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

Laboratory and Epidemiology Communications

Isolation of Influenza Virus Type AH3 from a Traveler Returningfrom Vietnam in July 2005 in Osaka, Japan
Tetsuo Kase*, Saeko Morikawa, Yoshinobu Okuno, Fusako Ito1 and Kiyosu Taniguchi2
Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka 537-0025,
1Osaka Prefectural Government, Osaka 540-8570 and
2
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
Communicated by Ichiro Kurane(Accepted October 25, 2005)
On 28 June 2005, the World Health Organization announced
that an additional case of human infection with AH5N1 avian
influenza was confirmed in Vietnam and that the new casebrought the total, in Vietnam, since mid-December 2004,
to 60 cases, of which 18 had been fatal. A man who visited
Vietnam from 4 July 2005 returned to Osaka, Japan, on 8July. As he felt a fever beginning on 7 July, he saw a doctor
soon after his flight to Japan. He was diagnosed with influenza type A infection using a rapid influenza test. Although
he declared that he was traveling around a coast area and thathe had not been near chickens in Vietnam, the doctor reported
this case to the Izumi Health Center of Osaka Prefecture,

because they could not completely deny the possibility that
his influenza was caused by highly pathogenic avian influenza (HPAI). On the afternoon of 8 July, the gargle from thepatient was transported to the Osaka Prefectural Institute ofPublic Health. The specimen was inoculated onto MadinDarby canine kidney (MDCK) cells. Simultaneously, viral
RNA was extracted with QIAamp Viral RNA Mini Kit(QIAGEN K.K., Tokyo, Japan) for analysis by RT-PCR. To
obtain an amplicon on a part of the HA1 region of the HA
gene in influenza A virus, we used primer pairs as follows:5´-CAGATGCAGACACAATATGT-3´ and 5´-AAACCGGC
AATGGCTCCAAA-3´ for H1, 5´-CAGATTGAAGTGACTAATGC-3´ and 5´-GTTTCTCTGGTACATTCCGC-3´ for H3and 5´-CATACCCAACAATAAAGAGG-3´ and 5´-GTGTTC
ATTTTGTTAATGAT-3´ for H5 (1,2). We carried out RT-PCRwith Ready-to-Go RT-PCR Beads (Amersham, Biosciences
UK Ltd., Buckinghamshire, UK) and by using a DNA
thermal cycler (Perkin-Elmer Corp., Norwalk, Conn., USA)

*Corresponding author: Mailing address: Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, 13-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan. Tel:+81-6-6972-1321, Fax: +81-6-6972-2393, E-mail: kase@iph.pref.
osaka.jp395

Table 1. The HI test results of isolated virus against refrence antisera
Antisera
A/Wyoming/
A/Panama/
03/2003
2007/99

A/Moscow/
13/98

A/New Caledonia/
20/99

1,280
nt
nt
nt
nt
nt

nt
320
nt
nt
nt
nt

nt
nt
2,560
nt
nt
nt

<10<101,280

Antigen
A/Moscow/13/98(H1N1)
A/New Caledonia/20/99(H1N1)
A/Wyoming/03/2003(H3N2)
A/Panama /2007/99(H3N2)
B/Brisbane/32/2002
B/Johannesburg/5/99
Isolated virus
A/Osaka/27/2005

B/Brisbane/
32/2002

B/Johannesburg/
5/99

nt
nt
nt
1,280
nt
nt

nt
nt
nt
nt
10,240
nt

nt
nt
nt
nt
nt
>20,480<10<10<10nt: not tested.
1 2

34561

2 345

A/Osaka/27/2005(H3)].
According to the Infectious Disease Surveillance Center,NIID, influenza epidemic seasons in Vietnam are from Juneto August and from December to March in usual years. InJuly 2005, an influenza epidemic occurred in Vietnam. As
there was no influenza virus activity in Osaka in this month,
it is concluded that the patient was infected with influenza
virus type AH3 in Vietnam. This is one of the cases in Japan.
Some papers have already reported that influenza virus
type AH3 was isolated from travelers when they came backto Japan from abroad (3-5). Since HPAI is spreading inVietnam and its neighbor countries now, it is importantthat we examine whether influenza of returnees from thesecountries is conventional human influenza or HPAI, even ifpatients have no history of contact with humans or chickens
infected with HPAI. Furthermore, the examination should
include detection of viral antigens, amplification of viral RNA
and viral isolation for definite diagnosis.

6500 bp

500 bp100 bp100 bp
ABFig. 1. RT-PCR for a part of the HA1 region of the HA gene in influenza A virus. (A) The RT-PCR products from the extract of the gargle
were electrophoresed in 1% agarose gel. Lane 1, marker; lane 2,positive control and primer pairs for H5; lane 3, primer pairs for
H1; lane 4, primer pairs for H3; lane 5, primer pairs for H5; lane 6,negative control and primer pairs for H5. (B) The RT-PCR products
from the extract of the culture fluid showing the CPE were electrophoresed in 1% agarose gel. Lane 1, marker; lane 2, negative control
and primer pairs for H5; lane 3, primer pairs for H1; lane 4, primer
pairs for H3; lane 5, primer pairs for H5; lane 6, positive control andprimer pairs for H5.

This article appeared in the Infectious Agents Surveillance
Report, vol. 26, p. 222, 2005 in Japanese.

consisting of 1 cycle at 42°C for 30 min, 94°C for 5 min,followed by 40 cycles of 94°C for 30 sec, 45°C for 30 sec,
and 72°C for 1 min. However, no PCR products were detectedfrom the extract of the gargle (Figure 1A).
On 11 July, as we observed the cytopathic effects (CPE) in
the MDCK cells inoculated with the specimen, we performeda hemagglutination (HA) test with the supernatant of viralculture using erythrocytes of chickens and humans (bloodgroup O). The fluid agglutinated human erythrocytes but notchicken ones. We peformed a hemagglutination inhibition (HI)test with human erythrocytes against the reference antisera
provided by the National Institute of Infectious Diseases(NIID). As shown in Table 1, the isolate was reacted with
anti-A/Wyoming/03/2003 (H3N2) serum only. We also
carried out RT-PCR with the culture fluid showing CPE by
the method described above. The RT-PCR products were specific for H3 (Figure 1B). The RT-PCR product was purified
in an agarose gel and sequenced directly with BigDye Terminator Cycle Sequencing Ready Reaction Kits with AmpliTaq
DNA Polymerase, FS (PE Applied Biosystems, Foster City,Calif., USA) by using an ABI PRISM 310 Genetic Analyzer(PE Applied Biosystems). A BLAST search showed thatthe sequence of the isolate possessed 98% homology with A/New York/10/2004 (H3N2). These results indicated that theisolated virus was a human influenza virus type AH3 [namedREFERENCES
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