Enterovirus 68 in Children with Acute Respiratory Tract Infections, Osaka, Japan

Enterovirus 68 strains were detected in 14 specimens from children with respiratory tract infections and 1 specimen from a child with febrile convulsions during 2010 in Osaka, Japan. These strains had deletions in the 5′ untranslated region and were genetically different from reported strains. This virus is associated with respiratory tract infections in Japan.

Because of its acid sensitivity and low optimum growth temperature (33°C), EV68 shares characteristics with human rhinovirus (HRV) (3,6) and is genetically and antigenically similar to HRV 87 (6,7). During 1970During -2005, only 26 EV68 strains were detected in the United States (5). Fourteen detections of EV68 were reported during 2006-2009 in Japan: 2 in 2006, 8 in 2007, and 4 in 2009 (8). EV68 is rarely detected in Japan, and no epidemics have been reported. We report deletions in genomes of EV68 strains detected in Japan.

The Study
During October 2009-October 2010, a total of 448 respiratory specimens were obtained from 448 patients (258 male patients and 190 female patients) with RTIs and fevers in a virus surveillance system in Osaka, Japan (9). The mean ± SD age of the patients was 41.4 ± 53.7 months (range <1-404 months), and 351 (78.3%) were <5 years of age.
Procedures for viral nucleic acid extraction and cDNA synthesis have been reported (9). PCR for detecting HRV and enterovirus was conducted by using EVP4 and OL68-1 primers, which detected HRV and human enterovirus, respectively, in amplicons of ≈530 and 650 bp, respectively (7).
EV68 was not detected in virus isolation tests with Vero and RD-18S cells. EV68 was detected during June-September 2010 (Figure 1). Characteristics of 15 EV68positive patients are shown in Table 1. Phylogenetic analysis using VP1 sequences (14 of 15 Osaka strains were sequenced) demonstrated that Osaka strains were clustered in 1 group and differed from previously reported strains ( Figure 2).
Regarding amino acids, <95% identity was observed in VP1, VP2, and VP3 in the Fermon strain, and no genes with <95% aa identity were found in the 37-99 strain in contrast with Osaka strains. Moreover, no integration or deletion of nucleotides was observed in VP4-3D sequences among Osaka, Fermon, and 37-99 strains.
To clarify why EV68 Osaka strain genomes were smaller than those of other enteroviruses and the EV68 Fermon strain (7), we aligned the partial 5′ UTR sequences (nt 541-820 corresponding to the Fermon strain) of 4 Osaka, Fermon, and 37-99 strains (online Appendix Figure, www.cdc.gov/EID/content/17/8/110028-appF.htm). Results showed that the Osaka and 37-99 strains had deletions at nt 681-704 and 717-727 in contrast with the Fermon strain. Moreover, a 1-nt deletion in Osaka strains was identifi ed at nt 641 in contrast with the Fermon and 37-99 strains. Only the JPOC10-378 strain had a 1-nt deletion at nt 670.

Conclusions
Because 14 patients with EV68 were detected during 2006-2009 (8), detection of 15 patients with EV68 during a 4-month period suggests an EV68 epidemic in the summer of 2010 in Japan. Phylogenetic analysis with VP1 sequences showed that Osaka strains differed genetically from previously reported strains.
For precise analysis of Osaka, Fermon, and 37-99 strains, nucleotide and amino acid sequences were  compared in all viral proteins. Results showed that Osaka strains more closely resembled the 37-99 strain than the Fermon strain. Alignment of partial 5′ UTR sequences showed that Osaka and 37-99 strains had deletions in 2 regions in contrast with the Fermon strain, and the amplicon was shorter than expected. Moreover, Osaka strains had 1-nt deletions in contrast with the 37-99 strain. The 5′ UTR of enterovirus contains an internal ribosome entry site (10) that is associated with translational effi ciency and virulence of the enterovirus (11,12). Deleted regions of Osaka strains appear to be in the fl anking region between the internal ribosome entry site and an open reading frame (1). Detection of EV68 in numerous patients was reported in France during 2008 (4) and Italy during 2008-2009 (13). Because this deletion was found in the 37-99 strain in 1998, recent detection of EV68 in Japan might be associated with this change in the viral genome. Smura et al. reported that serum samples from 281 pregnant women in Finland in 1983, 1993, and 2002 had high titers of neutralizing antibody against EV68 (14). This result indicates that EV68 has been in Finland since 1983.
All EV68-positive patients in this study were <5 years of age and had lower respiratory tract infl ammation. Seroepidemiologic studies in Finland showed that most adults might have been previously infected with EV68 and therefore might have neutralizing antibodies (14). Increased detection of EV68, especially in infants and children, will provide useful epidemiologic data.
Recent studies showed that EV68-infected human leukocytes produced infectious progeny virus (14). This result indicates that EV68 can replicate in blood and may damage the central nervous system. EV68 was detected in cerebrospinal fl uid of a young adult patient with acute fl accid paralysis (5). Epidemiologic data for EV68 are lacking, and little information is available regarding virologic characteristics. If one considers results of phylogenetic analyses and nucleotide and amino acid identities, evolutionary changes might have occurred in EV68. Our results show the potential role of EV68 infection in infants and children with RTIs.