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Enterovirus 71 Outbreak, Brunei

Enterovirus 71 Outbreak, Brunei also was isolated from stool or rectal swabs of 4 patients, of Enterovirus 71 whom 2 were coinfected with EV71; none had neurologic disease. Outbreak, Brunei Enteroviral RNA was extracted from cell cultures us- ing QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Ger- Sazaly AbuBakar, I-Ching Sam, Jaliha Yusof, many), and reverse transcription–PCR was performed to Meng Keang Lim, Suzana Misbah, amplify the viral capsid protein (VP1) gene at nt positions NorAziyah MatRahim, and Poh-Sim Hooi 31–861. The primers used were VP1F 5′-CAGGCTAGCA TGGGAGATAGGGTGGCAGATGTGATCGAGAGC-3′ Enterovirus 71 (EV71) outbreaks occur periodically in the Asia-Pacifi c region. In 2006, Brunei reported its fi rst and VP1R 5′-GGTGGATCCCAAAGGGTAGTAATGGC major outbreak of EV71 infections, associated with fatali- AGTACGACTAGTGCCGGT-3′. The 831-nt partial VP1 ties from neurologic complications. Isolated EV71 strains gene fragments were sequenced, and phylogenetic relations formed a distinct lineage with low diversity within subgeno- of the sequences were examined using selected enterovirus group B5, suggesting recent introduction and rapid spread reference strains obtained from GenBank (Table 2). Se- within Brunei. quences were aligned and phylogenetic trees were drawn using the neighbor-joining method (Figure), as described (12). Maximum-likelihood tree showed similar cluster- nterovirus 71 (EV71), a member of the family Picorna- ing and is not shown. The prototype coxsackievirus A16 Eviridae and the genus Enterovirus, is a common cause (CoxA16-G10) was used as the outgroup virus for con- of hand, foot, and mouth disease in children. Infection with struction of the phylogenetic tree. this virus is rarely complicated by severe neurologic dis- The phylogenetic tree, drawn on the basis of the align- ease, such as meningitis, brain stem encephalitis, neuro- ment of the VP1 gene sequences, showed 3 independent genic pulmonary edema, and acute fl accid paralysis. EV71 genogroups (A, B, and C) with the prototype BrCr strain was fi rst isolated in 1969 (1), and during the subsequent 30 as the only member of genogroup A (11). Within each of years, outbreaks were reported in the United States, Eu- genogroups B and C, 5 additional subgenogroups were rope, and Asia (2). Since 1997, several major outbreaks identifi ed, designated B1–B5 and C1–C5 (8,10). Although with deaths have occurred in the Asia-Pacifi c region, nota- no defi nitions have been established, generally there is nu- bly in Sarawak (East Malaysia), Peninsular Malaysia, Tai- cleotide variation of ≈16%–20% between genogroups and wan, Australia, Singapore, Japan, and Vietnam (3–10). differences of ≈6%–12% between subgenogroups within Brunei is situated on the island of Borneo (4°30′N, each genogroup (5,11). 114°E) and has a population of ≈370,000. From February All Brunei and Malaysia isolates from 2006 clustered through August 2006, Brunei experienced its fi rst reported into subgenogroup B5, except for 1 Brunei isolate, which major outbreak of EV71. More than 1,681 children report- grouped to subgenogroup B4. Nucleotide sequences of edly were affected, with 3 deaths resulting from severe the VP1 gene were highly similar (96%–100%) among all neurologic disease. We report the virologic fi ndings from strains in subgenogroup B5. All Brunei B5 isolates were this outbreak. clustered in an independent lineage within subgenogroup B5 (99.9% bootstrap support), separate from the estab- The Study lished Sarawak and Yamagata isolates from 2003 (8). During March through October 2006, samples from at Amino acid sequences were highly conserved among the least 100 patients from Brunei diagnosed with hand, foot, Brunei B5 isolates, with 99%–100% similarity. No amino and mouth disease or herpangina were received at the Uni- acid sequence changes were observed in the 2 isolates from versity Malaya Medical Center, Kuala Lumpur, Malaysia. patients who died. Samples were inoculated into Vero and A549 cell cultures for virus isolation. EV71 was isolated from 34 patients (in- Conclusions cluding 2 who died of severe neurologic complications), The different genogroups of EV71 are widely distrib- and an additional 7 isolates were obtained from Malaysian uted around the world (2). The continuing appearance of patients seen at the University Malaya Medical Center dur- new EV71 subgenogroups in recent years in the Asia-Pa- ing the outbreak period in Brunei (Table 1). Adenovirus cifi c region suggests that the virus is continuously evolving Author affi liations: University Malaya, Kuala Lumpur, Malaysia (S. (5,8,9). The annual rate of evolution is estimated at 1.35 AbuBakar, I-C. Sam, S. Misbah, N. MatRahim, P.-S. Hooi); and –2 ×10 substitutions per nucleotide, similar to poliovirus Raja Isteri Pengiran Anak Saleha Hospital, Bandar Seri Begawan, (11). In some countries, outbreaks occur in a cyclical pat- Brunei Darussalam (J. Yusof, M.K. Lim) tern every 3 years, predominantly caused by strains that are distinct from previous outbreaks (3,9). These strains often DOI: 10.3201/eid1501.080264 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 79 DISPATCHES have been detected in other countries in the region in years the concurrent spread of other genogroups, subgenogroups, preceding the outbreak. In some EV71 outbreaks, other or enteroviruses. enteroviruses cocirculate, particularly coxsackievirus A16 Other than its northern coastline, Brunei is surrounded or EV71 from a different subgenogroup (3,8,10). On the entirely by the East Malaysian state of Sarawak. In 2006, basis of the samples received in the study, the Brunei 2006 an outbreak of EV71 affected approximately 14,400 chil- EV71 outbreak was caused by subgenogroup B5 virus. dren in Sarawak (14). Thus, temporally and geographical- Apart from the single isolate from subgenogroup B4, no ly, the Brunei and Sarawak outbreaks were related, raising other enteroviruses were isolated, although 2 patients also the possibility that the same strains were involved. Sara- had adenovirus. Occasional EV71 and adenovirus co-infec- wak had experienced EV71 outbreaks every 3 years (1997, tion has been reported (13), also without association with 2000, and 2003), caused by subgenogroups B3, B4, and severe disease. The low sequence diversity and predomi- B5, respectively (3). However, no sequence results from the nance of the Brunei B5 isolates in this outbreak suggest Sarawak 2006 outbreak are available for comparison. All recent introduction and subsequent rapid spread, without subgenogroup B5 isolates reported seem to have diverged Table 1. Enterovirus 71 from Brunei and Malaysia isolated in 2006 Isolate GenBank accession no. Subgenogroup Specimen type Origin EV71/BRU/2006/33930 FM201328 B5 Rectalswab Brunei EV71/BRU/2006/34095 FM201329 B5 Rectalswab Brunei EV71/BRU/2006/34099 FM201330 B5 Rectalswab Brunei EV71/BRU/2006/34111 FM201331 B4 Skinswab Brunei EV71/BRU/2006/34235 FM201332 B5 Throatswab Brunei EV71/BRU/2006/34355 FM201333 B5 Throatswab Brunei EV71/BRU/2006/34456 FM201334 B5 Swab* Brunei EV71/BRU/2006/34597 FM201335 B5 Stool Brunei EV71/BRU/2006/34700 FM201336 B5 Stool Brunei EV71/BRU/2006/34701 FM201337 B5 Stool Brunei EV71/BRU/2006/35053 FM201338 B5 Rectalswab Brunei EV71/BRU/2006/35207 FM201339 B5 Stool Brunei EV71/BRU/2006/35245 FM201340 B5 Rectalswab Brunei EV71/BRU/2006/35247 FM201341 B5 Rectalswab Brunei EV71/BRU/2006/35334 FM201342 B5 Swab* Brunei EV71/BRU/2006/35335 FM201343 B5 Blisterswab Brunei EV71/BRU/2006/35338 FM201344 B5 Swab* Brunei EV71/BRU/2006/35341 FM201345 B5 Swab* Brunei EV71/BRU/2006/35379 FM201346 B5 Rectalswab Brunei EV71/BRU/2006/35479 FM201347 B5 Rectalswab Brunei EV71/BRU/2006/35640 FM201348 B5 Rectalswab Brunei EV71/BRU/2006/35641 FM201349 B5 Rectalswab Brunei EV71/BRU/2006/35643 FM201350 B5 Rectalswab Brunei EV71/BRU/2006/35645 FM201351 B5 Rectalswab Brunei EV71/BRU/2006/35646 FM201352 B5 Rectalswab Brunei EV71/BRU/2006/35649 FM201353 B5 Rectalswab Brunei EV71/BRU/2006/35652 FM201354 B5 Rectalswab Brunei EV71/BRU/2006/35653 FM201355 B5 Rectalswab Brunei EV71/BRU/2006/35728 FM201356 B5 Swab* Brunei EV71/BRU/2006/35730 FM201357 B5 Swab* Brunei EV71/BRU/2006/35731 FM201358 B5 Swab* Brunei EV71/BRU/2006/35732 FM201359 B5 Swab* Brunei EV71/BRU/2006/35754 FM201360 B5 Rectalswab Brunei EV71/BRU/2006/35755 FM201361 B5 Rectalswab Brunei EV71/MY/2006/1764281 FM201321 B5 Stool Malaysia EV71/MY/2006/1764283 FM201322 B5 Rectal swab Malaysia EV71/MY/2006/1764454 FM201323 B5 Nasopharyngeal swab Malaysia EV71/MY/2006/1764589 FM201324 B5 Stool Malaysia Stool Malaysia EV71/MY/2006/1764739 FM201325 B5 EV71/MY/2006/1765017 FM201326 B5 Stool Malaysia EV71/MY/2006/1765058 FM201327 B5 Stool Malaysia *Site of swab not known. 80 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 Enterovirus 71 Outbreak, Brunei Table 2. Reference Enterovirus 71 sequences used for phylogenetic analysis* Isolate GenBank accession no. Subgenogroup Origin Year Clinical details Reference BrCr-CA-70 U22521 A USA 1970 Encephalitis (11) S11051-SAR-98 AF376081 C1 Sarawak 1998 HFMD (6) 1M-AUS-12-00 AF376098 C1 Australia 2000 HFMD (6) 2M-AUS-3-99 AF376103 C2 Australia 1999 Myelitis (6) 2644-AUS-95 AF135949 C2 Australia 1995 NA (11) KOR-EV71-09 AY125973 C3 SouthKorea 2000 NA UD KOR-EV71-10 AY125974 C3 SouthKorea 2000 NA UD F2-CHN-00 AB115491 C4 China 2000 NA UD H26-CHN-00 AB115493 C4 China 2000 NA UD 1091S/VNM/05 AM490143 C5 Vietnam 2005 NA (10) 999T/VNM/05 AM490163 C5 Vietnam 2005 NA (10) 2609-AUS-74 AF135886 B1 Australia 1974 Meningitis (11) 2258-CA-79 AF135880 B1 USA 1979 Tremors (11) 7673-CT-87 AF009535 B2 USA 1987 NA (11) 2222-IA-88 AF009540 B2 USA 1988 Fever (11) MY104-9-SAR-97 AF376072 B3 Sarawak 1997 Cardiogenicshock (6) 26M-AUS-2-99 AF376101 B3 Australia 1999 HFMD (6) 1067-Yamagata-00 AB213625 B4 Japan 2000 HFMD (8) 2027-SIN-01 AF376111 B4 Singapore 1997 Acute flaccid paralysis (6) CN04104-SAR-00 AF376067 B4 Sarawak 2000 HFMD (6) 5511-SIN-00 AF376121 B5 Singapore 2000 HFMD (6) 2716-Yamagata-03 AB177816 B5 Japan 2003 HFMD (8) 2419-Yamagata-03 AB213647 B5 Japan 2003 HFMD (8) S19841-SAR-03 AY258310 B5 Sarawak 2003 NA UD SB12869-SAR-03 AY905545 B5 Sarawak 2003 NA (3) *HFMD, hand, foot, and mouth disease; NA, not available; UD, unpub. data. from an ancestral strain related to strain 5511/SIN/00 (Gen- Malaysia, and by grant FQ016-2007A from University Malaya, Bank accession no. AF376121), isolated in Singapore as Kuala Lumpur, Malaysia. early as 2000 (3). Subsequently, subgenogroup B5 emerged Dr AbuBakar is a professor and head of the Department of in Japan (8) and Sarawak (3) in 2003, before appearing in Medical Microbiology, Faculty of Medicine, University of Ma- Peninsular Malaysia and Brunei in 2006. The source of the laya, Malaysia. His research interests include pathogenesis and Brunei outbreak remains unclear, and it may not be one of emerging virus infections. these countries where subgenogroup B5 has already been reported. However, EV71 subgenogroup B5 clearly con- References tinues to diverge, and further subgenogroups are likely to 1. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus arise. isolated from patients with disease of the central nervous system. J In summary, the fi rst reported major outbreak of EV71 Infect Dis. 1974;129:304–9. in Brunei was caused by strains from subgenogroup B5 that 2. Bible JM, Pantelidis P, Chan PK, Tong CY. Genetic evolution of were distinct from other reported B5 isolates, suggesting enterovirus 71: epidemiological and pathological implications. Rev Med Virol. 2007;17:371–9. DOI: 10.1002/rmv.538 a recent introduction from an as-yet-unidentifi ed source. 3. Ooi MH, Wong SC, Podin Y, Akin W, del Sel S, Mohan A, et al. Hence, continued molecular surveillance of EV71 in Asia Human enterovirus 71 disease in Sarawak, Malaysia: a prospective is required to further our understanding of factors infl uenc- clinical, virological, and molecular epidemiological study. Clin In- ing the evolution of the virus and its association with emer- fect Dis. 2007;44:646–56. DOI: 10.1086/511073 4. AbuBakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam gence of outbreaks in the region. SK. Identifi cation of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalo- Acknowledgments myelitis in Malaysia. Virus Res. 1999;61:1–9. DOI: 10.1016/S0168- 1702(99)00019-2 We thank the Virology Laboratory, Department of Labora- 5. Kung SH, Wang SF, Huang CW, Hsu CC, Liu HF, Yang JY. Genetic tory Services, RIPAS Hospital, and the Disease Control Division, and antigenic analyses of enterovirus 71 isolates in Taiwan during Public Health Department, Ministry of Health of Brunei Darus- 1998–2005. Clin Microbiol Infect. 2007;13:782–7. DOI: 10.1111/ salam, for their work during the outbreak. j.1469-0691.2007.01745.x 6. McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa The study was funded in part by Top Down grant 36-02-03- MJ. Phylogenetic analysis of enterovirus 71 strains isolated during 6002 from the Ministry of Science, Technology and Innovation, linked epidemics in Malaysia, Singapore, and Western Australia. J Virol. 2001;75:7732–8. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 81 DISPATCHES 7. Singh S, Chow VTK, Chan KP, Ling AE, Poh CL. RT-PCR. Nucle- CoxA16-G10 otide, amino acid, and phylogenetic analyses of enterovirus type 71 BrCr-C A-70 A strains from Asia. J Virol Methods. 2000;88:193–204. DOI: 10.1016/ AF376081-S11051-SAR-98 96.9 C1 S0166-0934(00)00185-3 AF 376098-1M-AUS-12-00 AF376103-2M-AUS-3-99 10 0 8. Mizuta K, Abiko C, Murata T, Matsuzaki Y, Itagaki T, Sanjoh K, C2 AF135949-2644- AUS-95 et al. Frequent importation of enterovirus 71 from surrounding AY125973-KOR-EV71-09 C3 AY125974-KOR-EV71- 10 countries into the local community of Yamagata, Japan, between 100 AB115491-F2-CH N-00 1998 and 2003. J Clin Microbiol. 2005;43:6171–5. DOI: 10.1128/ C4 AB115493-H26-CH N-00 JCM.43.12.6171-6175.2005 AM490143-1091S-VNM-05 C5 AM490163-999T-VN M-05 9. Hosoya M, Kawasaki Y, Sato M, Honzumi K, Kato A, Hiroshima T, AF 135886- 2609-AUS-74 99.6 B1 et al. Genetic diversity of enterovirus 71 associated with hand, foot AF135880-2258-CA-79 10 0 AF009535-7673-CT -87 and mouth disease epidemics in Japan from 1983 to 2003. Pediatr In- 99 .6 B2 AF009540-2222-IA-88 fect Dis J. 2006;25:691–4. DOI: 10.1097/01.inf.0000227959.89339. AF376072-MY104-9-SAR -97 B3 c3 AF376101-26M-AUS-2-99 AB213625-1067-Yamagata-00 10. Tu PV, Thao NT, Perera D, Huu TK, Tien NT, Thuong TC, et al. Epi- 99 .6 AF376111-2027-SIN-01 B4 demiologic and virologic investigation of hand, foot, and mouth dis- AF 376067-CN04104-SAR -00 ease, Southern Vietnam, 2005. Emerg Infect Dis. 2007;13:1733–41. FM201331-34111-BR U-06* AF376121-5511-SIN-00 11. Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, Pallansch AB177816-2716-Yamagata-03 MA. Molecular epidemiology and evolution of enterovirus 71 AB213647-2419-Yamagata-03 AY258310-S19841-SAR-03 strains isolated from 1970 to 1998. J Virol. 1999;73:9969–75. AY905545-SB12869-SAR-03 12. AbuBakar S, Wong PF, Chan YF. Emergence of dengue virus type 4 99.8 FM201325-1764739-MY-06 FM201324-1764589-M Y-06 genotype IIA in Malaysia. J Gen Virol. 2002;83:2437–42. F M201322-1764283-MY-06 13. AbuBakar S, Shafee N, Chee HY. Adenovirus in EV71-associated F M201321-1764281-MY-06 † hand, foot, and mouth disease. Lancet. 2000;355:146. DOI: 10.1016/ FM 201323-1764454-MY-06 S0140-6736(05)72060-2 FM201327-1765058-MY-06 FM201326-1765017-MY-06 14. Sarawak Health Department/Ministry of Health Malaysia. Hand, FM 201341-35247-BR U-06* foot and mouth disease [cited 2008 Sep 1]. Available from http:// F M201346-35379-BRU-06* B5 FM201344-35338-BRU-06* www.sarawak.health.gov.my/hfmd.htm#INFO9 F M201339-35207-BRU-06* FM201329-34095-BRU-06* FM201332-34235-BRU-06* Address for correspondence: Sazaly AbuBakar, Department of Medical FM201330-34099-BRU-06* Microbiology, Faculty of Medicine, University Malaya, 50603 Kuala FM201328-33930-BRU-06* FM201334-34456-BRU-06* 99 .9 Lumpur, Malaysia; email: [email protected] FM201333-34355-BRU-06* F M201358-35731-BRU-06* FM 201359-35732-BR U-06* FM201348-35640-BR U-06* FM201347-35479-BRU-06* FM201343-35335-BRU-06* F M201345-35341-BR U-06* FM201351-35645-BRU-06* FM201342-35334-BRU-06* FM201338-35053-BRU-06* FM201337-34701-BRU-06* FM201336-34700-BRU-06* FM201335-34597-BRU-06* FM201356-35728-BRU-06* FM201349-35641-BRU-06* FM201340-35245-BRU-06* FM201352-35646-BRU -06* FM201350-35643-BRU -06* FM201354-35652-BRU-06* FM201353-35649-BRU-06* FM201355-35653-BRU-06* FM201357-35730-BRU-06* 0.05 FM201360-35754-BRU -06* FM201361-35755-BRU-06* Figure. Phylogenetic relationships of enterovirus 71 partial viral protein (VP1) gene sequences. The prototype coxsackievirus A16 (CoxA16-G10) was used as the outgroup virus. The phylogenetic tree shown was constructed by using the neighbor-joining method. Bootstrap values (>95%) are shown as percentages derived from 1,000 samplings at the nodes of the tree. Scale bar denotes number of nucleotide substitutions per site along the branches. Isolates from this study are indicated by * (Brunei) and † (Peninsular Malaysia). The opinions expressed by authors contributing to this journal do not necessarily refl ect the opinions of the Centers for Disease Con- trol and Prevention or the institutions with which the authors are affi liated. 82 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Emerging Infectious Diseases Pubmed Central

Enterovirus 71 Outbreak, Brunei

AbuBakar, Sazaly; Sam, I-Ching; Yusof, Jaliha; Lim, Meng Keang; Misbah, Suzana; ,; Hooi, Poh-Sim
Emerging Infectious Diseases , Volume 15 (1) – Jan 1, 2009
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Abstract

also was isolated from stool or rectal swabs of 4 patients, of Enterovirus 71 whom 2 were coinfected with EV71; none had neurologic disease. Outbreak, Brunei Enteroviral RNA was extracted from cell cultures us- ing QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Ger- Sazaly AbuBakar, I-Ching Sam, Jaliha Yusof, many), and reverse transcription–PCR was performed to Meng Keang Lim, Suzana Misbah, amplify the viral capsid protein (VP1) gene at nt positions NorAziyah MatRahim, and Poh-Sim Hooi 31–861. The primers used were VP1F 5′-CAGGCTAGCA TGGGAGATAGGGTGGCAGATGTGATCGAGAGC-3′ Enterovirus 71 (EV71) outbreaks occur periodically in the Asia-Pacifi c region. In 2006, Brunei reported its fi rst and VP1R 5′-GGTGGATCCCAAAGGGTAGTAATGGC major outbreak of EV71 infections, associated with fatali- AGTACGACTAGTGCCGGT-3′. The 831-nt partial VP1 ties from neurologic complications. Isolated EV71 strains gene fragments were sequenced, and phylogenetic relations formed a distinct lineage with low diversity within subgeno- of the sequences were examined using selected enterovirus group B5, suggesting recent introduction and rapid spread reference strains obtained from GenBank (Table 2). Se- within Brunei. quences were aligned and phylogenetic trees were drawn using the neighbor-joining method (Figure), as described (12). Maximum-likelihood tree showed similar cluster- nterovirus 71 (EV71), a member of the family Picorna- ing and is not shown. The prototype coxsackievirus A16 Eviridae and the genus Enterovirus, is a common cause (CoxA16-G10) was used as the outgroup virus for con- of hand, foot, and mouth disease in children. Infection with struction of the phylogenetic tree. this virus is rarely complicated by severe neurologic dis- The phylogenetic tree, drawn on the basis of the align- ease, such as meningitis, brain stem encephalitis, neuro- ment of the VP1 gene sequences, showed 3 independent genic pulmonary edema, and acute fl accid paralysis. EV71 genogroups (A, B, and C) with the prototype BrCr strain was fi rst isolated in 1969 (1), and during the subsequent 30 as the only member of genogroup A (11). Within each of years, outbreaks were reported in the United States, Eu- genogroups B and C, 5 additional subgenogroups were rope, and Asia (2). Since 1997, several major outbreaks identifi ed, designated B1–B5 and C1–C5 (8,10). Although with deaths have occurred in the Asia-Pacifi c region, nota- no defi nitions have been established, generally there is nu- bly in Sarawak (East Malaysia), Peninsular Malaysia, Tai- cleotide variation of ≈16%–20% between genogroups and wan, Australia, Singapore, Japan, and Vietnam (3–10). differences of ≈6%–12% between subgenogroups within Brunei is situated on the island of Borneo (4°30′N, each genogroup (5,11). 114°E) and has a population of ≈370,000. From February All Brunei and Malaysia isolates from 2006 clustered through August 2006, Brunei experienced its fi rst reported into subgenogroup B5, except for 1 Brunei isolate, which major outbreak of EV71. More than 1,681 children report- grouped to subgenogroup B4. Nucleotide sequences of edly were affected, with 3 deaths resulting from severe the VP1 gene were highly similar (96%–100%) among all neurologic disease. We report the virologic fi ndings from strains in subgenogroup B5. All Brunei B5 isolates were this outbreak. clustered in an independent lineage within subgenogroup B5 (99.9% bootstrap support), separate from the estab- The Study lished Sarawak and Yamagata isolates from 2003 (8). During March through October 2006, samples from at Amino acid sequences were highly conserved among the least 100 patients from Brunei diagnosed with hand, foot, Brunei B5 isolates, with 99%–100% similarity. No amino and mouth disease or herpangina were received at the Uni- acid sequence changes were observed in the 2 isolates from versity Malaya Medical Center, Kuala Lumpur, Malaysia. patients who died. Samples were inoculated into Vero and A549 cell cultures for virus isolation. EV71 was isolated from 34 patients (in- Conclusions cluding 2 who died of severe neurologic complications), The different genogroups of EV71 are widely distrib- and an additional 7 isolates were obtained from Malaysian uted around the world (2). The continuing appearance of patients seen at the University Malaya Medical Center dur- new EV71 subgenogroups in recent years in the Asia-Pa- ing the outbreak period in Brunei (Table 1). Adenovirus cifi c region suggests that the virus is continuously evolving Author affi liations: University Malaya, Kuala Lumpur, Malaysia (S. (5,8,9). The annual rate of evolution is estimated at 1.35 AbuBakar, I-C. Sam, S. Misbah, N. MatRahim, P.-S. Hooi); and –2 ×10 substitutions per nucleotide, similar to poliovirus Raja Isteri Pengiran Anak Saleha Hospital, Bandar Seri Begawan, (11). In some countries, outbreaks occur in a cyclical pat- Brunei Darussalam (J. Yusof, M.K. Lim) tern every 3 years, predominantly caused by strains that are distinct from previous outbreaks (3,9). These strains often DOI: 10.3201/eid1501.080264 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 79 DISPATCHES have been detected in other countries in the region in years the concurrent spread of other genogroups, subgenogroups, preceding the outbreak. In some EV71 outbreaks, other or enteroviruses. enteroviruses cocirculate, particularly coxsackievirus A16 Other than its northern coastline, Brunei is surrounded or EV71 from a different subgenogroup (3,8,10). On the entirely by the East Malaysian state of Sarawak. In 2006, basis of the samples received in the study, the Brunei 2006 an outbreak of EV71 affected approximately 14,400 chil- EV71 outbreak was caused by subgenogroup B5 virus. dren in Sarawak (14). Thus, temporally and geographical- Apart from the single isolate from subgenogroup B4, no ly, the Brunei and Sarawak outbreaks were related, raising other enteroviruses were isolated, although 2 patients also the possibility that the same strains were involved. Sara- had adenovirus. Occasional EV71 and adenovirus co-infec- wak had experienced EV71 outbreaks every 3 years (1997, tion has been reported (13), also without association with 2000, and 2003), caused by subgenogroups B3, B4, and severe disease. The low sequence diversity and predomi- B5, respectively (3). However, no sequence results from the nance of the Brunei B5 isolates in this outbreak suggest Sarawak 2006 outbreak are available for comparison. All recent introduction and subsequent rapid spread, without subgenogroup B5 isolates reported seem to have diverged Table 1. Enterovirus 71 from Brunei and Malaysia isolated in 2006 Isolate GenBank accession no. Subgenogroup Specimen type Origin EV71/BRU/2006/33930 FM201328 B5 Rectalswab Brunei EV71/BRU/2006/34095 FM201329 B5 Rectalswab Brunei EV71/BRU/2006/34099 FM201330 B5 Rectalswab Brunei EV71/BRU/2006/34111 FM201331 B4 Skinswab Brunei EV71/BRU/2006/34235 FM201332 B5 Throatswab Brunei EV71/BRU/2006/34355 FM201333 B5 Throatswab Brunei EV71/BRU/2006/34456 FM201334 B5 Swab* Brunei EV71/BRU/2006/34597 FM201335 B5 Stool Brunei EV71/BRU/2006/34700 FM201336 B5 Stool Brunei EV71/BRU/2006/34701 FM201337 B5 Stool Brunei EV71/BRU/2006/35053 FM201338 B5 Rectalswab Brunei EV71/BRU/2006/35207 FM201339 B5 Stool Brunei EV71/BRU/2006/35245 FM201340 B5 Rectalswab Brunei EV71/BRU/2006/35247 FM201341 B5 Rectalswab Brunei EV71/BRU/2006/35334 FM201342 B5 Swab* Brunei EV71/BRU/2006/35335 FM201343 B5 Blisterswab Brunei EV71/BRU/2006/35338 FM201344 B5 Swab* Brunei EV71/BRU/2006/35341 FM201345 B5 Swab* Brunei EV71/BRU/2006/35379 FM201346 B5 Rectalswab Brunei EV71/BRU/2006/35479 FM201347 B5 Rectalswab Brunei EV71/BRU/2006/35640 FM201348 B5 Rectalswab Brunei EV71/BRU/2006/35641 FM201349 B5 Rectalswab Brunei EV71/BRU/2006/35643 FM201350 B5 Rectalswab Brunei EV71/BRU/2006/35645 FM201351 B5 Rectalswab Brunei EV71/BRU/2006/35646 FM201352 B5 Rectalswab Brunei EV71/BRU/2006/35649 FM201353 B5 Rectalswab Brunei EV71/BRU/2006/35652 FM201354 B5 Rectalswab Brunei EV71/BRU/2006/35653 FM201355 B5 Rectalswab Brunei EV71/BRU/2006/35728 FM201356 B5 Swab* Brunei EV71/BRU/2006/35730 FM201357 B5 Swab* Brunei EV71/BRU/2006/35731 FM201358 B5 Swab* Brunei EV71/BRU/2006/35732 FM201359 B5 Swab* Brunei EV71/BRU/2006/35754 FM201360 B5 Rectalswab Brunei EV71/BRU/2006/35755 FM201361 B5 Rectalswab Brunei EV71/MY/2006/1764281 FM201321 B5 Stool Malaysia EV71/MY/2006/1764283 FM201322 B5 Rectal swab Malaysia EV71/MY/2006/1764454 FM201323 B5 Nasopharyngeal swab Malaysia EV71/MY/2006/1764589 FM201324 B5 Stool Malaysia Stool Malaysia EV71/MY/2006/1764739 FM201325 B5 EV71/MY/2006/1765017 FM201326 B5 Stool Malaysia EV71/MY/2006/1765058 FM201327 B5 Stool Malaysia *Site of swab not known. 80 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 Enterovirus 71 Outbreak, Brunei Table 2. Reference Enterovirus 71 sequences used for phylogenetic analysis* Isolate GenBank accession no. Subgenogroup Origin Year Clinical details Reference BrCr-CA-70 U22521 A USA 1970 Encephalitis (11) S11051-SAR-98 AF376081 C1 Sarawak 1998 HFMD (6) 1M-AUS-12-00 AF376098 C1 Australia 2000 HFMD (6) 2M-AUS-3-99 AF376103 C2 Australia 1999 Myelitis (6) 2644-AUS-95 AF135949 C2 Australia 1995 NA (11) KOR-EV71-09 AY125973 C3 SouthKorea 2000 NA UD KOR-EV71-10 AY125974 C3 SouthKorea 2000 NA UD F2-CHN-00 AB115491 C4 China 2000 NA UD H26-CHN-00 AB115493 C4 China 2000 NA UD 1091S/VNM/05 AM490143 C5 Vietnam 2005 NA (10) 999T/VNM/05 AM490163 C5 Vietnam 2005 NA (10) 2609-AUS-74 AF135886 B1 Australia 1974 Meningitis (11) 2258-CA-79 AF135880 B1 USA 1979 Tremors (11) 7673-CT-87 AF009535 B2 USA 1987 NA (11) 2222-IA-88 AF009540 B2 USA 1988 Fever (11) MY104-9-SAR-97 AF376072 B3 Sarawak 1997 Cardiogenicshock (6) 26M-AUS-2-99 AF376101 B3 Australia 1999 HFMD (6) 1067-Yamagata-00 AB213625 B4 Japan 2000 HFMD (8) 2027-SIN-01 AF376111 B4 Singapore 1997 Acute flaccid paralysis (6) CN04104-SAR-00 AF376067 B4 Sarawak 2000 HFMD (6) 5511-SIN-00 AF376121 B5 Singapore 2000 HFMD (6) 2716-Yamagata-03 AB177816 B5 Japan 2003 HFMD (8) 2419-Yamagata-03 AB213647 B5 Japan 2003 HFMD (8) S19841-SAR-03 AY258310 B5 Sarawak 2003 NA UD SB12869-SAR-03 AY905545 B5 Sarawak 2003 NA (3) *HFMD, hand, foot, and mouth disease; NA, not available; UD, unpub. data. from an ancestral strain related to strain 5511/SIN/00 (Gen- Malaysia, and by grant FQ016-2007A from University Malaya, Bank accession no. AF376121), isolated in Singapore as Kuala Lumpur, Malaysia. early as 2000 (3). Subsequently, subgenogroup B5 emerged Dr AbuBakar is a professor and head of the Department of in Japan (8) and Sarawak (3) in 2003, before appearing in Medical Microbiology, Faculty of Medicine, University of Ma- Peninsular Malaysia and Brunei in 2006. The source of the laya, Malaysia. His research interests include pathogenesis and Brunei outbreak remains unclear, and it may not be one of emerging virus infections. these countries where subgenogroup B5 has already been reported. However, EV71 subgenogroup B5 clearly con- References tinues to diverge, and further subgenogroups are likely to 1. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus arise. isolated from patients with disease of the central nervous system. J In summary, the fi rst reported major outbreak of EV71 Infect Dis. 1974;129:304–9. in Brunei was caused by strains from subgenogroup B5 that 2. Bible JM, Pantelidis P, Chan PK, Tong CY. Genetic evolution of were distinct from other reported B5 isolates, suggesting enterovirus 71: epidemiological and pathological implications. Rev Med Virol. 2007;17:371–9. DOI: 10.1002/rmv.538 a recent introduction from an as-yet-unidentifi ed source. 3. Ooi MH, Wong SC, Podin Y, Akin W, del Sel S, Mohan A, et al. Hence, continued molecular surveillance of EV71 in Asia Human enterovirus 71 disease in Sarawak, Malaysia: a prospective is required to further our understanding of factors infl uenc- clinical, virological, and molecular epidemiological study. Clin In- ing the evolution of the virus and its association with emer- fect Dis. 2007;44:646–56. DOI: 10.1086/511073 4. AbuBakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam gence of outbreaks in the region. SK. Identifi cation of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalo- Acknowledgments myelitis in Malaysia. Virus Res. 1999;61:1–9. DOI: 10.1016/S0168- 1702(99)00019-2 We thank the Virology Laboratory, Department of Labora- 5. Kung SH, Wang SF, Huang CW, Hsu CC, Liu HF, Yang JY. Genetic tory Services, RIPAS Hospital, and the Disease Control Division, and antigenic analyses of enterovirus 71 isolates in Taiwan during Public Health Department, Ministry of Health of Brunei Darus- 1998–2005. Clin Microbiol Infect. 2007;13:782–7. DOI: 10.1111/ salam, for their work during the outbreak. j.1469-0691.2007.01745.x 6. McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa The study was funded in part by Top Down grant 36-02-03- MJ. Phylogenetic analysis of enterovirus 71 strains isolated during 6002 from the Ministry of Science, Technology and Innovation, linked epidemics in Malaysia, Singapore, and Western Australia. J Virol. 2001;75:7732–8. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 81 DISPATCHES 7. Singh S, Chow VTK, Chan KP, Ling AE, Poh CL. RT-PCR. Nucle- CoxA16-G10 otide, amino acid, and phylogenetic analyses of enterovirus type 71 BrCr-C A-70 A strains from Asia. J Virol Methods. 2000;88:193–204. DOI: 10.1016/ AF376081-S11051-SAR-98 96.9 C1 S0166-0934(00)00185-3 AF 376098-1M-AUS-12-00 AF376103-2M-AUS-3-99 10 0 8. Mizuta K, Abiko C, Murata T, Matsuzaki Y, Itagaki T, Sanjoh K, C2 AF135949-2644- AUS-95 et al. Frequent importation of enterovirus 71 from surrounding AY125973-KOR-EV71-09 C3 AY125974-KOR-EV71- 10 countries into the local community of Yamagata, Japan, between 100 AB115491-F2-CH N-00 1998 and 2003. J Clin Microbiol. 2005;43:6171–5. DOI: 10.1128/ C4 AB115493-H26-CH N-00 JCM.43.12.6171-6175.2005 AM490143-1091S-VNM-05 C5 AM490163-999T-VN M-05 9. Hosoya M, Kawasaki Y, Sato M, Honzumi K, Kato A, Hiroshima T, AF 135886- 2609-AUS-74 99.6 B1 et al. Genetic diversity of enterovirus 71 associated with hand, foot AF135880-2258-CA-79 10 0 AF009535-7673-CT -87 and mouth disease epidemics in Japan from 1983 to 2003. Pediatr In- 99 .6 B2 AF009540-2222-IA-88 fect Dis J. 2006;25:691–4. DOI: 10.1097/01.inf.0000227959.89339. AF376072-MY104-9-SAR -97 B3 c3 AF376101-26M-AUS-2-99 AB213625-1067-Yamagata-00 10. Tu PV, Thao NT, Perera D, Huu TK, Tien NT, Thuong TC, et al. Epi- 99 .6 AF376111-2027-SIN-01 B4 demiologic and virologic investigation of hand, foot, and mouth dis- AF 376067-CN04104-SAR -00 ease, Southern Vietnam, 2005. Emerg Infect Dis. 2007;13:1733–41. FM201331-34111-BR U-06* AF376121-5511-SIN-00 11. Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, Pallansch AB177816-2716-Yamagata-03 MA. Molecular epidemiology and evolution of enterovirus 71 AB213647-2419-Yamagata-03 AY258310-S19841-SAR-03 strains isolated from 1970 to 1998. J Virol. 1999;73:9969–75. AY905545-SB12869-SAR-03 12. AbuBakar S, Wong PF, Chan YF. Emergence of dengue virus type 4 99.8 FM201325-1764739-MY-06 FM201324-1764589-M Y-06 genotype IIA in Malaysia. J Gen Virol. 2002;83:2437–42. F M201322-1764283-MY-06 13. AbuBakar S, Shafee N, Chee HY. Adenovirus in EV71-associated F M201321-1764281-MY-06 † hand, foot, and mouth disease. Lancet. 2000;355:146. DOI: 10.1016/ FM 201323-1764454-MY-06 S0140-6736(05)72060-2 FM201327-1765058-MY-06 FM201326-1765017-MY-06 14. Sarawak Health Department/Ministry of Health Malaysia. Hand, FM 201341-35247-BR U-06* foot and mouth disease [cited 2008 Sep 1]. Available from http:// F M201346-35379-BRU-06* B5 FM201344-35338-BRU-06* www.sarawak.health.gov.my/hfmd.htm#INFO9 F M201339-35207-BRU-06* FM201329-34095-BRU-06* FM201332-34235-BRU-06* Address for correspondence: Sazaly AbuBakar, Department of Medical FM201330-34099-BRU-06* Microbiology, Faculty of Medicine, University Malaya, 50603 Kuala FM201328-33930-BRU-06* FM201334-34456-BRU-06* 99 .9 Lumpur, Malaysia; email: [email protected] FM201333-34355-BRU-06* F M201358-35731-BRU-06* FM 201359-35732-BR U-06* FM201348-35640-BR U-06* FM201347-35479-BRU-06* FM201343-35335-BRU-06* F M201345-35341-BR U-06* FM201351-35645-BRU-06* FM201342-35334-BRU-06* FM201338-35053-BRU-06* FM201337-34701-BRU-06* FM201336-34700-BRU-06* FM201335-34597-BRU-06* FM201356-35728-BRU-06* FM201349-35641-BRU-06* FM201340-35245-BRU-06* FM201352-35646-BRU -06* FM201350-35643-BRU -06* FM201354-35652-BRU-06* FM201353-35649-BRU-06* FM201355-35653-BRU-06* FM201357-35730-BRU-06* 0.05 FM201360-35754-BRU -06* FM201361-35755-BRU-06* Figure. Phylogenetic relationships of enterovirus 71 partial viral protein (VP1) gene sequences. The prototype coxsackievirus A16 (CoxA16-G10) was used as the outgroup virus. The phylogenetic tree shown was constructed by using the neighbor-joining method. Bootstrap values (>95%) are shown as percentages derived from 1,000 samplings at the nodes of the tree. Scale bar denotes number of nucleotide substitutions per site along the branches. Isolates from this study are indicated by * (Brunei) and † (Peninsular Malaysia). The opinions expressed by authors contributing to this journal do not necessarily refl ect the opinions of the Centers for Disease Con- trol and Prevention or the institutions with which the authors are affi liated. 82 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009

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Published: Jan 1, 2009

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