Journal of Medical Microbiology (2014), 63, 1356–1362
DOI 10.1099/jmm.0.079699-0
Molecular epidemiology of enterovirus 71 strains isolated from children in Yamagata, Japan, between 1990 and 2013 Katsumi Mizuta,1 Yoko Aoki,1 Yohei Matoba,1 Kazue Yahagi,1 Tsutomu Itagaki,2 Fumio Katsushima,3 Yuriko Katsushima,3 Sueshi Ito,4 Seiji Hongo5 and Yoko Matsuzaki5 Correspondence Katsumi Mizuta [email protected]
1
Department of Microbiology, Yamagata Prefectural Institute of Public Health, Yamagata, Yamagata, Japan
2
Yamanobe Pediatric Clinic, Yamanobe, Yamagata, Japan
3
Katsushima Pediatric Clinic, Yamagata, Yamagata, Japan
4
Department of Pediatrics, Shonai Hospital, Tsuruoka, Yamagata, Japan
5
Department of Infectious Diseases, Yamagata University Faculty of Medicine, Yamagata, Yamagata, Japan
Received 11 June 2014 Accepted 20 July 2014
Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.
INTRODUCTION Enterovirus 71 (EV71) is a small, single-stranded, positivesense non-enveloped RNA virus belonging to the genus Enterovirus within the family Picornaviridae (McMinn, 2002; Solomon et al., 2010). Together with coxsackievirus A16 (CVA16), CVA10 and other CVA viruses, EV71 is known to be a causative agent of hand-foot-and-mouth disease (HFMD) (McMinn, 2002; Solomon et al., 2010). Although HFMD is typically a self-limiting illness in children, EV71 infection sometimes causes neurological manifestations such as brainstem encephalitis, and fatal cases in infants and children have been reported in the 20th–21st century, mainly across the Asia-Pacific region (McMinn, 2002; Solomon et al., Abbreviations: CVA, coxsackievirus A; EV71, enterovirus 71; HFMD, hand-foot-and-mouth disease. The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are AB936432–AB936561.
1356
2010). For example, in an outbreak of HFMD in Shangdong Province, China, in 2007, 1149 cases were reported, among which 11 children (0.96 %) presented with neurological complications and three died (mortality rate 0.26 %) (Zhang et al., 2009). EV71 infection has since become a major public issue in this area and there has been notable progress in studies of its epidemiology, identification of receptors, development of antiviral agents and vaccine development, all of which are necessary to control and ameliorate the severity of the disease (Liang et al., 2013; McMinn, 2002; Solomon et al., 2010; Wu et al., 2001; Zhu et al., 2013). Several epidemiological studies have clarified that EV71 is an active circulating agent capable of rapid evolution into several different genogroups and that EV71 consists of 14 subgenogroups (A, B1–5, C1–5, D, E and F), although no relationship between severity and genogroup has yet been confirmed (Liang et al., 2013; McMinn, 2002; Singh et al., 2002a; Solomon et al., 2010; Bessaud et al., 2014). The 079699 G 2014 The Authors
Printed in Great Britain
Longitudinal molecular epidemiology of enterovirus 71
development of a vaccine against EV71 has been pursued as a preventive measure and the main target region for vaccine development has been the VP1 region, which was defined as the neutralization determinant (McMinn, 2002; Oberste et al., 1999; Wu et al., 2001). As the number of studies involving antigenic analysis and measurement of neutralizing antibody against different subgenogroups targeting the VP1 region has been limited, we carried out a molecular epidemiological study of EV71 strains isolated in Yamagata, Japan, between 1990 and 2007 (Mizuta et al., 2009). Our results from a sequence analysis of the VP1 region, genotyping, a seroepidemiological survey and an antigenic analysis suggest that there is cross-antigenicity among the different EV71 subgenogroups circulating in the community and that it might be possible to prevent severe illnesses due to EV71 infections through the development of a vaccine that effectively induces neutralizing antibodies against EV71, as has been shown with the measles virus vaccine (Mizuta et al., 2009). In reality, studies related to vaccine development have suggested cross-protection among different EV71 subgenogroups, and it is recommended that future research focuses on strengthening the monitoring of prevalent strains and strain variations to check the cross-protective effects of vaccines, particularly if
genogroup replacement has occurred (Arita et al., 2007; Liang et al., 2013; Solomon et al., 2010). Furthermore, good surveillance programmes are needed in many different geographical regions to provide accurate and relevant information about EV71 transmission and evolution (Solomon et al., 2010). We have further analysed the EV71 Yamagata isolates, and here describe the longitudinal molecular epidemiology of EV71 between 1990 and 2013.
METHODS Virus isolation and identification were carried out by means of a microplate method using nasopharyngeal samples from children with HFMD, viral exanthematous disease or respiratory illnesses, as described previously (Mizuta et al., 2005, 2008, 2009). EV71 strains were isolated using human embryonic lung fibroblast (HEF), Vero, Vero E6, human rhabdomyosarcoma (RD)-18S and green monkey kidney (GMK) cell lines at the Virus Research Center, National Hospital Organization, Sendai Medical Center, Sendai, Japan, between 1990 and 1997, and thereafter at the Department of Microbiology, Yamagata Prefectural Institute of Public Health. The isolates were identified as EV71 based on a neutralization method. Sequence analysis for the complete VP1 region (891 nt) of the EV71 isolates was carried out as described previously (Mizuta et al., 2005, 2009), except that we also used primers 222 and R2 (Oberste et al., 2000; Singh et al., 2002b). Sequence data for the isolates from Yamagata had the following GenBank accession
20
Number of isolates
B2 B4
15
B5 C1
10
C2 C4
5
ND
0 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001 B2
20
Number of isolates
B4 B5
15
C1 C2
10
C4 ND
5
0 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Fig. 1. Monthly distribution (odd months numbered) and subgenogroups of EV71 strains isolated in Yamagata, Japan, between 1990 and 2013. The subgenogroups, C1, C2, B2, B4, C4 and B5, were grouped according to the phylogenetic analysis shown in Fig. 2. ND, Not done. http://jmm.sgmjournals.org
1357
K. Mizuta and others
BrCr-CA-70 U22521 81 100
100
100
99
99
99
0.02
100 99
100
1358
2604-AUS-74 AF135883 2232-NY-77 AF135871 7633-PA-87 AF009534 7673-USA-87 AF009535 Y90-3205 AB433863 Y93-2008 AB433866 MY16/1/SAR/97 AF376073 4350/SIN/98 AF376119 5536/SIN/00 AF376122 S21082/SAR/00 AF376084 738-Yamagata-00 AB213620 1067-Yamagata-00 AB213625 S110031-SAR-03 AY258307 S19741-SAR-03 AY258313 2419-Yamagata-03 AB213647 2542-Yamagata-03 AB177815 2950-Yamagata-2009 AB936506 1749-Yamagata-2012 AB936545 387-Yamagata-2013 AB936555 1499-Yamagata-2013 AB936557 2419-Yamagata-2012 AB936553 799-Yamagagta-2013 AB936556 1615-Yamagata-2012 AB936541 111-Yamagata-2013 AB936540 1701-Yamagata-2012 AB936531 1518-Yamagata-2012 AB936525 1842-Yamagata-2012 AB936536 MAD-72341-04 HG421068 1116-Yamagata-00 AB177810
Y97-865 AB433867 Y97-1134 AB433869 03750-MAA-97 AY207615 Y97-1055 AB433868 671-Yamagata-98 AB213614 705-Yamagata-98 AB213615 932-Yamagata-99 AB213616 1002-Yamagata-99 AB213619 2M-AUS-3-99 AF376103 NL3652000 AB491218 EP/5622/1999UK AM939583 CF1113FRA2000 FN598726 EP/7414/1999UK AM939585 ANG261FRA09 FN598777 NAN257FRA08 FN598776 NL2652008 AB491220 3246-Yamagata-2009 AB936502 2534-Yamagata-2009 AB936505 1613-Yamagata-2010 AB936520 1108-Yamagata-2010 AB936515 520-Yamagata-2010 AB936511 148-Yamagata-2010 AB936507 3215-Yamagata-2009 AB936501 OC091495-10 AB665731 OC091494-10 AB665730 1825-Yamagata-2009 AB936494 NL1550 AB524223 BRE324FRA07 FN598762 1416-FR-2007 FJ824734 NL07/ns/W/07 HQ676273 NL03/ns/W/07 HQ676270 NL25272007 AB491219 2008-Yamagata-2010 AB936523 813-Yamagata-2013 AB936558 1437-Yamagata-2013 AB936561 857-Yamagata-2013 AB936559 933-Yamagata-2013 AB936560 100 001-KOR-00 AY125966 013-KOR-00 AY125976 Y90-3761 AB433864 Y90-3896 AB433865 Y90-2913 AB433862 2246-NY-87 AF009542 NL11341991 AB491216 S11051/SAR/98 AF376081 EP/2063/2001UK AM939577 NL38782002 AB491217 EP/17728/1998UK AM939593 LYO28978FRA99 FN598721 CF1744FRA2000 FN598727 3254-TAI-98 AF286531 F2-CHN-00 AB115491 75-Yamagata-03 AB177813 452-Yamagata-03 AB213630 2779-Yamagata-02 AB213629 2321-Yamagata-03 AB213645 CF192013FRA2004 FN598741 ZJ-CHN-6-03 AY905619 082/NX/CHN/2008 HM212460 2147-Yamagata-06 AB433871 2928-Yamagata-06 AB433878 2753-Yamagata-06 AB433877 56-Yamagata-07 AB433879 1693-Yamagata-07 AB433886 1758-Yamagata-07 AB433889 1897-Yamagata-07 AB433891 100 1089T/VNM/05 AM490142 1301V/VNM/05 AM490149 CAF-NMA-03-008 JN255590 C08-146 JX307649
A B1 B2 B3 B4
B5
F
C2
C3
C1
C4
C5 E
Journal of Medical Microbiology 63
Longitudinal molecular epidemiology of enterovirus 71
3
Fig. 2. Phylogenetic tree for the complete (891 nt) VP1 region of representative enterovirus 71 (EV71) strains isolated in Yamagata, Japan, between 1990 and 2013 and reference strains. Subgenogroups B5, C2 and C4 strains repeatedly appeared in Yamagata with genomic variations with several years’ interval as shown by the different levels of grey shading. Yamagata and and 7, respectively. Branch lengths are proportional to the number of nucleotide European strains are indicated by differences. Numbers above the branches are the bootstrap probabilities (%). Bar, measurement of relative phylogenetic distance (0.02 nucleotide substitutions per site).
numbers: AB177809–AB177816, AB213614–AB213650 and AB433862– AB433892. Sequence data were analysed with CLUSTAL W (version 1.83), and a phylogenetic tree was reconstructed by the neighbour-joining method using the same software (Saitou & Nei, 1987).
A total of 240 EV71 strains were isolated in Yamagata between 1990 and 2013. The number of EV71 strains isolated in Yamagata by month is shown in Fig. 1. Although we did not measure the neutralizing titres for EV71 isolates after 2007, these isolates were neutralized with the identical antisera used in the previous study (Mizuta et al., 2009). A total of 223 of the 240 isolates were used for sequence analysis, and representative Yamagata strains were used for further phylogenetic analysis. Available EV71 strains that had not yet been typed in previous studies (Mizuta et al., 2005, 2009) were also sequenced in this study.
respectively. In 1990, three isolates were identified as belonging to subgenogroup C1 and one isolate was identified as belonging to B2. In 1993, one strain was identified as B2. Between 1997 and 1999, all analysed strains were typed as C2. In 2000, all analysed strains, except for one strain isolated in September and identified as C2, were typed as B4. All four isolates in 2001 were again typed as C2. Isolates between October 2002 and August 2003 were typed as C4, except for one strain isolated in June 2003 and identified as B5. C4 and B5 strains co-circulated in September, with only B5 observed thereafter until November 2003. All isolates between 2006 and 2007 were again typed as C4. All isolates, except for one strain (2950-Yamagata-2009 typed as B5), between 2009 and 2010 were typed as C2. The strain 2950-Yamagata-2009 was the only isolate from the Shonai area, which is located on the Japan Sea in Yamagata, whereas the other strains isolated between 2009 and 2010 were from inland areas. In 2012, all isolates were typed as B5, and B5 and C2 strains co-circulated in 2013.
The phylogenetic tree for the VP1 region and the monthly distribution of subgenogroups are shown in Figs 1 and 2,
C2, C4 and B5 strains appeared repeatedly in Yamagata. C2 strains with 93–100 % nucleotide similarity to each other
RESULTS
Australia Singapore Malaysia Taiwan China Yamagata, Japan France Netherlands Germany UK
Australia Singapore Malaysia Taiwan China Yamagata, Japan France Netherlands Germany UK
1990 1991 1992 C1 C1 C1
B2,C1 C1
2002 C1 C1,B4 C1 B4 C4 C4
1994 1995 1996 C1 C1,C2 C1
B2 C1
C1 C1
C1
2003 2004 C1 C4
2005
2006
C1
2007
1997
1998 C1 B3,B4 B3,C1 B3,B4,C1,C2 C1 B4,C2 C4 C2 C2 C1,C2 B2
2008
2009
B2 C1 2010
1999 B3,C2 B3 B4,C1 B4
2000 B4,C1 B4 B4,C1 B4 C4 C2 B4,C2 C1 C1,C2 C2 C2 C1 C1,C2 C1 2011
2012
2001 B4 B4 B4,C4 C4 C2 C1 C1 C1 2013
B5,C2 B5,C1 B4 C4 C4,B5 C1
C1,C2 C1 C1
1993
C4
C4
C5
B5,C5 C4 C4 C4 C1,C4 C1 C1,C2 C2 C1,C2 C1,C2 C1,C2 C4 C2 C2 C1 C1 C1,C2
B5,C4,C5 C4 C2 C2
B5,C2 C2
C2
B5
B5,C2
Fig. 3. Summary of EV71 genotypes circulating in the Asia-Pacific region (grey shading) and Europe since 1980 based on data from the following references: Bible et al. (2008); Cardosa et al. (2003); Diedrich et al. (2009); Herrero et al. (2003); Huang et al. (2008a, b, 2010); Kung et al. (2007); Ortner et al. (2009); Podin et al. (2006); Sanders et al. (2006); Schuffenecker et al. (2011); Wu et al. (2010); Van der Sanden et al. (2009); Zhang et al. (2009). http://jmm.sgmjournals.org
1359
K. Mizuta and others
circulated every year between 1997 and 2001, and again appeared between 2009 and 2010 with 94–100 % similarity. The nucleotide similarity between C2 strains from 1997 to 2001 and those from 2009 and 2010 was 90–95 %. C4 strains circulated between 2002 and 2003 with 98–100 % similarity, and then disappeared and reappeared between 2006 and 2007 with 97–100 % similarity. The nucleotide similarity between C4 strains from 2002 to 2003 and those from 2006 to 2007 was 95–96 %. B5 strains replaced C4 strains in September 2003 and circulated for 3 months with 98–100 % similarities and then disappeared. Only one B5 strain was isolated in November 2009 and there was another outbreak of B5 strains between 2012 and 2013 with 95–100 % similarity. The nucleotide similarity was 96, 94– 95 and 94–95 % between the B5 strains from 2003 and the one from 2009, the one from 2009 and those from 2012– 2013, and those from 2003 and those from 2012–2013, respectively.
DISCUSSION We showed the temporal distribution of EV71 subgenogroups over a 24-year period from 1990 to 2013, in Yamagata, Japan. Apart from a study undertaken in Sydney, Australia, between 1983 and 2011, to the best of our knowledge no such longitudinal analysis of the VP1 region of EV71 strains circulating in a local community has been reported (Sanders et al., 2006). Our longitudinal studies revealed several interesting epidemiological features, as reported previously based on the data obtained between 1990 and 2007 (Mizuta et al., 2005, 2009), and we further found several new findings in the last 6-year observation period. We found that six subgenogroups, B2, B4, B5, C1, C2 and C4, appeared one after another or concurrently during the study period. C1 and B2 disappeared by 1993 in Yamagata and these subgenogroup strains have not yet reappeared. However, the three other subgenogroups, excluding B4, have repeatedly appeared in Yamagata during the study period. Based on the data between 1990 and 2007 (Figs 1 and 2) (Mizuta et al., 2009), we previously reported that genogroup C strains circulated for longer periods with genomic variations, whereas genogroups B strains appeared for only one season. We observed genogroups B strains, B4 and B5, for only 5 and 6 months in 2000 and 2003, respectively, whereas C2 and C4 strains circulated for 5–6 years despite breaks (Mizuta et al., 2009). However, in this study, after the disappearance of B5 strains in 2003, we subsequently observed one B5 strain in 2009 and another outbreak of B5 strains between 2012 and 2013. Thus, these results suggested that not only genogroup C strains but also genogroup B strains have the potential to circulate repeatedly with active genomic variations. In Japan, the genotyping of EV71 strains has been reported recently based on partial VP1 and/or VP4-2 sequence 1360
analysis. Momoki et al. (2009) reported that one and three isolates between September and December in 2008 belonged to C2 and B5, respectively, and they suggested that, in 2008, C2 replaced C4, which had been in circulation between 2005 and 2007 in Yokohama City. Hosomi et al. (2010) reported that four strains in Kochi in 2010 belonged to C2 and Nakata et al. (2012) found that there was an outbreak of HFMD due to C2 strains in Osaka in 2010. Two isolates (OC091494 and OC091495) from Osaka City in 2010 showed 99 % sequence similarity with Yamagata isolates in 2010, apart from a 96 % similarity between the Osaka isolates and 2008-Yamagata-2010 (Fig. 2). Katsumi et al. (2013) reported the isolation of seven B5 strains and one C2 strain in 2012 in Sendai City. Kodama et al. (2013) reported that seven strains isolated in Ishikawa Prefecture in 2010 belonged to C2, and 14 strains isolated between 2012 and 2013 belonged to B5, although the investigation was carried out based on VP4–VP2 analysis. Taken together, these observations and our data from Yamagata suggest that the predominant subgenogroup changed from C4 to C2 across a wide area of Japan between 2007 and 2009. B5 strains probably caused a nationwide epidemic between 2012 and 2013, as reported from Sendai and Ishikawa (Katsumi et al., 2013; Kodama et al., 2013). Recent reports have also suggested that identical predominant subgenogroup strains have been in simultaneous circulation all over Japan. As we reported previously, the fact that the detected subgenogroups in Yamagata (B2, B4, B5, C1, C2 and C4) were also found in the Asia-Pacific region suggests the active circulation of EV71 in this area (Bible et al., 2008; Cardosa et al., 2003; Diedrich et al., 2009; Herrero et al., 2003; Huang et al., 2008a, b, 2010; Kung et al., 2007; Mizuta et al., 2005, 2009; Ortner et al., 2009; Podin et al., 2006; Sanders et al., 2006; Schuffenecker et al., 2011; Wu et al., 2010; van der Sanden et al., 2009; Zhang et al., 2009) (Fig. 3). On the other hand, several European countries, such as France, the Netherlands, Germany and the UK, recently reported the molecular epidemiology of EV71 (Bible et al., 2008; Diedrich et al., 2009; Schuffenecker et al., 2011; van der Sanden et al., 2009). When we compare the subgenogroups circulating in Asia-Pacific region and those in Europe, we can find several differences: (i) there was a co-circulation of genogroup B and C strains in the Asia-Pacific region throughout almost the entire study period, whereas only genogroup C strains were in circulation in Europe (except for subgenogroup B2 strains found in Germany between 1997 and 1998); (ii) B3, B4 and B5 strains have been reported only in the Asia-Pacific region; (iii) genogroup C, C1, C2 and C4 strains circulated as major subgenogroups in the Asia-Pacific region, whereas C1 and C2 strains were predominant in Europe between 1990 and 2009; and (iv) subgenogroup C5 strains are only observed in the Asia-Pacific region (Fig. 3). These observations suggest that EV71 circulates mainly in the Asia-Pacific region and in Europe, although this idea does not completely exclude the possible introduction of EV71 from Europe to Asia-Pacific region and vice versa. It is conceivable that the reappearance Journal of Medical Microbiology 63
Longitudinal molecular epidemiology of enterovirus 71
of C2 in Yamagata in 2009 might be the result of the introduction of EV71 from Europe or Singapore (Figs 1–3). Several capsid residues of EV71 that alter infectivity in the mouse model have been identified. For example, a novel lysine-to-glutamic acid substitution at position 244 in VP1 of B5 strain is critical for mouse adaptation and virulence, and a single mutation from glutamine to glutamic acid at residue 145 in VP1 of C4 strain generates a mouse-virulent phenotype (Zaini & McMinn, 2012; Zaini et al., 2012a). In Yamagata, all B5 strains had lysine at position 244, whereas all C4 strains had glutamic acid at residue 145 during the study period. It will also be necessary to clarify mechanisms of severity and pathogenicity in EV71 infections. Finally, to resolve the public health issue of EV71, we have to continue to analyse the longitudinal epidemiology of EV71 in local communities such as Yamagata, as well as on regional and global levels.
epidemiological and virological features. Scand J Infect Dis 40, 571– 574. Huang, Y. P., Lin, T. L., Kuo, C. Y., Lin, M. W., Yao, C. Y., Liao, H. W., Hsu, L. C., Yang, C. F., Yang, J. Y. & other authors (2008b). The
circulation of subgenogroups B5 and C5 of enterovirus 71 in Taiwan from 2006 to 2007. Virus Res 137, 206–212. Huang, Y. P., Lin, T. L., Hsu, L. C., Chen, Y. J., Tseng, Y. H., Hsu, C. C., Fan, W. B., Yang, J. Y., Chang, F. Y. & Wu, H. S. (2010). Genetic
diversity and C2-like subgenogroup strains of enterovirus 71, Taiwan, 2008. Virol J 7, 277. Katsumi, M., Chida, K., Niiki, Y., Sekine, M., Oguro, M., Kobayashi, M., Hasegawa, S. & Kayaba, J. (2013). Enteroviruses detected from
patients with hand-foot-mouth disease and herpangina in Sendai, Japan in 2012. Infect Agents Surv Rep 34, 9–10 (in Japanese). Kodama, H., Nariai, E. & Sakigawa, Y. (2013). Enteroviruses detected
from patients with hand-foot-mouth disease and herpangina between January 2010 and March 2013 in Ishikawa, Japan. Infect Agents Surv Rep 34, 202–204 (in Japanese). Kung, S. H., Wang, S. F., Huang, C. W., Hsu, C. C., Liu, H. F. & Yang, J. Y. (2007). Genetic and antigenic analyses of enterovirus 71 isolates
in Taiwan during 1998–2005. Clin Microbiol Infect 13, 782–787.
ACKNOWLEDGEMENTS We thank the medical staff and people of Yamagata Prefecture for their collaboration in specimen collection for the National Epidemiological Surveillance of Infectious Diseases, Japan. This work was partly supported by a grant from the Association for Research on Lactic Acid Bacteria. The authors declare they have no conflicts of interest.
Liang, Z. L., Mao, Q. Y., Wang, Y. P., Zhu, F. C., Li, J. X., Yao, X., Gao, F., Wu, X., Xu, M. & Wang, J. Z. (2013). Progress on the research and
development of inactivated EV71 whole-virus vaccines. Hum Vaccin Immunother 9, 1701–1705. McMinn, P. C. (2002). An overview of the evolution of enterovirus 71
and its clinical and public health significance. FEMS Microbiol Rev 26, 91–107.
REFERENCES
Mizuta, K., Abiko, C., Murata, T., Matsuzaki, Y., Itagaki, T., Sanjoh, K., Sakamoto, M., Hongo, S., Murayama, S. & Hayasaka, K. (2005).
Arita, M., Nagata, N., Iwata, N., Ami, Y., Suzaki, Y., Mizuta, K., Iwasaki, T., Sata, T., Wakita, T. & Shimizu, H. (2007). An attenuated
Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol 43, 6171–6175.
strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol 81, 9386–9395. Bessaud, M., Razafindratsimandresy, R., Nougaire`de, A., Joffret, M. L., Deshpande, J. M., Dubot-Pe´re`s, A., He´raud, J. M., de Lamballerie, X., Delpeyroux, F. & Bailly, J. L. (2014). Molecular
comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PLoS ONE 9, e90624. Bible, J. M., Iturriza-Gomara, M., Megson, B., Brown, D., Pantelidis, P., Earl, P., Bendig, J. & Tong, C. Y. (2008). Molecular epidemiology
of human enterovirus 71 in the United Kingdom from 1998 to 2006. J Clin Microbiol 46, 3192–3200. Cardosa, M. J., Perera, D., Brown, B. A., Cheon, D., Chan, H. M., Chan, K. P., Cho, H. & McMinn, P. (2003). Molecular epidemiology of
human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis 9, 461–468. Diedrich, S., Weinbrecht, A. & Schreier, E. (2009). Seroprevalence
and molecular epidemiology of enterovirus 71 in Germany. Arch Virol 154, 1139–1142. Herrero, L. J., Lee, C. S., Hurrelbrink, R. J., Chua, B. H., Chua, K. B. & McMinn, P. C. (2003). Molecular epidemiology of enterovirus 71 in
Mizuta, K., Abiko, C., Aoki, Y., Suto, A., Hoshina, H., Itagaki, T., Katsushima, N., Matsuzaki, Y., Hongo, S. & other authors (2008).
Analysis of monthly isolation of respiratory viruses from children by cell culture using a microplate method: a two-year study from 2004 to 2005 in Yamagata, Japan. Jpn J Infect Dis 61, 196–201. Mizuta, K., Aoki, Y., Suto, A., Ootani, K., Katsushima, N., Itagaki, T., Ohmi, A., Okamoto, M., Nishimura, H. & other authors (2009). Cross-
antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine 27, 3153–3158. Momoki, T., Saikusa, M., Kawakami, C. & Ikebuti, M. (2009).
Detection of enterovirus 71 in Yokohama city, Japan. Infect Agents Surv Rep 30, 72–73 (in Japanese). Nakata, K., Sakon, N., Yamazaki, K. & Kase, T. (2012).
Epidemiological and virological analysis of hand-foot-mouth disease cases between 2010 and 2011 in Osaka, Japan. Infect Agents Surv Rep 33, 57–58 (in Japanese). 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. Oberste, M. S., Maher, K., Flemister, M. R., Marchetti, G., Kilpatrick, D. R. & Pallansch, M. A. (2000). Comparison of classic and molecular
peninsular Malaysia, 1997–2000. Arch Virol 148, 1369–1385.
approaches for the identification of untypeable enteroviruses. J Clin Microbiol 38, 1170–1174.
Hosomi, T., Nabeshima, T., Geshi, I., Matsumoto, M. & Imai, A. (2010). Detection of enterovirus 71 in Kochi, Japan, in 2010. Infect
Ortner, B., Huang, C. W., Schmid, D., Mutz, I., Wewalka, G., Allerberger, F., Yang, J. Y. & Huemer, H. P. (2009). Epidemiology
Agents Surv Rep 31, 272–273 (in Japanese). Huang, K. Y., Zhang, X., Chung, P. H., Tsao, K. C., Lin, T. Y., Su, L. H. & Chiu, C. H. (2008a). Enterovirus 71 in Taiwan, 2004–2006:
http://jmm.sgmjournals.org
of enterovirus types causing neurological disease in Austria 1999– 2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol 81, 317–324. 1361
K. Mizuta and others
Podin, Y., Gias, E. L., Ong, F., Leong, Y. W., Yee, S. F., Yusof, M. A., Perera, D., Teo, B., Wee, T. Y. & other authors (2006). Sentinel
enterovirus 71 in the Netherlands, 1963 to 2008. J Clin Microbiol 47, 2826–2833.
surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years. BMC Public Health 6, 180.
Wu, C. N., Lin, Y. C., Fann, C., Liao, N. S., Shih, S. R. & Ho, M. S. (2001). Protection against lethal enterovirus 71 infection in newborn
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine 20, 895–904.
Sanders, S. A., Herrero, L. J., McPhie, K., Chow, S. S., Craig, M. E., Dwyer, D. E., Rawlinson, W. & McMinn, P. C. (2006). Molecular
Wu, Y., Yeo, A., Phoon, M. C., Tan, E. L., Poh, C. L., Quak, S. H. & Chow, V. T. (2010). The largest outbreak of hand; foot and mouth
epidemiology of enterovirus 71 over two decades in an Australian urban community. Arch Virol 151, 1003–1013.
disease in Singapore in 2008: the role of enterovirus 71 and coxsackievirus A strains. Int J Infect Dis 14, e1076–e1081.
Schuffenecker, I., Mirand, A., Antona, D., Henquell, C., Chomel, J. J., Archimbaud, C., Billaud, G., Peigue-Lafeuille, H., Lina, B. & Bailly, J. L. (2011). Epidemiology of human enterovirus 71 infections in
Zaini, Z. & McMinn, P. (2012b). A single mutation in capsid protein
France, 2000–2009. J Clin Virol 50, 50–56.
VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71 generates a mouse-virulent phenotype. J Gen Virol 93, 1935– 1940.
Singh, S., Poh, C. L. & Chow, V. T. (2002a). Complete sequence
Zaini, Z., Phuektes, P. & McMinn, P. (2012a). Mouse adaptation of a
analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000). Microbiol Immunol 46, 801–808. Singh, S., Chow, V. T., Phoon, M. C., Chan, K. P. & Poh, C. L. (2002b).
Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers. J Clin Microbiol 40, 2823–2827. 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. van der Sanden, S., Koopmans, M., Uslu, G., van der Avoort, H. & Dutch Working Group for Clinical Virology (2009). Epidemiology of
1362
sub-genogroup B5 strain of human enterovirus 71 is associated with a novel lysine to glutamic acid substitution at position 244 in protein VP1. Virus Res 167, 86–96. Zhang, Y., Tan, X. J., Wang, H. Y., Yan, D. M., Zhu, S. L., Wang, D. Y., Ji, F., Wang, X. J., Gao, Y. J. & other authors (2009). An outbreak of
hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong, China. J Clin Virol 44, 262– 267. Zhu, F. C., Meng, F. Y., Li, J. X., Li, X. L., Mao, Q. Y., Tao, H., Zhang, Y. T., Yao, X., Chu, K. & other authors (2013). 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 381, 2024–2032.
Journal of Medical Microbiology 63
DOI 10.1099/jmm.0.079699-0
Molecular epidemiology of enterovirus 71 strains isolated from children in Yamagata, Japan, between 1990 and 2013 Katsumi Mizuta,1 Yoko Aoki,1 Yohei Matoba,1 Kazue Yahagi,1 Tsutomu Itagaki,2 Fumio Katsushima,3 Yuriko Katsushima,3 Sueshi Ito,4 Seiji Hongo5 and Yoko Matsuzaki5 Correspondence Katsumi Mizuta [email protected]
1
Department of Microbiology, Yamagata Prefectural Institute of Public Health, Yamagata, Yamagata, Japan
2
Yamanobe Pediatric Clinic, Yamanobe, Yamagata, Japan
3
Katsushima Pediatric Clinic, Yamagata, Yamagata, Japan
4
Department of Pediatrics, Shonai Hospital, Tsuruoka, Yamagata, Japan
5
Department of Infectious Diseases, Yamagata University Faculty of Medicine, Yamagata, Yamagata, Japan
Received 11 June 2014 Accepted 20 July 2014
Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.
INTRODUCTION Enterovirus 71 (EV71) is a small, single-stranded, positivesense non-enveloped RNA virus belonging to the genus Enterovirus within the family Picornaviridae (McMinn, 2002; Solomon et al., 2010). Together with coxsackievirus A16 (CVA16), CVA10 and other CVA viruses, EV71 is known to be a causative agent of hand-foot-and-mouth disease (HFMD) (McMinn, 2002; Solomon et al., 2010). Although HFMD is typically a self-limiting illness in children, EV71 infection sometimes causes neurological manifestations such as brainstem encephalitis, and fatal cases in infants and children have been reported in the 20th–21st century, mainly across the Asia-Pacific region (McMinn, 2002; Solomon et al., Abbreviations: CVA, coxsackievirus A; EV71, enterovirus 71; HFMD, hand-foot-and-mouth disease. The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are AB936432–AB936561.
1356
2010). For example, in an outbreak of HFMD in Shangdong Province, China, in 2007, 1149 cases were reported, among which 11 children (0.96 %) presented with neurological complications and three died (mortality rate 0.26 %) (Zhang et al., 2009). EV71 infection has since become a major public issue in this area and there has been notable progress in studies of its epidemiology, identification of receptors, development of antiviral agents and vaccine development, all of which are necessary to control and ameliorate the severity of the disease (Liang et al., 2013; McMinn, 2002; Solomon et al., 2010; Wu et al., 2001; Zhu et al., 2013). Several epidemiological studies have clarified that EV71 is an active circulating agent capable of rapid evolution into several different genogroups and that EV71 consists of 14 subgenogroups (A, B1–5, C1–5, D, E and F), although no relationship between severity and genogroup has yet been confirmed (Liang et al., 2013; McMinn, 2002; Singh et al., 2002a; Solomon et al., 2010; Bessaud et al., 2014). The 079699 G 2014 The Authors
Printed in Great Britain
Longitudinal molecular epidemiology of enterovirus 71
development of a vaccine against EV71 has been pursued as a preventive measure and the main target region for vaccine development has been the VP1 region, which was defined as the neutralization determinant (McMinn, 2002; Oberste et al., 1999; Wu et al., 2001). As the number of studies involving antigenic analysis and measurement of neutralizing antibody against different subgenogroups targeting the VP1 region has been limited, we carried out a molecular epidemiological study of EV71 strains isolated in Yamagata, Japan, between 1990 and 2007 (Mizuta et al., 2009). Our results from a sequence analysis of the VP1 region, genotyping, a seroepidemiological survey and an antigenic analysis suggest that there is cross-antigenicity among the different EV71 subgenogroups circulating in the community and that it might be possible to prevent severe illnesses due to EV71 infections through the development of a vaccine that effectively induces neutralizing antibodies against EV71, as has been shown with the measles virus vaccine (Mizuta et al., 2009). In reality, studies related to vaccine development have suggested cross-protection among different EV71 subgenogroups, and it is recommended that future research focuses on strengthening the monitoring of prevalent strains and strain variations to check the cross-protective effects of vaccines, particularly if
genogroup replacement has occurred (Arita et al., 2007; Liang et al., 2013; Solomon et al., 2010). Furthermore, good surveillance programmes are needed in many different geographical regions to provide accurate and relevant information about EV71 transmission and evolution (Solomon et al., 2010). We have further analysed the EV71 Yamagata isolates, and here describe the longitudinal molecular epidemiology of EV71 between 1990 and 2013.
METHODS Virus isolation and identification were carried out by means of a microplate method using nasopharyngeal samples from children with HFMD, viral exanthematous disease or respiratory illnesses, as described previously (Mizuta et al., 2005, 2008, 2009). EV71 strains were isolated using human embryonic lung fibroblast (HEF), Vero, Vero E6, human rhabdomyosarcoma (RD)-18S and green monkey kidney (GMK) cell lines at the Virus Research Center, National Hospital Organization, Sendai Medical Center, Sendai, Japan, between 1990 and 1997, and thereafter at the Department of Microbiology, Yamagata Prefectural Institute of Public Health. The isolates were identified as EV71 based on a neutralization method. Sequence analysis for the complete VP1 region (891 nt) of the EV71 isolates was carried out as described previously (Mizuta et al., 2005, 2009), except that we also used primers 222 and R2 (Oberste et al., 2000; Singh et al., 2002b). Sequence data for the isolates from Yamagata had the following GenBank accession
20
Number of isolates
B2 B4
15
B5 C1
10
C2 C4
5
ND
0 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001 B2
20
Number of isolates
B4 B5
15
C1 C2
10
C4 ND
5
0 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911 1 3 5 7 911
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Fig. 1. Monthly distribution (odd months numbered) and subgenogroups of EV71 strains isolated in Yamagata, Japan, between 1990 and 2013. The subgenogroups, C1, C2, B2, B4, C4 and B5, were grouped according to the phylogenetic analysis shown in Fig. 2. ND, Not done. http://jmm.sgmjournals.org
1357
K. Mizuta and others
BrCr-CA-70 U22521 81 100
100
100
99
99
99
0.02
100 99
100
1358
2604-AUS-74 AF135883 2232-NY-77 AF135871 7633-PA-87 AF009534 7673-USA-87 AF009535 Y90-3205 AB433863 Y93-2008 AB433866 MY16/1/SAR/97 AF376073 4350/SIN/98 AF376119 5536/SIN/00 AF376122 S21082/SAR/00 AF376084 738-Yamagata-00 AB213620 1067-Yamagata-00 AB213625 S110031-SAR-03 AY258307 S19741-SAR-03 AY258313 2419-Yamagata-03 AB213647 2542-Yamagata-03 AB177815 2950-Yamagata-2009 AB936506 1749-Yamagata-2012 AB936545 387-Yamagata-2013 AB936555 1499-Yamagata-2013 AB936557 2419-Yamagata-2012 AB936553 799-Yamagagta-2013 AB936556 1615-Yamagata-2012 AB936541 111-Yamagata-2013 AB936540 1701-Yamagata-2012 AB936531 1518-Yamagata-2012 AB936525 1842-Yamagata-2012 AB936536 MAD-72341-04 HG421068 1116-Yamagata-00 AB177810
Y97-865 AB433867 Y97-1134 AB433869 03750-MAA-97 AY207615 Y97-1055 AB433868 671-Yamagata-98 AB213614 705-Yamagata-98 AB213615 932-Yamagata-99 AB213616 1002-Yamagata-99 AB213619 2M-AUS-3-99 AF376103 NL3652000 AB491218 EP/5622/1999UK AM939583 CF1113FRA2000 FN598726 EP/7414/1999UK AM939585 ANG261FRA09 FN598777 NAN257FRA08 FN598776 NL2652008 AB491220 3246-Yamagata-2009 AB936502 2534-Yamagata-2009 AB936505 1613-Yamagata-2010 AB936520 1108-Yamagata-2010 AB936515 520-Yamagata-2010 AB936511 148-Yamagata-2010 AB936507 3215-Yamagata-2009 AB936501 OC091495-10 AB665731 OC091494-10 AB665730 1825-Yamagata-2009 AB936494 NL1550 AB524223 BRE324FRA07 FN598762 1416-FR-2007 FJ824734 NL07/ns/W/07 HQ676273 NL03/ns/W/07 HQ676270 NL25272007 AB491219 2008-Yamagata-2010 AB936523 813-Yamagata-2013 AB936558 1437-Yamagata-2013 AB936561 857-Yamagata-2013 AB936559 933-Yamagata-2013 AB936560 100 001-KOR-00 AY125966 013-KOR-00 AY125976 Y90-3761 AB433864 Y90-3896 AB433865 Y90-2913 AB433862 2246-NY-87 AF009542 NL11341991 AB491216 S11051/SAR/98 AF376081 EP/2063/2001UK AM939577 NL38782002 AB491217 EP/17728/1998UK AM939593 LYO28978FRA99 FN598721 CF1744FRA2000 FN598727 3254-TAI-98 AF286531 F2-CHN-00 AB115491 75-Yamagata-03 AB177813 452-Yamagata-03 AB213630 2779-Yamagata-02 AB213629 2321-Yamagata-03 AB213645 CF192013FRA2004 FN598741 ZJ-CHN-6-03 AY905619 082/NX/CHN/2008 HM212460 2147-Yamagata-06 AB433871 2928-Yamagata-06 AB433878 2753-Yamagata-06 AB433877 56-Yamagata-07 AB433879 1693-Yamagata-07 AB433886 1758-Yamagata-07 AB433889 1897-Yamagata-07 AB433891 100 1089T/VNM/05 AM490142 1301V/VNM/05 AM490149 CAF-NMA-03-008 JN255590 C08-146 JX307649
A B1 B2 B3 B4
B5
F
C2
C3
C1
C4
C5 E
Journal of Medical Microbiology 63
Longitudinal molecular epidemiology of enterovirus 71
3
Fig. 2. Phylogenetic tree for the complete (891 nt) VP1 region of representative enterovirus 71 (EV71) strains isolated in Yamagata, Japan, between 1990 and 2013 and reference strains. Subgenogroups B5, C2 and C4 strains repeatedly appeared in Yamagata with genomic variations with several years’ interval as shown by the different levels of grey shading. Yamagata and and 7, respectively. Branch lengths are proportional to the number of nucleotide European strains are indicated by differences. Numbers above the branches are the bootstrap probabilities (%). Bar, measurement of relative phylogenetic distance (0.02 nucleotide substitutions per site).
numbers: AB177809–AB177816, AB213614–AB213650 and AB433862– AB433892. Sequence data were analysed with CLUSTAL W (version 1.83), and a phylogenetic tree was reconstructed by the neighbour-joining method using the same software (Saitou & Nei, 1987).
A total of 240 EV71 strains were isolated in Yamagata between 1990 and 2013. The number of EV71 strains isolated in Yamagata by month is shown in Fig. 1. Although we did not measure the neutralizing titres for EV71 isolates after 2007, these isolates were neutralized with the identical antisera used in the previous study (Mizuta et al., 2009). A total of 223 of the 240 isolates were used for sequence analysis, and representative Yamagata strains were used for further phylogenetic analysis. Available EV71 strains that had not yet been typed in previous studies (Mizuta et al., 2005, 2009) were also sequenced in this study.
respectively. In 1990, three isolates were identified as belonging to subgenogroup C1 and one isolate was identified as belonging to B2. In 1993, one strain was identified as B2. Between 1997 and 1999, all analysed strains were typed as C2. In 2000, all analysed strains, except for one strain isolated in September and identified as C2, were typed as B4. All four isolates in 2001 were again typed as C2. Isolates between October 2002 and August 2003 were typed as C4, except for one strain isolated in June 2003 and identified as B5. C4 and B5 strains co-circulated in September, with only B5 observed thereafter until November 2003. All isolates between 2006 and 2007 were again typed as C4. All isolates, except for one strain (2950-Yamagata-2009 typed as B5), between 2009 and 2010 were typed as C2. The strain 2950-Yamagata-2009 was the only isolate from the Shonai area, which is located on the Japan Sea in Yamagata, whereas the other strains isolated between 2009 and 2010 were from inland areas. In 2012, all isolates were typed as B5, and B5 and C2 strains co-circulated in 2013.
The phylogenetic tree for the VP1 region and the monthly distribution of subgenogroups are shown in Figs 1 and 2,
C2, C4 and B5 strains appeared repeatedly in Yamagata. C2 strains with 93–100 % nucleotide similarity to each other
RESULTS
Australia Singapore Malaysia Taiwan China Yamagata, Japan France Netherlands Germany UK
Australia Singapore Malaysia Taiwan China Yamagata, Japan France Netherlands Germany UK
1990 1991 1992 C1 C1 C1
B2,C1 C1
2002 C1 C1,B4 C1 B4 C4 C4
1994 1995 1996 C1 C1,C2 C1
B2 C1
C1 C1
C1
2003 2004 C1 C4
2005
2006
C1
2007
1997
1998 C1 B3,B4 B3,C1 B3,B4,C1,C2 C1 B4,C2 C4 C2 C2 C1,C2 B2
2008
2009
B2 C1 2010
1999 B3,C2 B3 B4,C1 B4
2000 B4,C1 B4 B4,C1 B4 C4 C2 B4,C2 C1 C1,C2 C2 C2 C1 C1,C2 C1 2011
2012
2001 B4 B4 B4,C4 C4 C2 C1 C1 C1 2013
B5,C2 B5,C1 B4 C4 C4,B5 C1
C1,C2 C1 C1
1993
C4
C4
C5
B5,C5 C4 C4 C4 C1,C4 C1 C1,C2 C2 C1,C2 C1,C2 C1,C2 C4 C2 C2 C1 C1 C1,C2
B5,C4,C5 C4 C2 C2
B5,C2 C2
C2
B5
B5,C2
Fig. 3. Summary of EV71 genotypes circulating in the Asia-Pacific region (grey shading) and Europe since 1980 based on data from the following references: Bible et al. (2008); Cardosa et al. (2003); Diedrich et al. (2009); Herrero et al. (2003); Huang et al. (2008a, b, 2010); Kung et al. (2007); Ortner et al. (2009); Podin et al. (2006); Sanders et al. (2006); Schuffenecker et al. (2011); Wu et al. (2010); Van der Sanden et al. (2009); Zhang et al. (2009). http://jmm.sgmjournals.org
1359
K. Mizuta and others
circulated every year between 1997 and 2001, and again appeared between 2009 and 2010 with 94–100 % similarity. The nucleotide similarity between C2 strains from 1997 to 2001 and those from 2009 and 2010 was 90–95 %. C4 strains circulated between 2002 and 2003 with 98–100 % similarity, and then disappeared and reappeared between 2006 and 2007 with 97–100 % similarity. The nucleotide similarity between C4 strains from 2002 to 2003 and those from 2006 to 2007 was 95–96 %. B5 strains replaced C4 strains in September 2003 and circulated for 3 months with 98–100 % similarities and then disappeared. Only one B5 strain was isolated in November 2009 and there was another outbreak of B5 strains between 2012 and 2013 with 95–100 % similarity. The nucleotide similarity was 96, 94– 95 and 94–95 % between the B5 strains from 2003 and the one from 2009, the one from 2009 and those from 2012– 2013, and those from 2003 and those from 2012–2013, respectively.
DISCUSSION We showed the temporal distribution of EV71 subgenogroups over a 24-year period from 1990 to 2013, in Yamagata, Japan. Apart from a study undertaken in Sydney, Australia, between 1983 and 2011, to the best of our knowledge no such longitudinal analysis of the VP1 region of EV71 strains circulating in a local community has been reported (Sanders et al., 2006). Our longitudinal studies revealed several interesting epidemiological features, as reported previously based on the data obtained between 1990 and 2007 (Mizuta et al., 2005, 2009), and we further found several new findings in the last 6-year observation period. We found that six subgenogroups, B2, B4, B5, C1, C2 and C4, appeared one after another or concurrently during the study period. C1 and B2 disappeared by 1993 in Yamagata and these subgenogroup strains have not yet reappeared. However, the three other subgenogroups, excluding B4, have repeatedly appeared in Yamagata during the study period. Based on the data between 1990 and 2007 (Figs 1 and 2) (Mizuta et al., 2009), we previously reported that genogroup C strains circulated for longer periods with genomic variations, whereas genogroups B strains appeared for only one season. We observed genogroups B strains, B4 and B5, for only 5 and 6 months in 2000 and 2003, respectively, whereas C2 and C4 strains circulated for 5–6 years despite breaks (Mizuta et al., 2009). However, in this study, after the disappearance of B5 strains in 2003, we subsequently observed one B5 strain in 2009 and another outbreak of B5 strains between 2012 and 2013. Thus, these results suggested that not only genogroup C strains but also genogroup B strains have the potential to circulate repeatedly with active genomic variations. In Japan, the genotyping of EV71 strains has been reported recently based on partial VP1 and/or VP4-2 sequence 1360
analysis. Momoki et al. (2009) reported that one and three isolates between September and December in 2008 belonged to C2 and B5, respectively, and they suggested that, in 2008, C2 replaced C4, which had been in circulation between 2005 and 2007 in Yokohama City. Hosomi et al. (2010) reported that four strains in Kochi in 2010 belonged to C2 and Nakata et al. (2012) found that there was an outbreak of HFMD due to C2 strains in Osaka in 2010. Two isolates (OC091494 and OC091495) from Osaka City in 2010 showed 99 % sequence similarity with Yamagata isolates in 2010, apart from a 96 % similarity between the Osaka isolates and 2008-Yamagata-2010 (Fig. 2). Katsumi et al. (2013) reported the isolation of seven B5 strains and one C2 strain in 2012 in Sendai City. Kodama et al. (2013) reported that seven strains isolated in Ishikawa Prefecture in 2010 belonged to C2, and 14 strains isolated between 2012 and 2013 belonged to B5, although the investigation was carried out based on VP4–VP2 analysis. Taken together, these observations and our data from Yamagata suggest that the predominant subgenogroup changed from C4 to C2 across a wide area of Japan between 2007 and 2009. B5 strains probably caused a nationwide epidemic between 2012 and 2013, as reported from Sendai and Ishikawa (Katsumi et al., 2013; Kodama et al., 2013). Recent reports have also suggested that identical predominant subgenogroup strains have been in simultaneous circulation all over Japan. As we reported previously, the fact that the detected subgenogroups in Yamagata (B2, B4, B5, C1, C2 and C4) were also found in the Asia-Pacific region suggests the active circulation of EV71 in this area (Bible et al., 2008; Cardosa et al., 2003; Diedrich et al., 2009; Herrero et al., 2003; Huang et al., 2008a, b, 2010; Kung et al., 2007; Mizuta et al., 2005, 2009; Ortner et al., 2009; Podin et al., 2006; Sanders et al., 2006; Schuffenecker et al., 2011; Wu et al., 2010; van der Sanden et al., 2009; Zhang et al., 2009) (Fig. 3). On the other hand, several European countries, such as France, the Netherlands, Germany and the UK, recently reported the molecular epidemiology of EV71 (Bible et al., 2008; Diedrich et al., 2009; Schuffenecker et al., 2011; van der Sanden et al., 2009). When we compare the subgenogroups circulating in Asia-Pacific region and those in Europe, we can find several differences: (i) there was a co-circulation of genogroup B and C strains in the Asia-Pacific region throughout almost the entire study period, whereas only genogroup C strains were in circulation in Europe (except for subgenogroup B2 strains found in Germany between 1997 and 1998); (ii) B3, B4 and B5 strains have been reported only in the Asia-Pacific region; (iii) genogroup C, C1, C2 and C4 strains circulated as major subgenogroups in the Asia-Pacific region, whereas C1 and C2 strains were predominant in Europe between 1990 and 2009; and (iv) subgenogroup C5 strains are only observed in the Asia-Pacific region (Fig. 3). These observations suggest that EV71 circulates mainly in the Asia-Pacific region and in Europe, although this idea does not completely exclude the possible introduction of EV71 from Europe to Asia-Pacific region and vice versa. It is conceivable that the reappearance Journal of Medical Microbiology 63
Longitudinal molecular epidemiology of enterovirus 71
of C2 in Yamagata in 2009 might be the result of the introduction of EV71 from Europe or Singapore (Figs 1–3). Several capsid residues of EV71 that alter infectivity in the mouse model have been identified. For example, a novel lysine-to-glutamic acid substitution at position 244 in VP1 of B5 strain is critical for mouse adaptation and virulence, and a single mutation from glutamine to glutamic acid at residue 145 in VP1 of C4 strain generates a mouse-virulent phenotype (Zaini & McMinn, 2012; Zaini et al., 2012a). In Yamagata, all B5 strains had lysine at position 244, whereas all C4 strains had glutamic acid at residue 145 during the study period. It will also be necessary to clarify mechanisms of severity and pathogenicity in EV71 infections. Finally, to resolve the public health issue of EV71, we have to continue to analyse the longitudinal epidemiology of EV71 in local communities such as Yamagata, as well as on regional and global levels.
epidemiological and virological features. Scand J Infect Dis 40, 571– 574. Huang, Y. P., Lin, T. L., Kuo, C. Y., Lin, M. W., Yao, C. Y., Liao, H. W., Hsu, L. C., Yang, C. F., Yang, J. Y. & other authors (2008b). The
circulation of subgenogroups B5 and C5 of enterovirus 71 in Taiwan from 2006 to 2007. Virus Res 137, 206–212. Huang, Y. P., Lin, T. L., Hsu, L. C., Chen, Y. J., Tseng, Y. H., Hsu, C. C., Fan, W. B., Yang, J. Y., Chang, F. Y. & Wu, H. S. (2010). Genetic
diversity and C2-like subgenogroup strains of enterovirus 71, Taiwan, 2008. Virol J 7, 277. Katsumi, M., Chida, K., Niiki, Y., Sekine, M., Oguro, M., Kobayashi, M., Hasegawa, S. & Kayaba, J. (2013). Enteroviruses detected from
patients with hand-foot-mouth disease and herpangina in Sendai, Japan in 2012. Infect Agents Surv Rep 34, 9–10 (in Japanese). Kodama, H., Nariai, E. & Sakigawa, Y. (2013). Enteroviruses detected
from patients with hand-foot-mouth disease and herpangina between January 2010 and March 2013 in Ishikawa, Japan. Infect Agents Surv Rep 34, 202–204 (in Japanese). Kung, S. H., Wang, S. F., Huang, C. W., Hsu, C. C., Liu, H. F. & Yang, J. Y. (2007). Genetic and antigenic analyses of enterovirus 71 isolates
in Taiwan during 1998–2005. Clin Microbiol Infect 13, 782–787.
ACKNOWLEDGEMENTS We thank the medical staff and people of Yamagata Prefecture for their collaboration in specimen collection for the National Epidemiological Surveillance of Infectious Diseases, Japan. This work was partly supported by a grant from the Association for Research on Lactic Acid Bacteria. The authors declare they have no conflicts of interest.
Liang, Z. L., Mao, Q. Y., Wang, Y. P., Zhu, F. C., Li, J. X., Yao, X., Gao, F., Wu, X., Xu, M. & Wang, J. Z. (2013). Progress on the research and
development of inactivated EV71 whole-virus vaccines. Hum Vaccin Immunother 9, 1701–1705. McMinn, P. C. (2002). An overview of the evolution of enterovirus 71
and its clinical and public health significance. FEMS Microbiol Rev 26, 91–107.
REFERENCES
Mizuta, K., Abiko, C., Murata, T., Matsuzaki, Y., Itagaki, T., Sanjoh, K., Sakamoto, M., Hongo, S., Murayama, S. & Hayasaka, K. (2005).
Arita, M., Nagata, N., Iwata, N., Ami, Y., Suzaki, Y., Mizuta, K., Iwasaki, T., Sata, T., Wakita, T. & Shimizu, H. (2007). An attenuated
Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol 43, 6171–6175.
strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol 81, 9386–9395. Bessaud, M., Razafindratsimandresy, R., Nougaire`de, A., Joffret, M. L., Deshpande, J. M., Dubot-Pe´re`s, A., He´raud, J. M., de Lamballerie, X., Delpeyroux, F. & Bailly, J. L. (2014). Molecular
comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PLoS ONE 9, e90624. Bible, J. M., Iturriza-Gomara, M., Megson, B., Brown, D., Pantelidis, P., Earl, P., Bendig, J. & Tong, C. Y. (2008). Molecular epidemiology
of human enterovirus 71 in the United Kingdom from 1998 to 2006. J Clin Microbiol 46, 3192–3200. Cardosa, M. J., Perera, D., Brown, B. A., Cheon, D., Chan, H. M., Chan, K. P., Cho, H. & McMinn, P. (2003). Molecular epidemiology of
human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis 9, 461–468. Diedrich, S., Weinbrecht, A. & Schreier, E. (2009). Seroprevalence
and molecular epidemiology of enterovirus 71 in Germany. Arch Virol 154, 1139–1142. Herrero, L. J., Lee, C. S., Hurrelbrink, R. J., Chua, B. H., Chua, K. B. & McMinn, P. C. (2003). Molecular epidemiology of enterovirus 71 in
Mizuta, K., Abiko, C., Aoki, Y., Suto, A., Hoshina, H., Itagaki, T., Katsushima, N., Matsuzaki, Y., Hongo, S. & other authors (2008).
Analysis of monthly isolation of respiratory viruses from children by cell culture using a microplate method: a two-year study from 2004 to 2005 in Yamagata, Japan. Jpn J Infect Dis 61, 196–201. Mizuta, K., Aoki, Y., Suto, A., Ootani, K., Katsushima, N., Itagaki, T., Ohmi, A., Okamoto, M., Nishimura, H. & other authors (2009). Cross-
antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine 27, 3153–3158. Momoki, T., Saikusa, M., Kawakami, C. & Ikebuti, M. (2009).
Detection of enterovirus 71 in Yokohama city, Japan. Infect Agents Surv Rep 30, 72–73 (in Japanese). Nakata, K., Sakon, N., Yamazaki, K. & Kase, T. (2012).
Epidemiological and virological analysis of hand-foot-mouth disease cases between 2010 and 2011 in Osaka, Japan. Infect Agents Surv Rep 33, 57–58 (in Japanese). 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. Oberste, M. S., Maher, K., Flemister, M. R., Marchetti, G., Kilpatrick, D. R. & Pallansch, M. A. (2000). Comparison of classic and molecular
peninsular Malaysia, 1997–2000. Arch Virol 148, 1369–1385.
approaches for the identification of untypeable enteroviruses. J Clin Microbiol 38, 1170–1174.
Hosomi, T., Nabeshima, T., Geshi, I., Matsumoto, M. & Imai, A. (2010). Detection of enterovirus 71 in Kochi, Japan, in 2010. Infect
Ortner, B., Huang, C. W., Schmid, D., Mutz, I., Wewalka, G., Allerberger, F., Yang, J. Y. & Huemer, H. P. (2009). Epidemiology
Agents Surv Rep 31, 272–273 (in Japanese). Huang, K. Y., Zhang, X., Chung, P. H., Tsao, K. C., Lin, T. Y., Su, L. H. & Chiu, C. H. (2008a). Enterovirus 71 in Taiwan, 2004–2006:
http://jmm.sgmjournals.org
of enterovirus types causing neurological disease in Austria 1999– 2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol 81, 317–324. 1361
K. Mizuta and others
Podin, Y., Gias, E. L., Ong, F., Leong, Y. W., Yee, S. F., Yusof, M. A., Perera, D., Teo, B., Wee, T. Y. & other authors (2006). Sentinel
enterovirus 71 in the Netherlands, 1963 to 2008. J Clin Microbiol 47, 2826–2833.
surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years. BMC Public Health 6, 180.
Wu, C. N., Lin, Y. C., Fann, C., Liao, N. S., Shih, S. R. & Ho, M. S. (2001). Protection against lethal enterovirus 71 infection in newborn
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine 20, 895–904.
Sanders, S. A., Herrero, L. J., McPhie, K., Chow, S. S., Craig, M. E., Dwyer, D. E., Rawlinson, W. & McMinn, P. C. (2006). Molecular
Wu, Y., Yeo, A., Phoon, M. C., Tan, E. L., Poh, C. L., Quak, S. H. & Chow, V. T. (2010). The largest outbreak of hand; foot and mouth
epidemiology of enterovirus 71 over two decades in an Australian urban community. Arch Virol 151, 1003–1013.
disease in Singapore in 2008: the role of enterovirus 71 and coxsackievirus A strains. Int J Infect Dis 14, e1076–e1081.
Schuffenecker, I., Mirand, A., Antona, D., Henquell, C., Chomel, J. J., Archimbaud, C., Billaud, G., Peigue-Lafeuille, H., Lina, B. & Bailly, J. L. (2011). Epidemiology of human enterovirus 71 infections in
Zaini, Z. & McMinn, P. (2012b). A single mutation in capsid protein
France, 2000–2009. J Clin Virol 50, 50–56.
VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71 generates a mouse-virulent phenotype. J Gen Virol 93, 1935– 1940.
Singh, S., Poh, C. L. & Chow, V. T. (2002a). Complete sequence
Zaini, Z., Phuektes, P. & McMinn, P. (2012a). Mouse adaptation of a
analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000). Microbiol Immunol 46, 801–808. Singh, S., Chow, V. T., Phoon, M. C., Chan, K. P. & Poh, C. L. (2002b).
Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers. J Clin Microbiol 40, 2823–2827. 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. van der Sanden, S., Koopmans, M., Uslu, G., van der Avoort, H. & Dutch Working Group for Clinical Virology (2009). Epidemiology of
1362
sub-genogroup B5 strain of human enterovirus 71 is associated with a novel lysine to glutamic acid substitution at position 244 in protein VP1. Virus Res 167, 86–96. Zhang, Y., Tan, X. J., Wang, H. Y., Yan, D. M., Zhu, S. L., Wang, D. Y., Ji, F., Wang, X. J., Gao, Y. J. & other authors (2009). An outbreak of
hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong, China. J Clin Virol 44, 262– 267. Zhu, F. C., Meng, F. Y., Li, J. X., Li, X. L., Mao, Q. Y., Tao, H., Zhang, Y. T., Yao, X., Chu, K. & other authors (2013). 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 381, 2024–2032.
Journal of Medical Microbiology 63
