Download Detection and Phylogenetic Analysis of Human Rhinoviruses in

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 OL68–1 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: nakamuma—pref.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, HRV­CO–1396, 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 59.3–64.6z, 56.4–64.8z, and
69.1–99z 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
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