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

Laboratory and Epidemiology Communications

A Diffuse Outbreak of Hepatitis E in Mie Prefecture, 2005
Yoko Nakano, Akinori Yamauchi, Takuya Yano, Osamu Nakayama, Haruko Sakai1,
Yuji Nagasaka2, Seiji Itaba3, Yoshiki Tabata3 and Akira Sugiyama*
Mie Prefectural Institute of Public Health and Environmental Sciences, Mie 512-1211;
Mie Prefectural Kuwana Health Center, Mie 511-8567; 2Mie Prefectural Yokkaichi Health Center,Mie 510-8511; and 3Division of Health and Welfare, Mie Prefecture Government, Mie 514-8570, Japan1

Communicated by Tatsuo Miyamura(Accepted February 7, 2006)
Patient 2, a 66-year-old female, suffered chills and loss ofappetite on May 14 and experienced general fatigue on May
16. She was examined at a local clinic on May 18 and, basedon indications of liver dysfunction observed at the clinic, wasexamined at the same hospital visited by Patient 1 on May
20. Her serum was also positive for HEV IgG and IgM antibodies, and she was diagnosed with hepatitis E.
Patient 3, a 59-year-old male, displayed hepatitis symptoms
in mid-May and was examined at Hospital Y in Yokkaichishi on May 17. His serum was positive for HEV-RNA and he
was diagnosed with hepatitis E.
Patient 4, a 54-year-old male, complained of loss of appetite and exhibited jaundice on June 5. This patient stated that
he had eaten raw liver from an animal of unknown species.
He was under treatment at Hospital Z in Kuwana-shi on June
10. His sera were positive for HEV IgG and IgM antibodies,and he was diagnosed with hepatitis E.
The patients had the following factors in common:(i) All patients had resided in Japan for the past several years.
Due to this absence of a history of foreign travel, the regionof infection was limited to Japan.
(ii) There was no obvious contact with HEV infected animals or other HEV vector. Although Patient 4 had eaten raw
liver, Patients 1, 2 and 3 had not eaten any wild animal flesh.
Thus, no causal food or drink was identified.
(iii) There was no illness in any cohabiting individuals orother such persons.
(iv) The source of drinking water in all cases was municipalwater. The residences of the patients all had flush toilets.
(v) None of these individuals kept pet animals in their homes.

Hepatitis E virus (HEV) causes acute hepatitis, which istransmitted primarily through oral contact with feces (1,2).
Pig meat is also a potential source of HEV infection and theconsumption of raw pig liver has been suggested to be one of
the main transmission routes between pigs and humans (3,4).
The virus has a 15- to 60-day incubation period and infected
people may be contagious for up to 2 weeks after symptoms
appear. The acute phase is self-limiting, but there is a 1 to
2% chance of developing sudden and severe liver damage,resulting in fulminant hepatitis (5).
In late June of 2005, four cases of hepatitis E were reportedby three hospitals in the northern district of Mie Prefecture,
Japan. To investigate the possible sources of infection in thesefour patients, we first examined their behavior and living environment. Virological analysis of stored sera was performedby reverse transcriptase-polymerase chain reaction (RT-PCR)
for HEV RNA. The amplification products of HEV RNA werethen sequenced and their sequences were compared to thegenotype I - IV sequences registered in the GenBank database.
Patient 1, a 55-year-old male, complained of generalfatigue and loss of appetite on May 13. He visited Hospital X
at Inabe-shi on May 16 and tested positive for HEV IgG andIgM antibodies; he was diagnosed with hepatitis E.
*Corresponding author: Mailing address: Department of Microbiology, Mie Prefectural Institute of Public Health and Environmental Sciences, 3690-1, Sakura-cho, Yokkaichi, Mie 5121211, Japan. Fax: +81-59-329-3004, E-mail: sugiya00@pref.
mie.jp136

(vi) None of the patients had any history of blood transfusion,
medical injection or contact with hepatitis patients during the2 months prior to the onset of illness.
(vii) There were no pork processing or storage plants within
a 1-km radius of the residences of these individuals.
Stored sera which had been collected at each hospital anddated as closely as possible to the date of onset were used as
investigational specimens. The open reading frame 1 (ORF1)
region of HEV was targeted for RT-PCR. RNA was extractedusing a QIAamp Viral RNA Mini Kit (Qiagen, Tokyo, Japan). Super Script II RnaseH RT (Invitrogen, Tokyo, Japan)
was used in RT reactions. TaKaRa Ex Taq (TaKaRa Bio, Inc.,Shiga, Japan) was used as a DNA polymerase. First PCR wascarried out using HE5-1 as a sense primer and a mixture of
HE5-4 and HE5-5 as an antisense primer. Nested PCR wasthen performed using HE5-2 as a sense primer and a mixtureof HE5-3 and HE5-6 as an antisense primer (6). The amplification conditions were 1 cycle of 1 min at 96°C, 30 cycles of
30 sec at 95°C, 45 sec at 55°C, 1 min at 72°C, and 1 cycle of7 min at 72°C. The amplification product was then ascertained using agarose gel electrophoresis. Amplification products with the expected sizes were obtained from Patients 1, 3
and 4, however, the HEV gene was not detected in the serumof Patient 2.
The amplification products of the ORF1 region were first
purified with a MiniElute PCR Purification Kit (Qiagen)and then sequenced using a SequiTherm EXCELTM II DNA
Sequencing Kit-LC (AR Brown Co., Ltd., Tokyo, Japan)
and a DNA sequencer LIC-4200 (Aloka, Tokyo, Japan).
The sequences were analyzed by the GENETYX version 7
computer software (Genetyx Corp., Tokyo, Japan). BLAST
screening was carried out for 326-bp for three specimens of
the amplification product (7-10). All three specimens wereidentified as HEV genotype III. Using Patient 1 as a reference case, Patient 3 showed 98.7% homology and Patient 4
showed 78.9% homology.
The sequences of the 3 specimens were compared to thegenotype I - IV sequences registered in the GenBank database.
The accession numbers of the reference sequences wereM80581, M73218, M74506, AF060668, AF060669,
AB074917, AB074918, AB074919, AB074920, AF110387,
AF110388, AF110389, AF279122, AF264009, AF264010,
AJ315768, AF195064, AF195065, AJ272108, AB097812,
AB080575 and AB108654 (6,11-23). The HEVs from Patients 1 and 3 were further differentiated from that of Patient
4 by their classification in a different cluster within genotype
III. The HEV from Patient 4 was closely related to genotype
III swJ570 (AB073912) isolated in Japan, while that fromPatients 1 and 3 was closely related to genotype III sequences
isolated in Europe (15,24). Figure 1 shows the analyticalresults of these strains.
Based on these results, Patients 1 and 3 were again interviewed at the health center, however, no common foods,
behavior patterns or other similarities were found; thus, neither the source nor the route of infection could be determined.
Given the observance of the extremely high homology of98.7% between the viruses detected in these two individuals,
some mutually unknown point of contact between them wasstrongly suspected. On this basis, we regarded these cases asa diffuse outbreak. We also attributed the inability to detect
the virus from Patient 2 to the long interval of 14 days fromthe onset of illness to specimen collection. The source ofinfection derived from our investigation can only be hypothesized to be “consumption of undercooked or raw meat fromFig. 1. Phylogenetic tree (neighbor-joining method).

an unknown animal species.”
This article appeared in the Infectious Agents Surveillance
Report (IASR), vol. 26, p. 267-269, 2005 in Japanese.
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Nucleotide sequences determined in the study were deposited in DDBJ under accession no. AB517742 toAB517744.

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