Jpn. J. Infect. Dis.

, 61, 2008

Laboratory and Epidemiology Communications

An Imported Dengue Fever Case by Dengue Virus 3 (DENV-3)

Infection in Gunma, Japan
Yukio Morita*, Hayato Kogure1, Mitsuru Sandoh1, Gihei Kawashima1, Yoshitake Sato1,
Sadao Nanba1, Yoshihiro Shoda2, Tomoyuki Suzuki, Masataka Shiono, Masae Shiobara,
Masahiko Kato, Kunihisa Kozawa, Masahiro Noda3, Nobuhiko Okabe4 and Hirokazu Kimura4
Gunma Prefectural Institute of Public Health and Environmental Sciences; 1Department of
Internal Medicine, General Ota Hospital, Society of Health Insurance of Fuji Heavy Industries, Ltd.;
2
Gunma Prefectural Ota Public Health Center, Gunma; and 3Department of Virology III and
4
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan
Communicated by Ichiro Kurane
(Accepted November 26, 2007)
Dengue virus (DENV) of the genus Flavivirus, family
Flaviviridae is an arthropod-borne virus that causes dengue
fever (DF) and dengue hemorrhage fever (DHF). DENV is
classified into four serotypes designated as DENV-1 to DENV4, and is endemic in more than 100 countries in tropical and
subtropical areas, South-East Asia, and the Western Pacific
areas being most seriously affected (1). According to the
World Health Organization, the global prevalence of DF has

dramatically increased in recent decades (http://www.who.int/

mediacentre/factsheets/fs117/en/). Thus, DF/DHF is a growing concern as one of the most important mosquito-borne
human infectious diseases (2). In Japan, relatively large epidemics of DF occurred between 1942 and 1944 in Nagasaki,
Kobe, and Osaka, originating from persons repatriating from
the tropics during World War II; these epidemics were eliminated in 1946 (1). About 10 to 50 cases of DF/DHF have
been reported annually in Japan in recent years, and all the
cases were imported as a travelers disease (3-6). The number of reported DF/DHF cases has shown an increasing trend
from the late 1990s (http://idsc.nih.go.jp/idwr/kansen/k04/
k04_50/k04_50.html [in Japanese]).

*Corresponding author: Mailing address: Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki,
Maebashi, Gunma 371-0052, Japan. Tel: +81-27-232-4881, Fax:
+81-27-234-8438, E-mail: morita-yu@pref.gunma.jp
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In September 2007, we experienced a patient with DENV3 infection who developed DF after returning to Gunma
Prefecture, Japan, from Ho Chi Minh City, Vietnam.
The case was a 26-year-old Japanese female who resided
in Gunma Prefecture. She had no previous history of chronic
or other diseases. She visited Ho Chi Minh City from August
22 to 24, 2007. She remembered clearly that she had been
bitten by some mosquitoes on her legs on the night of August
22. She then visited Siem Reap, Cambodia, on August 24.
On the morning of August 25, she developed a very high fever
(40C). She left Cambodia by plane and arrived in Japan in
the early morning of August 26. She had no apparent memory
of receiving mosquito bites while in Cambodia. After returning to Gunma Prefecture on August 27, she developed various
symptoms such as continuous high fever (39 to 40C), itching of the skin, profuse sweating, arthralgia, ague, diarrhea,
vomiting, and subcutaneous hemorrhage. She consulted a
physician in General Ota Hospital on August 27. On the first
day of the hospital visit (hospital day 1), the patients clinical
data were as follows: leukocyte count, 2.9 103/L (normal range, 3.5 to 9.0 103/L); platelet count, 1.3 105/
L (normal range, 1.5 to 3.0 105/L); and C-reactive
protein level, 1.5 mg/dL (normal range, <0.3 mg/dL). Ectopic
enzymes (i.e., aspartate aminotransferase [AST], alanine
aminotransferase [ALT], and lactate dehydrogenase [LDH])
and total bilirubin were within the normal range. She was
treated with antibiotics (levofloxacin [300 mg/day, days 1 to
5], azithromycin [500 mg/day, days 3 to 5], cefepime [2,000
mg/day, days 3 to 6], and ciprofloxacin [600 mg/day, days 7
to 9]) and intravenous fluids for 9 days. On hospital days 3
and 4, 50 g of granulocyte colony-stimulating factor (GCSF) was intravenously administered to correct leukocytopenia. On hospital day 4, concentrated platelets (20 U/day) were
administered to treat thrombocytopenia. High fever (<39C)
persisted through hospital day 5 and subsided on hospital day
7. On hospital days 1 to 8, a non-steroidal anti-inflammatory
drug (acetaminophen, 400 mg/day, days 1 to 7), -globulin
(5 g/day, days 2 to 4), gabexate mesilate (FOY, 2,000 mg/
day, days 3 to 5, or 1,800 mg/day, days 6 to 8), and ulinastatin
(300,000 U/day, days 3 to 5) were intravenously administered to reduce inflammation and disseminated intravascular
coagulation (DIC). On day 8, ectopic enzyme levels (AST,
267 IU/L; ALT, 211 IU/L; and LDH, 1,048 IU/L) were significantly elevated. The total bilirubin was within the normal
range. Based on the symptoms and travel history, the examining physician suspected DENV infection.
A blood sample was collected on hospital day 3 and examined by reverse transcription-polymerase chain reaction (RTPCR), using cross-reactive primers of DENV covering CPrM (GenBank accession no. AB362209) and the DENV-3specific primers covering E-NS1 (AB362210) (7). The DNA
sequences of E-NS1 (320 nucleotides) regions were aligned.
Homology analysis of the amplified E-NS1 product was performed by the ClustalW program. A phylogenetic tree was
constructed by the neighbor-joining technique, specifically
Kimuras two-parameter method (neighbor-joining [N-J]
method), using the Tree View (Win32) (ver. 1.6.6) software.
The reliability of the tree was estimated using 1,000 bootstrap replications (8). Blood specimens were cultured for
viruses or bacteria isolation using various cell lines (Vero,
RD, HEp-2, and HEL cells) and agars, respectively; however,
only the E-NS1 amplicon of DENV-3 was detected by RTPCR from the blood samples.
The phylogenetic tree was constructed based on the E-NS1

Fig. 1. Phylogenetic tree based on the E-NS1 sequences of DENV-3.

Phylogenetic distance was calculated using Kimuras two-parameter
method, and the tree was plotted using the neighbor-joining method.
Numbers at each branch indicate the bootstrap values of the clusters
supported by that branch. Inscriptions indicate the country where the
dengue virus gene was detected, GenBank accession numbers, and
collection year.

sequences of DENV-3 (Fig. 1). Approximately 5% genetic

diversity was detected among 16 strains that were isolated in
Vietnam (10 strains), Thailand (4 strains), East Timor (1
strain), and Taiwan (1 strain). The sequence [E-NS1
(AB362210)] determined in the present study was located in
a cluster with those that were isolated in Vietnam, and this
sequence was most closely related to that of DENV detected
in Aedes albopictus in Vietnam in 1996 (AF400029).
DF is an important infectious disease even in the countries
in the temperate zone including Japan, as well as in tropical
and subtropical countries. There is no vaccine available for
human use. In addition, as shown in the present case, relatively severe clinical manifestations such as continuous high
fever, hepatic disorder, leukocytopenia, and thrombocytopenia can develop (2). There is no evidence of domestic DENV
transmission in Japan. However, Ae. albopictus, one of the
main vectors, is widely distributed except for Hokkaido in
Japan (9). Thus, it is possible that an outbreak of DF/DHF
may occur in Japan, once the virus enters the country (1).
Moreover, a large number of overseas travelers are at risk for
DENV infection. The present case reconfirms the importance
of laboratory diagnosis of DF in Japan.
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