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

Laboratory and Epidemiology Communications

Isolation of Influenza A/H3 and B Viruses from an Influenza Patient:
Confirmation of Co-Infection by Two Influenza Viruses
Shoichi Toda*, Reiko Okamoto, Tomoko Nishida, Toshiki Nakao,
Masatoshi Yoshikawa, Eitaro Suzuki1 and Shigenori Miyamura
Division of Biology, Yamaguchi Prefectural Research Institute of Public Health, Yamaguchi 753-0821, and1Suzuki Pediatric Clinic, Yamaguchi 755-0151, Japan
Communicated by Ichiro Kurane(Accepted February 13, 2006)
Influenza A/H3 and B viruses co-circulated in the 2004 2005 epidemic season in Japan, and we confirmed a case ofco-infection by both viruses. The two influenza viruses wereisolated from a single patient by the plaque purification technique, and co-infection was confirmed.
The patient was a 4-year-old girl whose throat swab specimens were positive for both influenza A and B viruses by
a quick diagnostic agent. The virus was isolated from thethroat swab by inoculation onto Madin-Darby canine kidney(MDCK) cells. The first culture sample (MDCK1) responded
to B/Johannesburg/5/99 antiserum in a hemagglutination
inhibition (HI) test using antisera provided by the National
Institute of Infectious Diseases (NIID), Tokyo (Table 1). HA
genes of both influenza A/H3 and B viruses were detected byreverse transcriptase-polymerase chain reaction (RT-PCR) in
the first culture sample. These results suggest that the A/H3and B influenza viruses co-existed in sample MDCK1. Virus
cloning was performed by plaque purification after neutralization with each antiserum (Fig. 1). The influenza A/H3 andB viruses were cloned. The influenza A/H3 virus responded
only to A/Wyoming/03/2003 antiserum, and the B virus
responded to B/Johannesburg/5/99 antiserum. Thus, we conclude that this patient was co-infected with A/Wyoming/03/
2003-like and B/Johannesburg/5/99-like influenza viruses.
In another possible case of co-infection, a 9-year-old girlwas diagnosed with influenza at a sentinel surveillance clinic
by a rapid antigen detection kit which gave a positive resultfor influenza A virus but did not test for influenza B virus.
The patient’s throat swab specimen was inoculated onto
MDCK cells and the first culture sample was tested by HI
assay. The virus did not respond to any of the tested antisera(data not shown), however, HA genes of both influenza
A/H3 and B viruses were detected by RT-PCR in the virus
sample. Unfortunately, plaque cloning of the influenza viruses
was not performed in this case.
It is generally believed that co-infection by two influenza
viruses is a rare event. In the present study, we confirmed acase of co-infection in a patient who visited her local clinic
during the peak period of epidemic caused by influenza A/H3and B viruses. Although there was also a second case of suspected co-infection with influenza A and B viruses, the firstculture sample did not respond to any of the tested antisera inan HI assay. Paired serum samples were not available fromthese two cases and it was not possible to serologically confirm co-infection. The present study demonstrates the possibility of co-infection by different types of influenza viruses whenan epidemic is caused by multiple types of influenza viruses.
This article appeared in the Infectious Agents Surveillance
Report (IASR), vol. 26, p. 297-298, 2005 in Japanese.

Table 1. Antigenic analysis of the isolated viruses1)
Virus antigens
Reference antigen
A/Moscow/13/98 (H3N2)
A/New Caledonia/20/99 (H1N1)
A/Wyoming/03/2003 (H3N2)
B/Brisbane/32/2002 (Victoria)
B/Johannesburg/5/99 (Yamagata)
Isolated viruses
first cultured virus (MDCK1)
cloned A/H3 virus (MDCK5)
cloned B virus (MDCK5)

A/Moscow/13/98

HI titers with Japanese standard antisera for influenza severance in 2004/052)
A/New Caledonia/20/99 A/Wyoming/03/2003 B/Brisbane/32/2002 B/Johannesburg/5/99640
80<10<10<1040320<10<10<10<10<10640<10<10<10<10<1032040<10<10<10401,280<10<10<10<10<10<10<10640<1010<10<101,280<102,5601): HI titers were tested by using 0.75% guinea pig red blood cell.
: Reference antigen and antisera were provided by NIID for influenza pathogen surveillance.

2)*Corresponding author: Mailing address: Division of Biology,
Yamaguchi Prefectural Research Institute of Public Health, Aoi
2-5-67, Yamaguchi 753-0821, Japan. Tel: +81-83-922-7630, Fax:+81-83-922-7632, E-mail: toda.shouichi@pref.yamaguchi.lg.jp142

First cultured virus (MDCK1): co-existence with A/H3 and B↓

Create a series of 10-fold dilutions of MDCK1 (10–1-10–5)↓

Neutralize with each antiserum (anti-A/H3 or anti-B)1)
Inoculate onto MDCK cells sheeted on 6 well plates2)
Incubate at 35°C for 4 - 5 days in the Agar culture medium3)
Harvest plaque formed in the well of lowest dilution4)↓↓

Plaque from 10–4 MDCK1
neutralized with anti-A/H3(MDCK2)

Plaque from 10–3 MDCK1
neutralized with anti-B(MDCK2)↓↓

Increase a quantity of each virus of plaque on MDCK cells5)↓↓

1st cloned B type virus(MDCK3)

1st cloned A/H3 type virus(MDCK3)↓↓

Repeat the plaque purification and virus culture at once↓↓

2nd cloned B type virus(MDCK5)

2nd cloned A/H3 type virus(MDCK5)

Fig. 1. Plaque purification was carried out twice and the success of cloning was confirmed by RT-PCR.
1): Antiserum (anti-A/Wyoming/03/2003 or anti-B/Johannesburg/5/99) were used at 10–1 dilution, and were neutralized withsame quantity of diluted viruses at 4°C, 3 h.
2)
: In each plate, 5 wells were used for 10–1 - 10–5 virus dilutions and 1 well for cell control.
3): Two mililiters of 0.8% Agar medium with 5 μg/ml Trypsin and 0.01% DEAE-Dextran was added to each well. Afteragar became solid (15 min at room temperature), plates were set into CO2 incubator.
4): Plaques were observed as white spots without staining, and harvested by sucking with micropipette tip.
5)
: Harvested plaques were suspended in the maintenance medium and inoculated according to the usual method.

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