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

Laboratory and EpidemiOlogy CoTnmunications●Genetic Analysts Of Outbreak of Hepatitis A Virus Infection among

HIVI Seropositive Men
Tomohiko Koibuchi I, TateruIshida2, Tetsuya Nakamural , Atsushi Ajisawa3, Masayoshi Negishi3,Tetsuya Kashiyama4, Akiko Takechi4 and Aikichi Iwamotol･2*LDepartment of Infectious Disease and Applied lmmunology.I2DepaT･tment Infectious Diseases, Institute of Medical Science, UniversltyOf T0秒0,

Shirokanedai 4‑6‑1 , Minato‑ku, Tokyo,
3De･partment lnjTectious Diseases, 7Tokyo Metropolitan Komagome Hospital,
Honko〝∽gome 3‑1 8122, Bunkyo‑ku, 7Tokyo and4DepartmenE of Internal Medicine, To砂o Metropolitan Ohkubo Hospital,

Kabukicho 2‑44‑ 1 , Shinjuku‑ku, Tokyo
Communicated by Aikichi lwamoto(Accepted January 5, 2000)

We previously reported an outbreak of hepatitis A virus with men (MSM) (I). HAV is classified into seven genotypeS(HAV) infection among HIV‑ I seropositive men who had sex in terms of nucleotide sequence differences of the VPl/2A

region (nucleotide 3024 to 3 191) (2). Weperformed a genetic*Corresponding author: Fax: +8 I ‑3‑5449‑5427, E‑mail: aikichi @analysis of HAV to elucidate the chain of virus transmission.

Thirteen serum samples at the acute phase of HAV infec‑ims･ u‑tokyo･ acJp249

Jpn. J. Infect. Dis., 52, 1999
cy145 PhilippizleJI
JXSS IzLdoAeJLia
Zl‑122

SvedezL

CP‑53PrazLCe

SLF88
AQK27

5ierra LeozLe
ⅩezLya

捕‑53 Japan
S6 JAp一nA‑167 JapazL

A‑261 JApan
X5‑1 tISA
A‑149 JApaA
mO31 Jap姐ュ2 ■ ▲一正一67 0 ' l0

X‑138 Japan
M‑15tlSA

A‑129 Jap乱■
523‑1 JAP乱■
エー20l SotLth AAericA

0.01Figure･ Phylogenetic tree based onthe VPl/2A reglOn Of HAY genomes･

10 from the present study and I 8 from references (2)･ Romannumerals I to VIl designatcthc respective genotypeS, whereas A
and B designate sub‑genotypcs･ The scale at the bottom indicates thepercentage of divergenceper length･

Table. Primers for nested PCR of VP2/lA reglOn
Primers

Nucleotide

Position

Sequence

HAY‑ 1289 I ‑29 14

5㌧GGTTrCTXTrCAGATTGCAAATIA‑3 I

HAY‑23375‑33 98

5 '‑AGTAAAAACTCCA GCATCCAmC13 'HAV‑32905‑2925

5 ㌧TTGCAAmCAATCAITCTG l3 'HAV‑433 57 13377

5 '‑TTCAAGAGTCCACACACTTCrl3 'tion were collected. RNA from 140 Lil of serum was extracted

in 10 of 13 patients. All 10 patients'VPl/2Areg10nS hadtheusing the QIAamp viralRNA kit (Qiagen Inc. , Ⅶencia, Calif.)

same sequences. The figure shows a phylogenetic tree of theseand subjected to reverse transcription uslng 5 LLM random

primer pairs shown in Table (the extemal primer pal一s Were

10 sequences together with 18 HAY clones described inthe
literature (2). All 10 patients were grouped into genotype I A
which isthe most common HAV genotype in the world･The striking homogenelty in the HAV isolates indicates

HAV‑ I and HAV‑2, and the intemal pnmer palrS Were HAV‑

that possibly one HAV clone spread among a high‑risk popula‑

3 and HAY‑4). The first amplification,using 20 LLl of CDNA,
was performed in a volume of 50LLI with I x Ex‑Taq buffer,
0･2 mM dNTP, 0.5 JIM primers, and I.25U Ex‑Taq Crakaration in a short time. These results reinforcethe importance ofeducational and preventive measures for MSM against HAV･primer (Takara Co., Ltd., Kyoto). The VPl/2A region wasamplified by polymerase chain reaction (PCR) uモing theCo., Ltd.), while amplification cycles were 35 of 30 see at94oC, 30 see at 55oC,and 30 see at 720C with a finalextension

REFERENCES

for 7 min at 72.C. Nested PCR wasperformed with 10 LLl of1. Ishida, T., Nakamura, T., Ajisawa, A., Negishi, M･,

the first reaction product in 50 LLl underthe same conditions
at the first PCR. Amplified DNA fragments were sequenced
by a direct sequenclng method using a Cycle sequence withdye termination chemistry (ABI Prism Big Dye terminator

Kashiyama, T., Takechi, A. and Iwamoto, A･ (1999):
Outbreak of hepatitis A virus infection among HIV‑1
seropositive men who had sex withmen･ Jpn･ J･ Infect･
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A., Nainan, 0. V, Siegl, G., Wdell,A., Margolis, H･ S･,

tree was constructed using the unweighted pair group method

Isomura, S., Ito, K., Ishizu, T., Moritsugu, Y and Lemon,
S. M. (1992): Genetic relatedness of hepatitis A virus

with an arithmetic mean (UPGMA) procedure using
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Gen. Viro1., 73, 1365‑1377.

The VPl/2A region could be amplified by the nested PCR250