Jpn. J. Infect. Dis., 52 (6), 249-250, 1999

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Laboratory and Epidemiology Communications

Genetic Analysis of Outbreak of Hepatitis A Virus Infection among HIV-1 Seropositive Men

Tomohiko Koibuchi¹, Tateru Ishida², Tetsuya Nakamura¹, Atsushi Ajisawa³, Masayoshi Negishi³, Tetsuya Kashiyama⁴, Akiko Takechi⁴ and Aikichi Iwamoto^1,2＊

¹Department of Infectious Disease and Applied Immunology, ²Department Infectious Diseases, Institute Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo, ³Department Infectious Diseases, Tokyo Metropolitan Komagome Hospital, Honkomagome 3-18-22, Bunkyo-ku, Tokyo and ⁴Department of Internal Medicine, Tokyo Metropolitan Ohkubo Hospital, Kabukicho 2-44-1, Shinjuku-ku, Tokyo

Communicated by Aikichi Iwamoto

(Accepted January 5, 2000)

We previously reported an outbreak of hepatitis A virus (HAV) infection among HIV-1 seropositive men who had sex with men (MSM) (1) . HAV is classified into seven genotypes in terms of nucleotide sequence differences of the VP1/2A region (nucleotide 3024 to 3191) (2) . We performed a genetic analysis of HAV to elucidate the chain of virus transmission.

Thirteen serum samples at the acute phase of HAV infection were collected. RNA from 140μl of serum was extracted using the QIAamp viral RNA kit (Qiagen Inc., Valencia, Calif.) and subjected to reverse transcription using 5μM random primer (Takara Co., Ltd., Kyoto). The VP1/2A region was amplified by polymerase chain reaction (PCR) using the primer pairs shown in Table (the external primer pairs were HAV-1 and HAV-2, and the internal primer pairs were HAV-3 and HAV-4). The first amplification, using 20μl of cDNA, was performed in a volume of 50 μl with 1×Ex-Taq buffer, 0.2 mM dNTP, 0.5μM primers, and 1.25U Ex-Taq (Takara Co., Ltd.), while amplification cycles were 35 of 30 sec at 94℃, 30 sec at 55℃, and 30 sec at 72℃ with a final extension for 7 min at 72℃. Nested PCR was performed with 10μl of the first reaction product in 50 μl under the same conditions at the first PCR. Amplified DNA fragments were sequenced by a direct sequencing method using a cycle sequence with dye termination chemistry (ABI Prism Big Dye terminator sequencing Ready Reaction Kit, Perkin-Elmer, Foster City, Calif.) on a Perkin-Elmer ABI-377 sequencer. A phylogenetic tree was constructed using the unweighted pair group method with an arithmetic mean (UPGMA) procedure using GENETYX-MAC Version 8.5 (Software Development Co., Tokyo).

The VP1/2A region could be amplified by the nested PCR in 10 of 13 patients. All 10 patients' VP1/2A regions had the same sequences. The figure shows a phylogenetic tree of these 10 sequences together with 18 HAV clones described in the literature (2). All 10 patients were grouped into genotype IA which is the most common HAV genotype in the world.

The striking homogeneity in the HAV isolates indicates that possibly one HAV clone spread among a high-risk population in a short time. These results reinforce the importance of educational and preventive measures for MSM against HAV.

REFERENCES

1. Ishida, T., Nakamura, T., Ajisawa, A., Negishi, M., Kashiyama, T., Takechi, A. and Iwamoto, A. (1999): Outbreak of hepatitis A virus infection among HIV-1 seropositive men who had sex with men. Jpn. J. Infect. Dis., 52, 131-132.
2. Robertson, B.H., Jansen, R.W., Khanna, B., Totsuka, A., Nainan, O.V., Siegl, G., Widell, A., Margolis, H.S., Isomura, S., Ito, K., Ishizu, T., Moritsugu, Y. and Lemon, S.M. (1992): Genetic relatedness of hepatitis A virus strains recovered from different geographical regions. J. Gen. Virol., 73, 1365-1377.

*Corresponding author: Fax: +81-3-5449-5427, E-mail: aikichi＠ims.u-tokyo.ac.jp

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