Jpn. J. Infect. Dis., 58, 2005

Laboratory and Epidemiology Communications

Prevalence of Coxsackievirus A5, A6, and A10 in Patientswith Herpangina in Aichi Prefecture, 2005
Teruo Yamashita*, Miyabi Ito, Akiko Taniguchi and Kennji Sakae
Department of Microbiology, Aichi Prefectural Institute of Public Health, Aichi 462-8576, Japan
Communicated by Tatsuo Miyamura(Accepted October 11, 2005)
CV-A2*

Herpangina is caused primarily by coxsackievirus A (CVA) 1, 2, 3, 4, 5 (CV-A5), 6 (CV-A6), 8, 10 (CV-A10), and 22(1) in the genus Enterovirus of the family Picornaviridae.
The illness is characterized by an acute onset of fever andsore throat (2). In Aichi Prefecture, located near the center ofJapan’s main island of Honshu, the number of case reportsof herpangina increased from the 23rd week of 2005 andresulted in occurrence exceeding 12 cases per sentinel in the27th week of 2005 (a diagram is available at http://www.
pref.aichi.jp/eiseiken/herpan_graph.html). The number was
twice as great as the average of the last 5 years. We detected
CV-A5, -A6, and -A10 from these patients.
Feces and/or throat swabs were collected from 64
herpangina patients who visited 9 pediatric clinics in Aichi
Prefecture between April and July 2005. Their ages ranged
from 4 months to 7 years. Their samples were suspended in
Veal infusion broth and centrifuged at 10,000 × g for 20 min.
The supernatants were inoculated onto RD cells and used for
RT-PCR, as previously described (3). Briefly, the supernatants
were mixed with TRIzol and followed by isopropanol
precipitation. The pellet was suspended in RNase-free water.
RT-PCR was performed using Access Quick RT-PCR System (Promega, Madison, Wis., USA). Primers OL68-1(+) andMD91(-), designed by Olive et al. (4) and Rotbart et al. (5),were used for RT-PCR. Primers OL68-1(+) and EVP4(-),
designed by Rotbart et al. (5), were used for the nested PCR.
Following the PCR, amplified products were purified andintroduced into a pGEM-T vector (Promega). The DNA

CV-A14*
CV-A4*
CV-A7*
CV-A8*
CV-A12*05-10099
05-10208
03-1008400-10719
01-10142
02-00160
04-10062
CV-A10*00-10093
CV-A6*
05-10051
05-1013303-10032
01-10260
02-10059
CV-A5*

Table 1. Detection of coxsackievirus A from patients withherpangina between 2000 and 2005

03-10093
05-00141

Virus
CV-A 2
CV-A 4CV-A 5
CV-A 6
CV-A 8
CV-A10
CV-A12
CV-A16

2000
120

2001
51

11115
3

172

123

Year/No. patients
2002
2003
40
40
2521

2004
77

21
1
144
34

1
42

2
1
1

CV-A3*

2005
64

EV-76*
EV-89*
EV-90*

1
10EV-91*
CV-A16*2
1

19

EV-71*
0.1

Fig. 1. Phylogenetic relationship among prototype strain ofcoxsackievirus type A and isolates from patients with herpangina
based on nucleotide sequences of the VP4 region. The sequences ofthis year (05-10099, 05-10208, 05-10051, 05-10133, and 05-00141)
clustered with CV-A10, CV-A6, or CV-A5, and were most closely
related with the isolates in 2003 (03-10084, 03-10032, or 03-10093).
*: prototype strain.

*Corresponding author: Mailing address: Department of Microbiology, Aichi Prefectural Institute of Public Health, 7-6, Nagare,
Tsujimachi, Kita-ku, Nagoya, Aichi 462-8576, Japan. Tel:+81-52-910-5674, Fax: +81-52-913-3641, E-mail: tyamashita@
hi-ho.ne.jp390Jpn. J. Infect. Dis., 58, 2005
that our PCR was as sensitive as the use of suckling mice (6).
Therefore, we have used PCR for identification of CV-A since
2004.

sequence was determined by a Model-4000 automated DNA
sequencer (Li-cor, Lincoln, Nebr., USA). Serotype was determined from the sequence by comparison to a database of allenterovirus VP4 sequences using the Genetyxi program(Genetix, New Milton, Hampshire, UK).
No cytopathic effect (CPE) was observed in any of theinoculated RD cells, but the virus RNA could be detected in
30 of 64 (46.9%) patients using PCR. Of 30 positive samples,the sequences of 19 samples were most closely related to thatof CV-A10, 10 to CV-A6, and one to CV-A5 (Table 1). The
sequences in the same group of this year were at least 99%
identical to one another. CV-A10 isolates were about 79%
identical to the prototype strain and 87% to the isolated strain
in 2003. CV-A6 isolates were about 80% identical to the prototype strain and 95% to the isolates in 2003. These pairwisecomparisons were consistent with the results of phylogenetic
reconstruction (Fig. 1). The sequences of CV-A10 isolates inthese last 6 years cluster into 4 major phylogenetic groups.
The sequences from this year were most closely related tothe isolates from 2003. The sequences of CV-A6 from thisyear were most closely related to that from 2003 (Fig. 1).
The patients with CV-A10 ranged in age from 4 months to 7years, and those with CV-A6 were up to 3 years old.
In 1994, CV-A10 was the most prevalent serotype inpatients with herpangina in Aichi Prefecture. Since then, thenumber of isolates has become fewer. It could be isolated
every season between 2000 and 2004, but the number was
not as high as another serotype that was the most prevalent ineach year (Table 1). CV-A6 was the most prevalent serotype
in 2000 and 2003. This year, CV-A10 and -A6 were moreprevalent, and resulted in a larger number of patients.
Until 2003, we used suckling mice for isolation of viruses
from patients with herpangina. However, this method from apractical standpoint is difficult, and we were able to confirm

This article appeared in the Infectious Agents Surveillance
Report, vol. 26 no. 7, p. 178, 2005 in Japanese.
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