Jpn. J. Infect. Dis., 69, 60–65, 2016
Original Article
Serologic and Virologic Studies of an Imported Dengue Case Occurring in 2014 in Okinawa, Japan Mika Saito1*, Maki Tamayose2, Kazuya Miyagi2, Hiroo Takaragawa1,3, Masao Tateyama2, Masayuki Tadano1, and Jiro Fujita2 1Department
of Microbiology and Oncology, Graduate School of Medicine, 2Department of Infectious, Respiratory, and Digestive Medicine, Control and Prevention of Infectious Diseases (First Department of Internal Medicine), Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215; and 3Crop Science Laboratory, Subtropical Agriculture Course, Graduate School of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan SUMMARY: After returning from Bali, Indonesia, in February 2014, a 72-year-old man was hospitalized in Okinawa owing to a high fever and rash. Dengue was clinically suspected, and the patient tested positive for IgM against dengue using a commercial kit. Serologically, the patient showed secondary seroreactivity. Significant increases in neutralization titers (N-titers) against all 4 serotypes of dengue virus (DENV) and Japanese encephalitis virus (JEV) strains were recognized in convalescent-phase sera comparing to acute phase sera. The N-titer against DENV serotype 1 (DENV-1) was the highest among all DENV serotypes. Interestingly, the N-titers against JEV strains were significantly higher than those against all types of DENV comparing to acute phase sera. The virus was isolated from the acute-phase serum and identified as DENV-1 and designated RD14/Okinawa. The patient's symptoms were due to DENV-1 infection. Phylogenetic sequencing analysis indicated that the isolate RD14/Okinawa belonged to genotype I of DENV-1, which is closely related to the Southeast Asian strains and isolates found during the dengue outbreak in Japan in 2014. We should undertake control measures against dengue in Okinawa, which is a subtropical area with Aedes albopictus activity throughout year. modes of transportation, as well as the distribution of vector mosquitoes due to global warming (3,5). An outbreak of dengue domestic infection of more than 150 patients occurred suddenly in Japan in August 2014 after a period 69 years with no outbreaks (6). Aedes albopictus is thought to have been responsible for this outbreak. In western Japan, a dengue epidemic had previously occurred in 1942–1945, during the Second World War (7). It is believed that military personnel who had contracted dengue in other parts of Asia brought it back on military ships, and ports such as Nagasaki, Kobe, and Yokohama had focal dengue outbreaks (8). Okinawa had at least 10 epidemics recorded from 1893–1955, when the last dengue epidemic was reported in the Yaeyama region, although this was not recorded officially in a Japanese national report (9); Aedes aegypti, the principle vector of dengue, was also identified on Ishigaki Island until 1970 (10). A retrospective serosurvey revealed that the dengue epidemics in Okinawa had been due to DENV-1 and DENV-2 (11). Recently, 11 dengue cases were reported between 1999 and 2014 in Okinawa, and all seemed to have been infected overseas. There are cross-reactive antigens within the DENV serotypes and also among different serogroups of the flaviviruses, such as JEV, which make serodiagnosis difficult in regions where 2 or more different serogroups coexist. In the case of primary infection by a flavivirus, a simple reaction is obtained for the causative agent, but in the case of secondary infection, the presence of a preexisting antibody and immunologic memories mean that seroreactivity is too broad for the DENV subgroup and also for the flavivirus subgroup to identify the causative virus of the more recent infection, even by a
INTRODUCTION Dengue is a mosquito-borne viral disease caused by infection with 1 of 4 different serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), which belong to the family Flaviviridae, genus Flavivirus, which includes different serogroups such as Japanese encephalitis virus (JEV). Previously, DENV infection was classified according to severity as dengue fever or dengue hemorrhagic fever (1), but the World Health Organization (WHO) introduced new classifications in 2012: dengue with or without warning signs and severe dengue (2). We use the term ``dengue'' in accordance with this newer classification. Dengue is endemic in more than 100 countries in Southeast Asia, the Americas, the western Pacific, Africa, and the eastern Mediterranean region (3), and recently it has been estimated that 390 million cases of dengue infection occur per year, of which 96 million manifest apparent symptoms (4). Dengue is one of the most important emerging and reemerging infectious diseases not only because of its high incidence, which has increased 30-fold in the past 50 years, and the disease severity, but also because of the expansion of its geographical distribution, mainly via human Received February 5, 2015. Accepted April 9, 2015. J-STAGE Advance Publication June 12, 2015. DOI: 10.7883/yoken.JJID.2015.061 *Corresponding author: Mailing address: Department of Microbiology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207, Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: ＋81-98-895-1132, Fax: ＋81-98-895-1410, E-mail: mikas＠med.u-ryukyu.ac.jp 60
Imported Dengue Case in Okinawa, 2014
cells and that of the Vero cell culture onto Vero cells, involving so-called blind passages. The supernatants were then inoculated onto BHK-21 cells and incubated for 3 days. The presence of the virus was examined with an immunostaining assay using mouse polyclonal antibodies against DENV-1, DENV-2, DENV-3, and DENV-4. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted using the specific primers for DENV-1, DENV-2, DENV-3, DENV-4, and chikungunya virus as described previously (14–16). Sequences and nucleic acid sequence analysis: A total of 1,485 nucleotides of the envelope (E) gene region of isolate RD14/Okinawa in this study were sequenced directly using the specific primers as described previously (17). Multiple alignments and the phylogenetic analysis were performed with the neighbor-joining (NJ) method using Clustal-X (18), and phylogenetic trees were drawn using the NJplot program (19). The DENV-1 used for phylogenetic analysis in this study is listed in Table 1. Serologic test: For serologic analysis, 4 prototype strains of DENV-1 (Hawaiian), DENV-2 (New Guinea B), DENV-3 (H-87), and DENV-4 (H-241); the isolate in this study (RD14/Okinawa); and 2 JEV strains (Nakayama: vaccine, genotype 3; Beijing-1: vaccine, genotype 3) were used for 50z focus-reduction neutralization tests, as described previously (20,21). An indirect IgG enzyme-linked immunosorbent assay (ELISA) was
neutralization test, which is the most specific test for distinguishing the etiology (12). In this study, we conducted serologic and virologic tests on an imported dengue case that occurred in February 2014 in Okinawa. We report here the seroreactivity of the patient and genetic analysis of the virus isolated in this study. We also discuss dengue control measures for robustly preparing against future epidemics in Okinawa. MATERIALS AND METHODS Sampling from the patient: Serum samples were obtained from a 72-year-old man who was clinically suspected of having dengue. Sampling was conducted 3 times: once to obtain acute-phase serum on February 21 and twice to obtain convalescent-phase sera on February 28 and March 5, 2014. Before serologic testing, sera were inactivated at 569 C for 30 minutes. Isolation and identification of viruses: Acute-phase serum was tested for the virus and viral genome. The virus isolation and identification procedure were as described previously (13). Briefly, serum was inoculated onto clone C6/36 mosquito A. albopictus cells and clone Vero African green monkey kidney cells and incubated for 7 days; the supernatant of the C6/36 cell culture was further inoculated chronologically onto C6/36
Table 1. Dengue virus serotype 1 strains used for analysis in this study Strain
Yr
Location
Genotype
Accession number
Mochizuki Hawaii TH-SMAN 16007 IBH28328 P72-1244 WestPac PUO 359 GZ/80 AUS HCS1 765101 A88 DEI 0151 LaoCH323 BeH 584526 98901530 DF DV-1 Abidjan 02SA079 D1/hu/Seychelles/NIID41/2003 Fj231 SC01 257/04 Malaysia2008 SG(EHI)D1/16174Y09 SG(EHI)D1/37966Y10 SKB-CM002 SKB-CM013 SKB-CM046 RD14/Okinawa D1/Hu/Tokyo/NIID106/2014 D1/Hu/Saitama/NIIH100/2014
1943 1945 1954 1964 1968 1972 1974 1980 1980 1983 1987 1988 1991 1996 1997 1998 1999 2002 2003 2004 2004 2004 2008 2009 2010 2012 2012 2012 2014 2014 2014
Japan Hawaii, USA Thailand Thailand Nigeria Malaysia Nauru Thailand China Australia Taiwan Indonesia Peru Laos Brazil Indonesia Ivory Coast Philippines Seychells China Indonesia Reunion Malaysia Singapore Singapore Java, Indonesia Java, Indonesia Java, Indonesia Okinawa, Japan Tokyo, Japan Saitama, Japan
I I II II V III IV I I IV I IV V I V IV V IV IV I IV IV I I I I IV IV I I I
AB074760 AF425619 D10513 AF180817 AF425625 AF425622 U88535 AF425630 AF350498 AF425611 AF425628 AB074761 AF425626 AB003090 AF425614 AB189121 AF298807 AY422783 AB195673 DQ193572 AY858983 DQ285560 JN415512 JF960216 JF960228 KF052647 KF052648 KF052649 KP2980041) LC006123 LC011945
1):
sequenced in this study. 61
Serologic diagnosis was established when the IgM antibody was detected and a significant (4-fold or greater) rise in antibody titer in the convalescent-phase serum was observed. A positive result by any one of these laboratory tests was defined as DENV infection (12,14).
conducted using a JEV vaccine for animals (Beijing-1, Kaketsuken, Kumamoto, Japan) as the ELISA antigen; anti-dengue antibodies could be detected by cross-reactivity between JEV and DENV. Laboratory diagnosis: A clinical diagnosis of dengue was made based on the clinical manifestations and general hematologic data. Detection of the DENVspecific genome by RT-PCR and virus isolation in C6/36 cells were carried out using acute-phase sera.
RESULTS A man experienced a high fever in Okinawa, with the
Fig. 1. Phylogenetic tree based on a 1,485-nucleotide sequence of the entire envelope gene region, drawn using NJplot program. Phylogenetic analysis of DENV-1 strains was conducted with DENV-2 (AB609589), DENV-3 (AB189125), and DENV-4 (AF326573) as outgroups. The bootstrap probabilities of each node were calculated using 1,000 replicates. *The isolate in this study is indicated in a box with a double line. Japanese isolates obtained during the outbreak in 2014 are indicated in a box with a dotted line. The isolate obtained from Java, Indonesia, in 2012 is indicated in a box with a solid line. The numbers on the nodes are bootstrap values. Countries of isolation are described after the strain. INDO, Indonesia; SING, Singapore; JPN, Japan; MALA, Malaysia; CHIN, China; TPN, Taiwan; LAO, Lao PDR; THAI, Thailand; BRA, Brazil; COTE, Ivory Coast; NIGE, Nigeria; PHIL, Philippines; NAU, Nauru; REU, Reunion; SYC, Seychelles; AUS, Australia. 62
Imported Dengue Case in Okinawa, 2014
served in ELISA titers and in N-titers against DENV-2, DENV-4, RD14/Okinawa, and JEV Nakayama and Beijing-1 strains were recognized in both acute- and convalescent-phase sera (Table 2). The N-titers against DENV-1 and isolate RD14/Okinawa were the highest among those against all serotypes of DENV, and interestingly, those against JEV Nakayama and Beijing-1 strains were significantly higher than those against DENV-1 and RD14/Okinawa. In the case presented here, the patient showed a secondary immune response, and the causative virus was not identified merely by the neutralization test, but by IgM antibody detection, as described above.
onset dated to February 19, 2014, 4 days after returning from Bali, Indonesia. During his stay for nearly 1 month, he had been bitten by mosquitoes. His clinical manifestations were fever, joint pain, skin rash, vomiting, nausea, sore throat, and bloodshot eyes. His platelet count fell from 70,000 to 35,000/mL 6 days after onset and then recovered to 125,000/mL 23 days after onset, whereas his C-reactive protein level was 1.03 mg/L in the acute phase but decreased to an undetectable level. Aspartate transaminase and alanine aminotransferase levels, indicators of liver function, were slightly elevated, and a tourniquet test for hemorrhagic tendency yielded negative results. IgM antibody against DENV was detected in the serum 6 days after onset at a health center using a commercial kit (Dengue Virus IgM Capture DxSelectTM, Focus Diagnostics; Cypress, CA, USA). For further etiologic study, the viral genome was tested for 4 serotypes of DENV and chikungunya virus in acute-phase serum using RT-PCR and nested PCR, with negative results. After inoculation of acute-phase serum onto C6/36 and Vero cells, the supernatant was tested sequentially for viral antigens via immunostaining and RT-PCR. As a result, the supernatant from 7 days after the blind passage via C6/36 was positive for viral antigen, but that after the passage via Vero was always negative. The DENV-1 polyclonal antibody strongly stained for the viral antigen. In addition, the shape of the focus of the isolate differed from the Hawaii strain of DENV-1 used as a positive control, indicating that the isolate was not the result of contamination. The supernatant was further tested using RT-PCR with specific primers for each serotype of DENV, and a positive result for DENV-1 was obtained. Therefore, together with the background of the patient's history of a trip overseas, the serologic results mentioned below, and his physical signs, it was concluded that the patient's symptoms were due to DENV-1 infection with a recently wellrecognized serotype from Southeast Asia, including Indonesia. This isolate, designated as RD14/Okinawa, was further analyzed genetically based on 1,485 nucleotides of the entire E region and compared to published strains. The results indicate that the isolate belongs to genotype 1 of DENV-1 (Fig. 1). In addition, the isolate is closely related to the Singapore strains isolated in 2009 and 2010, and the cluster to which the isolate belongs includes the Indonesia Java strain SKBCM002/12/INDO isolated in 2012 and Japanese isolates from patients infected at Yoyogi Park, Tokyo, and Saitama during a focal outbreak in 2014. For serologic testing, significant increases in N-titers against DENV-1 and DENV-3 and seroconversion ob-
DISCUSSION An imported case of dengue, in which the patient had a history of recent travel to Bali, Indonesia, was confirmed in Okinawa in February 2014; infection with DENV-1 was proven by virus isolation, together with clinical manifestations and the results of serologic testing. The isolate belongs to genotype I of DENV-1 and is closely related to the Southeast Asian strains and isolates identified in Japan during the dengue outbreak in 2014. Two genotypes of DENV-1, I and IV, have been circulating in Surabaya, Indonesia, since 2008 (22) and Java, Indonesia, since 2012 (23). The isolate in this study belonging to genotype I of DENV-1 was closely related to the Indonesian strains, which coincides with the destination of the patient's recent trip. Kotaki et al. (22) speculated that genotype I in Indonesia might have been introduced into Surabaya from other Southeast Asian countries, including Thailand, Singapore, Vietnam, and Malaysia, within the last 15 years. The dengue outbreak in Japan in 2014 was also caused by genotype I of DENV-1. Okinawa thus also has a risk of imported dengue and domestic infection. The N-titers of DENV-1 (Hawaii) and DENV-3 were recognized in acute-phase sera. Serologically, the patient had a secondary immune response for dengue, suggesting his exposure to DENV. It is believed that an N-titer of more than 10 can protect against homotypic infection. However, DENV-1 was responsible for the symptoms of the patient in this study, and the antibody allowed homotypic infection and virus isolation. These phenomena were also reported previously (24). The Ntiter against RD14/Okinawa was less than 10 in acutephase sera, but increased to 590 in convalescent-phase serum, which is the highest titer among the DENVs. A possible explanation for this phenomenon is the different antigenicity between the Hawaii strain used in this
Table 2. Serologic test on sera from an imported dengue patient in Okinawa IgG Date of sampling
ELISA
140221 140228 140305
＜100 8,000 12,000
JEV A C1 C2
Neutralization test DENV DEN1
DEN2
30 280 280
＜10 22 27
JEV
Isolate
DEN3
DEN4
Nakayama
Beijing-1
RD14
40 160 nd
＜10 54 64
＜10 1,100 740
＜10 6,400 32,000
＜10 115 590
nd, not done. 63
neutralization test and RD14/Okinawa, which may allow escape from neutralization, despite the 2 strains belonging to the same genotype. Fourteen amino acid differences in the entire envelope protein between RD14/Okinawa and the Hawaii strain were observed, including 2 sites (I324V and I380V) in domain III where the neutralization epitope exists (25); 4 sites (S155T, T161I, T171S, and T277K) in the domain I–II hinge region, one of the principal determinants in type-specific neutralizing antibodies in humans (26); and 8 other sites (S8N, S227P, Q234E, V251A, F402L, V461I, T473A, and L484M). Notably, residue 277 in the kl loop is important for variability of conformation (26), which might be responsible for this antigenic difference. For laboratory diagnosis, use of the RD14/Okinawa strain can be appropriate for neutralization testing to reflect recent circumstances. Interestingly, the N-titer against JEV Beijing-1 increased from less than 10 to 32,000, which was significantly higher than that against DENV. It is also known that a secondary response by sequential infection can occur not only for dengue serotypes but also for other flaviviruses such as JEV and yellow fever virus; this phenomenon is understood in the wider concept of ``original antigenic sin'' (12). The patient, aged 72, had probably previously been exposed to JEV by natural infection, because vaccinations of JEV with the Nakayama strain had been started in Okinawa in 1972, but at the time, the patient would have been 30 years old, which was not the target age for vaccination. Vaccinations with the Beijing-1 strain were started in 1989. The immune status to the Beijing-1 and Nakayama strains would have decreased to an undetectable level. It is also known that there is a variety of antigenicity for JEV strains. In the case presented here, diagnosis of DENV-1 infection by serologic testing was not possible. Makino et al. (12), interestingly, mentioned that a group of cases with a significant increase in antibodies against both JEV and DENV in the convalescent phase had the highest rate of DENV isolation and detection of the virus genome. Further study of additional cases is needed for more in-depth investigation in the future. Approximately 200 imported dengue cases are reported every year in Japan. Even if no domestic infection and few imported cases were reported in Okinawa, the cases might be underestimated and overlooked, because of the lack of doctors with clinical experience with dengue. Okinawa is a risk area for establishing a transmission cycle as it lies in a subtropical region in which A. albopictus can survive throughout the year (27) and for viral invasion as it is also a tourist destination for visitors from within Japan (approximately 6 million) and from other countries (approximately 550,000). At present, there is no specific treatment or vaccine for dengue; therefore, risk assessment through robust monitoring of cases of fever and vector mosquitoes is very important. In order to prepare for epidemics practically and effectively, control measures can be implemented immediately, for example, providing education in communities regarding controlling mosquito breeding sites through cleaning campaigns, avoiding mosquito bites, and warning against the use of aspirin during a fever.
Acknowledgments We express appreciation to Department of Microbiology and Oncology, Graduate School of Medicine, University of the Ryukyus. We thank the help of the research assistants Ms. Mutsumi Isa, Mr. Yuta Tamashiro, and Mr. Tetsu Kinoshita. This study was partly supported by a Grant-in-Aid for Scientific Research (No. 23510030), from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Unit Center for Researcher Support and Development, University of the Ryukyus.
Conflict of interest None to declare. REFERENCES 1. Halstead SB. Dengue. Lancet. 2007;370:1644-52. 2. World Health Organization and Special Programme for Research and Training in Tropical Diseases. Handbook for Clinical Management of Dengue. Geneva: WHO; 2012. Available at . Accessed December 24, 2014. 3. Guzman MG, Harris E. Dengue. Lancet. 2015;385:453-65. 4. Bhatt S, Gething PW, Brady OJ, et al. The global distribution and burden of dengue. Nature. 2013;496:504-7. 5. Nihei N, Komagata O, Mochizuki K, et al. Geospatial analysis of invasion of the Asian tiger mosquito Aedes albopictus: competition with Aedes japonicus japonicus in its northern limit area in Japan. Geospat Health. 2014;8:417-27. 6. Sakamoto N. Domestic dengue virus infection. J Jap Soc Int Med. 2014;103:2653-6. 7. Hotta S. Dengue epidemics in Japan, 1942–1945. J Trop Med Hyg. 1953;56:83. 8. Schlesinger RW. Dengue viruses. Virol Monogr. 1977;16:1-132. 9. Ura M. Epidemiology of dengue fever in Okinawa. Annu Rep Okinawa Pref Inst Pub Health. 1978;12:104-14. Japanese. 10. Tanaka K. Mosquitoes in Ryukyu Islands. Med Entomol Zool. 1971;22:80. Japanese. 11. Tadano M, Okuno Y, Fukunaga T, et al. Retrospective serological studies on dengue epidemics in Osaka and Okinawa. Biken J. 1983;26:165-7. 12. Makino Y, Tadano M, Saito M, et al. Studies on serological cross-reaction in sequential flavivirus infections. Microbiol Immunol. 1994;38:951-5. 13. Igarashi A, Chiowanich P, Leechanachai P, et al. Virological and epidemiological studies on encephalitis in Chiang Mai area, Thailand, in the year of 1982: III. Virus isolation from clinical materials. Trop Med. 1983;25:149-54. 14. National Institute of Infectious Diseases. Laboratory Manual for Dengue Virus. Available at . Japanese. 15. Morita K, Tanaka M, Igarashi A. Rapid identification of dengue virus serotypes by using polymerase chain reaction. J Clin Microbiol. 1991;29:2107-10. 16. National Institute of Infectious Diseases. Laboratory manual for chikungunya virus : Ver.1.1. Available at . Japanese. 17. Osman O, Fong MY, Sekaran SD. Genetic characterization of dengue virus type 1 isolated in Brunei in 2005–2006. J Gen Virol. 2009;90:678-86. 18. Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25: 4876-82. 19. Perriere G, Gouy M. WWW-query: an on-line retrieval system for biological sequence banks. Biochimie. 1996;78:364-9. 20. Saito M, Taira K, Itokazu K, et al. Recent change of the antigenicity and genotype of Japanese encephalitis viruses distributed on Okinawa Island, Japan. Am J Trop Med Hyg. 2007;77:737-46. 21. Ishimine T, Tadano M, Fukunaga T, et al. An improved micromethod for infectivity assays and neutralization tests of dengue viruses. Biken J. 1987;30:39-44. 22. Kotaki T, Yamanaka A, Mulyatno KC, et al. Continuous dengue type 1 virus genotype shifts followed by co-circulation, clade shifts and subsequent disappearance in Surabaya, Indonesia, 2008–2013. Infect Genet Evol. 2014;28:48-54. 23. Nusa R, Prasetyowati H, Meutiawati F, et al. Molecular surveillance of dengue in Sukabumi, West Java province, Indonesia. J Infect Dev Ctries. 2014;8:733-41.
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Imported Dengue Case in Okinawa, 2014 24. Sirivichayakul C, Sabchareon A, Limkittikul K, et al. Plaque reduction neutralization antibody test does not accurately predict protection against dengue infection in Ratchaburi cohort, Thailand. Virol J. 2014;11:48. 25. Zulueta A, Martin J, Hermida L, et al. Amino acid changes in the recombinant dengue 3 envelope domain III determine its antigenicity and immunogenicity in mice. Virus Res. 2006;121:65-73.
26. Fibriansah G, Tan JL, Smith SA, et al. A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface. EMBO Mol Med. 2014;6:358-71. 27. Toma T, Miyagi I. Seasonal changes in the hatchability of Aedes albopictus (Diptera: Culicidae) eggs in Okinawajima, Ryukyu Archipelago, Japan. Jpn J Sanit Zool. 1990;41:195-204.
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Original Article
Serologic and Virologic Studies of an Imported Dengue Case Occurring in 2014 in Okinawa, Japan Mika Saito1*, Maki Tamayose2, Kazuya Miyagi2, Hiroo Takaragawa1,3, Masao Tateyama2, Masayuki Tadano1, and Jiro Fujita2 1Department
of Microbiology and Oncology, Graduate School of Medicine, 2Department of Infectious, Respiratory, and Digestive Medicine, Control and Prevention of Infectious Diseases (First Department of Internal Medicine), Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215; and 3Crop Science Laboratory, Subtropical Agriculture Course, Graduate School of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan SUMMARY: After returning from Bali, Indonesia, in February 2014, a 72-year-old man was hospitalized in Okinawa owing to a high fever and rash. Dengue was clinically suspected, and the patient tested positive for IgM against dengue using a commercial kit. Serologically, the patient showed secondary seroreactivity. Significant increases in neutralization titers (N-titers) against all 4 serotypes of dengue virus (DENV) and Japanese encephalitis virus (JEV) strains were recognized in convalescent-phase sera comparing to acute phase sera. The N-titer against DENV serotype 1 (DENV-1) was the highest among all DENV serotypes. Interestingly, the N-titers against JEV strains were significantly higher than those against all types of DENV comparing to acute phase sera. The virus was isolated from the acute-phase serum and identified as DENV-1 and designated RD14/Okinawa. The patient's symptoms were due to DENV-1 infection. Phylogenetic sequencing analysis indicated that the isolate RD14/Okinawa belonged to genotype I of DENV-1, which is closely related to the Southeast Asian strains and isolates found during the dengue outbreak in Japan in 2014. We should undertake control measures against dengue in Okinawa, which is a subtropical area with Aedes albopictus activity throughout year. modes of transportation, as well as the distribution of vector mosquitoes due to global warming (3,5). An outbreak of dengue domestic infection of more than 150 patients occurred suddenly in Japan in August 2014 after a period 69 years with no outbreaks (6). Aedes albopictus is thought to have been responsible for this outbreak. In western Japan, a dengue epidemic had previously occurred in 1942–1945, during the Second World War (7). It is believed that military personnel who had contracted dengue in other parts of Asia brought it back on military ships, and ports such as Nagasaki, Kobe, and Yokohama had focal dengue outbreaks (8). Okinawa had at least 10 epidemics recorded from 1893–1955, when the last dengue epidemic was reported in the Yaeyama region, although this was not recorded officially in a Japanese national report (9); Aedes aegypti, the principle vector of dengue, was also identified on Ishigaki Island until 1970 (10). A retrospective serosurvey revealed that the dengue epidemics in Okinawa had been due to DENV-1 and DENV-2 (11). Recently, 11 dengue cases were reported between 1999 and 2014 in Okinawa, and all seemed to have been infected overseas. There are cross-reactive antigens within the DENV serotypes and also among different serogroups of the flaviviruses, such as JEV, which make serodiagnosis difficult in regions where 2 or more different serogroups coexist. In the case of primary infection by a flavivirus, a simple reaction is obtained for the causative agent, but in the case of secondary infection, the presence of a preexisting antibody and immunologic memories mean that seroreactivity is too broad for the DENV subgroup and also for the flavivirus subgroup to identify the causative virus of the more recent infection, even by a
INTRODUCTION Dengue is a mosquito-borne viral disease caused by infection with 1 of 4 different serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), which belong to the family Flaviviridae, genus Flavivirus, which includes different serogroups such as Japanese encephalitis virus (JEV). Previously, DENV infection was classified according to severity as dengue fever or dengue hemorrhagic fever (1), but the World Health Organization (WHO) introduced new classifications in 2012: dengue with or without warning signs and severe dengue (2). We use the term ``dengue'' in accordance with this newer classification. Dengue is endemic in more than 100 countries in Southeast Asia, the Americas, the western Pacific, Africa, and the eastern Mediterranean region (3), and recently it has been estimated that 390 million cases of dengue infection occur per year, of which 96 million manifest apparent symptoms (4). Dengue is one of the most important emerging and reemerging infectious diseases not only because of its high incidence, which has increased 30-fold in the past 50 years, and the disease severity, but also because of the expansion of its geographical distribution, mainly via human Received February 5, 2015. Accepted April 9, 2015. J-STAGE Advance Publication June 12, 2015. DOI: 10.7883/yoken.JJID.2015.061 *Corresponding author: Mailing address: Department of Microbiology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207, Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: ＋81-98-895-1132, Fax: ＋81-98-895-1410, E-mail: mikas＠med.u-ryukyu.ac.jp 60
Imported Dengue Case in Okinawa, 2014
cells and that of the Vero cell culture onto Vero cells, involving so-called blind passages. The supernatants were then inoculated onto BHK-21 cells and incubated for 3 days. The presence of the virus was examined with an immunostaining assay using mouse polyclonal antibodies against DENV-1, DENV-2, DENV-3, and DENV-4. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted using the specific primers for DENV-1, DENV-2, DENV-3, DENV-4, and chikungunya virus as described previously (14–16). Sequences and nucleic acid sequence analysis: A total of 1,485 nucleotides of the envelope (E) gene region of isolate RD14/Okinawa in this study were sequenced directly using the specific primers as described previously (17). Multiple alignments and the phylogenetic analysis were performed with the neighbor-joining (NJ) method using Clustal-X (18), and phylogenetic trees were drawn using the NJplot program (19). The DENV-1 used for phylogenetic analysis in this study is listed in Table 1. Serologic test: For serologic analysis, 4 prototype strains of DENV-1 (Hawaiian), DENV-2 (New Guinea B), DENV-3 (H-87), and DENV-4 (H-241); the isolate in this study (RD14/Okinawa); and 2 JEV strains (Nakayama: vaccine, genotype 3; Beijing-1: vaccine, genotype 3) were used for 50z focus-reduction neutralization tests, as described previously (20,21). An indirect IgG enzyme-linked immunosorbent assay (ELISA) was
neutralization test, which is the most specific test for distinguishing the etiology (12). In this study, we conducted serologic and virologic tests on an imported dengue case that occurred in February 2014 in Okinawa. We report here the seroreactivity of the patient and genetic analysis of the virus isolated in this study. We also discuss dengue control measures for robustly preparing against future epidemics in Okinawa. MATERIALS AND METHODS Sampling from the patient: Serum samples were obtained from a 72-year-old man who was clinically suspected of having dengue. Sampling was conducted 3 times: once to obtain acute-phase serum on February 21 and twice to obtain convalescent-phase sera on February 28 and March 5, 2014. Before serologic testing, sera were inactivated at 569 C for 30 minutes. Isolation and identification of viruses: Acute-phase serum was tested for the virus and viral genome. The virus isolation and identification procedure were as described previously (13). Briefly, serum was inoculated onto clone C6/36 mosquito A. albopictus cells and clone Vero African green monkey kidney cells and incubated for 7 days; the supernatant of the C6/36 cell culture was further inoculated chronologically onto C6/36
Table 1. Dengue virus serotype 1 strains used for analysis in this study Strain
Yr
Location
Genotype
Accession number
Mochizuki Hawaii TH-SMAN 16007 IBH28328 P72-1244 WestPac PUO 359 GZ/80 AUS HCS1 765101 A88 DEI 0151 LaoCH323 BeH 584526 98901530 DF DV-1 Abidjan 02SA079 D1/hu/Seychelles/NIID41/2003 Fj231 SC01 257/04 Malaysia2008 SG(EHI)D1/16174Y09 SG(EHI)D1/37966Y10 SKB-CM002 SKB-CM013 SKB-CM046 RD14/Okinawa D1/Hu/Tokyo/NIID106/2014 D1/Hu/Saitama/NIIH100/2014
1943 1945 1954 1964 1968 1972 1974 1980 1980 1983 1987 1988 1991 1996 1997 1998 1999 2002 2003 2004 2004 2004 2008 2009 2010 2012 2012 2012 2014 2014 2014
Japan Hawaii, USA Thailand Thailand Nigeria Malaysia Nauru Thailand China Australia Taiwan Indonesia Peru Laos Brazil Indonesia Ivory Coast Philippines Seychells China Indonesia Reunion Malaysia Singapore Singapore Java, Indonesia Java, Indonesia Java, Indonesia Okinawa, Japan Tokyo, Japan Saitama, Japan
I I II II V III IV I I IV I IV V I V IV V IV IV I IV IV I I I I IV IV I I I
AB074760 AF425619 D10513 AF180817 AF425625 AF425622 U88535 AF425630 AF350498 AF425611 AF425628 AB074761 AF425626 AB003090 AF425614 AB189121 AF298807 AY422783 AB195673 DQ193572 AY858983 DQ285560 JN415512 JF960216 JF960228 KF052647 KF052648 KF052649 KP2980041) LC006123 LC011945
1):
sequenced in this study. 61
Serologic diagnosis was established when the IgM antibody was detected and a significant (4-fold or greater) rise in antibody titer in the convalescent-phase serum was observed. A positive result by any one of these laboratory tests was defined as DENV infection (12,14).
conducted using a JEV vaccine for animals (Beijing-1, Kaketsuken, Kumamoto, Japan) as the ELISA antigen; anti-dengue antibodies could be detected by cross-reactivity between JEV and DENV. Laboratory diagnosis: A clinical diagnosis of dengue was made based on the clinical manifestations and general hematologic data. Detection of the DENVspecific genome by RT-PCR and virus isolation in C6/36 cells were carried out using acute-phase sera.
RESULTS A man experienced a high fever in Okinawa, with the
Fig. 1. Phylogenetic tree based on a 1,485-nucleotide sequence of the entire envelope gene region, drawn using NJplot program. Phylogenetic analysis of DENV-1 strains was conducted with DENV-2 (AB609589), DENV-3 (AB189125), and DENV-4 (AF326573) as outgroups. The bootstrap probabilities of each node were calculated using 1,000 replicates. *The isolate in this study is indicated in a box with a double line. Japanese isolates obtained during the outbreak in 2014 are indicated in a box with a dotted line. The isolate obtained from Java, Indonesia, in 2012 is indicated in a box with a solid line. The numbers on the nodes are bootstrap values. Countries of isolation are described after the strain. INDO, Indonesia; SING, Singapore; JPN, Japan; MALA, Malaysia; CHIN, China; TPN, Taiwan; LAO, Lao PDR; THAI, Thailand; BRA, Brazil; COTE, Ivory Coast; NIGE, Nigeria; PHIL, Philippines; NAU, Nauru; REU, Reunion; SYC, Seychelles; AUS, Australia. 62
Imported Dengue Case in Okinawa, 2014
served in ELISA titers and in N-titers against DENV-2, DENV-4, RD14/Okinawa, and JEV Nakayama and Beijing-1 strains were recognized in both acute- and convalescent-phase sera (Table 2). The N-titers against DENV-1 and isolate RD14/Okinawa were the highest among those against all serotypes of DENV, and interestingly, those against JEV Nakayama and Beijing-1 strains were significantly higher than those against DENV-1 and RD14/Okinawa. In the case presented here, the patient showed a secondary immune response, and the causative virus was not identified merely by the neutralization test, but by IgM antibody detection, as described above.
onset dated to February 19, 2014, 4 days after returning from Bali, Indonesia. During his stay for nearly 1 month, he had been bitten by mosquitoes. His clinical manifestations were fever, joint pain, skin rash, vomiting, nausea, sore throat, and bloodshot eyes. His platelet count fell from 70,000 to 35,000/mL 6 days after onset and then recovered to 125,000/mL 23 days after onset, whereas his C-reactive protein level was 1.03 mg/L in the acute phase but decreased to an undetectable level. Aspartate transaminase and alanine aminotransferase levels, indicators of liver function, were slightly elevated, and a tourniquet test for hemorrhagic tendency yielded negative results. IgM antibody against DENV was detected in the serum 6 days after onset at a health center using a commercial kit (Dengue Virus IgM Capture DxSelectTM, Focus Diagnostics; Cypress, CA, USA). For further etiologic study, the viral genome was tested for 4 serotypes of DENV and chikungunya virus in acute-phase serum using RT-PCR and nested PCR, with negative results. After inoculation of acute-phase serum onto C6/36 and Vero cells, the supernatant was tested sequentially for viral antigens via immunostaining and RT-PCR. As a result, the supernatant from 7 days after the blind passage via C6/36 was positive for viral antigen, but that after the passage via Vero was always negative. The DENV-1 polyclonal antibody strongly stained for the viral antigen. In addition, the shape of the focus of the isolate differed from the Hawaii strain of DENV-1 used as a positive control, indicating that the isolate was not the result of contamination. The supernatant was further tested using RT-PCR with specific primers for each serotype of DENV, and a positive result for DENV-1 was obtained. Therefore, together with the background of the patient's history of a trip overseas, the serologic results mentioned below, and his physical signs, it was concluded that the patient's symptoms were due to DENV-1 infection with a recently wellrecognized serotype from Southeast Asia, including Indonesia. This isolate, designated as RD14/Okinawa, was further analyzed genetically based on 1,485 nucleotides of the entire E region and compared to published strains. The results indicate that the isolate belongs to genotype 1 of DENV-1 (Fig. 1). In addition, the isolate is closely related to the Singapore strains isolated in 2009 and 2010, and the cluster to which the isolate belongs includes the Indonesia Java strain SKBCM002/12/INDO isolated in 2012 and Japanese isolates from patients infected at Yoyogi Park, Tokyo, and Saitama during a focal outbreak in 2014. For serologic testing, significant increases in N-titers against DENV-1 and DENV-3 and seroconversion ob-
DISCUSSION An imported case of dengue, in which the patient had a history of recent travel to Bali, Indonesia, was confirmed in Okinawa in February 2014; infection with DENV-1 was proven by virus isolation, together with clinical manifestations and the results of serologic testing. The isolate belongs to genotype I of DENV-1 and is closely related to the Southeast Asian strains and isolates identified in Japan during the dengue outbreak in 2014. Two genotypes of DENV-1, I and IV, have been circulating in Surabaya, Indonesia, since 2008 (22) and Java, Indonesia, since 2012 (23). The isolate in this study belonging to genotype I of DENV-1 was closely related to the Indonesian strains, which coincides with the destination of the patient's recent trip. Kotaki et al. (22) speculated that genotype I in Indonesia might have been introduced into Surabaya from other Southeast Asian countries, including Thailand, Singapore, Vietnam, and Malaysia, within the last 15 years. The dengue outbreak in Japan in 2014 was also caused by genotype I of DENV-1. Okinawa thus also has a risk of imported dengue and domestic infection. The N-titers of DENV-1 (Hawaii) and DENV-3 were recognized in acute-phase sera. Serologically, the patient had a secondary immune response for dengue, suggesting his exposure to DENV. It is believed that an N-titer of more than 10 can protect against homotypic infection. However, DENV-1 was responsible for the symptoms of the patient in this study, and the antibody allowed homotypic infection and virus isolation. These phenomena were also reported previously (24). The Ntiter against RD14/Okinawa was less than 10 in acutephase sera, but increased to 590 in convalescent-phase serum, which is the highest titer among the DENVs. A possible explanation for this phenomenon is the different antigenicity between the Hawaii strain used in this
Table 2. Serologic test on sera from an imported dengue patient in Okinawa IgG Date of sampling
ELISA
140221 140228 140305
＜100 8,000 12,000
JEV A C1 C2
Neutralization test DENV DEN1
DEN2
30 280 280
＜10 22 27
JEV
Isolate
DEN3
DEN4
Nakayama
Beijing-1
RD14
40 160 nd
＜10 54 64
＜10 1,100 740
＜10 6,400 32,000
＜10 115 590
nd, not done. 63
neutralization test and RD14/Okinawa, which may allow escape from neutralization, despite the 2 strains belonging to the same genotype. Fourteen amino acid differences in the entire envelope protein between RD14/Okinawa and the Hawaii strain were observed, including 2 sites (I324V and I380V) in domain III where the neutralization epitope exists (25); 4 sites (S155T, T161I, T171S, and T277K) in the domain I–II hinge region, one of the principal determinants in type-specific neutralizing antibodies in humans (26); and 8 other sites (S8N, S227P, Q234E, V251A, F402L, V461I, T473A, and L484M). Notably, residue 277 in the kl loop is important for variability of conformation (26), which might be responsible for this antigenic difference. For laboratory diagnosis, use of the RD14/Okinawa strain can be appropriate for neutralization testing to reflect recent circumstances. Interestingly, the N-titer against JEV Beijing-1 increased from less than 10 to 32,000, which was significantly higher than that against DENV. It is also known that a secondary response by sequential infection can occur not only for dengue serotypes but also for other flaviviruses such as JEV and yellow fever virus; this phenomenon is understood in the wider concept of ``original antigenic sin'' (12). The patient, aged 72, had probably previously been exposed to JEV by natural infection, because vaccinations of JEV with the Nakayama strain had been started in Okinawa in 1972, but at the time, the patient would have been 30 years old, which was not the target age for vaccination. Vaccinations with the Beijing-1 strain were started in 1989. The immune status to the Beijing-1 and Nakayama strains would have decreased to an undetectable level. It is also known that there is a variety of antigenicity for JEV strains. In the case presented here, diagnosis of DENV-1 infection by serologic testing was not possible. Makino et al. (12), interestingly, mentioned that a group of cases with a significant increase in antibodies against both JEV and DENV in the convalescent phase had the highest rate of DENV isolation and detection of the virus genome. Further study of additional cases is needed for more in-depth investigation in the future. Approximately 200 imported dengue cases are reported every year in Japan. Even if no domestic infection and few imported cases were reported in Okinawa, the cases might be underestimated and overlooked, because of the lack of doctors with clinical experience with dengue. Okinawa is a risk area for establishing a transmission cycle as it lies in a subtropical region in which A. albopictus can survive throughout the year (27) and for viral invasion as it is also a tourist destination for visitors from within Japan (approximately 6 million) and from other countries (approximately 550,000). At present, there is no specific treatment or vaccine for dengue; therefore, risk assessment through robust monitoring of cases of fever and vector mosquitoes is very important. In order to prepare for epidemics practically and effectively, control measures can be implemented immediately, for example, providing education in communities regarding controlling mosquito breeding sites through cleaning campaigns, avoiding mosquito bites, and warning against the use of aspirin during a fever.
Acknowledgments We express appreciation to Department of Microbiology and Oncology, Graduate School of Medicine, University of the Ryukyus. We thank the help of the research assistants Ms. Mutsumi Isa, Mr. Yuta Tamashiro, and Mr. Tetsu Kinoshita. This study was partly supported by a Grant-in-Aid for Scientific Research (No. 23510030), from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Unit Center for Researcher Support and Development, University of the Ryukyus.
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