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Genetic Diversity of Enterovirus 71 Associated With Hand, Foot and Mouth Disease Epidemics in Japan from 1983 to 2003

Hosoya, Mitsuaki MD*; Kawasaki, Yukihiko MD*; Sato, Masatoki MD*; Honzumi, Ken*; Kato, Asako*; Hiroshima, Toyomasa; Ishiko, Hiroaki PhD; Suzuki, Hitoshi MD*

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The Pediatric Infectious Disease Journal: August 2006 - Volume 25 - Issue 8 - p 691-694
doi: 10.1097/01.inf.0000227959.89339.c3



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Human enterovirus 71 (EV71) is one of the major etiologic agents of hand, foot and mouth disease (HFMD). The surveillance data indicate that EV71 infection follows an epidemic mode of transmission, causing large outbreaks and then becoming quiescent for a few years.

Molecular epidemiology of EV71 strains isolated in the United States and 5 other countries had been described by Brown et al.1 In their study, the prototype strain, BrCr-CA-70, isolated in California in 1970, was the sole member of genogroup A. Strains isolated in the United States and Australia from 1972 to 1988 was all members of genogroup B, and the group has not been isolated in the United States from 1988. Genogroup C was isolated in 1985 or later in the United States, Canada, Australia and the Republic of China. The study showed that EV71 was a genetically evolving virus.

EV71 is also associated with cases of acute neurologic diseases, including poliomyelitis-like paralysis, encephalitis and aseptic meningitis. In 1997, deaths associated with epidemics of EV71-associated HFMD in Sarawak, Malaysia, followed by outbreaks with high mortality in Taiwan in 1998 and 2000, have raised considerable public concern about the virulence of this virus.2 Several groups3,4 have attempted to describe the molecular epidemiology of recent EV71 isolates in the Asia-Pacific region. In Sarawak, in 1997, all isolates from fatal and nonfatal cases belonged to subgroup B3. Viruses belonging to B3 were also isolated in Singapore in 1998 and in Perth in 1999. Some EV71 strains isolated from children with severe neurologic disease during Perth epidemics in 1999 belonged to subgroup C2. Another recently described subgroup B cluster (B4) was identified in Singapore in 1997 and continued to circulate there in 2000 through 2002. Viruses from B4 were also identified as the primary cause of a large outbreak in Sarawak in 2000. These studies suggested that dominant EV71 strains causing recent epidemics were genetically changed and the divergent strains were transmitted in the Asia-Pacific region.

However, the relationship of repeated HFMD outbreaks caused by EV71 with the genetic diversity of the virus strains has not been fully described. We attempted to provide a more complete picture of the relationship between the longitudinal EV71 epidemics for more than 20 years in a restricted area, that is, Fukushima Prefecture, Japan, and the genetic diversity of the EV71 strains, and the transversal genetic relationship between the EV71 strains isolated in Fukushima and those isolated in the world using phylogenetic analysis constructed using the neighbor-joining method on the basis of the VP4 sequence.


Pharyngeal swab and/or rectal swab samples were collected from patients with HFMD in Fukushima Prefecture for virus surveillance and transferred to Fukushima Institute of Public Health for virus isolation. HEp-2, Vero and RD-18 cells were used for the isolation of enteroviruses. Confluent cell cultures were seeded in microplate wells and inoculated with 100 μL of maintenance medium and 50 μL of pharyngeal swab samples. The cell culture were then incubated at 34°C in 5% CO2/95% air and observed for 7 days to check for cytopathic effects. Virus isolates were identified by a neutralization test using anti-EV71 polyclonal antibodies provided from National Institute of Infectious Diseases in Japan. A total of 186 EV71 strains were isolated and identified from 1983 to 2003. Those isolates were stored at −80°C.

Selected isolates (35 of 100 strains) from 1983 to 1998 and all isolates (86 strains) from 1999 to 2003 were used for further genetic analysis. All strains were isolated from patients with HFMD. There was no case with fatal outcome or severe neurologic complications. All strains were isolated from pharyngeal swab, except 3 strains isolated from rectal swabs (Fukushima/1448–2/96, Fukushima/9990/03 and Fukushima/10029/03).

The methods of molecular diagnosis of enteroviruses by nested reverse transcription (RT)–polymerase chain reaction (PCR) and phylogeny-based classification using the VP4 sequences are described elsewhere.5 Briefly, viral RNA was directly extracted from 100 μL of the stock virus samples using the Smitest R kit (Genome Science Laboratories) according to the manufacturer's instructions. The RNA was dissolved with 10 μL of RNase-free distilled water containing 40 U of ribonuclease inhibitor (RNasin; Promega) and 50 pmol of a reverse primer, OL68-1 (nt 1178–1197, 5′-GGTAA(C/T)TTCCACCACCA[A/G/C/T]CC-3′). The positions of the primers for RT-PCR were numbered according to the complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain.6 The RNA was subjected to heat denaturation for 15 seconds at 100°C followed by snap-cooling in an ice-water bath. To each RNA sample, we added 10 μL of a reaction mixture, 200 U of Moloney murine leukemia virus reverse transcription (Life Technology), 2.5 mM dNTPs and 40 U of RNasin (Promega). cDNA synthesis was performed for 1 hour at 37°C. In total, 5 μL of the cDNA reaction mixture was added to 45 μL of 1× Taq buffer containing 12.5 pmol of a forward primer, MD91 (nt 444–468, 5′-′CCTCCGGCCCCTGAATGCGGCTAAT-3′) and 2.5 U of TaqDNA polymerase (Roche Diagnostic Systems). Seminested PCR was performed using 5 μL of the PCR product with a pair of primers, EVP4 (nt 541–560, 5′-CTACTTTGGGTGTCCGTGTT-3′) and OL68-1. After initial denaturation at 94°C for 5 minutes, 40 cycles of amplification were performed using the GeneAmp PCR System 9600 (PE-Applied Biosystems). Each cycle consisted of denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds and an extension reaction at 72°C for 1 minute followed by a final extension at 72°C for 7 minutes. The PCR products, including entire VP4 sequences, were separated in 1% agarose gels and purified with a QIA quick-gel extraction kit (Qiagen). The nucleotide sequence was determined with a 373A DNA autosequencer (PE-Applied Biosystems) with fluorescent dideoxy chain terminations (PE-Applied Biosystems) and EVP4 and OL68-1 primers.

The VP4 nucleotide sequences of the 121 EV71 strains isolated in Fukushima were used for phylogeny-based analysis along with those of 64 prototype enterovirus strains (Table 1 [online only]). We estimated the evolutionary distances using the Kimura 2-parameter method7 and constructed unrooted phylogenetic trees with the neighbor-joining method.8 Bootstrap analysis was performed by resampling the data sets 1000 times. Bootstrap values greater than 70% were considered to be statistically significant for the grouping. The VP4 sequences of representative 43 EV71 strains isolated in Fukushima were also compared with those of 65 strains from other regions in Japan and 99 strains from other countries, including Taiwan, Malaysia, Korea, China, Australia, the United Kingdom and the United States taken from international databases (Genbank) using phylogenetic analysis (Table 2 [online only]). Available clinical information on those cases was obtained from the literature, and the cases with fatal prognosis or severe illness such as encephalitis, paralysis and Guillain-Barré syndrome were indicated in figures.


We observed HFMD outbreaks in Fukushima in 1984, 1987, 1990, 1993, 1997 and 2000, and a more recent, larger outbreak in 2003 (Fig. 1). During 21 epidemics, we observed no HFMD case with encephalitis or fatal prognosis in Fukushima Prefecture. A nested RT-PCR assay was performed for the detection of enteroviral genome sequences and a positive PCR result was obtained in all 121 samples. Detected enterovirus strains were identified as EV71 using phylogeny-based analysis. Phylogenetic reconstruction of EV71 strains isolated in Fukushima from 1983 to 2001 demonstrated 6 genetically distinct subgroups, which were previously designated as B-1, B-2 and 3, B-4, C-1, C-2, and C-3 and other 2 indistinct subgroups, which belonged to genogroup C but were not classified into previously designated subgroups. The later 2 subgroups were named as C-U1 and C-U2, because their independence was not supported by bootstrap values. Of those subgroups, B-1, C-U1, C-U2, C-2 and B4 dominantly related to epidemics that occurred in the years 1984, 1987 and 1990, 1993, 1997 and 2000, respectively (Fig. 2 [online only]).

Numbers of enterovirus 71 (EV71) isolates in Fukushima Prefecture and in Japan between 1983 and 2003. Numbers of EV71 isolates in Fukushima (closed circle) and in Japan (open circle) are expressed as reported to the Infectious Disease Surveillance Center in Japan by prefectural and municipal public health institutes through the Japanese infectious agents’ surveillance program.

EV71 strains isolated from 2002 to 2003 in Fukushima were grouped to 4 distinct clusters. Of those subgroups, 2 were classified into subgroups C-1 and B-4, and the other 2 belonged to group B and group C but were distinct from the previously designated subgroups. The later 2 subgroups were very recently designated as B-59 and C-4,10 respectively. Subgroups B5 and C4 were dominant in 2003.

VP4 sequences detected from representative 43 EV71 strains isolated in Fukushima were compared with those from international databases (Genbank), which included 65 strains isolated in other parts of Japan, 68 strains in Asia-Pacific region, 17 strains in the United States and 14 strains in Europe (Fig. 3 A, B [online only]). Subgroup B-1 included 7 EV71 strains isolated in Fukushima from 1983 to 1985, 12 strains in other parts of Japan from 1970 to 1984, 4 strains in Asia from 1973 to 1986, 6 strains in the United States from1976 to 1987 and 2 strains in Europe from 1975 to 1978 (Fig. 3A online). Subgroup C-2 included 13 EV71 strains isolated in Fukushima from 1997 to 2001, 10 strains in other parts of Japan from 1997 to 2000, 12 strains in Taiwan in 1998, 5 strains in the United Kingdom from 1996 to 1999 and 3 strains in Australia in 1999 (Fig. 3B [online only]). Newly designated subgroup B-5 included 22 EV71 strains isolated in Fukushima in 2003, 3 strains in other parts of Japan in 2003 and one strain in Malaysia in 2003.9 Subgroup C-4 included 49 EV71 strains isolated in Fukushima from 2002 to 2003 and 15 strains in China from 1998 to 2005. In general, EV71 isolates derived from each outbreak occurred in Fukushima made a single cluster with those isolated during almost the same time period in another area of Japan and in other countries.

Available clinical information on the cases was obtained from the literature. The cases with fatal prognoses or neurologic complications were indicated in Figures 3A and 3B. We could not find any association of serious cases with certain subgenogroups in the cluster analysis.


EV71 causes large outbreaks of HFMD worldwide. Enterovirus surveillance data in Fukushima Prefecture from 1983 to 2003 indicate that the annual proportion of EV71 isolates relative to total enterovirus isolates fluctuates widely, from 0% in 1988, 1991, 1992, 1998 and 1999 to 29.0% in 2003. Peaks of EV71 isolations from HFMD occurred in the years 1984, 1987, 1990, 1993, 1997, 2000 and 2003. This epidemic pattern is very similar to that observed in Japanese surveillance data (Fig. 1).11 Those observations indicate that EV71 follows an epidemic mode of transmission, causing large outbreaks and then becoming quiescent for a few years. Quiescence between outbreaks is probably the result of the development of population immunity that occurs during a high-infection-rate epidemic.

Phylogeny-based classification by use of the VP4 sequence is useful for the identification of human enteroviruses.12,13 The method takes advantage of the detection of the divergence in VP4 sequences both between and within serotypes, and thus is also of use for global epidemiologic studies of enteroviruses.4,5 We investigated the genetic diversity of EV71 associated with HFMD outbreaks in Fukushima Prefecture, Japan, from 1983 to 2003 and compared their genetic relation with those isolated in other regions of Japan and in other countries using the same method. The VP1 dendrograms provide great confidence by high bootstrap values when elucidating new genogroups.1 In the present study, EV71 genogroups A, B and C were designated based on the VP4 sequences by differing at 12.1 to 24.2% at the nucleotide level, and the differing was similar to that based on the VP1 gene analysis.

Phylogenetic reconstruction of EV71 strains isolated in Fukushima from 1983 to 2003 demonstrated at least 8 genetically distinct clusters, which included 6 subgroups previously designated as B-1, B-2 and 3, B-4, C-1, C-2 and C-3, and 2 subgroups newly designated as B-5 and C-4. Other 2 indistinct clusters belonged to genogroup C and were named C-U1 and C-U2. Of those subgroups, B-1, C-U1, C-U2, C-2, B4, and B-5 and C-4 dominantly related to epidemics that occurred in the years 1984, 1987 and 1990, 1993, 1997, 2000 and 2003, respectively. Our results showed that EV71 strains causing HFMD epidemics in Fukushima, Japan, had been genetically changed, and the repeated large outbreaks might be caused by the introduction of genetically divergent EV71 strains. EV71 isolates derived from each outbreak in Fukushima made a single cluster with those isolated during almost the same time period in another area of Japan and in other countries. Those results demonstrated that the genetically divergent EV71 strains might be transmitted from other regions in the world to Japan, be predominant for a few years and disappear.

In clinical viewpoints, HFMD cases with neurologic complications or fatal prognosis were genetically, chronologically and geographically widely distributed. This indicates that the virulent EV71 genogroups might not relate to the cases with severe illness.

In conclusion, our results suggest that the large repeated EV71-related HFMD outbreaks in the world might be the result of worldwide transmission of newly introduced genetically divergent EV71 strains as well as a large cohort of nonimmune individuals. To confirm the aspect, a worldwide surveillance system for HFMD and genetic analysis of isolated EV71 strains is necessary.


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enterovirus 71; genetic diversity; phylogenetic analysis

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