Jpn. J. Infect. Dis., 59, 2006

Laboratory and Epidemiology Communications

Virological Analysis of a Case of Dual Infection byInfluenza A (H3N2) and B Viruses
Shin’ichi Shimada*, Kenji Sadamasu1, Takayuki Shinkai1, Osamu Kakuta2, Yoshinori Kikuchi,
Michiyo Shinohara, Kazue Uchida, Rie Doi, Kyoko Kohmoto, Miho Shimizu and Setsuko Nakajima3
Department of Viral Diseases, Saitama Institute of Public Health, Saitama 338-0824;
1
Division of Virology, Department of Microbiology, Tokyo Metropolitan Institute ofPublic Health, Tokyo 169-0073; 2Kakuta Pediatric Clinic, Saitama 360-0023; and3
Department of Virology, Medical School, Nagoya City University, Nagoya 467-8601, Japan
Communicated by Ichiro Kurane(Accepted January 6, 2006)
AH1-HA, AN1-NA and AH5-HA genes were not. The results
suggested that the patient was infected simultaneously by bothinfluenza A (H3N2) and B viruses.
The levels of AH3-HA and B-HA genes in the original
specimen were assessed by real-time quantitative PCR. Briefly,
viral RNA was extracted from the specimen as describedabove and 50 μl of complementary DNA (cDNA) wassynthesized using SuperScriptTM II reverse transcriptase(Invitrogen, Carlsbad, Calif., USA) from 20 μl of RNA solution. Real-time PCR was performed at Tokyo Metropolitan
Institute of Public Health as reported previously (2) with somemodifications on primers and probes. The numbers of copies
of influenza AH3-HA and B-HA genes in 10 μl of cDNA
were estimated to be 1.2 × 105 and 1.2 × 101, respectively.
This result suggested that the amount of influenza AH3 virus
was 10,000 times higher than that of influenza B virus in theoriginal specimen.
Viral isolation was attempted again using MDCK cells in
Eagle’s MEM with or without trypsin, in order to separate A(H3N2) and B viruses (3,4). The subtype of proliferating
virus in each medium was determined by the rapid diagnosis
test (ESPLINE influenza A & B-N). In the presence of trypsin,
only influenza A virus was isolated, whereas in the absenceof trypsin both influenza A and B viruses were isolated. They
were passaged twice at a restricted dilution, and named A/
Saitama/26/2005 and B/Saitama/37/2005, respectively. They
reacted to antisera to A/Wyoming/03/2003(H3N2) and B/Johannesburg/5/99, respectively, in an HI test (Table 1).
In recent years, influenza epidemics were caused by bothinfluenza A (H1N1 or/and H3N2) and B viruses, but thepredominant virus differed year by year. There have beenreports of simultaneous infection by different subtypes of influenza viruses (4-6). In addition, we sometimes encounteredsimultaneous infections by influenza virus and adenovirus orenterovirus. It is possible that differences in the sensitivity ofcell lines to different influenza type or subtype viruses leadto the isolation of only predominant type/subtype viruses.
Although CaCo-2 cells were previously reported to be sensitive to influenza viruses (7,8), they supported the propagationof influenza A virus only in the present study. In the analysis
of a dual infection in Chiba Prefecture, Japan, only influenza
A (H3N2) virus was isolated in MDCK cells, while onlyinfluenza B virus was isolated in HeLa cells, which were also
reported to be sensitive to influenza viruses (9) (personalcommunication, T. Ogawa, Chiba Prefectural Institute ofThe influenza epidemic in the 2004/2005 season wasrelatively large compared to those in other seasons in Saitama
Prefecture, Japan. Thirty-seven influenza A (H3N2) and 38
influenza B virus isolates were obtained at Saitama Instituteof Public Health (SIPH). Influenza A (H1N1) virus, a minor
subtype in this season, was isolated from only one specimen
at the inception of the epidemic, December 2004 (1).
Influenza A (H3N2) and B viruses were simultaneously
isolated from one individual, a 2-year-old boy. This boy
developed slight influenza symptoms on March 2, 2005.
A rapid diagnostic test (ESPLINE influenza A & B-N;
FUJIREBIO Inc., Tokyo, Japan) was positive for both A and
B influenza antigens at the clinic. At the same time, his motherand sister were diagnosed with influenza A and B, respectively, by the same test kit. A nasal specimen was collected
only from the boy for further virological examination andsent to SIPH a week later.
The specimen was positive only for influenza A by therapid diagnosis test (ESPLINE influenza A & B-N) at SIPH.
Subsequently, the specimen was inoculated onto several
cell lines. An obvious influenza-like cytopathic effect wasobserved 3 days after inoculation to MDCK and CaCo-2 cells.
The culture supernatant (the crude isolate) from inoculated
MDCK cells was examined by a hemagglutination inhibition(HI) test using standard antisera (Table 1). The sample was
not neutralized by any of the used antisera. Influenza A and
B antigens were detected by the rapid diagnosis test kit(ESPLINE influenza A & B-N), influenza A antigen being
much stronger than influenza B antigen. On the other hand,
only influenza A antigen was detected in CaCo-2 supernatant.
In the next series of experiments, the hemagglutinin (HA)
and neuraminidase (NA) genes were analyzed by reverse
transcription-polymerase chain reaction (RT-PCR). Viral RNA
was extracted from the culture supernatant using the QIAamp
Viral RNA Mini Kit (QIAGEN, Germantown, Md., USA),according to the manufacturer’s instructions. RT and PCR
were performed consecutively in one tube by the Access RTPCR system (Promega, Madison, Wis., USA), according tothe manufacturer’s recommendations. Influenza AH3-HA,
AN2-NA and B-HA genes were detected, whereas influenza*Corresponding author: Mailing address: Department of Viral
Diseases, Saitama Institute of Public Health, Kami-Ohkubo,
Sakura-ku, Saitama 338-0824, Japan. Tel: +81-48-853-5034,
Fax: +81-48-840-1041, E-mail: a0182785@pref.saitama.lg.jp
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Table 1. Results of hemagglutination inhibition (HI) assay for the isolated viruses
Standard antigens
or isolates

A/New Caledonia/20/99(H1N1)

HI titer against ferret antisara1)
A/Moscow/13/98 A/Wyoming/03/2003B/Johannesburg/5/99(H1N1)(H3N2)

B/Brisbane/32/2002

A/New Caledonia/20/99
A/Moscow/13/98
A/Wyoming/03/2003
B/Johannesburg/5/99
B/Brisbane/32/2002

160–––––2)
1,280
–––––
640
–––––
1,280–––––
1,280

The crude isolate
A/Saitama/26/2005
B/Saitama/37/2005<10<10<10<10<10<10<10160<10<10<10640<10<10<10Standard antigens and ferret antisera were provided by National Influenza Surveillance Center, National Institute of Infectious Diseases.
1): 0.5% turkey red blood cell was used.
2): Not done.

Public Health). The factors that are responsible for dual
infection with two types of influenza viruses have not been
determined; they may include the immune system of thehost, virus properties, or other factors. Interestingly, in thepresent study, the level of influenza A virus was 10,000 times
higher than that of influenza B virus in the clinical specimen.
However, similar levels of influenza A (H3N2) and B viruses
were detected in the clinical specimen from a case of dual
infection by the direct plaque formation culture methodin Yokohama City, Japan (personal communication, C.
Kawakami, Yokohama City Institute of Health). Further study
is needed to elucidate the mechanism of dual influenza virus
infection.
In conclusion, a case of dual infection by influenza A(H3N2) and B viruses was described. The presence of dual
infection should always be kept in mind in the virologicalanalysis of influenza cases.

acute respiratory syndrome (SARS). Ann. Rep. Tokyo
Metr. Inst. Pulic Health, 55, 25-29 (in Japanese).
3. Tobita, K. (1976): Isolation of influenza viruses in MDCKcells. Clin. Virol., 4, 58-61 (in Japanese).
4. Matsuura, K., Katori, K., Nagai, Y., Masaki, A. andTanaka, Y. (2002): A case of mixed infection due to influenza virus type A (H3) and type B. Ann. Rep. ToyamaInst. Health, 25, 172-175 (in Japanese).
5. Yamane, N., Arikawa, J., Odagiri, T., Sukeno, N. andIshida, N. (1978): Isolation of three different influenza
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6. Kendal, A. P., Lee, J. T., Parish, H. S., Raines, D., Noble,
G. R. and Dowdle, W. R. (1979): Laboratory-based
surveillance of influenza A virus in the United States during the winter of 1977 - 1978. II. Isolation of a mixtureof A/Victoria- and A/USSR-like viruses from a single
person during an epidemic in Wyoming, USA, January
1978. Am. J. Epidemiol., 110, 462-468.
7. Yoshino, S., Yamamoto, S. and Kawabata, N. (1998):
Use of CaCo-2 cells for isolation of influenza virus. J.
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8. Shimada, S., Uchida, K., Shinohara, M. and Gotoh, A.
(1998): Isolation of influenza viruses in CaCo-2 cells. J.
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This article appeared in the Infectious Agents Surveillance
Report (IASR), vol. 26, p. 152-153, 2005 in Japanese.
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