Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Jpn. J. Infect. Dis., 63, 2010 Laboratory and Epidemiology Communications Detection and Phylogenetic Analysis of Human Rhinoviruses in Okinawa, Japan Masaji Nakamura*, Kiyomasa Itokazu, Katsuya Taira, Tatsuyoshi Kawaki1, Jun Kudaka, Minoru Nidaira, Sho Okano, Hirokazu Kimura2, and Masahiro Noda3 Department of Biological Sciences, Okinawa Prefectural Institute of Health and Environment, Okinawa 9011202; 1Aozora Pediatric Clinic, Okinawa 9011302; and 2Infectious Disease Surveillance Center and 3Department of Virology III, National Institute of Infectious Diseases, Tokyo 2080011, Japan Communicated by Ichiro Kurane (Accepted April 21, 2010) swabs by using a QIAamp Viral RNA Mini kit (Qiagen, Valencia, Calif., USA) and suspended in DNase/ RNasefree water. After RNA extraction, cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif., USA) and random hex amer primers (Takara, Shiga, Japan), and PCR was performed using the primers E2 and OL681 as de scribed previously (7,8). Amplicons were purified using a QIAquick PCR Purification kit (Qiagen) and the nucleotide sequences were determined by direct se quencing. Partial nucleotide sequences (393 nt) of the VP4/VP2 region of HRV were phylogenetically ana lyzed using the Molecular Evolutionary Genetics Analy sis (MEGA) software version 4 (9). Evolutionary dis tances were estimated using Kimura's twoparameter method, and phylogenetic trees were constructed using the neighborjoining (NJ) method (10). The reliability of the tree was estimated using 1,000 bootstrap replica tions. In the present study, 13 HRV strains were detected by RTPCR in patients with ARIs and other viral infec tions. Figure 1 shows a phylogenetic tree based on the VP4/VP2 sequences including the present strains and reference strains. Of the 13 new strains, 4 (31z) were classified into HRVA, 3 (23z) into HRVB, and 6 (46z) into HRVC. Human rhinoviruses (HRVs) are the cause of com mon colds and asthmatic exacerbation (1). Phylogenetic analysis of the VP4/VP2 sequences of HRVs has re vealed that all HRV serotypes except serotype 87 belong to 2 different species, HRVA and HRVB (2). Recent ly, several groups have reported the presence of a new HRV species, HRVC (3,4). Although HRVC cannot be cultured, it is distributed worldwide and is found in association with community outbreaks of acute respira tory infections (ARIs) (4,5). In Japan, HRVA isolated from patients with ARIs in Yamagata Prefecture has been phylogenetically analyzed (6). However, the molecular epidemiology of HRVs from Okinawa Pre fecture is not well known. Therefore, we performed phylogenetic analysis of the VP4/VP2 sequences of HRVs detected in patients with ARIs and other viral in fections in Okinawa Prefecture from June 2008 to Janu ary 2010. Viral RNA was extracted from the nasopharyngeal *Corresponding author: Mailing address: Department of Biological Sciences, Okinawa Prefectural Institute of Health and Environment, 2085 Ozato, Nanjoshi, Okina wa 9101202, Japan. Tel: {81989450785, Fax: {81 989459366, Email: nakamumapref.okinawa.lg.jp 221 Fig. 1. Phylogenetic tree based on the VP4/VP2 coding region sequences (393 nt) of the 41 human rhinoviruses (HRVs) including the present strains and reference strains. The present strains are shown as bold letters. Numbers in parentheses indicate the Genbank accession number. The numbers at each branch indicate the bootstrap value for the clusters. (URTI) and 1 was diagnosed with pneumonia. The 3 patients with HRVB infection were separately diag nosed with a lower respiratory tract infection (LRTI), pneumonia, and viral myocarditis. Finally, of the 6 patients with HRVC infection, 2 had URTI, 3 had LRTI, and 1 had viral meningitis. However, we could not estimate the relevance of pathogenicity with HRV species or strains because of the small number of sam ples in this study. In conclusion, our results suggest that genetically diverse HRVs, including those belonging to HRVC (a new species), are distributed in Okinawa. However, ad ditional epidemiological and molecular epidemiological studies may be needed to better understand HRV infec tion in Okinawa Prefecture. The 4 present strains belonging to HRVA were locat ed in 4 distinct subclusters formed by the serotype known reference strains (HRV 59, HRV 85, HRV 36, and HRV58). The 3 present strains belonging to HRVB were located in 2 distinct subclusters formed by the sero type known reference strains (HRV 35 and HRV 91). The 6 present strains belonging to HRVC also segregat ed into 6 distinct subclusters formed by the reference strains (HRVC 025, PNC86275, Resp3266/06, HRVC 024, HRVCO1396, and PNC43211). These Okinawa strains analyzed in this study were also similar to other strains (PUMCH2452, N37, and PUMCH3926 from China, Resp3917 and Resp2659 from the United King dom, PNC89019 and PNC90314 from Finland, RV265 and RV459 from the USA, and S03970 from Spain). The nucleotide sequences of the present strains belong ing to HRVC were 59.364.6z, 56.464.8z, and 69.199z identical to HRVA, HRVB, and HRVC reference strains, respectively. These results suggest that HRVs from Okinawa have diverse genetic variations. Of the 4 patients with HRVA infection, 3 were clini cally diagnosed with an upper respiratory tract infection This work was supported in part by Research on Emerging and Reemerging Infectious Diseases, Labour and Welfare Programs of the Ministry of Health, Labour and Welfare of Japan (H21Shinkouippan013). 222 REFERENCES 1. Wos, M., Sanak, M., Soja, J., et al. (2008): The presence of rhinovirus in lower airways of patients with bronchial asthma. Am. J. Respir. Crit. Care Med., 177, 10821089. 2. Savolainen, C., Blomqvist, S., Mulders, M.N., et al. (2002): Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70. J. Gen. Virol., 83, 333340. 3. Lamson, D., Renwick, N., Kapoor, V., et al. (2006): MassTag polymerasechainreaction detection of respiratory pathogens, in cluding a new rhinovirus genotype, that caused influenzalike ill ness in New York State during 20042005. J. Infect. Dis., 194, 13981402. 4. Lau, S.K., Yip, C.C., Tsoi, H.W., et al. (2007): Clinical features and complete genome characterization of a distinct human rhinovirus (HRV) genetic cluster, probably representing a previ ously undetected HRV species, HRVC, associated with acute respiratory illness in children. J. Clin. Microbiol., 45, 36553664. 5. Briese, T., Renwick, N., Venter, M., et al. (2008): Global distri 6. 7. 8. 9. 10. 223 bution of novel rhinovirus genotype. Emerg. Infect. Dis., 14, 944947. Mizuta, K., Hirata, A., Suto, A., et al. (2010): Phylogenetic and cluster analysis of human rhinovirus species A (HRVA) isolated from children with acute respiratory infections in Yamagata, Japan. Virus Res., 14, 265274. Chapman, N.M., Tracy, S., Gauntt, C.J., et al. (1990): Molecu lar detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J. Clin. Microbiol., 28, 843850. Olive, D.M., AlMulla, S., Khan M.A., et al. (1990): Detection and differentiation of picornaviruses in clinical samples following genomic amplification. J. Gen. Virol., 71, 21412147. Tamura, K., Dudley, J., Nei, M., et al. (2007): MEGA4: Molecu lar Evolutionary Genetics Analysis (MEGA) Software version 4.0. Mol. Biol. Evol., 24, 15961599. Saitou, N. and Nei, M. (1987): The neighborjoining method: a new method for reconstructing phylogenetic trees. Biol. Evol., 4, 406425.