Japanese Encephalitis

Updated September, 2010

Kaushik Bharati, Ph.D.

Virology

               Japanese encephalitis is the most important form of viral encephalitis that affects mostly children and young adolescents in the tropics of Asia. This disease is so named because it was originally detected in Japan in the 1870’s. Japanese encephalitis was originally called “Japanese type B encephalitis” to differentiate it from the type A encephalitis or von Economo’s encephalitis or encephalitis lethargica, which occurred during winter months and had a different clinical presentation. The designation “type B” was later dropped. Though outbreaks of encephalitis attributed to Japanese encephalitis virus were reported in Japan as early as 1871, it was not until 1924 that Japanese encephalitis virus was isolated from a clinical case in the first recorded epidemic of Japanese encephalitis from Japan. The celebrated “Nakayama strain” was isolated in 1935 from the brain of a dying Japanese encephalitis patient. Moreover, the mode of transmission, by Culicine mosquitoes, was not elucidated till 1950 (65) (Table 1).

               The disease is endemic in most parts of Southeast Asia, China, India and Oceania. The disease is continuously expanding its geographical territory. Over the last decade or so, it has spread to hitherto unaffected regions, such as the western parts of India (75, 15), Karachi, Pakistan (36), western provinces of Papua New Guinea (38) and the Torres Strait islands of northern Australia (26, 27, 56). There is a realistic possibility that Japanese encephalitis could spread further (57). The geographical distribution of Japanese encephalitis is presented in Figure 1.

Japanese Encephalitis Virus

               Japanese encephalitis virus, responsible for causing Japanese encephalitis is an arthropod-borne virus (arbovirus) belonging to the family Flaviviridae and genus Flavivirus. The prototype virus belonging to this genus is the yellow fever virus (Latin flavus: yellow). The other closely related viruses that belong to this genus and cause human disease include dengue virus, St. Louis encephalitis virus , Murray Valley encephalitis virus, West Nile virus and tick-borne encephalitis virus. Other lesser known flaviviruses include Omsk hemorrhagic fever virus, Kunjin virus, Kyasanur Forest disease virus, Alfuy, Cacipacore, Yaounde, Koutango and Ustusu viruses. Hepatitis C virus (HCV), which belongs to the genus Hepacivirus, is also grouped under the family Flaviviridae (99) (Table 2). Japanese encephalitis virus is spread by the bite of infected Culicine mosquitoes, predominantly Culex tritaeniorhynchus. The major amplifying vertebrate hosts are domestic pigs and ardeid wading birds, such as herons and egrets. Humans become infected coincidentally when they encroach this enzootic cycle between mosquitoes, birds and pigs. Viral titers are so low in humans that further transmission does not occur. For this reason, humans are regarded as dead-end hosts. Other vertebrate animals that are naturally infected with Japanese encephalitis virus include donkeys, chicken, ducks, water buffalos, cattle, sheep, mice, snakes and frogs, although their role in further transmission of Japanese encephalitis virus is not firmly established. However, bats could play an important role in overwintering (93). Moreover, the demonstration of transplacental transmission in bats could be an effective mechanism for viral persistence (94). It has been suggested that bats, migratory birds as well as wind-blown mosquitoes that have been infected with Japanese encephalitis virus, may be responsible for introducing the virus to hitherto unaffected geographical regions (104). The life-cycle of Japanese encephalitis in nature is depicted in Figure 2.

Japanese Encephalitis Virus Molecular Structure

               Japanese encephalitis virus is spherical and is approximately 50 nm in diameter. Its nucleocapsid core is surrounded by an envelope. Its single-stranded, positive sense RNA genome contains a single long open reading frame  flanked by 5’- and 3’-untranslated regions, which have secondary structures that are essential for the initiation of translation and for replication. The 5’ end of the genome has a type 1 cap, while the 3’ end lacks a poly-A tail. The viral genome is approximately 11,000 base pairs long, and codes for a single polyprotein of approximately 3400 amino acids that is cleaved into 3 structural and 7 non-structural proteins. The structural proteins are capsid (C), envelope (E), and membrane (M). The non-structural proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (7).

The Structural Proteins: The structural component of the virion nucleocapsid is formed by the C protein. The M protein is formed by furin-mediated cleavage and removal of the N-terminal segment from its precursor, the pre-membrane protein (prM). The E protein is a typical membrane glycoprotein, consisting of a C-terminal membrane-spanning domain and forms the outer structural protein component of the virus. It is the major virion antigen responsible for a number of important processes that include virion assembly, receptor binding, and membrane fusion. The E protein is the most important viral protein from a vaccine development standpoint, as it possesses most of the neutralizing epitopes that are targets for neutralizing antibodies, the primary mediators of immunity (23, 30, 41).

The Non-Structural Proteins: The NS1 protein is also a glycosylated protein that is believed to be involved in the assembly and release of virions (52). This protein is found on the cell surface and in the culture medium of infected cells (6). During the course of infection, this protein evokes a strong antibody response that protects the host against challenge with flavivirus, presumably through a complement-mediated pathway (83) or by antibody-dependent cell cytotoxicity (ADCC) (84). NS2A and NS2B are low molecular-weight proteins that are thought to be involved in the processing of other viral proteins (71). NS3 protein is conserved among flaviviruses and has protease and nucleotide triphosphatase/ helicase activities (2). NS4A and NS4B are small proteins whose functions are not clear, although they may be involved in the membrane localization of NS3 and NS5 through protein-protein interactions (7), or in the formation of the genomic RNA replication complex (106). NS5 protein is the largest and most conserved protein and is the viral RNA-dependent RNA-polymerase (RdRp) (59). The genome organization of Japanese encephalitis virus is depicted in Figure 3.

Japanese Encephalitis Virus Genotypes

               Four distinct genetic subtypes or genotypes of Japanese encephalitis virus have been identified on the basis of nucleotide sequence data of C/PrM and E genes and phylogenetic analysis (10, 11). Genotype I includes isolates from Northern Thailand, Cambodia and Korea. Genotype II includes isolates from Southern Thailand, Malaysia, Indonesia and Northern Australia. Genotype III includes isolates from mostly temperate regions of Asia, including Japan, China, Taiwan, the Philippines and the Asian subcontinent. Genotype IV includes some isolates from Indonesia. In addition, based on phylogenetic evidence, the Muar strain of Japanese encephalitis virus isolated in Singapore in 1952 from a patient who originated in Muar (Malaysia), may represent a fifth genotype (103). Since Genotypes I and III are found predominantly in the northern epidemic-prone areas of Asia, and Genotypes II and IV circulate in the southern endemic areas, it was initially suggested that the genotype distribution could explain the differences in the clinico-epidemiological pattern of disease. However, after all the five genotypes had been fully sequenced, it was evident that the ancestral virus evolved and spread from a central location in Malaysia-Indonesia region (where all the five genotypes are present) to newer geographic locations (91). However, why the first Japanese encephalitis epidemics appeared in Japan (where only genotypes 1 and 3 are present) and not in the Malaysia-Indonesia region (where all five genotypes are present) remains a mystery; though a possible explanation could be that epidemics might have occurred in Malaysia-Indonesia during ancient times, before the advent of recorded history. All the genotypes differ from each other by about 10-20% at the nucleotide level and 2-6% at the amino acid level, and belong to the same serotype and are similar in terms of virulence and host preference. As a consequence, any Japanese encephalitis virus strain used for the development of a vaccine antigen may be expected to confer protection against all other genotypes. Current Japanese encephalitis virus vaccines are based on genotype III strains and it has been demonstrated that these vaccines confer protection against heterologous Japanese encephalitis virus strains, although the neutralizing antibody titers in these instances are lower than those against homologous strains (50).

Japanese Encephalitis Virus Transmission Cycle

               Japanese encephalitis virus transmission revolves around mosquitoes, pigs and ardeid wading birds. This basic cycle had been elucidated way back in the mid sixties in a comprehensive study carried out on Honshu Island, Japan (43). Usually after two 4-day amplification cycles in pigs, approximately 20% of the pigs become seroconverted. Mosquitoes become infected by feeding on the viremic pigs. After this, the virus undergoes a 7-14 day extrinsic incubation period in mosquitoes, where it multiplies in various organs and body compartments. This extrinsic incubation period is temperature dependent. An optimum temperature is required for virus replication and low temperatures can severely reduce transmission rates. After the extrinsic incubation period, mosquitoes infect other pigs, as a result of which almost 100% of the swine population becomes seroconverted. Blood meal from these highly viremic pigs is followed by another round of extrinsic incubation period, following which, the virus spills over to the human population and clinical cases start to appear (Table 3).

Epidemiology

               Japanese encephalitis virus is distributed throughout the temperate and tropical regions of southern and eastern Asia. Two major epidemiological patterns of disease are observed. These can be classified as either sporadic cases occurring in endemic areas or epidemics that are confined to specific geographical areas. In the northern temperate areas, such as northern Vietnam, northern Thailand, Korea, Japan, Taiwan, China, Nepal, and northern India, Japanese encephalitis occurs in the form of epidemics during the summer/monsoon months. The southern tropical areas, such as southern Vietnam, southern Thailand, Indonesia, Malaysia, Philippines, Sri Lanka and southern India are endemic for Japanese encephalitis and cases occur sporadically throughout the year with a peak after the onset of the monsoon season. In regions where the tropical and temperate climes intermingle, the disease pattern also becomes superimposed. The annual disease burden has been estimated to be around 175,000 cases (102), though this may be a gross underestimate when considering the fact that there is significant underreporting of Japanese encephalitis cases; for example, only around 50,000 cases are reported annually. This underreporting stems from the fact that the South-East Asia region is lacking efficient surveillance and monitoring systems, coupled with a weak health infrastructure (Table 4). This is predominantly a disease of children and serological tests have shown that when the children reach adulthood, all have been exposed to the virus and therefore exhibit neutralizing (Nt) antibody titers. However, non-immune adults coming from non-endemic regions of the world and staying in endemic areas for more than a month are likely to get infected. Another instance when adults become infected is when the virus spreads to new geographical locations, as was the case in Nepal (110) and Australia (27). Immunocompromised adults are also susceptible to Japanese encephalitis virus infection, as are the elderly, possibly due to waning immunity. Japanese encephalitis is essentially a disease of rural areas, although populations residing in peri-urban areas are now also being afflicted. This evidently stems from the fact that urban constructions are slowly encroaching on agricultural areas where there is a preponderance of mosquito breeding sites such as rice fields, as a result of which the burgeoning human population is being affected. Moreover, climate change will definitely have a drastic effect on the epidemiology of Japanese encephalitis in the coming decades, as it is likely to alter the mosquito breeding patterns. Most Japanese encephalitis virus infections are asymptomatic (105), the ratio of symptomatic to asymptomatic ranging anywhere between 1:25 to 1:1000, in Americans posted for a short period in Korea (25) and Chinese children (34) respectively. Of the 50,000 cases of Japanese encephalitis that are reported annually, 10,000 prove to be fatal. Those who survive the disease are very often left with neurological and psychiatric problems, which may include frank motor deficits, mental retardation, convulsions and behavioral problems (47). Japanese encephalitis, therefore, is a serious public health problem, even though its severity is often understated in the literature.

Erlanger TE, et al. Past, Present and Future of Japanese Encephalitis.  Emerging Infectious Diseases 2009;15(1):1-7.

Clinical Manifestations Pertinent to Treatment

               As has been highlighted above, Japanese encephalitis infections are most often asymptomatic or there might be a mild non-specific febrile illness. According to the World Health Organization (WHO), approximately 1 in 300 cases of Japanese encephalitis result in symptomatic disease. This, by and large depends on 4 major factors; (1) route of entry, (2) titer of virus, (3) neurovirulence of virus and (4) host factors, such as age, health, genetic make-up, and pre-existing immunity. The first signs of infection appear after an incubation period of 1-6 days, but may take as long as 15 days to manifest.

Disease Onset and Progression

               The onset of the disease may be acute or gradual. The disease may be crudely divided into three stages: (i) a prodromal stage, before central nervous system (CNS) involvement occurs, (ii) CNS stage, where the virus infects the CNS and (iii) a convalescent stage, where either marked improvement occurs or the CNS symptoms may persist. The prodromal stage is marked by fever above 38˚C, chills, myalgia, malaise, headaches accompanied by nausea, vomiting and abdominal pains similar to those found in an acute abdominal syndrome. Besides these common symptoms, gastric hemorrhage, thrombocytopenia and liver dysfunction have also been observed in the 2005 Indian Japanese encephalitis epidemic (49). These non-specific signs may continue for 2-4 days or the patient’s condition may deteriorate rapidly. CNS involvement (day 3-5) is marked by a progressive decline in alertness, often leading to coma. CNS infection can result in encephalitis, meningitis or myelitis, or a combination of all the three, in the form of meningoencephalomyelitis.

Movement Disorders

               A Parkinson’s like syndrome, characterized by dull mask-like facies, tremors and cogwheel rigidity have been observed in Japanese encephalitis cases (61, 62). Convulsions may be experienced in up to 87% of patients. The meningeal syndrome predominates with painful neck stiffness. Motor paralysis including hemiplegia and tetraplegia may also be present. A poliomyelitis-like acute flaccid paralysis has also been observed in Vietnamese children (90). Other movement disorders include opisthotonus (Figure 4), dystonia (Figure 5), oro-facial dyskinesias (e.g. lip-smacking), hemiparesis (Figure 6), choreoathetosis and gaze palsy (Figure 7). Abulia is another striking feature observed in Japanese encephalitis patients.

Clinical Correlates

               The clinician should look out for early clinical signs of CNS involvement, such as abnormal oculocephalic reflexes, acute onset hemiparesis with hypertonia and decorticate and decerebrate posturing, which help in the early clinical identification of intracranial hypertension. Effective management of intracranial hypertension will dictate whether the patient survives or dies.

Neuroimaging

               Neuroimaging techniques such as computed tomography (CT) scan and magnetic resonance imaging (MRI) scan carried out in Japanese encephalitis patients have revealed extensive bilateral thalamic lesions (Figure 8). In a Japanese encephalitis endemic region, bilateral lesions of the thalamus are indicative of Japanese encephalitis. It has been suggested that the movement disorders are the clinical correlates of damage to the thalamus and other parts of the brain (63). Lesions to sites such as the former, as well as that to the lentiform nucleus and basal ganglia could result in Parkinsonism. Other complications such as abulia could be due to thalamic lesions disrupting the prefrontal cortex-caudate-pallido-thalamocortical circuit. Dystonia results from putaminal and possibly thalamic lesions (44). MRI can effectively differentiate between Japanese encephalitis and Herpes simplex encephalitis. The former is associated with thalamic, basal ganglia, and brainstem involvement, whereas the latter is associated with medial temporal and baso-frontal cortical involvement. Another powerful technology is the single-photon emission computed tomography (SPECT). SPECT may be useful as a diagnostic tool in the early stages of Japanese encephalitis (40). Studies have been carried out to investigate the prognostic value of high-end techniques such as MRI and SPECT in relation to Japanese encephalitis (Figure 9). However, findings indicate that these techniques do not correlate well with 6-month outcome in Japanese encephalitis patients (64).

Outcome

               Fatality is seen in 20-30% of the cases (92), with signs of acute cerebral edema or severe respiratory distress from pulmonary edema. Children who survive the disease usually regain neurological function over the following weeks to months. However, almost half of the survivors are left with serious neuropsychiatric sequelae. These include a persistently altered sensorium, epileptic seizures and severe mental retardation. These neurological complications are very often understated in the medical literature. One should understand that the patients with neurological deficits are very often in their early childhood and hence, have to bear with these complications for the rest of their lives, given the fact that most of them cannot afford treatment. Moreover, even those who have a good recovery, often have minor sequelae like learning and behavioral problems, giving rise to stigmatization. Hence, the human face of the disease burden is enormously more than what the mere statistics suggest.

Ooi MH, Lewthwaite P, et al. The Epidemiology, Clinical Features, and Long-Term Prognosis of Japanese Encephalitis in Central Sarawak, Malaysia, 1997-2005. Clin Infect Dis. 2008 Aug 15;47:458-68.

Laboratory Diagnosis

Differential Diagnosis

               The diagnosis of Japanese encephalitis viral infection should be made within an epidemiological context. Because of clinical, biological and epidemiological similarities, three other viral diseases should be considered in the differential diagnosis. These are (1) Herpes simplex virus (HSV) encephalitis, (2) Dengue and (3) West Nile encephalitis. Moreover, other CNS infections should also be kept in the differential diagnosis. For example, bacterial and fungal meningitis, tuberculosis, cerebral malaria, leptospirosis, tetanus and typhoid encephalopathy. Moreover, enteroviruses, paramyxoviruses, rabies virus, Chikungunya virus and Nipah virus should also be kept in mind. Some non-infectious diseases that exhibit CNS manifestations include tumors, cerebrovascular accidents, Reye’s syndrome, toxic and alcoholic encephalopathies, and epilepsy. Hence, it is of the utmost importance to spend some time in taking down the case history. However, in case of epidemics, occurring in Japanese encephalitis endemic areas, it is often easier to rule out the other causes from the differential diagnosis.

Physical Examination

               A quick yet thorough physical examination is crucial. The level of consciousness should be established with a quantitative scale such as the Glasgow Coma Scale (GCS) and any seizures arising out of the infection should be immediately treated. Any other mental and behavioural abnormalities should be documented. The examination should search for any other causes of the altered level of consciousness (89).

Lumbar Puncture

               Although there is some controversy with regard to carrying out a lumbar puncture (42), if the condition of the patient permits, a lumbar puncture should be performed. This is because the initial CSF findings, such as opening pressure, cell count, glucose and protein levels are crucial for initially establishing a CNS infection. The CSF findings will also show whether the infection is viral or bacterial in origin. Moreover, downstream laboratory testing, by techniques such as MAC ELISA or PCR will pin-point the incriminating organism, and thus provide a guide for treatment and management. JE should be suspected if biochemical investigations reveal the following: (1) high CSF opening pressure (>250 mm), (2) moderate CSF pleocytosis (10-100 cells/mm3), (3) mildly increased CSF protein (50-200 mg/dL), but (4) normal levels of CSF glucose.

Confirmation of the Diagnosis

               Confirmation of a suspected case of Japanese encephalitis requires laboratory diagnosis. The etiological diagnosis of Japanese encephalitis  is based on virus isolation or demonstration of virus specific antigen or antibody in the CSF or serum (Box 1). The humoral immune response to Japanese encephalitis virus infection involves early production of IgM antibodies in both serum and CSF, followed by IgG production (4). Hence, the IgM-capture ELISA (MAC ELISA), which detects specific IgM in the CSF or in the serum of almost all patients within 7 days of onset of disease has become the practical standard for the diagnosis of Japanese encephalitis (13). Three MAC ELISA kits are being manufactured commercially. These are (1) The JE-Dengue IgM Combo ELISA Test (Panbio Limited), (2) The JE IgM ELISA (InBios International, Inc.), and (3) The JEV CheX kit (XCyton Diagnostics Ltd.). All three tests use a cell culture-derived recombinant particulate Japanese encephalitis antigen; the Panbio test also uses recombinant dengue 1-4 antigens. These three kits have been compared head-to-head in a rigorous study conducted by PATH (37). In this study, a panel of well-characterized sera was used to assess the performance of the kits. Sera, as opposed to CSF, were used in the study due to the fact that more often sera are available for analysis in clinical settings, and because cross-reactivity is more of a concern in case of sera samples rather than CSF samples. Since the problem of cross-reactivity while testing for antibodies to flaviviruses is well-recognized (102), the Panbio kit has the advantage when used in settings where dengue co-circulates. The Panbio kit was the only kit, out of the three tested, with at least 90% agreement to the USAMC-AFRIMS (United States Army Medical Component – Armed Forces Research Institute of Medical Sciences) standard (the internationally accepted standard diagnostic test for Japanese encephalitis) when both Japanese encephalitis and dengue IgM-positive samples were considered in the analysis. In this situation, sensitivity was good for the InBios and XCyton kits, but lower for the Panbio kit. However, specificity was low for both the InBios and XCyton kits as a result of cross-reactivity with dengue antibodies. When dengue cross-reactivity was eliminated, all the three kits had specificities of 96% or above. If pricing is competitive, then the Panbio kit would have a distinct advantage over the other two. A viral genome amplification RT-PCR technique (98) allows rapid detection of viral RNA in the CSF of Japanese encephalitis patients. Another molecular technique, the reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is a rapid real-time detection system for Japanese encephalitis virus, and the results can be obtained within 30 minutes under isothermal conditions at 63°C (73). This test could become useful in low resource settings as it does not require a thermocycler. However, these molecular tests are not currently used for routine diagnostic purposes.

Pathogenesis

               Entry into the CNS As has been highlighted above, Japanese encephalitis virus transmission occurs by the bite of infected Culicine mosquitoes. Following the mosquito bite, the virus initially replicates locally, after which it spreads to the blood stream, causing a transient viremic phase. The virus also multiplies in the regional lymph nodes. Studies indicate that in normal circumstances, the virus enters the CNS by passage across the cerebrovascular endothelium, rather than across the olfactory membrane, where the blood-brain barrier is scanty or lacking (66). Moreover, a recent study indicates that the permeability of the blood-brain barrier is differentially altered in response to Japanese encephalitis virus infection, leading to entry of the virus particles into the cerebrum, as the initial site of virus entry into the CNS (54). Co-factors that can disrupt the blood-brain barrier in Japanese encephalitis virus infections include co-infection by the parasite Taenia solium. In fact, this particular parasitic infection appears to be very common in fatal Japanese encephalitis virus cases (14). The author feels, from personal experience, that the olfactory route of transmission is important in case of laboratory workers, especially during administration of injections into mice with the live virus, where aerosols can easily enter the nasal cavity if appropriate measures (masks) are not taken. Japanese encephalitis virus has also been reported to infect the developing fetus transplacentally and cause abortions (9). Mechanism of neuronal damage Within the CNS, Japanese encephalitis virus replicates in the neurons, more specifically within their secretory system, involving the rough endoplasmic reticulum and Golgi apparatus, eventually leading to their destruction as the virus matures and the infection spreads to other neighboring neurons (29). Although there is no direct evidence from humans, neuronal apoptosis has been demonstrated in flavivirus disease models, both in vitro, as well as in vivo. It has been demonstrated that activation of microglial cells lead to production of proinflammatory cytokines that may elicit neuronal death (21). It has been suggested that an elevation in proinflammatory cytokine levels in the cerebro spinal fluid (CSF) indicates a poor prognosis (69). Other mechanisms of neuronal damage that have been suggested include astrocyte activation (20) and nitric oxide-mediated damage. The latter has received particular attention due to the fact the Japanese encephalitis virus has been found to induce the expression of inducible nitric oxide synthase (iNOS), the major enzyme in nitric oxide synthesis, thought to be a key component of the host innate immune response (81). Elucidation of the mechanism of neuronal damage may lead to identification of targets for drug intervention.

SUSCEPTIBILITY IN VITRO AND IN VIVO

               Although there are currently no antiviral agents against Japanese encephalitis virus for use in the clinic, there are quite a few approaches under laboratory development against this virus. Investigators have demonstrated that Japanese encephalitis virus, as well as other flaviviruses that share a similar genome organization, are susceptible to a whole spectrum of agents. These may be broadly classified into chemical compounds, natural products, siRNAs and DNAzymes.

Chemical Compounds

               Quite a number of chemical compounds have been evaluated in vitro as well as in vivo to assess their anti-Japanese encephalitis virus activity. The chemical compound furanonaphthoquinone and its derivatives such as 2-methylnaphtho [2,3-b]furan-4,9-dione (FNQ3) have been observed to possess antiviral effects against Japanese encephalitis virus (97). FNQ3 was found to inhibit viral replication in vitro by inhibiting the expression of the Japanese encephalitis virus E and NS3 proteins. Diethyldithiocarbamate (DDTC), a low molecular weight dithiol has been found to prolong the average survival time of mice infected with a lethal dose of Japanese encephalitis virus and delay the progression of disease (82). Some ring-expanded (“fat”) nucleoside and nucleotide analogues (RENs) have been biochemically screened for their antiviral activity against the NTPases/helicases of a number of flaviviruses, including Japanese encephalitis virus. In vitro studies indicate that the most sensitive virus that is susceptible to these RENs is the WNV, with HCV being the least sensitive, and Japanese encephalitis virus being intermediate (109). Dehydroepiandrosterone (DHEA), a precursor of both estrogenic and androgenic steroids, has been recently found to suppress Japanese encephalitis virus replication and virus-induced apoptosis in murine neuroblastoma (N18) cells (8). DHEA suppressed Japanese encephalitis virus replication and reduced Japanese encephalitis virus-induced cytotoxicity in vitro, through a non-genomic steroid-hormone mechanism, involving activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. A recent report has indicated that heparan sulfate mimetics, such as suramin, pentosan polysulfate (PPS) and PI-88 have antiviral effects in vitro against a number of flaviviruses, including DEN, WNV, MVE, and Japanese encephalitis virus (51). However, in vivo mouse model studies indicated that only PI-88 demonstrated a significant beneficial effect in disease outcome. An anilidoquinoline derivative, 2-(2-methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide, has been evaluated in vitro and in vivo for anti-Japanese encephalitis virus activity. In vitro studies of the compound showed antiviral and antiapoptotic activities, while in vivo studies showed a prolongation of survival time in Japanese encephalitis virus-infected mice (22). Minocycline, a semisynthetic second-generation tetracycline antibiotic has been recently evaluated in vitro as well as in vivo, in a mouse model of Japanese encephalitis (60). Preliminary findings indicate that minocycline could be pursued further as an anti-Japanese encephalitis virus agent. A recent study evaluated the anti-Japanese encephalitis virus action of pentoxifylline in vitro and in vivo (85). Pentoxifylline is a known inhibitor of TNF-α, the elevation of which in Japanese encephalitis patients has been correlated with a poor outcome (78). Pentoxifylline was found to inhibit Japanese encephalitis virus replication in vitro, probably by interfering with virion assembly and release. The drug, at concentrations of 100mg/kg and 200mg/kg body weight, was able to completely protect mice against a lethal challenge with 50 median lethal doses (50 LD50) of Japanese encephalitis virus. It was observed that all the mice that survived the infection failed to demonstrate replicating virus. These findings are quite promising and warrant further studies in larger animal models of Japanese encephalitis.

Natural Products

               A few natural products have been evaluated to test their anti-Japanese encephalitis virus activity in vitro and in vivo. Rosmarinic acid (RA), a phenolic compound found in Labiatae herbs has recently been evaluated in a mouse model of Japanese encephalitis (95). RA significantly increased the survival of Japanese encephalitis virus-infected mice, which was correlated with decreased viral load and proinflammatory cytokine levels in these animals. Moreover, in vitro studies using a mouse microglial cell line BV-2 infected with Japanese encephalitis virus, confirmed that RA decreases the level of proinflammatory cytokines such as IL-12, TNF-α, IFN-γ, MCP-1 and IL-6. These findings warrant further studies with RA in Japanese encephalitis. Arctigenin (AR), a naturally occurring plant lignan, chemically known as phenylpropanoid dibenzylbutyrolactone, has recently been evaluated in vitro and in vivo. AR decreased viral load and viral replication within the brain of BALB/c mice infected with Japanese encephalitis virus. Other indicators of viral pathogenesis were also decreased by AR treatment (96). More recently, Curcumin, a naturally occurring phenolic compound extracted from Curcuma longa, has been reported to possess anti-Japanese encephalitis virus activity when tested in vitro in Neuro2a cell line (17).

siRNAs

               The Japanese encephalitis field has not lagged far behind in employing the powerful technology of RNA interference to find a therapeutic solution to this viral disease. In spite of the fact that only a handful of studies have been published till date, the results are quite promising with respect to the applicability of this strategy in inhibition of Japanese encephalitis virus replication both in vitro as well as in vivo. In the majority of these studies, the siRNA constructs have targeted the non-structural genes of Japanese encephalitis virus. This technology has been used to target NS1 (55), NS3 (68) and NS5 genes (77), with varying degrees of inhibition. Moreover, it has recently been demonstrated that a single siRNA that targets a highly conserved region of the viral E gene, can protect mice against both Japanese encephalitis virus- and WNV-induced encephalitis (45). This has far-reaching implications in the clinical setting where infections can occur due to more than one flavivirus, especially in regions where the geographical domains of these viruses overlap. The delivery of siRNAs, particularly to anatomical regions such as the brain, which is protected by the blood-brain barrier, has been is tremendous problem. Traditionally these agents have been delivered by intracerebral injection. But in the real-life situation, this is not possible. This problem has recently been solved in an ingenious way by employing a short peptide derived from rabies virus glycoprotein (RVG) to which nine arginine residues were fused at the carboxyl terminus (RVG-9R) in order to bind the siRNA. The RVG-9R efficiently delivered siRNAs to neuronal cells in vitro, resulting in efficient gene silencing. Moreover, intravenous administration of RVG-9R-bound antiviral siRNA efficiently penetrated the BBB and afforded robust protection against fatal viral encephalitis in mice (46).

DNAzymes

               DNAzymes (Dzs) are single-stranded oligodeoxynucleotides (ODNs) with Mg++-dependent enzymatic activity capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions (80). In a recent study, Dz-mediated inhibition of Japanese encephalitis virus has been described (1). Nuclease-resistant Dzs containing phosphorothioate linkages were efficiently taken up by mouse neuronal and glial cells, and addition of a continuous stretch of 10 guanosine residues (poly-(G)10) to the 3’-end of a Dz led to its enhanced delivery to cells containing scavenger receptors (ScRs). These Dzs inhibited Japanese encephalitis virus replication in cultured mouse cells of neuronal (Neuro2a) and macrophage (J774E) origin. Intracerebral administration of a poly-(G)10-tethered, phosphorothioated Dz in Japanese encephalitis virus-infected mice led to >99.99% inhibition of virus replication in the mouse brain, leading to extended life-span or complete recovery in a dose-dependent manner. This is the first report of the use of Dzs for inhibition of virus replication in vivo in mouse model and warrants further studies to explore and expand upon the therapeutic possibilities of this novel technology.

ANTIVIRAL THERAPY

               There is currently no antiviral therapy for Japanese encephalitis. Ribavirin, an antiviral agent, has been evaluated for therapeutic applicability in Japanese encephalitis patients. A randomized, controlled trial of oral ribavirin was carried out in children in the state of Uttar Pradesh (India), where Japanese encephalitis epidemics are highly prevalent, but this antiviral agent did not have any effect in reducing early mortality associated with Japanese encephalitis (48). IFN-α has been evaluated for its therapeutic efficacy in JE patients. A study in two patients in Thailand indicated that recombinant IFN-α could be an effective and promising agent for the treatment of Japanese encephalitis, following complete recovery of these patients (28). However, a randomized, double-blind, placebo-controlled trial of IFN-α2a did not improve the outcome of Japanese encephalitis patients (88). A recent clinical case study in an Italian Japanese encephalitis patient, returning from a 3-week trip to rural Vietnam, has indicated that intravenous immunoglobulin administration could be beneficial, following complete recovery of the patient (5). However, further in-depth clinical evaluation of this therapy is required before anything concrete can be said about this treatment approach.

Targeted Flavivirus Drug Discovery Efforts

               The members of the flavivirus family all follow a similar replication strategy. So, there is a high possibility that viral inhibitors can be discovered that have a broad-spectrum of antiviral activity. The targeted approach towards drug discovery involves, first identifying the targets, and then designing molecules that could act as inhibitors of these selected molecular targets.

Polymerase Inhibitors: The polymerases are the most promising targets for development of antiviral agents (101). The RdRp is a multimeric complex and is essential for viral replication. Moreover, since this molecular complex is encoded by the viral genome, and has no cellular counterpart, there is no chance of cellular toxicity (58).

Protease Inhibitors: Another attractive target for drug development is the viral protease. Viral protease inhibitors are already being used in clinical practice. The HIV-protease inhibitors are examples of this class of drugs that are components of the highly active anti-retroviral therapy (HAART). The flavivirus proteases could also be targeted in a similar fashion. However, it needs to be investigated whether these viral serine proteases are sufficiently different from the cellular serine proteases, so that toxicity issues do not arise (33).

Virus Entry Inhibitors: Virus entry inhibitors are ideal antiviral agents. These agents are in many ways superior to the polymerase or protease inhibitors as issues of toxicity and emergence of resistant strains does not occur in this class of antiviral agents. In fact, virus entry inhibitors have been approved for use against HIV-1 (79).

Capping Inhibitors: The flaviviruses, like other RNA viruses, replicate in the cytoplasm, and have evolved their own capping enzymes that are independent of the host. Since 5’ capping is an essential component of viral replication, the methyltransferase enzyme, which is an essential component of the capping machinery, is an attractive target for antiviral drug development (16).

Helicase Inhibitors: The NS3 is the helicase of flaviviruses. It is an essential component for viral replication. It is involved in unwinding the double-stranded RNA intermediate during genome replication and acts in a multimeric complex with NS5. This enzyme is also an attractive target for antiviral drug development (18, 53).

ADJUNCTIVE THERAPY

               Adjunctive therapy essentially involves treatment measures directed at controlling both the immediate complications of infection, including seizures and raised intracranial pressure, and the longer-term consequences of neurologic impairment, such as limb contractures and bed sores (87) (Figure 10). Corticosteroids have been investigated in the treatment of Japanese encephalitis. Although methylprednisolone showed some beneficial effects when used in non-controlled pulse therapy (70), high-dose dexamethasone therapy failed to show any beneficial effects in acute Japanese encephalitis cases (32). Hence, treatment is essentially symptomatic and supportive. The major adjunctive therapy strategies that are broadly divided into management during the acute phase, and management during the recovery phase, are discussed below.

Management During the Acute Phase

               Management of patients during the acute phase of the disease is of the utmost importance because the treatment given in this period decides whether the patients live or die. Here, the knowledge, experience, skills and dexterity of the clinical staff is called into play. This period is a real testing time for the health infrastructure of the hospital where the patients are being managed. Everything, from laboratory diagnostics to the actual treatment administered, has to be well coordinated for the patients’ survival.

Management of Fever: One of the major clinical signs of children being brought to hospital would be fever. Management of fever is a clinical priority, mainly because of the fact that high fever can lead to increase in intracranial pressure by increasing cerebral metabolism and cerebral blood flow, which can lead to cerebral edema (12). Antipyretics such as paracetamol may be used along with sponging in order to lower the temperature.

Management of Seizures: Seizures are very common in Japanese encephalitis patients, particularly in children. Seizures may be generalized tonic-clonic seizures, or more subtle, such as twitching of a digit, or twitching around the lips or eyes. Uncontrolled seizures can lead to raised intracranial pressure, leading to increased metabolism and other physiological changes that can further aggravate the intracranial hypertension, operating in a positive-feedback vicious cycle, often leading to brain herniation. Seizures can often be managed with phenytoin and benzodiazepines; diazepam and lorazepam being first-line choices. If not, intubation and artificial ventilation should be initiated so that higher doses of sedatives and anticonvulsants can be administered. The patient should be monitored by carrying out an electro-encephalogram (EEG) from time to time (89).

Management of Raised Intracranial Pressure: From the foregoing discussion, it is evident that raised intracranial pressure is one the most life-threatening complications of Japanese encephalitis infection. Management of raised intracranial pressure is interrelated with other management strategies, as will also be evident from above. The first thing to do is to keep the patient’s head in a midline position, keeping it tilted at ~30^o. Keeping the head tilted improves the cerebral perfusion pressure, a key determinant in cerebral circulation, influenced by the intracranial pressure. Moreover, keeping the head elevated increases CSF drainage and maximizes cerebral venous return. It should be stressed that the head should lie on the midline in order to prevent any obstruction in the venous drainage through the jugular vein (24). Hyperventilation can reduce the intracranial pressure by bringing about a decrease in cerebral blood flow, which in its turn is brought about by a decrease in the CO₂ tension (PCO₂) caused by the hyperventilation itself. Intracranial pressure begins to fall within seconds to minutes, stabilizes in about 30 minutes, and returns back to the original value in about an hour. Although hyperventilation is useful for bringing down the intracranial pressure, prolonged hyperventilation can, in fact, be harmful, and may worsen the outcome. Hyperventilation should always be withdrawn gradually (67). Mannitol is commonly used to control raised intracranial pressure. The action of mannitol is dependent of the rate of administration – higher infusion rates lead to a rapid fall in intracranial pressure that is short-lived, while slower infusion rates bring about a sustained decrease in the intracranial pressure (72). However, overdosing with mannitol should be avoided at all costs, as this can aggravate cerebral edema (39). Loop diuretics such as frusemide can be used alone, or in combination with mannitol to lower the elevated intracranial pressure. Frusemide alone causes slow reduction in intracranial pressure, but when combined with mannitol, the fall in pressure is rapid and much more sustained than when either drug is used alone (107).

Management During the Recovery Phase

               Management during the recovery phase is no less important than that during the acute phase. This phase is often neglected, because the danger of death has passed. But it is in this phase that the real struggle for the surviving patients begins. It may be recalled that ~50% of the survivors, usually small children, have severe neuropsychiatric sequelae, which are likely to be life-long. A thorough neurological and neuropsychiatric examination of the patients is required, soon after discharge from hospital. This will help assess the condition of the patient for leading a normal life. Intellectual and behavioral problems are common in children recovering from Japanese encephalitis. These conditions should be treated properly and promptly as these can lead to absenteeism from school and thus hamper normal social functioning. Hence, management of neurological deficits is a challenging task, and requires a lot of patience, encouragement and tender loving care, both on the part of the healthcare provider, as well as the family of the affected child. Moreover, treatment of neurological problems is a costly affair and is particularly difficult in the resource-poor developing countries of Asia, where Japanese encephalitis is most prevalent.

ENDPOINTS FOR MONITORING THERAPY

               Monitoring should be done continuously during the acute phase of the disease. EEG should be carried out after administration of anticonvulsants and should be continued until seizures subside. Coma should be monitored by the GCS score. Other parameters such as blood pressure, urine output, serum osmolality, oxygen saturation and central venous pressure should be monitored until the condition of the patient improves.

VACCINES 

Indications

               Japanese encephalitis vaccine is recommended for native and expatriate residents of endemic areas, laboratory workers potentially exposed to the virus, and for travelers spending 1 month or more in endemic areas. It should be borne in mind that vaccination against Japanese encephalitis in endemic areas will be a continuous process as approximately 3 billion people currently live in Japanese encephalitis-endemic regions, where more than 70 million children are born each year. It should be noted that given the mostly infrequent occurrence of Japanese encephalitis in early infancy and the likely interference with passively acquired maternal antibodies during the first months of life, vaccination is not recommended for children below the age of 6 months (3).

Doses and Schedules

               There are three types of Japanese encephalitis vaccines currently in circulation. These are:

               • Mouse brain-derived inactivated vaccine.

               • Cell culture-derived inactivated vaccines.

               • Cell culture-derived live-attenuated vaccine.

Mouse Brain-Derived Inactivated Vaccine

               The mouse brain-derived inactivated vaccine is produced by growing the virus by inoculating the brain of suckling mice pups and subsequent virus purification and inactivation with formaldehyde. Both the Nakayama and Beijing-1 strains have been used to manufacture the vaccine, though studies have shown that the latter strain produces a higher antigenic yield in cultured mouse brain tissue.

               The primary vaccination is done between the ages of 1 and 3 years at doses of 0.5 ml and 1 ml subcutaneously. The dose regimen consists of one injection on day 0, day 7 and day 30 with a booster after 1 year and thereafter every 3 years until the child attains 10 years of age. The protective efficacy is above 90% (31). The only contraindication of the mouse brain-derived inactivated Japanese encephalitis vaccine is a history of hypersensitivity reactions to a previous dose. However, pregnant women should be vaccinated only when at high risk of exposure to the infection. This vaccine has been given safely in various sates of immunodeficiency, including HIV infection (76). The vaccine used to be manufactured by BIKEN (Japan) and marketed as JE-VAX®. The production of this vaccine has been discontinued. Current stocks from another manufacturer (Green Cross) are also likely to be exhausted soon.

Cell Culture-Derived Inactivated Vaccines

               A cell culture-derived inactivated vaccine, manufactured by propagating the Beijing P-3 strain in primary hamster kidney (PHK) cells is available exclusively in China. However, since primary hamster kidney cells are not approved by WHO as a vaccine-production substrate, this Chinese vaccine has not received international acceptance. Other cell culture-derived inactivated Japanese encephalitis vaccines that have been accepted internationally involve the Vero cell platform (100). These are being manufactured in accordance with international norms following GMP and GCP criteria and are in various stages of development. One such vaccine, IXIARO®, is manufactured by Intercell, an Austrian company. After extensive clinical trials, the vaccine was approved by the US Food and Drug Administration (USFDA) on March 30, 2009. It has also been approved by the European Commission and the Australian Therapeutic Goods Administration (TGA). The commercial launch of this vaccine in the USA is slated to occur later this year.

Cell Culture-Derived Live-Attenuated Vaccine

               This vaccine is based on the genetically stable, neuro-attenuated SA 14-14-2 strain of Japanese encephalitis that is derived from serial passage of the SA 14 strain of the virus in primary hamster kidney cells. It elicits broad-spectrum immunity against heterologous Japanese encephalitis strains. Reversion to neurovirulence is considered highly unlikely. As the vaccine is produced on primary cells, the manufacturing process includes detailed screening for endogenous and adventitious viruses. This single-dose vaccine has been licensed for use in China since 1988, where over 200 million children have been successfully immunized so far, with a brilliant safety record. The Republic of Korea subsequently acquired a commercial license to manufacture this vaccine. This vaccine is also widely used in Nepal, Sri Lanka and India. A case-control study conducted in Nepal indicated that five years after administration of a single dose of SA 14-14-2 provided a protective efficacy of 96% and the persistence of neutralizing antibody titer was 63.8% (86). In the Indian context, the imported SA 14-14-2 vaccine has been used to immunize over 9.3 million children (aged between 1 and 15 years) in the summer of 2006 in 4 states. An expert committee reported that the 65 serious adverse events that were reported, including 22 fatalities, were not related to the vaccine (108).

               A recent study in the Philippines has revealed that the SA 14-14-2 vaccine can be safely administered along with measles vaccine in 9 month old infants without any loss of immunogenicity in either of the vaccines (19). This study is very promising from an implementation viewpoint in that the SA 14-14-2 vaccine can, in the future, be incorporated into the routine childhood immunization programs of various Japanese encephalitis endemic countries. A major advantage of this vaccine is that it is inexpensive and hence would be affordable by the economically weaker countries of Asia where Japanese encephalitis is endemic.

Genetically Engineered Japanese Encephalitis Vaccines

               Besides the above three types of Japanese encephalitis vaccines that are commercially available, quite a few innovative genetically engineered third generation Japanese encephalitis vaccines are in various stages of development. The most promising of these that warrants mention is the Chimeri-Vax-JE(TM) developed by Acambis, UK. This recombinant DNA vaccine is based on the Yellow Fever (YF) 17D vaccine strain as a backbone, but with the envelope and pre-membrane protein genes of YF virus replaced by those of Japanese encephalitis virus. Clinical trials are currently ongoing. The results in terms of immunological response and side-effects have thus far been very favorable. An update on the current vaccines against Japanese encephalitisis given in Table 5.

Adverse Effects

               Mouse brain-derived inactivated vaccine Adverse events that have been reported from European, American, and Australian vaccine recipients include itching, urticaria, and occasionally angio-edema of the face – sometimes requiring admission to hospital and corticosteroid therapy (74). Other adverse effects, particularly the possibility of occurrence of acute disseminated encephalomyelitis (ADEM) temporally linked to the vaccine (35), has led to discontinuation of this vaccine.

Cell Culture-Derived Inactivated and Live-Attenuated Vaccines

               Mild local reactions such as injection site redness and swelling have been reported. Mild systemic reactions include fever and headache in rare cases.

Guidelines:  ACIP: Japanese Encephalitis Vaccines.  MMWR, March 2010.

PREVENTION OR INFECTION CONTROL MEASURES

               Japanese encephalitis control measures may be three-pronged, namely (i) changes in pig rearing techniques, (ii) vector control, and (iii) prophylactic vaccination of susceptible human populations. Since the former two approaches have their limitations, it is the third that has to be relied upon to keep this disease at bay. However, it must be stressed that the other approaches should not be neglected altogether. In the Southeast Asian countries, where pig-rearing is widely practiced, the pigsties should be located far away from human dwellings. However, this is not always practicable. Vaccination of swine has also been suggested and practiced in countries like Japan. But a universal and sustained swine vaccination effort is likely to be a costly affair, and thus, out of reach of most resource-poor JE-endemic countries. If pig-rearing is practiced in large pig-farms, located far away from human dwellings, and managed by co-operatives instead of having a individual family-based pig-rearing business, then this problem could possibly be overcome. Mosquito control by insecticides has largely been found to be ineffective, impractical and costly [104]. However, mosquito larva control by biological means such as keeping larvivorous fish in the paddy fields is an alternative and eco-friendly approach which may be adopted. Avoiding mosquito bites by wearing full-sleeved shirts, restricting outdoor activities in the evenings, and using mosquito repellents, could dramatically reduce the incidence of JE. Hence, what is truly required for JE control is the adoption of an integrated approach, including improved agricultural practices, improved living standards, greater health awareness, as well as sustained mass childhood vaccination programs. It should however be noted that unlike smallpox and polio, for which humans are the only host, JE is a zoonotic disease with large animal reservoirs and hence cannot be totally wiped out from the face of the Earth.

CONTROVERSIES, CAVEATS, OR COMMENTS

               The Japanese encephalitis problem, unlike many other Public Health problems, is multifaceted and complex. This complexity stems from the fact that we are still not totally aware of the true magnitude of the problem. This, in turn, stems from the lack of good quality disease burden data. In many countries, such as Bangladesh, Cambodia, Indonesia, North Korea, Laos, Myanmar, Papua New Guinea, and Pakistan, there is definite lack of data regarding the distribution and Public Health importance of Japanese encephalitis. Until and unless solid disease burden data is available, National Regulatory Authorities in the respective Japanese encephalitis-endemic countries cannot be convinced about the importance of implementation of a childhood immunization program for Japanese encephalitis. Hence, much more aggressive and thorough epidemiological studies are required. This is especially true in areas like Pakistan and possibly, beyond. We know that Japanese encephalitis cases have been documented from Karachi. But what about countries like Afghanistan and places further west? In these places, Japanese encephalitis surveillance systems are non-existent, but the virus could well be circulating due to presence of vectors. This is a challenge for the epidemiologists. Proper surveillance is strongly linked to good-quality diagnostics. Without good-quality yet affordable diagnostics, Japanese encephalitis can be confused with other diseases, such as Nipah and Chandipura virus outbreaks.

               Two main prerequisites for controlling Japanese encephalitis are (i) greater political will, and (ii) sound financial resources. With a good combination of these two factors, initiation and sustaining of childhood vaccination programs would become much easier. There is ample evidence from the wealthier Japanese encephalitis-endemic Asian countries like Japan, South Korea and Taiwan, what a good vaccination program can achieve. This type of vaccination program is urgently required in countries like India, which has a high disease burden, and thereby contributes a large share of the Japanese encephalitis burden to the Asian region. Vaccination programs have been initiated in two states, but a national immunization program is required. For implementation of any vaccination program, adequate trained staff, a functional cold-chain, good roads for transportation of vaccines and a good campaigning mechanism, for educating the masses, all contribute to acceptability of the vaccine.

               In the near future, greater challenges are likely to occur. These may stem from newer issues of this day and age. Climate change could well be one such issue. Changing weather patterns, especially rainfall patterns, could lead to altering agricultural practices, which would lead to unpredictable changes in mosquito breeding patterns. Moreover, changing bird migration patterns brought about by climate change, could lead to introduction of Japanese encephalitis to new geographical locations. However, there is still hope. A sustained and integrated approach would definitely lead to proper management of Japanese encephalitis in the near future, given the fact that good quality and effective vaccines are on the horizon.

TABLES AND FIGURES

Figure 1 : Global distribution pattern of Japanese encephalitis. The areas shaded in yellow are Japanese encephalitis risk-prone regions. The areas encircled in red, such as Karachi (Pakistan) and Torres Strait islands (Australia) and parts of the northern Australian mainland are newer areas affected by Japanese encephalitis.

Figure 2: Japanese Encephalitis Virus Life-Cycle

Figure 3: A schematic representation of the Japanese encephalitis virus genome. The genome is a single-stranded, plus-sense RNA molecule containing a long open reading frame (ORF) encoding the viral polyprotein with 5’ and 3’ untranslated regions (UTRs). The encoded proteins subsequently self-assemble into complete Japanese encephalitis virus particles. See text for a detailed discussion of the structural and non-structural proteins encoded by the Japanese encephalitis virus genome.

Figure 4 : Opisthotonus in a boy with JE (Photo: Courtesy Dr. P. Nagabhushana Rao)

Figure 5 : Dystonia in the right hand of a boy with JE (Photo: Courtesy Dr. P. Nagabhushana)

Figure 6:  Left hemiparesis in a child with JE (Photo: Courtesy Dr. P. Nagabhushana Rao)

Figure 7:  Right gaze palsy in a child with JE (Photo: Courtesy Dr. P. Nagabhushana Rao)

Figure 8: (A) CT scan showing thalamic hypodensity; (B) MRI scan showing hyperintense thalami (Photos: Courtesy Dr. P. Nagabhushana Rao)

Figure 9: (A) MRI T2WI of a JE patient showing bilateral thalamic hyperintensity; (B) SPECT showing thalamic and left frontal hypoperfusion in a child with JE who had mouth open dystonia (Photos: Courtesy Dr. U.K. Misra)

Figure 10: Bed sore on the head of a JE patient (Photo: Courtesy Dr. P. Nagabhushana Rao)

Table 1:  Japanese Encephalitis : An Historical Timeline

Table 2: Major Human Flaviviruses and their Endemic Areas

Table 3: Japanese Encephalitis – Facts & Figures

Table 4: JE-Related Statistics from Major JE-Endemic Countries

Table 5: Vaccines Against Japanese Encephalitis

Box 1: Diagnostic Tests for Japanese Encephalitis

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