Molecular Epidemiology of Human Enterovirus 71 Strains and Recent Outbreaks in the Asia-Pacific Region: Comparative Analysis of the VP1 and VP4 Genes

Mary Jane Cardosa, David Perera, Betty A. Brown, Doosung Cheon, Hung Ming Chan, Kwai Peng Chan, Haewol Cho, Peter McMinn

Emerging Infectious Diseases. 2003;9(4) 

In This Article


Although several studies have attempted to describe the phylogenetic origins of HEV71 strains recently circulating in the Asia-Pacific region,[11,15,17,18,19,20,21,22] no single study has included representative virus strains from all of the HEV71 epidemics recorded in the Asia-Pacific region since 1997 in a comprehensive analysis. We attempted to provide a more complete picture of the phylogenetic relationships between the HEV71 strains circulating in recent outbreaks and to place them in the context of strains isolated earlier and in other parts of the world since this virus was first described. The different genetic typing strategies and nomenclatures adopted by different groups have led to much confusion about the molecular epidemiology of recent HEV71 strains. Briefly summarizing these studies and linking the data presented into a single unifying picture are thus necessary.

Two very limited phylogenetic studies focused on short segments of the 5' UTR. The first compared 13 strains of HEV71 isolated from fatal and nonfatal cases in Malaysia during 1997[20] and showed that at least two lineages of this enterovirus circulated in Peninsular Malaysia during that period. HEV71 strains isolated from a pair of siblings, one with a fatal illness and one with uncomplicated HFMD, belonged to different genetic lineages. Wang et al.[16] also examined the 5' UTR of 36 isolates from the large Taiwanese epidemic in 1998 and reported that most of the isolates clustered into a single genetic lineage, with two exceptions. Both of these studies are of limited value, not only because of the narrow focus on isolates from a single country in a single year, but also because the highly conserved 5' UTR is not a suitable region upon which to base phylogenetic analysis of enteroviruses.[31]

More recently, Wang et al.[11] examined the 5' UTR and the VP1 gene of 58 Taiwan strains isolated in 1998-2000 and showed that the major genogroup of HEV71 circulating in Taiwan changed from genogroup C in 1998 to genogroup B in 1999-2000. In this study, we reexamined nine HEV71 strains reported by Wang et al. and found that the major genogroup circulating during the 1998 Taiwan epidemic was C2, as we had shown previously.[22] More importantly, our analysis shows that the major genogroup circulating in Taiwan changed from C2 in 1998 to B4 in 2000.

Two other phylogenetic analyses of HEV71 focus on the VP4 gene region. Shimizu et al.[15] examined a 420-nt region in the VP4/VP2 gene region of 16 isolates from Malaysia and Japan in 1997 and 13 isolates from Taiwan in 1998. These recent isolates were distributed into two major genogroups, which these researchers labeled genotypes A and B. (These genotype designations are not equivalent to genogroups A and B, originally described by Brown et al.[21]). Chu et al.[19] sequenced a 207-nt region of the VP4 gene of 23 Taiwanese strains. Twenty of the strains used in this study were isolated from fatal and nonfatal cases during the 1998 epidemic. Three strains isolated during the 1986 outbreak of HEV71-associated HFMD in Taiwan were included; this study confirmed that most 1998 Taiwanese strains were from a single genogroup, although several isolates were genetically distinct from the main group. We reexamined the VP4 genetic sequences from the HEV71 strains isolated in Taiwan during 1986 and found that they belonged to genogroup B1.

Use of the VP1 gene has been reported in four phylogenetic studies of HEV71 strains derived from recent HFMD epidemics in the Asia-Pacific region. Two of these studies appear to be of limited value, having analyzed only short VP1 gene sequences and limited numbers of HEV71 isolates within restricted geographic areas. Singh et al.[18] presented a dendrogram based on a 341-nt region of the VP1 gene and concluded that several recent Southeast Asian strains belonged to two completely new genotypes. However, this finding appears to be flawed, as closer examination of the sequences used in construction of the dendrogram shows that they include a mixture of partial VP1 gene sequences and 5' UTR sequences from (occasionally) the same strain. Shih et al.[17] examined partial VP1 gene sequences from 16 isolates from fatal and nonfatal cases in Taiwan during 1998; Shih's study provided further confirmation that most isolates from the 1998 Taiwan outbreak belonged to a single genogroup. Shih et al.[17] described this predominant genogroup as "genotype B" and the minor genogroup as "genotype C" but labeled the isolates in their dendrograms in the reverse order. If one assumes that the dendrogram labeling is correct, their findings are consistent with the nomenclature of Brown et al..[21]

In our study, we used all available published data in addition to new sequence data from the VP4 gene of 51 recent strains and the VP1 gene of 11 Korean strains and 1 Sarawak strain of HEV71 to provide a comprehensive picture of the molecular epidemiology of HEV71 in the Asia-Pacific region since 1997. Our study shows that the first recent HEV71 outbreak recognized in the region occurred in Sarawak in 1997 and was associated with the previously undescribed genogroup B3. Viruses belonging to this genogroup were also circulating in Singapore and Japan in 1997, continued to circulate in Singapore in 1998, and were the primary cause of the epidemic in Western Australia in 1999. Since 1999, genogroup B3 viruses have not been identified anywhere within the region. In 1998, viruses of the C2 genogroup were the primary cause of the Taiwanese epidemic; strains from a distinct lineage within the same genogroup also circulated in Western Australia during 1999. Viruses belonging to the C1 genogroup appear to have undergone low-level endemic circulation in Malaysia, Singapore, and Perth between 1997 and 2002 and have not been associated with large-scale epidemics to date.

Our analysis shows that a great diversity of HEV71 strains circulate in the region and elsewhere and that no particular genogroup is specifically associated with severe disease. Since we were hampered by the fact that many studies failed to provide an accurate diagnosis for the cause of death in fatal cases of HEV71 infection, we suggest that identification of cases of encephalitis, poliomyelitis-like paralysis, or both is likely to provide a more accurate endpoint in the determination of disease severity associated with particular HEV71 genogroups. At this time, insufficient published data are available relating to cause of death to perform this analysis. Currently, the best available data on disease severity are from our previous study,[22] in which viruses isolated from children with severe neurologic disease belonged exclusively to genogroup C2 and possessed a unique amino acid substitution in VP1 at position 170(A®V).

Genogroup B4 strains were isolated occasionally in Peninsular Malaysia, Singapore, and Taiwan from 1997 to 1999. In 2000, viruses from this genogroup caused large epidemics in Sarawak, Singapore, and Taiwan; however, in Korea, an outbreak of HFMD was caused by the new C3 genogroup. Table 3 shows the major HEV71 genotypes circulating in countries within the region since 1997. Clearly, HEV71 is a major emerging virus in the region. Because of this virus' potential for causing severe neurologic disease, we need to understand the factors that have led to the spread of this virus and the genetic factors that contribute to its neurovirulence and epidemic potential.

In conclusion, VP1 and VP4 gene sequences both provide similar phylogenetic information, but the higher bootstrap values seen in the VP1 dendrograms provide greater confidence, particularly when elucidating new genotypes. Thus, the use of the shorter VP4 gene may be helpful for HEV71 surveillance, but the VP1 gene should still be used for molecular epidemiologic research and for confirming data obtained with VP4-based analysis. Virus identification and classification have been reliant on antigenic methods for serotypic identification, and VP1 gene sequence data have been shown to infer serotype.[32] Furthermore, the protein encoded by the VP1 gene is the most exposed and immunodominant of the capsid proteins[33] and is likely to give the most useful information in molecular epidemiologic investigations, unlike untranslated sequences or genes, which encode products not found exposed on the virion surface.

Sarawak is currently experiencing a large outbreak of HEV71 that began in February 2003. Most cases have uncomplicated HFMD, but a small number include neurologic disease. With methods described in this article, more than 70 strains isolated this year have been sequenced in the VP4 gene region. The results show that 80% of the strains belong to genogroup C1(which has been seen sporadically in the Asia-Pacific region since 1997) and suggest that the next big outbreak of HEV71 in the region will likely be due to strains from this genogroup. The remaining strains belong to a distinct cluster within the B genogroup and may represent an emerging new subgenogroup.


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