Enterovirus 71 Outbreak, Brunei

Enterovirus 71 (EV71) outbreaks occur periodically in the Asia-Pacific region. In 2006, Brunei reported its first major outbreak of EV71 infections, associated with fatalities from neurologic complications. Isolated EV71 strains formed a distinct lineage with low diversity within subgenogroup B5, suggesting recent introduction and rapid spread within Brunei.


The Study
During March through October 2006, samples from at least 100 patients from Brunei diagnosed with hand, foot, and mouth disease or herpangina were received at the University Malaya Medical Center, Kuala Lumpur, Malaysia. Samples were inoculated into Vero and A549 cell cultures for virus isolation. EV71 was isolated from 34 patients (including 2 who died of severe neurologic complications), and an additional 7 isolates were obtained from Malaysian patients seen at the University Malaya Medical Center during the outbreak period in Brunei (Table 1). Adenovirus also was isolated from stool or rectal swabs of 4 patients, of whom 2 were coinfected with EV71; none had neurologic disease.
Enteroviral RNA was extracted from cell cultures using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), and reverse transcription-PCR was performed to amplify the viral capsid protein (VP1) gene at nt positions 31-861. The primers used were VP1F 5′-CAGGCTAGCA TGGGAGATAGGGTGGCAGATGTGATCGAGAGC-3′ and VP1R 5′-GGTGGATCCCAAAGGGTAGTAATGGC AGTACGACTAGTGCCGGT-3′. The 831-nt partial VP1 gene fragments were sequenced, and phylogenetic relations of the sequences were examined using selected enterovirus reference strains obtained from GenBank ( Table 2). Sequences were aligned and phylogenetic trees were drawn using the neighbor-joining method ( Figure), as described (12). Maximum-likelihood tree showed similar clustering and is not shown. The prototype coxsackievirus A16 (CoxA16-G10) was used as the outgroup virus for construction of the phylogenetic tree.
The phylogenetic tree, drawn on the basis of the alignment of the VP1 gene sequences, showed 3 independent genogroups (A, B, and C) with the prototype BrCr strain as the only member of genogroup A (11). Within each of genogroups B and C, 5 additional subgenogroups were identifi ed, designated B1-B5 and C1-C5 (8,10). Although no defi nitions have been established, generally there is nucleotide variation of ≈16%-20% between genogroups and differences of ≈6%-12% between subgenogroups within each genogroup (5,11).
All Brunei and Malaysia isolates from 2006 clustered into subgenogroup B5, except for 1 Brunei isolate, which grouped to subgenogroup B4. Nucleotide sequences of the VP1 gene were highly similar (96%-100%) among all strains in subgenogroup B5. All Brunei B5 isolates were clustered in an independent lineage within subgenogroup B5 (99.9% bootstrap support), separate from the established Sarawak and Yamagata isolates from 2003 (8). Amino acid sequences were highly conserved among the Brunei B5 isolates, with 99%-100% similarity. No amino acid sequence changes were observed in the 2 isolates from patients who died.

Conclusions
The different genogroups of EV71 are widely distributed around the world (2). The continuing appearance of new EV71 subgenogroups in recent years in the Asia-Pacifi c region suggests that the virus is continuously evolving (5,8,9). The annual rate of evolution is estimated at 1.35 ×10 -2 substitutions per nucleotide, similar to poliovirus (11). In some countries, outbreaks occur in a cyclical pattern every 3 years, predominantly caused by strains that are distinct from previous outbreaks (3,9). These strains often have been detected in other countries in the region in years preceding the outbreak. In some EV71 outbreaks, other enteroviruses cocirculate, particularly coxsackievirus A16 or EV71 from a different subgenogroup (3,8,10). On the basis of the samples received in the study, the Brunei 2006 EV71 outbreak was caused by subgenogroup B5 virus. Apart from the single isolate from subgenogroup B4, no other enteroviruses were isolated, although 2 patients also had adenovirus. Occasional EV71 and adenovirus co-infection has been reported (13), also without association with severe disease. The low sequence diversity and predominance of the Brunei B5 isolates in this outbreak suggest recent introduction and subsequent rapid spread, without the concurrent spread of other genogroups, subgenogroups, or enteroviruses.
Other than its northern coastline, Brunei is surrounded entirely by the East Malaysian state of Sarawak. In 2006, an outbreak of EV71 affected approximately 14,400 children in Sarawak (14). Thus, temporally and geographically, the Brunei and Sarawak outbreaks were related, raising the possibility that the same strains were involved. Sarawak had experienced EV71 outbreaks every 3 years (1997, 2000, and 2003 In summary, the fi rst reported major outbreak of EV71 in Brunei was caused by strains from subgenogroup B5 that were distinct from other reported B5 isolates, suggesting a recent introduction from an as-yet-unidentifi ed source. Hence, continued molecular surveillance of EV71 in Asia is required to further our understanding of factors infl uencing the evolution of the virus and its association with emergence of outbreaks in the region.  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).
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