 64, 940-943. Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D., Brown, F., 1989. The threedimensional structure of foot-and-mouth disease virus at 2.9 A resolution. Nature 337,

ro of

709-716. Belnap, D.M., McDermott, B.M., Jr., Filman, D.J., Cheng, N., Trus, B.L., Zuccola,

-p

H.J., Racaniello, V.R., Hogle, J.M., Steven, A.C., 2000. Three-dimensional structure

re

of poliovirus receptor bound to poliovirus. Proc Natl Acad Sci U S A 97, 73-78.

lP

Bian, L., Wang, Y., Yao, X., Mao, Q., Xu, M., Liang, Z., 2015. Coxsackievirus A6: a new emerging pathogen causing hand, foot and mouth disease outbreaks worldwide.

na

Expert Rev Anti Infect Ther 13, 1061-1071.

ur

Blomqvist, S., Klemola, P., Kaijalainen, S., Paananen, A., Simonen, M.L., Vuorinen,

Jo

T., Roivainen, M., 2010. Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland. J Clin Virol 48, 49-54. Cabrerizo, M., Tarrago, D., Munoz-Almagro, C., Del Amo, E., Dominguez-Gil, M., Eiros, J.M., Lopez-Miragaya, I., Perez, C., Reina, J., Otero, A., Gonzalez, I., Echevarria, J.E., Trallero, G., 2014. Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain. Clin Microbiol Infect 20, O150-O156.

19

Journal Pre-proof

Centers for Disease, C., Prevention, 2012. Notes from the field: severe hand, foot, and mouth disease associated with coxsackievirus A6 - Alabama, Connecticut, California, and Nevada, November 2011-February 2012. MMWR Morb Mortal Wkly Rep 61, 213-214. Chang, L.Y., Hsia, S.H., Wu, C.T., Huang, Y.C., Lin, K.L., Fang, T.Y., Lin, T.Y.,

1998 to 2002. Pediatr Infect Dis J 23, 327-332.

ro of

2004. Outcome of enterovirus 71 infections with or without stage-based management:

-p

Chang, S.C., Li, W.C., Chen, G.W., Tsao, K.C., Huang, C.G., Huang, Y.C., Chiu,

re

C.H., Kuo, C.Y., Tsai, K.N., Shih, S.R., Lin, T.Y., 2012. Genetic characterization of

lP

enterovirus 71 isolated from patients with severe disease by comparative analysis of complete genomes. J Med Virol 84, 931-939.

na

Chen, K.T., Chang, H.L., Wang, S.T., Cheng, Y.T., Yang, J.Y., 2007. Epidemiologic

ur

features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in

Jo

Taiwan, 1998-2005. Pediatrics 120, e244-e252. Chen, M., Zuo, X., Tan, Y., Ju, Y., Bi, F., Wang, H., Chen, M., 2019. Six amino acids of VP1 switch along with pandemic of CV-A6-associated HFMD in Guangxi, southern China, 2010-2017. J Infect 78, 323-337. Di, B., Zhang, Y., Xie, H., Li, X., Chen, C., Ding, P., He, P., Wang, D., Geng, J., Luo, L., Bai, Z., Yang, Z., Wang, M., 2014. Circulation of Coxsackievirus A6 in handfoot-mouth disease in Guangzhou, 2010-2012. Virol J 11, 157.

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Fujimoto, T., Iizuka, S., Enomoto, M., Abe, K., Yamashita, K., Hanaoka, N., Okabe, N., Yoshida, H., Yasui, Y., Kobayashi, M., Fujii, Y., Tanaka, H., Yamamoto, M., Shimizu, H., 2012. Hand, foot, and mouth disease caused by coxsackievirus A6, Japan, 2011. Emerg Infect Dis 18, 337-339. Hewat, E.A., Neumann, E., Conway, J.F., Moser, R., Ronacher, B., Marlovits, T.C.,

ro of

Blaas, D., 2000. The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view. EMBO J 19, 6317-6325.

-p

Jubelt, B., Lipton, H.L., 2014. Enterovirus/picornavirus infections. Handb Clin

re

Neurol 123, 379-416.

lP

Li, J., Chen, F., Liu, T., Wang, L., 2012. MRI findings of neurological complications in hand-foot-mouth disease by enterovirus 71 infection. Int J Neurosci 122, 338-344.

na

Li, J., Sun, Y., Du, Y., Yan, Y., Huo, D., Liu, Y., Peng, X., Yang, Y., Liu, F., Lin, C.,

ur

Liang, Z., Jia, L., Chen, L., Wang, Q., He, Y., 2016. Characterization of

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Coxsackievirus A6- and Enterovirus 71-Associated Hand Foot and Mouth Disease in Beijing, China, from 2013 to 2015. Front Microbiol 7, 391. Lin, W., Su, Y., Jiang, M., Liu, J., Zhang, Y.Y., Nong, G.M., 2017. Clinical features for 89 deaths of hand, foot and mouth disease in Guangxi, China, 2014. Int J Infect Dis 64, 15-19. Lo, S.H., Huang, Y.C., Huang, C.G., Tsao, K.C., Li, W.C., Hsieh, Y.C., Chiu, C.H., Lin, T.Y., 2011. Clinical and epidemiologic features of Coxsackievirus A6 infection

21

Journal Pre-proof

in children in northern Taiwan between 2004 and 2009. J Microbiol Immunol Infect 44, 252-257. Logotheti, M., Pogka, V., Horefti, E., Papadakos, K., Giannaki, M., Pangalis, A., Sgouras, D., Mentis, A., 2009. Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer

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of 2007. J Clin Virol 46, 270-274. Lu, H.K., Lin, T.Y., Hsia, S.H., Chiu, C.H., Huang, Y.C., Tsao, K.C., Chang, L.Y.,

-p

2004. Prognostic implications of myoclonic jerk in children with enterovirus

re

infection. J Microbiol Immunol Infect 37, 82-87.

lP

Mirand, A., Henquell, C., Archimbaud, C., Ughetto, S., Antona, D., Bailly, J.L., Peigue-Lafeuille, H., 2012. Outbreak of hand, foot and mouth disease/herpangina

na

associated with coxsackievirus A6 and A10 infections in 2010, France: a large

ur

citywide, prospective observational study. Clin Microbiol Infect 18, E110-E118.

Jo

Mizuta, K., Tanaka, S., Komabayashi, K., Aoki, Y., Itagaki, T., Katsushima, F., Katsushima, Y., Yoshida, H., Ito, S., Matsuzaki, Y., Ikeda, T., 2019. Phylogenetic and antigenic analyses of coxsackievirus A6 isolates in Yamagata, Japan between 2001 and 2017. Vaccine 37, 1109-1117. Montes, M., Artieda, J., Pineiro, L.D., Gastesi, M., Diez-Nieves, I., Cilla, G., 2013. Hand, foot, and mouth disease outbreak and coxsackievirus A6, northern Spain, 2011. Emerg Infect Dis 19, 676–678. Nigrovic, L.E., 2013. Aseptic meningitis. Handb Clin Neurol 112, 1153-1156.

22

Journal Pre-proof

Oberste, M.S., Maher, K., Kilpatrick, D.R., Pallansch, M.A., 1999. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 73, 1941-1948. Ooi, M.H., Wong, S.C., Lewthwaite, P., Cardosa, M.J., Solomon, T., 2010. Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol 9, 1097-1105.

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Ooi, M.H., Wong, S.C., Mohan, A., Podin, Y., Perera, D., Clear, D., del Sel, S., Chieng, C.H., Tio, P.H., Cardosa, M.J., Solomon, T., 2009. Identification and

-p

validation of clinical predictors for the risk of neurological involvement in children

re

with hand, foot, and mouth disease in Sarawak. BMC Infect Dis 9, 3.

lP

Osterback, R., Vuorinen, T., Linna, M., Susi, P., Hyypia, T., Waris, M., 2009. Coxsackievirus A6 and hand, foot, and mouth disease, Finland. Emerg Infect Dis 15,

na

1485-1488.

ur

Prager, P., Nolan, M., Andrews, I.P., Williams, G.D., 2003. Neurogenic pulmonary

Jo

edema in enterovirus 71 encephalitis is not uniformly fatal but causes severe morbidity in survivors. Pediatr Crit Care Med 4, 377-381. Puenpa, J., Chieochansin, T., Linsuwanon, P., Korkong, S., Thongkomplew, S., Vichaiwattana, P., Theamboonlers, A., Poovorawan, Y., 2013. Hand, foot, and mouth disease caused by coxsackievirus A6, Thailand, 2012. Emerg Infect Dis 19, 641-643. Richter, J., Koptides, D., Tryfonos, C., Christodoulou, C., 2006. Molecular typing of enteroviruses associated with viral meningitis in Cyprus, 2000-2002. J Med Microbiol 55, 1035-1041.

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Journal Pre-proof

Solomon, T., Lewthwaite, P., Perera, D., Cardosa, M.J., McMinn, P., Ooi, M.H., 2010. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10, 778-790. Tan, X., Li, L., Zhang, B., Jorba, J., Su, X., Ji, T., Yang, D., Lv, L., Li, J., Xu, W., 2015. Molecular epidemiology of coxsackievirus A6 associated with outbreaks of

ro of

hand, foot, and mouth disease in Tianjin, China, in 2013. Arch Virol 160, 1097-1104. Tee, K.K., Lam, T.T., Chan, Y.F., Bible, J.M., Kamarulzaman, A., Tong, C.Y.,

-p

Takebe, Y., Pybus, O.G., 2010. Evolutionary genetics of human enterovirus 71:

re

origin, population dynamics, natural selection, and seasonal periodicity of the VP1

lP

gene. J Virol 84, 3339-3350.

Xing, W., Liao, Q., Viboud, C., Zhang, J., Sun, J., Wu, J.T., Chang, Z., Liu, F., Fang,

na

V.J., Zheng, Y., Cowling, B.J., Varma, J.K., Farrar, J.J., Leung, G.M., Yu, H., 2014.

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Hand, foot, and mouth disease in China, 2008-12: an epidemiological study. Lancet

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Infect Dis 14, 308-318.

Xu, L., Zheng, Q., Li, S., He, M., Wu, Y., Li, Y., Zhu, R., Yu, H., Hong, Q., Jiang, J., Li, Z., Li, S., Zhao, H., Yang, L., Hou, W., Wang, W., Ye, X., Zhang, J., Baker, T.S., Cheng, T., Zhou, Z.H., Yan, X., Xia, N., 2017. Atomic structures of Coxsackievirus A6 and its complex with a neutralizing antibody. Nat Commun 8, 505. Yang, F., Yuan, J., Wang, X., Li, J., Du, J., Su, H., Zhou, B., Jin, Q., 2014. Severe hand, foot, and mouth disease and coxsackievirus A6-Shenzhen, China. Clinical Infectious Diseases 59, 1504-1505.

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Yen, F.B., Chang, L.Y., Kao, C.L., Lee, P.I., Chen, C.M., Lee, C.Y., Shao, P.L., Wang, S.C., Lu, C.Y., Huang, L.M., 2009. Coxsackieviruses infection in northern Taiwan--epidemiology and clinical characteristics. J Microbiol Immunol Infect 42, 38-46. Zeng, H., Huang, W., Wen, F., Wang, Y., Gan, Y., Zeng, W., Chen, R., He, Y., Liu,

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Z., Liang, C., Wong, K.K., 2016. MRI signal intensity differentiation of brainstem encephalitis induced by Enterovirus 71: a classification approach for acute and

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convalescence stages. Biomed Eng Online 15, 25.

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Zeng, H., Lu, J., Yang, F., Liu, L., Zheng, H., Ke, C., Song, T., Li, H., Sun, L.,

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Guangdong Provincial, H.S.G., 2018. The increasing epidemic of hand, foot, and mouth disease caused by coxsackievirus-A6, Guangdong, China, 2017. J Infect 76,

na

220-223.

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Zhang, Y., Zhu, Z., Yang, W., Ren, J., Tan, X., Wang, Y., Mao, N., Xu, S., Zhu, S.,

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Cui, A., Zhang, Y., Yan, D., Li, Q., Dong, X., Zhang, J., Zhao, Y., Wan, J., Feng, Z., Sun, J., Wang, S., Li, D., Xu, W., 2010. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol J 7, 94.

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Figure Legends Figure 1. Distribution of Coxsackievirus A6 (CA6) and non-CA6 associated with HFMD children hospitalized in 2017, Guangzhou, China. During 2017, a total of 358 children with hand-foot-and mouth disease (HFMD) in Guangzhou, China were hospitalized at the Guangdong Women and Children hospital and enrolled. Among

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them, 261(55 severe cases and 206 mild cases) were infected with CA6, and 97 were

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infected with other enteroviruses.

isolates. The CA6

isolates (GZ/CHN/2017/01~

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Coxsackievirus A6 (CA6)

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Figure 2. Neighbor-joining tree of the complete VP1 gene (915 nucleotides) of the

GZ/CHN/2017/16) obtained from those with severe hand-foot-and mouth disease

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(HFMD) in this study were shown in red, and the other isolates (GZ/CHN/2017/17~

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GZ/CHN/2017/32) obtained from the mild disease patients were shown in blue. The

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scale bars indicate the number of nucleotide substitutions per site. Bootstrap values were calculated on 1,000 replicates. Phylogenetic nodes with bootstrap values over 80 were marked as purple lines.

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Supplemental Material Supplemental File 1 Amino acid sequences alignment analysis of the VP1 coding region of Coxsackievirus A6 (CA6) strains. Eighty randomly selected CA6 strains of

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lineage E2 and six representative strains of each CA6 genotypes were evaluated.

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The authors declare no conflict of interest in this study.

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Table 1: Demographic characteristics and clinical features of Coxsackievirus A6associated HFMD Severe HFMD

Total number

N=206

N=55

Age (months)

20.6(7.3~33.8)

19.3(6.7~31.9)

0.520

178(86.4)

53(96.4)

0.05

<36 months

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Mild HFMD

p

113(54.9)

41(74.5)

0.008

Duration of hospitalization (Days)

6.2(4.1~8.3)

9.0(4.2~13.8)

<0.001

Days of fever

2.4(1.1~3.6)

3.1(1.2~4.9)

0.009

1(0.5%)

0(0)

1

46(22.3%)

12(21.8%)

1

159(77.2%)

43(78.2%)

1

100(100%)

100(100%)

N.D.

193(93.7%)

51(92.7%)

0.762

Face

18(8.7%)

3(5.5%)

0.606

Extremities

204(99%)

52(94.5%)

0.065

Trunk

44(21.4%)

10(18.2%)

0.709

Hands and/or feet

191(92.7%)

52(94.6%)

0.772

Decreased oral intake

87(37.4%)

34(61.8%)

0.010

Tonsil enlargement

108(52.4%)

41(74.6%)

0.003

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Body temperature (°C) <37.0

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37.0~38.9

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≥39.0

Oral

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Region with skin rashes/ulcers

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11(5.3%)

8(14.6%)

0.035

Coughing

83(40.3%)

26(47.3%)

0.360

Vomiting

14(6.8%)

18(32.7%)

<0.001

Local lymph node enlargement

2(1.0%)

3(5.5%)

0.065

Pruritus

3(1.5%)

2(3.6%)

0.284

Neurologic complications

0

55(100%)

N.D.

Febrile seizures

0

Headache

0

N.D.

20(36.4%)

N.D.

2(3.6%)

N.D.

29(52.7%)

N.D.

0

2(3.6%)

N.D.

0

1(1.8%)

N.D.

0

6(10.9%)

N.D.

0

brainstem encephalitis

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Aseptic meningitis

Pulmonary edema

38(69.1%)

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Diarrhea

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N.D.: not done; HFMD: hand-foot-and-mouth disease

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Table 2: Laboratory tests results of HFMD patients with Coxsackievirus A6 infection Severe HFMD

Total numbers

N=206

N=55

WBC(4-10×109/L)

14.48(8.24~20.71)

14.73(9.75~19.72)

0.748

RBC(4.0-4.5×109/L)

4.38(3.98~4.78)

4.34(3.91~4.77)

0.548

PLT(125-350×109/L)

336(236~435)

356(258~454)

0.180

ALT(9-40 U/L)

28(2~54)

AST(15-40 U/L)

41(29~53)

Ur(3.1-8.0 mmol/L)

3.6(1.4~5.7)

Cr(25-100 μmol/L)

25.0(18.7~31.4)

UA(208-428 μmol/L) LDH(313-618 U/L)

Blood glucose rising

0.292

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47(17~77)

0.917

285.0(200.0~370.0)

286.3(209.4~363.3)

0.606

594(346~841)

638(218~856)

0.222

120(42~199)

158(113~271)

0.005

126(61.2%)

37(67.3%)

0.437

N=184

N=55

16(8.7%)

21(38.2%)

N=163

N=50

28(17.2%)

26(52.0%)

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Blood glucose

0.390

25.8(17.5~34.0)

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Positive FOBT result

31(14~45)

0.464

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FOBT

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4.0(5.1~9.0)

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CK(40-200 U/L) CRP rising

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Mild HFMD

<0.001

<0.001

HFMD: head-foot-and-mouth disease; FOBT: fecal occult-blood test; PLT: platelet; CRP: C-reactive protein; RBC: red blood cell; WBC: white blood cell; ALT: alanine

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aminotransferase; AST: aspartate aminotransferase; Ur: urea; CR: creatinine; UA: uric acid; LDH: lactate dehydrogenase; CK: creatine kinase.

CA6 has become the major pathogen both in mild and severe HFMD.

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CA6 can lead to severe systemic disorders mainly include aseptic meningitis and pulmonary edema.

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Amino acids changes of VP1, V174I and T283A, may be associated with the severity of CA6 infection.

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These findings could raise awareness of the clinical importance of CA6 infection among practitioners.

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