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    A 184 basepair ns5 nucleotide sequence fragment from Bandung, Indonesia (sample HTV236-01) was aligned with other West Nile virus (WNV) sequences from GenBank using BioEdit. The maximum likelihood tree was constructed by using software MEGA5. Bootstrap values are expressed as a percentage of 1,000 replicates.

  • 1.

    Rosenberg R, Johansson MA, Powers AM, Miller BR, 2013. Search strategy has influenced the discovery rate of human viruses. Proc Natl Acad Sci USA 110: 1396113964.

    • Search Google Scholar
    • Export Citation
  • 2.

    Punjabi NH, Taylor WR, Murphy GS, Purwaningsih S, Picarima H, Sisson J, Olson JG, Baso S, Wangsasaputra F, Lesmana M, Oyofo BA, Simanjuntak CH, Subekti D, Corwin AL, Richie TL, 2012. Etiology of acute, non-malaria, febrile illnesses in Jayapura, northeastern Papua, Indonesia. Am J Trop Med Hyg 86: 4651.

    • Search Google Scholar
    • Export Citation
  • 3.

    Kosasih H, Ibrahim IN, Wicaksana R, Alisjahbana B, Hoo Y, Yo IH, Antonjaya U, Widjaja S, Winoto I, Williams M, Blair PJ, 2011. Evidence of human hantavirus infection and zoonotic investigation of hantavirus prevalence in rodents in western Java, Indonesia. Vector Borne Zoonotic Dis 11: 709713.

    • Search Google Scholar
    • Export Citation
  • 4.

    Lanciotti RS, 2003. Molecular amplification assays for the detection of flaviviruses. Adv Virus Res 61: 6799.

  • 5.

    Smithburn KC, Hughes TP, Burke AW, Paul JH, 1940. A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg 20: 471492.

    • Search Google Scholar
    • Export Citation
  • 6.

    Mackenzie JS, Williams DT, 2009. The zoonotic flaviviruses of southern, south-eastern and eastern Asia, and Australasia: the potential for emergent viruses. Zoonoses Public Health 56: 338356.

    • Search Google Scholar
    • Export Citation
  • 7.

    Chaintoutis SC, Chaskopoulou A, Chassalevris T, Koehler PG, Papanastassopoulou M, Dovas CI, 2013. West Nile virus lineage 2 strain in Greece, 2012. Emerg Infect Dis 19: 827829.

    • Search Google Scholar
    • Export Citation
  • 8.

    Bakonyi T, Ferenczi E, Erdelyi K, Kutasi O, Csorgo T, Seidel B, Weissenbock H, Brugger K, Ban E, Nowotny N, 2013. Explosive spread of a neuroinvasive lineage 2 West Nile virus in Central Europe, 2008/2009. Vet Microbiol 165: 6170.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zeller HG, Schuffenecker I, 2004. West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas. Eur J Clin Microbiol Infect Dis 23: 147156.

    • Search Google Scholar
    • Export Citation
  • 10.

    Monini M, Falcone E, Busani L, Romi R, Ruggeri FM, 2010. West Nile virus: characteristics of an African virus adapting to the third millennium world. Open Virol J 4: 4251.

    • Search Google Scholar
    • Export Citation
  • 11.

    Sambri V, Capobianchi M, Charrel R, Fyodorova M, Gaibani P, Gould E, Niedrig M, Papa A, Pierro A, Rossini G, Varani S, Vocale C, Landini MP, 2013. West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention. Clin Microbiol Infect 19: 699704.

    • Search Google Scholar
    • Export Citation
  • 12.

    Colpitts TM, Conway MJ, Montgomery RR, Fikrig E, 2012. West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25: 635648.

    • Search Google Scholar
    • Export Citation
  • 13.

    Olson JG, Ksiazek TG, Gubler DJ, Lubis SI, Simanjuntak G, Lee VH, Nalim S, Juslis K, See R, 1983. A survey for arboviral antibodies in sera of humans and animals in Lombok, Republic of Indonesia. Ann Trop Med Parasitol 77: 131137.

    • Search Google Scholar
    • Export Citation
  • 14.

    Lindsey NP, Staples JE, Lehman JA, Fischer M, Centers for Disease Control and Prevention (CDC), 2010. Surveillance for human West Nile virus disease—United States, 1999–2008. MMWR Surveill Summ 59: 117.

    • Search Google Scholar
    • Export Citation

 

 

 

 

West Nile Virus Documented in Indonesia from Acute Febrile Illness Specimens

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  • Eijkman Institute for Molecular Biology, Jakarta, Indonesia; Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia; Universitas Padjadjaran, Bandung, Indonesia; Centers for Disease Control and Prevention, Fort Collins, Colorado; Bogor Agricultural University, Darmaga Campus, Bogor 16680, Indonesia

We report the presence of West Nile virus in a cryopreserved, dengue-negative serum specimen collected from an acute fever case on Java in 2004–2005. The strain belongs to genotype lineage 2, which has recently been implicated in human outbreaks in Europe.

The Indonesian archipelago has been predicted to be a “hotspot” for the emergence of zoonotic and vector-borne pathogens.1 Dengue (DENV), chikungunya (CHIKV), and Japanese encephalitis (JEV) are some of the arthropod-borne viruses (arboviruses) known to cause febrile illness in Indonesia; however it is suspected that other relatively uncommon arboviruses are also causing disease. Currently, there is only limited data on the etiology of febrile illnesses in Indonesia.2 As part of an effort to build capacity to detect the etiologies of underlying acute febrile illnesses in Indonesia, we have begun an analysis of cryopreserved specimens collected in an earlier study of acute febrile illness, which had previously tested negative for hantaviruses and DENV.

Archived samples were selected from an acute febrile illness study that enrolled hospitalized suspected hantavirus patients at two hospitals in Bandung, West Java, Indonesia during 2004–2005.3 Samples were collected from patients ≥ 10 years of age with fever of unknown etiology and at least one of the following symptoms: 1) hemorrhagic manifestations, 2) platelet count < 100,000/mm3, 3) renal insufficiency, 4) liver dysfunction, or 5) non-cardiogenic pulmonary edema. Serum samples were collected from patients at admission to the hospital and at discharge. Collections were made under institutional review board approvals from the National Institutes of Health Research and Development, Indonesian Ministry of Health, and the U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia. The samples were originally tested for hantavirus and dengue using reverse transcription-polymerase chain reaction (RT-PCR) and in-house and commercial immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) tests (Focus Diagnostics, Cypress, CA).

Of 406 cases enrolled, 249 had evidence of recent DENV infection and one had evidence of hantavirus infection. Infecting etiologies for the remaining 157 cases were negative for both DENV and hantavirus. We tested 154 acute specimens from these cases, which had been preserved at −80°C, for other arboviruses. Initial testing was performed by using group/family-specific primers in conventional RT-PCR assays followed by gel electrophoresis of the resulting amplicons.4 The flavivirus group primer testing resulted in four positive samples (265-bp amplicon products), which were further tested with virus-specific RT-PCR reactions and immunofluorescent assay of Vero cells infected with either DENV or JEV. The purified nucleic acid of one flavivirus-positive sample that was negative for both DENV and JEV in the additional testing was subjected to nucleotide sequencing at the Eijkman Institute with the same primers used to generate the PCR product. A 242 basepair sequence from the NS5 gene was generated from the original 265 bp PCR amplicon. Genetic comparisons revealed the closest match (99% nucleotide identity) with the first West Nile virus (WNV) strain isolated (B956), an isolate from Uganda within lineage 2.5 Phylogenetic analyses confirmed the relationship of the Indonesian strain with other lineage 2 WNV sequences (Figure 1).

Figure 1.
Figure 1.

A 184 basepair ns5 nucleotide sequence fragment from Bandung, Indonesia (sample HTV236-01) was aligned with other West Nile virus (WNV) sequences from GenBank using BioEdit. The maximum likelihood tree was constructed by using software MEGA5. Bootstrap values are expressed as a percentage of 1,000 replicates.

Citation: The American Society of Tropical Medicine and Hygiene 90, 2; 10.4269/ajtmh.13-0445

The WNV positive sample came from a 15-year-old boy admitted for systemic febrile illness with epistaxis, gastrointestinal symptoms, elevated serum transaminases, leucopenia, and thrombocytopenia. No neurological symptoms were reported and the patient was discharged after full recovery. Culture of the serum sample was attempted; however, the sample did not produce cytopathology in Vero cells propagated for 10 days.

West Nile virus, a zoonotic, mosquito-transmitted arbovirus belonging to the Flaviviridae family, is reported to be the most common cause of epidemic viral encephalitis in the United States. Phylogenetic analysis has supported the presence of two major genetic lineages. The lineage 1 viruses have typically been associated with large outbreaks and thus are considered to be more virulent than the lineage 2 strains.6 However, several recent outbreaks in Europe have been caused by lineage 2 WNV strains.79 Thus, it is possible that more recent lineage 2 strains are emerging with increased levels of virulence. The Indonesian strain might follow this trend but data regarding neurovirulence from Indonesia are lacking. The close phylogenetic relationship of the Indonesian strain with those from Uganda rather than strains from Australia is somewhat unexpected as there is relatively less movement of people and goods between Africa and Indonesia. Grouping of isolates, however, does not necessarily correlate with the geographic distribution of the virus10 and may be a further indicator of the recent widespread movement of pathogenic arboviruses.

WNV is considered a serious threat to public health and known to cause large outbreaks of epidemic encephalitis in Europe and North America with significant morbidity and mortality.11,12 WNV has not been previously isolated in Indonesia, but a serological study detected the presence of antibody to this virus in Lombok, Indonesia.13 Our study associates detection of WNV nucleic acid in Indonesia with human illness. Given that these samples were collected several years ago, the possibility that WNV may be currently circulating in Indonesia warrants further study. The clinical spectrum caused by WNV varies dramatically from inapparent infection (∼80% of cases) to non-neurological febrile illness (∼20% of cases), to the most severe form including neuroinvasive disease (< 1%).14 Neuroinvasive disease cases have a mortality rate of about 10% and often display chronic manifestations.

There has been limited laboratory capacity in Indonesia to detect arboviruses other than dengue and the role of CHIKV, WNV, JEV, and other vector-borne viruses, such as Zika virus, as causes of febrile illness or more serious encephalitis has likely been underestimated. Development of rapid and simple molecular diagnostic tests combined with the establishment of dedicated research facilities in Indonesia will lead to an increased understanding of both endemic and emerging pathogens. With this recent detection of WNV in a febrile human in Indonesia, it is clear that WNV should be considered a serious threat to public health in Southeast Asia. Enhanced surveillance studies in humans, vectors and animals, and epidemiological surveys are warranted in Indonesia.

ACKNOWLEDGMENTS

We acknowledge Universitas Padjadjaran, Bandung, the National Institute of Health Research and Development, Ministry of Health; and U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia for their support in conduction of research and specimen archiving.

  • 1.

    Rosenberg R, Johansson MA, Powers AM, Miller BR, 2013. Search strategy has influenced the discovery rate of human viruses. Proc Natl Acad Sci USA 110: 1396113964.

    • Search Google Scholar
    • Export Citation
  • 2.

    Punjabi NH, Taylor WR, Murphy GS, Purwaningsih S, Picarima H, Sisson J, Olson JG, Baso S, Wangsasaputra F, Lesmana M, Oyofo BA, Simanjuntak CH, Subekti D, Corwin AL, Richie TL, 2012. Etiology of acute, non-malaria, febrile illnesses in Jayapura, northeastern Papua, Indonesia. Am J Trop Med Hyg 86: 4651.

    • Search Google Scholar
    • Export Citation
  • 3.

    Kosasih H, Ibrahim IN, Wicaksana R, Alisjahbana B, Hoo Y, Yo IH, Antonjaya U, Widjaja S, Winoto I, Williams M, Blair PJ, 2011. Evidence of human hantavirus infection and zoonotic investigation of hantavirus prevalence in rodents in western Java, Indonesia. Vector Borne Zoonotic Dis 11: 709713.

    • Search Google Scholar
    • Export Citation
  • 4.

    Lanciotti RS, 2003. Molecular amplification assays for the detection of flaviviruses. Adv Virus Res 61: 6799.

  • 5.

    Smithburn KC, Hughes TP, Burke AW, Paul JH, 1940. A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg 20: 471492.

    • Search Google Scholar
    • Export Citation
  • 6.

    Mackenzie JS, Williams DT, 2009. The zoonotic flaviviruses of southern, south-eastern and eastern Asia, and Australasia: the potential for emergent viruses. Zoonoses Public Health 56: 338356.

    • Search Google Scholar
    • Export Citation
  • 7.

    Chaintoutis SC, Chaskopoulou A, Chassalevris T, Koehler PG, Papanastassopoulou M, Dovas CI, 2013. West Nile virus lineage 2 strain in Greece, 2012. Emerg Infect Dis 19: 827829.

    • Search Google Scholar
    • Export Citation
  • 8.

    Bakonyi T, Ferenczi E, Erdelyi K, Kutasi O, Csorgo T, Seidel B, Weissenbock H, Brugger K, Ban E, Nowotny N, 2013. Explosive spread of a neuroinvasive lineage 2 West Nile virus in Central Europe, 2008/2009. Vet Microbiol 165: 6170.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zeller HG, Schuffenecker I, 2004. West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas. Eur J Clin Microbiol Infect Dis 23: 147156.

    • Search Google Scholar
    • Export Citation
  • 10.

    Monini M, Falcone E, Busani L, Romi R, Ruggeri FM, 2010. West Nile virus: characteristics of an African virus adapting to the third millennium world. Open Virol J 4: 4251.

    • Search Google Scholar
    • Export Citation
  • 11.

    Sambri V, Capobianchi M, Charrel R, Fyodorova M, Gaibani P, Gould E, Niedrig M, Papa A, Pierro A, Rossini G, Varani S, Vocale C, Landini MP, 2013. West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention. Clin Microbiol Infect 19: 699704.

    • Search Google Scholar
    • Export Citation
  • 12.

    Colpitts TM, Conway MJ, Montgomery RR, Fikrig E, 2012. West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25: 635648.

    • Search Google Scholar
    • Export Citation
  • 13.

    Olson JG, Ksiazek TG, Gubler DJ, Lubis SI, Simanjuntak G, Lee VH, Nalim S, Juslis K, See R, 1983. A survey for arboviral antibodies in sera of humans and animals in Lombok, Republic of Indonesia. Ann Trop Med Parasitol 77: 131137.

    • Search Google Scholar
    • Export Citation
  • 14.

    Lindsey NP, Staples JE, Lehman JA, Fischer M, Centers for Disease Control and Prevention (CDC), 2010. Surveillance for human West Nile virus disease—United States, 1999–2008. MMWR Surveill Summ 59: 117.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Khin Saw Aye Myint, Eijkman Institute of Molecular Biology, 69 Diponegoro, Jakarta 10430, Indonesia. E-mails: khinsawying@hotmail.com or myintk@eijkman.go.id

Financial support: This study was supported by the USAID Emerging Pandemic Threat (EPT) Program and the U.S. Centers for Disease Control and Prevention.

Authors' addresses: Khin Saw Aye Myint, Eijkman Institute of Molecular Biology, Jakarta, Indonesia, E-mail: khinsawying@hotmail.com. Herman Kosasih, Mita Puspita, and Bachti Alisjahbana, Universitas Padjadjaran, Faculty of Medicine, Bandung, Indonesia, E-mails: herman_kosasih@yahoo.com, mitapuspita@gmail.com, and b.alisjahbana@gmail.com. I. Made Artika, Aditya Perkasa, and Chairin Nisa Ma'roef, Eijkman Institute for Molecular Biology, Emerging Virus Research Unit, Jakarta, Indonesia, E-mails: imart@eijkman.go.id, adhit@eijkman.go.id, and nami@eijkman.go.id. Ungke Antonjaya, Eijkman-Oxford Clinical Research Unit, Microbiology, Jakarta, Indonesia, E-mail: uantonjaya@eocru.org. Jeremy P. Ledermann and Ann M. Powers, Centers for Disease Control and Prevention, Division of Vector Borne Infectious Diseases, Fort Collins, CO, E-mails: bpj7@cdc.gov and apowers@cdc.gov.

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