Arch Virol DOI 10.1007/s00705-014-2067-6

BRIEF REPORT

Genetic characterization of emerging coxsackievirus A12 associated with hand, foot and mouth disease in Qingdao, China Xiaolin Liu • Naiying Mao • Weisen Yu • Qing Chai • Hui Wang • Weidong Wang • Lijuan Wang • Zhaoguo Wang • Wenbo Xu

Received: 27 December 2013 / Accepted: 22 March 2014 Ó Springer-Verlag Wien 2014

Abstract To characterize the genetic properties of coxsackievirus A12 (CVA12) strains isolated from hand, foot and mouth disease (HFMD) patients in Qingdao during 2008-2011, the complete genome and VP1 coding region were sequenced and analyzed. Phylogenetic analysis showed that all strains from China clustered into three different branches, suggesting multiple lineages of CVA12 co-circulating in Asia. Sequence analysis indicated a monophyletic group only when the P1 region was examined, indicating possible recombination between CVA12 and other HEV-A serotypes. The emergence of CVA12 involved in an HFMD outbreak in China is a public-health issue.

Hand, foot and mouth disease (HFMD) has remained a significant public-health challenge in the Western Pacific Region in recent years, especially in Southeast Asian countries [1–3]. In 2012, more than 2 million HFMD infections, and over five hundred deaths, were reported in mainland China [4]. HFMD is caused by human enteroviruses (HEVs), which are members of the family X. Liu and N. Mao contributed equally to this work. X. Liu  W. Yu  Q. Chai  H. Wang  W. Wang  L. Wang  Z. Wang (&) Qingdao Center for Disease Control and Prevention, Qingdao 266033, Shandong, People’s Republic of China e-mail: [email protected] N. Mao  W. Xu (&) National Measles Laboratory, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Room 607, Beijing 102206, People’s Republic of China e-mail: [email protected]

Picornaviridae and is most commonly associated with enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) [5]. Although EV71 and CVA16 are the major causative agents of HFMD and are distributed widely, other HEVs, including CVA2, 4, 5, 6, 9 and 10, have been associated with HFMD outbreaks [6–9]. However, the pathogen spectrum of HFMD and the circulation of HEVs have not been fully investigated because of the lack of molecular diagnostic methods in most clinical virology laboratories in China. Qingdao is a coastal city located on the southern tip of the Shandong Peninsula, and severe HFMD epidemics have persisted there since 2007. From 2008 to 2011, a total of 4,806 clinical specimens from the HFMD surveillance system were screened by real-time RT-PCR for HEVs as described previously [10]. The results revealed that EV71 and CVA16 were the predominant HEVs for HFMD epidemics in Qingdao and accounted for 48.6 % and 22.5 %, respectively, of the total viral agents. However, for HFMD cases not associated with EV71 or CVA16, molecular genotyping revealed that coxsackievirus A12 (CVA12) was one of most frequently identified enteroviruses (Table 1). Twenty stool specimens typed as CVA12 positive were then inoculated to two cell lines; human rhabdomyosarcoma (RD) and human larynx carcinoma (HEp-2) cells. In total, six CVA12 strains were isolated from patients under 5 years old and stored at -70 °C. We used the specific primer pair CVA12-2406S (50 -CTCAAATTGTGCCG GGACACAGAATC-30 ) and CVA12-3351Q (50 -TTCAC CACTCTGTAATTTCCCACAT-30 ) to amplify the complete VP1 genome region. The complete VP1 sequences (888 nt in length) of six Qingdao CVA12 isolates were sequenced. Based on the genetic divergence of the VP1 gene (Fig. 1), two strains, QD-LXH535/SD/CHN/2009 and QDHDH507/SD/CHN/2011 were selected as representative

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X. Liu et al. Table 1 Serotype distribution of non-HEV71 and non-CVA16 HEV in 50 PCR-positive HFMD specimens during 2008-2011 in Qingdao

HEV

2008

2009

2010

2011

CVA4

-

-

2

-

2

CVA5

1

-

-

-

1

CVA6

2

-

3

-

5

CVA9

-

1

-

-

1

CVA10

1

-

12

-

13

CVA12

2

11

-

9

22

CVB2

-

1

-

1

2

CVB4

-

-

2

-

2

Echovirus 9

2

-

-

-

Total

8

13

19

10

Fig. 1 (a) Phylogenetic analysis based on partial VP1 gene sequences (275 bp) of Qingdao strains and other strains of CAV12 from different geographic origins. (b) Phylogenetic analysis based on complete VP1 gene (888 bp) sequences of two Qingdao strains and prototype strain Texas-12. The neighbor-joining method was used to

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Total

2 50

construct the trees. Numbers at the nodes represent the percentage of 1,000 bootstrap pseudoreplicates. The symbol d indicates Qingdao CVA12 isolates, and m indicates the prototype strain Texas-12. The scale bar is in units of nucleotide substitutions per site

Coxsackievirus A12 associated with HFMD in China Table 2 Nucleotide sequences of primers for amplification of the whole genome sequence of CVA12. The positions of the primers are relative to the genome of CVA12 prototype strain Texas-12 (AF081302.1) Primer

Position (nt)

0001S48

Sequence (50 -30 )

Orientation

GGGGACAAGTTTGTACAAAAAAGCA-GGCTTTAAAACAGCTCTGGGGTT

Forward

CVA12-943A

943-962

CCTCCGCACTAGGTGACTTC

Reverse

CVA12-890S

871-890

GGATTTCACCCAAGATCCAA

Forward

CVA12-1772A

1772-1791

CCAGGTAAAATGGGAGCTGA

Reverse

CVA12-1650S

1631-1650

GCCACAACAGCCATACCAAT

Forward

CVA12-2677A

2277-2296

CCAAGCCTGATCGAGAGAAG

Reverse

CVA12-2511S CVA12-3599A

2492-2511 3599-3618

ACCACTCAAACCCACCAAAC AGCATGAGGTGGGATTGGTA

Forward Reverse

CVA12-3464S

3445-3464

GTCATCCACGACAGCTCAAG

Forward

CVA12-4555A

4536-4555

GTGGTCTGGGTCTGGAGGTA

Reverse

CVA12-4462S

4443-4462

TCATCAGAGGCTCTCCAGGT

Forward

CVA12-5547A

5547-5566

CACCAATTCTACAGCGTCCA

Reverse

CVA12-5405S

5384-5405

CCTCGATTTCGCTCTGTCTC

Forward

CVA12-6373A

6373-6392

TGGAGTAGGGAAGGTCCAAA

Reverse

CVA12-6213S

6194-6213

ATCGACACCTCCCAGATGAG

Forward

CVA12-7146A

7146-7165

GTCTTGAGTGTTGCGTGCAT

Reverse

CVA12-6884

6865-6884

ATTCAAGGGCATTGATCTGG

7500A

GGGGACCACTTTGTACAAGAAAGCTGGG(T)

Forward 24

Reverse

Table 3 Clinical information for Qingdao CVA12 strains isolated from HFMD patients Lab no.

Gender

Age (years)

Date of onset

Date of sample collection

Sample

GenBank accession no.

QD-LXH535

Male

4

25-4-2009

28-4-2009

Stool

KF422142

QD-HDH507

Male

4

11-5-2011

14-5-2011

Stool

KF422143

QD-PDHS345

Female

4

16-5-2011

18-5-2011

Stool

KF422144

QD-JZH622

Male

3

8-6-2011

8-6-2011

Stool

KF422145

QD-SFHS189

Male

5

21-6-2011

21-6-2011

Stool

KF422146

QD-CYHS249

Female

3

30-6-2011

2-7-2011

Stool

KF422147

strains for further genetic characterization by sequencing the entire genome using laboratory-designed primers (Table 2). PCR products were sequenced in both directions to resolve possible sequence ambiguities. The two complete genome sequences, with a length of 7396 nt, and four complete VP1 nucleotide sequences of Qingdao CVA12 strains were deposited in the GenBank database under accession numbers KF422142-47 (Table 3). Alignment of the nucleotide sequences of CVA12 strains was performed using BioEdit sequence alignment editor software (Version 7.0.9; Tom Hall, North Carolina State University, Raleigh, NC, USA) [13]. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 [14]. After alignment, similarity plot and bootscan analyses were performed using the Simplot program (Version 3.5.1; Stuart Ray, Johns Hopkins University, Baltimore, MD, USA) [15].

The sequences of the VP1 coding region of six Qingdao strains displayed a relatively high level of similarity to each other at both the nucleotide and amino acid level (96.6 %-100 % and 98.3 %-100 %, respectively), and only 81.1 %-82.0 % nucleotide and 94.9 %95.2 % amino acid sequence identity to the prototype strain Texas-12 was observed. Phylogenetic analysis based on a partial sequence of VP1 region revealed that all CVA12 strains can be divided into four clusters (A, B, C and D), with at least 6.3 % diversity between clusters (Fig. 1). Qingdao strains were grouped into cluster D, while 18 other strains from China, isolated from 2006 to 2011, were distributed into three distinct clusters (B, C, and D) in the phylogenetic dendrograms, implying multiple transmission chains of CVA12 in mainland China. Additionally, one Chinese strain (PUMCH3700) was grouped together with three Japanese

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Fig. 2 Unrooted trees representing the phylogenetic relationships among Qingdao CVA12 strains and other HEV-A prototype strains. Phylogenetic trees based on nucleotide sequences of the 50 UTR (a), P1 gene (b), P2 gene (c), and P3 gene (d) were constructed from the nucleotide sequence alignment using the neighbor-joining algorithm

of the MEGA 5.0 software. Numbers at nodes indicate bootstrap support for that node (percent of 1000 bootstrap pseudoreplicates). The scale bar represents the genetic distance, and all unrooted trees have the same scale. The scale bar is in units of nucleotide substitutions per site

strains in cluster C, suggesting a possibility of transmission between countries. Genomic recombination plays a critical role in the evolution of a given enterovirus serotype [16]. To investigate the genetic relationships and possible recombination between the Qingdao strains and other prototype HEV-A strains whose sequences are available in GenBank, phylogenetic trees based on the 50 UTR, P1, P2, and P3 regions of the genome were constructed (Fig. 2). The phylogenetic tree of the P1 region showed that Qingdao strains clustered in the same group with the CVA12 prototype Texas-12 strain, with 80.8 % in nucleotide sequence identity and were segregated from the other prototype HEV-A strains,

with 62.8 %-71.4 % nucleotide sequence identity. The Qingdao strains grouped together with CVA4, CVA5, CVA14 and CVA16 in the coding region for the nonstructural proteins P2 and P3. When combined with similarity plot and bootscan data (Fig. 3), this showed that CVA12 strains had a monophyletic pattern only in the P1 region, indicating possible recombination between CVA12 and other HEV-A types. CVA12 belongs to species Enterovirus A, together with 16 other serotypes and is rarely reported [11]. The prototype strain (Texas-12) was first isolated in the United States in 1948, and its complete genome sequence is the only one available in the GenBank database [12]. There are limited

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Coxsackievirus A12 associated with HFMD in China

Fig. 3 Nucleotide sequence recombination analysis of the whole genome of Qingdao CVA12 using the software Simplot. The structural organization (A), similarity plots (B), and bootscan analysis (C) of complete HEV-A genomes are shown. The QD-HDH507/SD/ CHN/2011 isolate was used as a query sequence. The following default parameter settings for the SimPlot software were used for

analysis: window size, 200 nucleotides (nt); step size, 20 nt; replicates used, n=100; gap stripping, (on); distance model, Kimura 2-parameter; tree model, neighbor-joining. For each bootscan analysis, the name of the virus from which the query sequence was obtained is indicated in the box

numbers of CVA12 sequences available in the GenBank database. The CVA12 strains were isolated only from three countries during 1948-2011, including the USA, Japan and China, indicating either that CVA12 infection may have a limited geographical distribution, especially in Asia, or that it is only involved in mild infections. CVA12 was continuously circulating in mainland China and frequently

isolated from acute respiratory infection and HFMD patients during 2006-2011. It is not known whether CVA12 plays an important role in the pathogenesis of the HFMD outbreak in China and other Asian countries due to the lack of HEV surveillance data. However, CVA12 is still considered an important emerging pathogen causing HFMD because of its potential to recombine with CVA16, EV71

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and other HEV-A serotypes as a result of co-infection during an HFMD epidemic. This is the first report to describe the phylogeny of CVA12 based on the complete VP1 gene and whole genome sequences. Our comprehensive study on the complete genome of CVA12 demonstrates the need for etiological diagnosis and surveillance of HFMD in the world, especially in countries with a high HFMD disease burden. Acknowledgments This study was supported by the National Infectious Disease Surveillance Program of the Ministry of Science and Technology of the People’s Republic of China (project no. 2012ZX10004201-003 and 2013ZX10004202), and the National Science and Technology Major Project for Creation of Major New Drugs (project no. 2013ZX09304101-006),and HFMD Surveillance Program of Qingdao Municipal Science and Technology Bureau (project no.08-2-1-1-nsh). We thank Dr. Dustin Yang for his critical comments and excellent editorial assistance.

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