Jpn. J. Infect. Dis., 52, 1999

Laboratory and Epidemiology Communications

Detection and Identification of Adenovirus from Ophthalmological Specimens by Virus Isolation and PCR

Jumboku Kajiwara * , Mitsuhiro Hamasaki, Ryoichi Mori and Shinobu Oniki ¹

Fukuoka Prefectural Institute of Health and Environmental Sciences, 39 Mukaizano, Dazaifu City,
Fukuoka 818-0135 and 1 Oniki Eye Clinic, 712 Futsukaichi, Chikushino City, Fukuoka 818-0051

Communicated by Ryoichi Mori

(Accepted February 1, 1999)

     Most epidemic kerato-conjunctivitises (EKCs) are caused by human adenovirus infections. 
Adenovirus is classified into 6 subgenera and 49 serotypes (1). Adenovirus types 3, 4, 8, 11, 
19 and 37 are responsible for most eye infections. In laboratory diagnosis of adenoviruses, 
virus isolation using tissue culture is routine, but requires more than 2 weeks for isolation 
and typing. More recently, diagnosis by combination of polymerase chain reaction (PCR)
coupled with restriction fragment length polymorphism (RFLP) has been introduced for 
identification of adenoviruses in the conjunctiva swabs.
     In this study, we used PCR and RFLP to detect and identify adenoviruses on swabs from
conjunctiva of 73 patients who had visited an eye clinic in Chikushino City in Fukuoka Prefec-
ture and been diagnosed with EKC (71 cases), pharyngo-conjunctivitis (1 case) or acute 
conjunctivitis (1 case). The swab specimens were dissolved in 3 ml Hanks' solution 
containing 0.5% gelatin and centrifuged at 10,000xg for 10 min. The supernatant was stock 
frozen at -80 C till the examination.
     For virus isolation, Vero, FL, RD-18s and Hep-2 cells cultured in 96-well microplates were 
infected with the specimen in a volume of 25 μl/well and cultured for 1 week. The cultures 
negative for CPE were subcultured further. Specimens that failed to induce CPE after 4 sub-
cultures were considered negative. Isolated adenoviruses were typed by using antisera provided
by the National Institute of Infectious Diseases, Japan and the DENKA SEIKEN Co., Ltd., Tokyo.
     PCR was conducted according to the method of Saito and Imagawa, (3). 200 μl of each 
specimen was centrifuged at 12,000xg for 30 min. The pellet was dissolved in 100 μl of 
0.31 mg/ml proteinase K, 0.5% Tween 20, 1 mM EDTA, and 10 mM Tris-HCl (pH 8.3), then 
incubated at 55 C for 1 hr. After the samples were heated at 95 C for 10 min, DNA was 
extracted.  Nested PCR using the primers shown in Table 1 (external primer pairs were AdTU7 
and AdTU4',  and internal pairs were AdnU-S' and AdnU-A) was conducted. Both the first and 
second PCR amplifications consisted of 36 cycles of denaturation at 94 C for 1 min, annealing 
at 50 C for 1 min and elongation at 72 C for 2 min; after the second amplification the samples 
were incubated at 72 C for 7 min for completion of the elongation reaction. The amplified 
materials were digested with EcoT14I, HaeIII, or HinfI and electrophoresed in 3% agarose gel 
(Fig. 1).
     The results are summarized in Table 2. Virus isolation in culture and serotyping identified 
adenoviruses from 28 specimens: eight Ad3, one Ad8, one Ad11, fifteen Ad19, and three Ad37
specimens.  PCR-RFLP identified adenoviruses from 54 specimens: nine Ad3, eleven Ad8, 
one Ad11, twentyfour Ad19, and nine Ad37 specimens.  The data on all specimens agreed 
perfectly between the two methods performed. The PCR-RFLP method required 3 days for 
a complete analysis. The positivity was 74% (54/73) for PCR-RFLP, and 38.4% (28/73) for 
virus isolation and serotyping. For B subgenera Ad3 and Ad11, the sensitivities of the two 
methods were comparable, while for D subgenera Ad8, Ad19 and Ad37 (particularly for Ad8)
the virus isolation in culture was very inefficient. In neutralization, Ad19 and Ad37 showed 
cross reactivity, but in PCR-RFLP the two viruses were clearly distinguished (Fig. 1). Our data
thus demonstrated the usefulness of PCR-RFLP for the laboratory diagnosis of adenoviruses. 
However, we should also be aware of the limitations of the PCR method, e.g., PCR detects 
only a fragment of the virus genome and detects viruses only if they have a sequence suitable
for the primers. Because virus isolation and PCR-RFLP provide complementary information,
both should be used for the virus surveillance.

     The eye swabs used in this study were collected by the National Epidemiological 
Surveillance of Infectious Diseases.

REFERENCES
1. Schnurr, D. and Dondero, M.E. (1993): Two new candidate adenovirus serotypes. 
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2. Aoki, K., Isobe, K. and Ohono, S. (1994): Nationwide surveillance program of epidemic
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3. Saitoh-Inagawa, W., Oshima, A., Aoki, K., Itoh, N., Isobe, K., Uchino, E., Ohono, S., 
    Nakazima, H., Hata, K. and Ishiko, H. (1996): Rapid diagnosis of adenoviral conjunctivitis
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* Corresponding author : Fax : +81-92-928-1203

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