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Emergence of Japanese encephalitis in Australia: a diagnostic perspective

  • David Pham
    Correspondence
    Address for correspondence: Dr David Pham, Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW 2145, Australia.
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia
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  • Annaleise R. Howard-Jones
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia

    Faculty of Medicine and Health, University of Sydney, NSW, Australia
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  • Linda Hueston
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia
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  • Neisha Jeoffreys
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia
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  • Stephen Doggett
    Affiliations
    Department of Medical Entomology, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia
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  • Rebecca J. Rockett
    Affiliations
    Faculty of Medicine and Health, University of Sydney, NSW, Australia

    Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, Australia
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  • John-Sebastian Eden
    Affiliations
    Faculty of Medicine and Health, University of Sydney, NSW, Australia

    Centre for Virus Research, Westmead Institute for Medical Research, Westmead, NSW, Australia
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  • Vitali Sintchenko
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia

    Faculty of Medicine and Health, University of Sydney, NSW, Australia

    Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, Australia
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  • Sharon C-A. Chen
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia

    Faculty of Medicine and Health, University of Sydney, NSW, Australia

    Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, Australia
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  • Matthew V. O'Sullivan
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia

    Faculty of Medicine and Health, University of Sydney, NSW, Australia

    Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, Australia
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  • Susan Maddocks
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia
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  • Dominic E. Dwyer
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia

    Faculty of Medicine and Health, University of Sydney, NSW, Australia
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  • Jen Kok
    Affiliations
    Centre for Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia

    Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, Australia
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Open AccessPublished:August 19, 2022DOI:https://doi.org/10.1016/j.pathol.2022.07.001

      Summary

      The unprecedented emergence of Japanese encephalitis (JE) in mainland Australia represents an outbreak of high clinical and public health significance. JE is a zoonosis spread by mosquitoes and is one of the most important causes of endemic viral encephalitis in South-East Asia and the Indian subcontinent. While occasional cases of human Japanese encephalitis virus (JEV) infection have occurred in far north Australia, its detection in pigs and the substantial number of locally acquired human cases across multiple jurisdictions in early 2022 prompted the declaration of this outbreak as a Communicable Disease Incident of National Significance. Laboratory testing for JEV is complex, and most cases are diagnosed by serology, for which interpretation is difficult. This review provides a comprehensive outline of currently available methods for JEV diagnosis including serology, nucleic acid amplification testing, virus isolation, sequencing and metagenomics. The relative advantages and disadvantages of the diagnostic tests are presented, as well as their value in clinical and public health contexts. This review also explores the role of mosquito, veterinary and human surveillance as part of the laboratory response to JEV. As JEV may become endemic in Australia, a collaborative and coordinated One Health approach involving animal, human and environmental health is required for optimal disease response and control.

      Key words

      Introduction

      Japanese encephalitis virus (JEV) is a positive-sense, single stranded, enveloped ribonucleic acid (RNA) virus of the genus Flavivirus, family Flaviviridae.
      • Gould E.
      • Solomon T.
      Pathogenic flaviviruses.
      It is closely related to other flaviviruses, including West Nile virus (WNV) and West Nile virus subtype KUNV (WNVKUNV), dengue virus, yellow fever virus, Zika virus and Murray Valley encephalitis virus (MVEV). Five JEV genotypes have been identified based on phylogenetic analyses of the viral envelope gene (Fig. 1): G1 to G5, each with distinct, but sometimes overlapping, geographic variations.
      • Solomon T.
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      Origin and evolution of Japanese encephalitis virus in Southeast Asia.
      JEV occurs as a single serotype.
      • Tsarev S.A.
      • Sanders M.L.
      • Vaughn D.W.
      • Innis B.L.
      Phylogenetic analysis suggests only one serotype of Japanese encephalitis virus.
      Fig. 1
      Fig. 1Japanese encephalitis virus genome. C, capsid; E, envelope; NS, non-structural; prM, premembrane.
      Since 1995, JEV has occasionally been detected in far northern Australia, including the Torres Strait Islands, Cape York and most recently the Tiwi Islands in 2021.
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      • Ritchie S.A.
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      An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995.
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      Short report: the first isolation of Japanese encephalitis virus from mosquitoes collected from mainland Australia.
      Endemic JEV transmission has not previously been described elsewhere in Australia.
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      Arboviruses associated with human disease in Australia.
      ,
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      Japanese encephalitis virus in Australia: from known known to known unknown.
      However, as of March 2022, multiple cases of JE have been diagnosed in four Australian states (New South Wales, Victoria, South Australia and Queensland), primarily in residents or visitors to the Murray River. At least three deaths have been reported, and the outbreak was declared a Communicable Disease Incident of National Significance (CDINS) on 4 March 2022.
      Australian Government Department of Health
      Japanese encephalitis virus situation declared a communicable disease incident of national significance.
      Although Australian JEV cases in the 1990s were of genotype G2, the current JEV outbreak is caused by G4. This is the least common genotype worldwide, and until 2017 was found only in Indonesia and Papua New Guinea, suggesting a new introduction in recent years, possibly via migratory birds or mosquitoes.
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      The ecology and evolution of Japanese encephalitis virus.
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      Changing geographic distribution of Japanese encephalitis virus genotypes, 1935–2017.
      • Liang G.D.
      • Huanyu W.
      Epidemiology of Japanese encephalitis: past, present, and future prospects.
      This review outlines currently available methods for JEV diagnosis including serology, nucleic acid testing, virus isolation, sequencing and metagenomics (Table 1). The relative advantages and disadvantages of these approaches in clinical and public health contexts are presented. This review also explores the role of mosquito, veterinary and human surveillance as part of JEV responses. Given potential future endemicity of JEV in Australia, robust laboratory frameworks are important to manage seasonal peaks and inform future public health responses.
      Table 1Key characteristics, advantages and disadvantages of available assays for diagnosis of Japanese encephalitis virus infection
      MethodsSample typeTesting windowAdvantagesDisadvantagesComments
      Serology
       Pan-flavivirus IgG/IgMCSF, serumWithin first 2 weeks of infection, convalescent samples may be requiredScreening assayNon-specific; reactive test will need reflexive confirmation
       JEV IgGCSF, serumFrom 7–10 days after infection, convalescent samples often required after 2–4 week intervalCan be useful in serosurveillanceSignificant cross-reactivity with other flaviviruses

      Delay in IgG seroconversion
      Commercial ELISA and IF assays available
       JEV IgMCSF, serumFrom 7–10 days of infectionAppears early

      Very sensitive, especially in CSF
      Some cross-reactivity with other flavivirusesCommercial ELISA and IF assays available
       JEV total AbCSF, serumWithin first week of infectionCompetitive ELISA allows for non-human serosurveillanceUsed in sentinel chicken flock testing and serosurveillances
       JEV env/NS1 IgG/IgMCSF, serumSpecific epitope targets allow for differentiation between recombinant vaccination and natural infectionNot useful to differentiate between inactivated vaccination or natural infectionSpecialised testing may only be available at reference laboratories
       JEV neutralisationCSF, serumMost specific serological assay for diagnosisLaborious, slow turnaround timeSpecialised testing may only be available at reference laboratories
       Other flavivirus serologies e.g., DENV, MVEV, WNVCSF, serumWithin first 2 weeks of infection, convalescent samples often required after 2–4 week intervalShould be run alongside JEV testing to more specifically document seroconversion and mitigate issues of cross-reactivity in serological diagnoses
      NAAT
       Pan-flavivirus NAATCSF, blood, brain tissue, urineFirst week of infectionAssists in syndromic detection in arbovirus-endemic regionsNon-specific, may require amplicon sequencing or specific NAAT to confirm
       JEV NAATCSF, blood, brain tissue, urineFirst week of infectionHighly specificSmall window period for testing, low sensitivityPan-genotypic and genotype-specific primers available for G1–G5
       CultureCSF, blood, brain tissue, urineUsually first week of infection, but may have prolonged shedding in urine and persistence in tissueHighly specific

      Allows for sequencing for genotyping
      Requires specialised expertise and equipment for viral culture
       MetagenomicsCSF, blood, brain tissue, urineUsually first week of infection, but may persist in tissue and aid in postmortem diagnosisHypothesis-free testingLargely research use only, requires specialised expertise and equipment
       Whole genome sequencingAmplicons from NAAT or cultured virusHigh resolution, ability to type virusRequires specialised expertise
      Ab, antibodies; CSF, cerebrospinal fluid; DENV, dengue virus; ELISA, enzyme linked immunosorbent assay; IF, immunofluorescence; JEV, Japanese encephalitis virus; MVEV, Murray Valley encephalitis; NAAT, nucleic acid amplification testing; WNV, West Nile virus.

      Clinical features and epidemiology

      JEV is highly neurotropic and is an important cause of viral encephalitis in South-East Asia and the Western Pacific where it is estimated to cause 70,000 clinical cases and 20,000 deaths annually (Fig. 2A).
      • Campbell G.
      • Hills S.
      • Fischer M.
      • et al.
      Estimated global incidence of Japanese encephalitis.
      Centers for Disease Control and Prevention
      Geographic distribution of Japanese encephalitis virus.
      The elderly and young are most susceptible to severe encephalitis. The incubation period for human JEV infection is between 5–15 days and asymptomatic infection is the most common clinical course.
      • Rosen L.
      The natural history of Japanese encephalitis virus.
      Typical manifestations of non-encephalitic disease include an acute febrile illness with headache, myalgia and/or rash. Encephalitis is characterised by focal neurological disease, particularly seizures, Parkinsonian features, and/or altered consciousness.
      • Solomon T.
      Flavivirus encephalitis.
      Other features include abnormal brain computed tomogram (CT), magnetic resonance imaging (MRI), electroencephalogram (EEG) and/or pleocytosis in cerebrospinal fluid (CSF) examination. Polio-like acute flaccid paralysis and Guillain-Barré syndrome have been rarely described.
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      Acute flaccid paralysis as the initial manifestation of Japanese encephalitis: a case report.
      Whilst meningoencephalitis occurs in ≤1% of cases, mortality ranges from 5–50% in case series, averaging 18% over the past 30 years.
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      Japanese encephalitis - the prospects for new treatments.
      It also has high morbidity with 44% of survivors experiencing long-term neurological sequelae including quadriplegia, ventilator dependence, and locked-in syndrome.
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      Japanese encephalitis - the prospects for new treatments.
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      ‘More than devastating’ - patient experiences and neurological sequelae of Japanese encephalitis.
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      The epidemiology, clinical features, and long-term prognosis of Japanese encephalitis in Central Sarawak, Malaysia, 1997–2005.
      Fig. 2
      Fig. 2(A) Geographical distribution of Japanese encephalitis virus (JEV) infection (adapted from the US Centers for Diseases Control and Prevention).
      Centers for Disease Control and Prevention
      Geographic distribution of Japanese encephalitis virus.
      Ovals delineate the rough geographical distributions of different JEV genotypes.
      • Gao X.
      • Liu H.
      • Li X.
      • et al.
      Changing geographic distribution of Japanese encephalitis virus genotypes, 1935–2017.
      G1, red; G2, orange; G3, green; G4, blue; G5, purple. (B) JEV transmission cycle including mosquito (vectors), wading birds (natural hosts), pigs (amplifying hosts) and humans (dead end hosts).
      • Mulvey P.
      • Duong V.
      • Boyer S.
      • et al.
      The ecology and evolution of Japanese encephalitis virus.
      ,
      • le Flohic G.
      • Porphyre V.
      • Barbazan P.
      • Gonzalez J.P.
      Review of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology.
      JEV is transmitted between birds and swine in enzootic cycles via mosquito vectors, mainly Culex spp. (Fig. 2B).
      • Mulvey P.
      • Duong V.
      • Boyer S.
      • et al.
      The ecology and evolution of Japanese encephalitis virus.
      ,
      • le Flohic G.
      • Porphyre V.
      • Barbazan P.
      • Gonzalez J.P.
      Review of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology.
      Wading birds are thought to be reservoirs responsible for maintaining JEV circulation via mosquitoes, as well as spread to new geographic areas via migratory patterns or opportunistic movements with the prevailing climatic conditions (such as widespread rainfall). Due to substantial viraemia, pigs serve as amplifying hosts and the key intermediate host in human JEV transmission. Despite this, JEV rarely causes clinical disease in these natural hosts other than spontaneous abortion and congenital malformations in pigs.
      • Mansfield K.L.
      • Hernández-Triana L.M.
      • Banyard A.C.
      • Fooks A.R.
      • Johnson N.
      Japanese encephalitis virus infection, diagnosis and control in domestic animals.
      Humans and horses are dead-end hosts. Viraemia is typically brief and low-level; human transmission outside of the rare vertical, blood transfusion and solid organ transplant associated transmission have not been documented.
      • Cheng V.C.C.
      • Sridhar S.
      • Wong S.C.
      • et al.
      Japanese encephalitis virus transmitted via blood transfusion, Hong Kong, China.
      ,
      • Chaturvedi U.C.
      • Mathur A.
      • Chandra A.
      • Das S.K.
      • Tandon H.O.
      • Singh U.K.
      Transplacental Infection with Japanese encephalitis virus.
      Veterinary surveillance has confirmed JEV is now widespread in commercial piggeries in NSW, with detections also reported in South Australia, Queensland and Victoria. It is likely endemic in feral and backyard pig populations, though the extent of zoonotic infection is not yet established. Data are not yet available from bird populations in Australia. For several months, mosquito populations increased in rural areas following extensive rainfall and flooding associated with the La Niña weather patterns of 2021–22. There are six mosquito species in Australia capable of transmitting JEV, including the key species Culex tritaeniorhynchus recently detected in the Northern Territory.
      • Lessard B.D.
      • Kurucz N.
      • Rodriguez J.
      • Carter J.
      • Hardy C.M.
      Detection of the Japanese encephalitis vector mosquito Culex tritaeniorhynchus in Australia using molecular diagnostics and morphology.
      However, this species is uncommon and limited to very small populations collected in the Northern Territory. The most likely Australian vector is C. annulirostris, which is both abundant and widespread in all mainland states, and the major vector of local flaviruses.
      • Hall-Mendelin S.
      • Jansen C.C.
      • Cheah W.Y.
      • et al.
      Culex annulirostris (Diptera: Culicidae) host feeding patterns and Japanese encephalitis virus ecology in Northern Australia.
      While there are two effective, safe and well tolerated JE vaccines available in Australia, vaccination is only recommended for laboratory workers that may be exposed to JEV, travellers spending one or more months in JEV endemic areas or people who live or work on the outer islands of the Torres Strait.,
      • Islam N.
      • Lau C.
      • Leeb A.
      • Mills D.
      • Furuya-Kanamori L.
      Safety profile comparison of chimeric live attenuated and Vero cell-derived inactivated Japanese encephalitis vaccines through an active surveillance system in Australia.
      Therefore, the Australian population is largely susceptible to JEV infection.

      Serology

      Serology is the primary diagnostic method of JEV infection. As part of the immune response to JEV infection, neutralising antibodies are rapidly produced, leading to short duration, low level viraemia. Detection of JEV from clinical specimens by nucleic acid amplification tests (NAAT) or virus isolation is often unsuccessful. Therefore, most cases of JE are diagnosed by serological methods in conjunction with a compatible exposure and clinical history. Interpretation of serological assays for JEV requires knowledge of patient demographics, clinical features of infection, timing of potential exposure, and any prior exposure and infection to, or vaccination against other flaviviruses that may lead to cross-reactive antibodies.
      The World Health Organization (WHO) recommends testing for JEV-specific immunoglobulin M (IgM) in CSF or serum.,
      • World Health Organization
      WHO Manual for the laboratory diagnosis of Japanese encephalitis virus infection.
      After the first 9–10 days of illness, the presence of anti-JEV IgM in the CSF has a sensitivity and specificity of >95% for CNS infection.
      • Burke D.S.
      • Nisalak A.
      • Ussery M.A.
      • Laorakpongse T.
      • Chantavibul S.
      Kinetics of IgM and IgG responses to Japanese encephalitis virus in human serum and cerebrospinal fluid.
      ,
      • Han X.Y.
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      • Xu Z.Y.
      • Tsai T.F.
      Serum and cerebrospinal fluid immunoglobulins M, A, and G in Japanese encephalitis.
      False negatives may occur prior to this time. In sera, approximately 75% of patients have anti-JEV IgM by 4 days after onset, but most patients will have IgM antibody 7–10 days after onset (Fig. 3).
      • Chanama S.
      • Sukprasert W.
      • Sa-ngasang A.
      • et al.
      Detection of Japanese encephalitis (JE) virus-specific IgM in cerebrospinal fluid and serum samples from JE patients.
      Anti-JEV IgM can persist for 30–90 days but longer durations have been documented.
      • Roehrig J.T.
      • Nash D.
      • Maldin B.
      • et al.
      Persistence of virus-reactive serum immunoglobulin M antibody in confirmed West Nile virus encephalitis cases.
      Detection of anti-JEV IgM may occasionally reflect a recent infection or vaccination rather than acute disease. Anti-JEV IgG rises more slowly, with about 80% of patients having detectable IgG in serum and/or CSF by 7 days after onset.
      • Burke D.S.
      • Nisalak A.
      • Ussery M.A.
      • Laorakpongse T.
      • Chantavibul S.
      Kinetics of IgM and IgG responses to Japanese encephalitis virus in human serum and cerebrospinal fluid.
      Anti-JEV IgG peaks in serum and CSF on day 30. By 6 months, anti-JEV IgG is no longer detectable in serum in 50% of cases, but anti-JEV IgG in CSF may persist. There may be a strong and persistent CSF-to-serum anti-JEV IgM and IgG gradient.
      Fig. 3
      Fig. 3Time course of expected viral load, IgM and IgG responses as correlated to clinical manifestations in severe Japanese encephalitis infection (adapted from World Health Organization).
      • World Health Organization
      WHO Manual for the laboratory diagnosis of Japanese encephalitis virus infection.
      • Burke D.S.
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      • Chantavibul S.
      Kinetics of IgM and IgG responses to Japanese encephalitis virus in human serum and cerebrospinal fluid.
      • Han X.Y.
      • Ren Q.W.
      • Xu Z.Y.
      • Tsai T.F.
      Serum and cerebrospinal fluid immunoglobulins M, A, and G in Japanese encephalitis.
      Historically, the haemagglutination inhibition assay was widely used to measure anti-JEV antibodies.
      • Clarke D.H.
      • Casals J.
      Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses.
      While easily performed with minimal training, practical limitations include low throughput, suboptimal sensitivity and specificity, and the necessity of paired sera, potentially delaying diagnosis for weeks. Complement fixation methods have similar issues.
      • Casals J.
      • Palacios R.
      Complement-fixation in encephalitis and rabies virus infections.
      In the 1980s, anti-JEV IgM and IgG capture enzyme linked immunosorbent assays (ELISAs) were developed and have since entered mainstream use for JEV diagnosis. A competitive ELISA technique can be used for seroprevalence testing, especially in animals, as it does not require specific anti-human antibodies.
      • Yang D.K.
      • Kim B.H.
      • Lim S.I.
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      Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine.
      ,
      • Dhanze H.
      • Bhilegaonkar K.N.
      • Rawat S.
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      Seasonal seroprevalence of Japanese encephalitis in swine using indirect IgG ELISA.
      The anti-JEV IgM antibody class capture assay (ACCA) is the methodology recommended by the WHO.
      • Burke D.S.
      • Nisalak A.
      • Ussery M.A.
      Antibody capture immunoassay detection of Japanese encephalitis virus immunoglobulin M and G antibodies in cerebrospinal fluid.
      ,
      • Bundo K.
      • Igarashi A.
      Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients.
      Several commercial assays are available based on ACCA, including JE Detect (InBios International, USA), JE-Dengue IgM combo (Panbio, Australia), and JEV CheX (Xcyton, India).
      • Biggerstaff B.J.
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      Evaluation of three commercially available Japanese encephalitis virus IgM enzyme-linked immunosorbent assays.
      • Cuzzubbo A.J.
      • Endy T.P.
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      Evaluation of a new commercially available immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Japanese encephalitis infections.
      • Featherstone D.
      • Russell B.J.
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      Evaluation of IgM antibody capture enzyme-linked immunosorbent assay kits for detection of IgM against Japanese encephalitis virus in cerebrospinal fluid samples.
      These commercial kits perform with relatively high specificity but low sensitivity. A nitrocellulose membrane-based IgM capture dot ELISA rapid diagnostic test has also been developed.
      • Solomon T.
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      • et al.
      Rapid diagnosis of Japanese encephalitis by using an immunoglobulin M dot enzyme immunoassay.
      Defined epitope blocking assays is another useful ELISA format, as they can be developed against specific targets that may differentiate between immunisation or infection.
      • Konishi E.
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      Detection by ELISA of antibodies to Japanese encephalitis virus nonstructural 1 protein induced in subclinically infected humans.
      ,
      • Konishi E.
      • Shoda M.
      • Ajiro N.
      • Kondo T.
      Development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to Japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses.
      Immunofluorescence (IF) techniques permit antibody class distinction and titres, and the advent of monoclonal antibodies has further increased the specificity of IF. Pre-fixed slides can be obtained, and a biosafety-level 3 (BSL-3) facility is not required to perform the assay. The anti-JEV IgG/IgM Indirect Immunofluorescence Test (IIFT) assay from Euroimmun (Germany) is based on immunofluorescence.
      • Litzba N.
      • Klade C.S.
      • Lederer S.
      • Niedrig M.
      Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination.
      Evaluation of commercial serological assays against reference methods is important before such assays are recommended.
      A wide range of in-house assays also exist, often utilising ELISA, IF and/or neutralisation. These in-house assays have largely been developed and utilised by reference laboratories and can provide reliable diagnostic performance once appropriately validated. The gold standard of serological diagnosis remains neutralisation, which can be utilised by reference laboratories as a confirmatory test. This is the most sensitive and specific serological test, but is labour-intensive and costly, with low throughput and long turnaround times.
      • Maeki T.
      • Tajima S.
      • Ikeda M.
      • et al.
      Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis.
      Furthermore, it requires stocks of viable virus, a BSL-3 facility, and JEV-vaccinated staff for handling of live JEV, although chimaeric viruses may represent safer diagnostic antigens without the need for high-level biocontainment.
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      • Harrison J.J.
      • Watterson D.
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      A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus.
      There are specific criteria that need to be satisfied to confirm JEV infection by serology, including seroconversion or a four-fold or greater rise between acute and convalescent sera in the absence of recent JEV vaccination or detection of anti-JEV IgM in CSF.
      • Singh K.P.
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      • Jain P.
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      Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area: co-infection or cross reactivity?.
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      Concurrent dengue virus and Japanese encephalitis virus infection of the brain: is it co-infection or co-detection?.
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      Cross-reactive IgM responses in patients with dengue or Japanese encephalitis.
      • Dubot-Pérès A.
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      • Chanthongthip A.
      • Newton P.N.
      • de Lamballerie X.
      How many patients with anti-JEV IgM in cerebrospinal fluid really have Japanese encephalitis?.
      • Suntayakorn S.
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      • Hoke C.H.
      • et al.
      An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate.
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      Flavivirus-induced antibody cross-reactivity.
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      • Park J.Y.
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      A diagnostic algorithm to serologically differentiate West Nile virus from Japanese encephalitis virus Infections and its validation in field surveillance of poultry and horses.
      Serological diagnosis of JEV may be complicated by significant cross-reactivity to many antigenically-related flaviviruses in Australia (MVEV, WNVKUN) and elsewhere (e.g., WNV, dengue). These co-circulating endemic flaviviruses have similar clinical presentations, and as such, serological testing to exclude those viruses is essential where JEV antibodies are detected.
      • Singh K.P.
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      • Jain P.
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      Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area: co-infection or cross reactivity?.
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      • Kumar N.
      • Jain A.
      Concurrent dengue virus and Japanese encephalitis virus infection of the brain: is it co-infection or co-detection?.
      • A-Nuegoonpipat A.
      • Panthuyosri N.
      • Anantapreecha S.
      • et al.
      Cross-reactive IgM responses in patients with dengue or Japanese encephalitis.
      • Dubot-Pérès A.
      • Sengvilaipaseuth O.
      • Chanthongthip A.
      • Newton P.N.
      • de Lamballerie X.
      How many patients with anti-JEV IgM in cerebrospinal fluid really have Japanese encephalitis?.
      • Suntayakorn S.
      • Nimmannitya S.
      • Hoke C.H.
      • et al.
      An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate.
      A positive serological result for JEV should not be confirmed until other flavivirus titres have been assessed, alongside consideration of patient-specific risk factors, exposures and clinical history. Anti-JEV IgM appears to be somewhat less cross-reactive than anti-JEV IgG. A multiplex microsphere immunoassay can assist with streamlining the diagnostic progress by conducting multiple serological assays in parallel.
      • Basile A.J.
      • Horiuchi K.
      • Panella A.J.
      • et al.
      Multiplex microsphere immunoassays for the detection of IgM and IgG to arboviral diseases.
      The availability of commercial ELISAs and IF assays allow non-reference laboratories to perform JEV testing without specialised expertise or on-site viral culture. However, due to pitfalls related to specificity, sensitivity and interpretation, an initial positive result should be referred for confirmatory testing at a reference laboratory where a wide range of potentially cross-reacting viruses can be excluded.

      Direct virus detection

      Nucleic acid amplification testing (NAAT)

      The use of NAAT is diagnostically appealing in facilitating a rapid and scalable testing platform with the capacity for multiplexing with other pathogens of significance. NAAT also provides specificity not afforded by cross-reacting serological approaches. However, since JEV viraemia is short-lived and often of low amplitude, the sensitivity and testing window and hence, clinical utility, of NAAT may be limited.
      • Bharucha T.
      • Sengvilaipaseuth O.
      • Vongsouvath M.
      • et al.
      Development of an improved RT-qPCR Assay for detection of Japanese encephalitis virus (JEV) RNA including a systematic review and comprehensive comparison with published methods.
      Nonetheless, a range of NAAT approaches have been applied to JEV diagnosis in human and porcine populations, including reverse transcription polymerase chain reaction (RT-PCR), utilising traditional TaqMan and molecular beacon-based approaches. Nested PCR formats provide enhanced sensitivity [0.1 plaque forming units (PFU)/mL] in porcine blood, however this is specific for genotype G1.
      • Jeong Y.E.
      • Jeon M.J.
      • Cho J.E.
      • et al.
      Development and field evaluation of a nested RT-PCR kit for detecting Japanese encephalitis virus in mosquitoes.
      In-house assays have used a range of targets encompassing conserved regions across JEV genotypes, most commonly M, NS1, NS2, NS3 or E genes, some of which can provide genotype-specific differentiation. Pan-flavivirus primers may also be used. Multiplexed NAAT assays, combining JEV targets with other flavivirus targets (with or without a broader panel of zoonotic pathogens) have been shown to be effective.
      • Shao N.
      • Li F.
      • Nie K.
      • et al.
      TaqMan real-time RT-PCR Assay for detecting and differentiating Japanese encephalitis virus.
      • Wang H.-Y.
      • Wu S.-Q.
      • Jiang L.
      • et al.
      Establishment and optimization of a liquid bead array for the simultaneous detection of ten insect-borne pathogens.
      • Barros S.C.
      • Ramos F.
      • Zé-Zé L.
      • et al.
      Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR.
      In low resource settings, cheaper loop-mediated isothermal amplification (LAMP) methodologies may be employed and have equivalent sensitivity to RT-PCR.
      • Chen Z.
      • Liao Y.
      • Ke X.
      • et al.
      Comparison of reverse transcription loop-mediated isothermal amplification, conventional PCR and real-time PCR assays for Japanese encephalitis virus.
      Whole blood, CSF and urine provide suitable specimen types for NAAT with viral persistence most marked in blood and urine (out to 28 days in some studies).
      • Huang G.K.L.
      • Tio S.Y.
      • Caly L.
      • et al.
      Prolonged detection of Japanese encephalitis virus in urine and whole blood in a returned short-term traveler.
      Viral levels in plasma and serum are lower than whole blood and hence these specimen types should generally be avoided. A pilot study demonstrated that throat swabs may also be a useful, less invasive sampling site.
      • Bharucha T.
      • Sengvilaipaseuth O.
      • Seephonelee M.
      • et al.
      Detection of Japanese encephalitis virus RNA in human throat samples in Laos – a pilot study.
      NAAT on brain tissue may be informative but data are thus far insufficient to validate the assay on this specimen type and sensitivity is unknown, particularly if brain tissue is obtained post-mortem. Following collection, it is imperative that brain samples are stored (–70 to –80oC) and transported appropriately to optimise diagnostic yield.

      Viral culture

      In specialised centres, viral culture in specific cell lines may be used for JEV diagnosis and characterisation. Viral culture may be attempted from CSF, whole blood, urine or brain tissue. Samples must be handled in BSL-3 facilities by laboratory workers vaccinated against JEV. Cell lines that have been used in the past for flavivirus culture include mosquito derived cell lines (such as C6/36) and Vero cells, although JEV has been successfully isolated in human hepatoma derived HuH-7, HuH-7.5, HuH-7.5.1, adapted human embryonic kidney 293 (HEK-293), human microglial and porcine cell lines.
      • Saito K.
      • Fukasawa M.
      • Shirasago Y.
      • et al.
      Comparative characterization of flavivirus production in two cell lines: human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero.
      ,
      • Lannes N.
      • Neuhaus V.
      • Scolari B.
      • et al.
      Interactions of human microglia cells with Japanese encephalitis virus.
      As with mosquito derived cell lines, cytopathic effect may not be present, so alternate methods such as RNA quantitation by NAAT or immunostaining are required to identify growth.
      • Broom A.K.
      • Hall R.A.
      • Johansen C.A.
      • et al.
      Identification of Australian arboviruses in inoculated cell cultures using monoclonal antibodies in ELISA.
      ,
      • O’Brien C.A.
      • Harrison J.J.
      • Colmant A.M.G.
      • et al.
      Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.
      Substantial rises in viral RNA levels may be detected as early as 9–12 h post-inoculation.

      Whole genome sequencing

      Additional genotypic information including phylogenetic relatedness and transmission networks (including between humans, pigs, birds, and mosquito populations) can be gleaned from whole genome sequencing (WGS) of samples that have tested positive by NAAT.
      • Pyke A.T.
      • Choong K.
      • Moore F.
      • et al.
      A case of Japanese encephalitis with a fatal outcome in an Australian who traveled from Bali in 2019.
      This approach has largely supplanted earlier single target PCR methodologies for genotyping due to the added value of phylogenetic analysis. The nucleotide diversity between JEV genotypes will require development of genotype specific whole genome amplification techniques, particularly for the current G4 cases. However phylogenetic cluster analysis of emerging human, pig and potentially mosquito strains will be critical to unravelling the transmission pathways for the emerging G4 cluster in Australia and the detection of other possible circulating JEV genotypes.

      Metagenomics

      In cases of unexplained encephalitis where targeted investigations have been non-revealing, metagenomics may have a key role.
      • Simon-Loriere E.
      • Faye O.
      • Prot M.
      • et al.
      Autochthonous Japanese encephalitis with yellow fever coinfection in Africa.
      ,
      • Mai N.T.H.
      • Phu N.H.
      • Nhu L.N.T.
      • et al.
      Central nervous system infection diagnosis by next-generation sequencing: a glimpse into the future?.
      The agnostic nature of this approach enables unexpected pathogens to be revealed including emerging viruses or divergent genotypes of known pathogens that evade directed assays.
      • Li X.
      • Li J.
      • Wu G.
      • Wang M.
      • Jing Z.
      Detection of Japanese encephalitis by metagenomic next-generation sequencing of cerebrospinal fluid: a case report and literature review.
      ,
      • Olausson J.
      • Brunet S.
      • Vracar D.
      • et al.
      Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics.
      Appropriate sample types include brain tissue or CSF (or blood or urine in particular circumstances) in the context of a suggestive clinical history. While expense and technical constraints dictate that metagenomics still largely functions as a research tool, future advances in the field including dedicated funding streams should see this approach adopted into mainstream diagnostics for cases of unexplained encephalitis.
      • Annand E.J.
      • Horsburgh B.A.
      • Xu K.
      • et al.
      Novel Hendra virus variant detected by sentinel surveillance of horses in Australia.

      Histopathology

      Histopathology can be performed on brain tissue samples, usually obtained post-mortem. Typical features of neuronal invasion include perivascular cuffing, inflammatory cell infiltration and phagocytosis of infected cells.
      • Miyake M.
      The pathology of Japanese encephalitis.
      The predominant T cell is CD4+, which can be found in the perivascular cuff and CSF. JEV antigen can be detected via cytoplasmic staining of neurons, and distribution is multifocal.
      • Johnson R.T.
      • Burke D.S.
      • Elwell M.
      • et al.
      Japanese encephalitis: immunocytochemical studies of viral antigen and inflammatory cells in fatal cases.
      • Iwasaki Y.
      • Zhao J.X.
      • Yamamoto T.
      • Konno H.
      Immunohistochemical demonstration of viral antigens in Japanese encephalitis.
      • Wong K.T.
      • Ng K.Y.
      • Ong K.C.
      • et al.
      Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA.
      In patients that die rapidly, there may be no histological signs of inflammation, but immunohistochemical studies can detect viral antigen in otherwise morphologically normal neurons. This may explain the normal CSF findings in a proportion of patients with Japanese encephalitis.

      Electron microscopy

      While not a mainstream diagnostic modality, electron microscopy has been used to structurally characterise JEV and other flaviviruses, as well as to elucidate the subcellular mitochondrial damage induced by the virus.
      • Zhang J.
      • Han W.
      • Xie C.
      • et al.
      Autophagy inhibitors alleviate Japanese encephalitis virus-induced cerebral inflammation in mice.
      This modality is available in specialised centres only and does not have a role in routine diagnostics due to expense and accessibility constraints.

      Quality assurance

      An external quality assurance program for arbovirus diagnostics was piloted by the WHO Regional Office for the Western Pacific in 2011. This has now grown into a global program. Currently, external quality assurance of JEV laboratory testing is provided through the Royal College of Pathologists of Australasia Quality Assurance Program.
      • Abdad M.Y.
      • Squires R.
      • Cognat S.
      • Oxenford C.J.
      • Konings F.
      External quality assessment for arbovirus diagnostics in the World Health Organization Western Pacific Region, 2013–2016: improving laboratory quality over the years.
      Available modules include serology and molecular programs for flavivirus, both of which have two surveys annually.
      • The Royal College of Pathologists of Australia Quality Assurance Program
      Specimen exchange with international laboratories, such as the Centers for Disease Control (USA), can also occur for less common flaviviruses.

      JEV surveillance

      Serosurveillance

      Sentinel chicken flocks can be used for flavivirus surveillance, although these are not undertaken in all Australian states.
      • Broom A.K.
      • Azuolas J.
      • Hueston L.
      • et al.
      Australian encephalitis: sentinel chicken surveillance programme.
      These programs are funded by State health departments and accompanied by contingency plans implemented if one or more chickens in a flock seroconverts. Sentinel chickens are typically young birds not infected with arboviruses previously and must test negative before being deployed into the sentinel program. The chickens are placed in outdoor cages in various locations, usually near inland waterways or major population centres, and are bled regularly for detection of arbovirus antibodies. Historically, the monitored arboviruses have been MVEV and WNVKUNV. JEV has not been monitored due to lack of endemicity in Australia, but retrospective analyses for JEV antibodies in stored serum may assist in unveiling recent trends.
      As part of the response to the Communicable Disease Incident of National Significance (CDINS) declaration, testing of piggery workers for JEV-specific antibodies before vaccination was performed to assess seroprevalence in this at-risk group. Two JEV vaccines are available in Australia: IMOJEV (Sanofi Pasteur) and JESPECT (Seqirus). IMOJEV is single-dose live attenuated, recombinant viral vaccine based on the 17D-204 yellow fever vaccine virus, but with replacement of the JEV premembrane (prM) and envelope (E) genes.
      • Appaiahgari M.B.
      • Vrati S.
      Clinical development of IMOJEV® - a recombinant Japanese encephalitis chimeric vaccine (JE-CV).
      As such, serological assays targeting different JEV epitopes might discriminate a vaccination response from natural infection. By contrast, JESPECT is an adsorbed two-dose, inactivated vaccine where serological testing may not adequately differentiate between vaccination or infection.
      • Kaltenböck A.
      • Dubischar-Kastner K.
      • Schuller E.
      • Datla M.
      • Klade C.S.
      • Kishore T.S.A.
      Immunogenicity and safety of IXIARO® (IC51) in a Phase II study in healthy Indian children between 1 and 3 years of age.
      However, JESPECT can be given to immunocompromised or pregnant/breastfeeding patients, while IMOJEV is contraindicated as a live attenuated vaccine. The utility of serology in determining vaccine responsiveness or a surrogate marker of protection also remains undefined.

      Mosquito monitoring

      State-based mosquito surveillance programs have been active for many years analysing mosquito ‘grinds’ or honey-baited preservation cards by NAAT for known circulating arboviruses.
      • Doggett S.
      • Haniotis J.
      • Clancy J.
      • et al.
      New South Wales arbovirus surveillance program annual report 2018-2019.
      • Knope K.E.
      • Doggett S.L.
      • Jansen C.C.
      • et al.
      Arboviral diseases and malaria in Australia, 2014–15: annual report of the national arbovirus and Malaria advisory committee.
      • Johnson B.J.
      • Kerlin T.
      • Hall-Mendelin S.
      • et al.
      Development and field evaluation of the sentinel mosquito arbovirus capture kit (SMACK).
      • Hall-Mendelin S.
      • Ritchie S.A.
      • Johansen C.A.
      • et al.
      Exploiting mosquito sugar feeding to detect mosquito-borne pathogens.
      Until this year, JEV was not included in targeted mosquito testing protocols due to its non-endemicity. This mosquito-derived data is compiled by the National Arbovirus and Malaria Advisory Committee (NAMAC) with reference to state-by-state case numbers of notifiable diseases including arboviruses and malaria. Retrospective analyses of mosquito grinds will inform historical and previously undetected JEV activity.

      Discussion

      Japanese encephalitis (JE) is a potentially debilitating disease with complex diagnostics. The short viraemia and difficulties associated with culture and metagenomics dictates that most diagnoses are made retrospectively by serological analyses. Serology interpretation is further confounded by significant cross-reactivity with other flaviviruses.
      The management of JE is largely supportive with no pathogen-specific therapies.
      • Turtle L.
      • Solomon T.
      Japanese encephalitis - the prospects for new treatments.
      As such, diagnostic confirmation is rarely clinically urgent, but may obviate further unnecessary testing and treatments. More common causes of encephalitis, such as those caused by human herpesvirus infections, which may have overlapping clinical syndromes but specific treatments, should be excluded initially. However, it is critical to define the extent of JEV outbreaks, inform epidemiology and transmission pathways (including the identification of novel vectors, natural reservoirs and amplifying hosts) and to plan appropriate public health responses. Additionally, ongoing surveillance efforts in sentinel animals and mosquitoes will provide early warnings of JEV circulation in new areas. Australian diagnostic networks in animal, human and environmental health are well positioned to present a unified, coordinated, One Health response to JEV outbreaks. The existing laboratory networks provide the optimal framework for development and validation of diagnostic assays to guide public health responses across the different health sectors at the state and national levels.
      Although global JEV incidence has been declining over the last 30 years, there is marked variability at country levels.
      • Erlanger T.E.
      • Weiss S.
      • Keiser J.
      • Utzinger J.
      • Wiedenmayer K.
      Past, present, and future of Japanese encephalitis.
      The identification of G4 as the current circulating Australian JEV genotype may indicate that this genotype is now more widespread globally than previously appreciated. Ongoing international collaborative efforts will be critical to inform these broader transmission patterns and their possible drivers. Moreover, much of the existing data on diagnostic tools for JEV, as well as vaccine efficacy, has been established in settings with predominant G1 and G3 genotypes.
      The emergence of JEV within Australia is unprecedented and highly concerning, particularly if endemicity is already established. Factors contributing to spillover of JEV into humans in Australia and its ongoing transmission include escalating population growth, intensified agricultural practices, expanding domestic and feral pig populations, and low levels of vaccination and disease-specific surveillance.
      • Erlanger T.E.
      • Weiss S.
      • Keiser J.
      • Utzinger J.
      • Wiedenmayer K.
      Past, present, and future of Japanese encephalitis.
      The impact of climate change in providing possible suitable ecological niches for expanding mosquito populations and altering bird migration patterns should not be underestimated as a potential trigger for this outbreak. Current models of climate change are suggesting that the current JEV affected areas will be drier in the future. This could limit mosquito breeding. However, the same models are suggesting that mosquito seasons may be extended by warmer climates. More data on the geographical and numerical extent of this JEV outbreak in animals, humans and the environment are required before future epidemiological patterns in Australia can be reliably predicted, but ongoing endemicity in both temperate and tropical climates with seasonal peaks during the summer months and rainy season, respectively, is postulated.
      Given ongoing climate change and the evolution of the animal-human interface, the emergence and spread of infectious diseases, including zoonoses, remain critical public health considerations. A One Health approach with strong alliances amongst key stakeholders in animal, human and environmental health are paramount in containing the current emergence of JEV as well as identifying and responding to future emerging pathogens.

      Conflicts of interest and sources of funding

      The authors state that there are no conflicts of interest to disclose. No special funding was received by the authors of this review.

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