Epidemiologic Reviews Copyright © 1992 by The Johns Hopkins University School of Hygiene and Public Health All rights reserved
Vol. 14,1992 Printed in U.S.A.
The Epidemiology of Japanese Encephalitis: Prospects for Prevention
David W. Vaughn and Charles H. Hoke, Jr.
In Japan, minor epidemics of "summer encephalitis," presumed to be due in large part to Japanese encephalitis, were recorded every year between 1873 and 1968. Major epidemics occurred in 1924, 1935, and 1948 (1). Japanese encephalitis is now recognized throughout much of Asia and is considered the most important mosquito-borne viral encephalitis, with an estimated worldwide incidence of 45,000 cases each year primarily in children. Approximately 25 percent of cases die and 50 percent develop permanent neurologic and psychiatric sequelae (2). The magnitude of the problem is even more impressive when it is considered that the disease often occurs in epidemics that are somewhat predictable and that Japanese encephalitis is a vaccine preventable disease. CLASSIFICATION SYSTEMS/VIROLOGY
Japanese encephalitis virus was first isolated in Japan in 1935 from the brain of a patient dying from encephalitis (3). It was originally called type B encephalitis to differentiate it from the type A encephalitis (von Economo encephalitis or encephalitis lethargica) which occurred during winter months with a different clinical presentation. Encephalitis epidemics in 1933 and 1937 in the United States were initially thought to be caused by the same virus (type From the Department of Virus Diseases, The Walter Reed Army Institute of Research, Washington, D.C. Reprint requests to Dr. Charles H. Hoke, Jr., Department of Virus Diseases, The Waiter Reed Army Institute of Research, Washington, DC 20307-5100. The authors thank Dr. John Gingrich for his assistance in reviewing the known vectors of Japanese encephalitis virus and Dr. Rebecca Bent for her time spent editing this manuscript.
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B); cross-challenge and cross-neutralization studies in mice demonstrated that the virus from North America (St. Louis encephalitis virus) was, in fact, distinct from the virus of Asia, designated the "Japanese" type B encephalitis virus (4). The designation "type B" was later dropped. Although the virus had been classified with other flaviviruses as a group B arbovirus in the Togavirus family (5), it has more recently been classified into a separate family, the family Flaviviridae (6). Yellow fever is the prototype, and all members of the family are antigenically related, having similar genomic organization and structure. Characteristics of this family include virion size of 40-50 nanometers, a lipid envelope, three structural proteins (C, M, and E), and a single-stranded (positive sense) RNA genome of approximately 10 kilobases in length. Sequences of several isolates are available (7). The virus grows in a variety of primary and continuous cell cultures including those of hamster, pig, chicken, monkey, and mosquito origin (8). The virus is most commonly isolated from mosquito pools in areas of transmission, from brain tissue obtained from fatal cases, and from the blood of non-immune sentinel pigs (pigs known to be Japanese encephalitis virus non-immune placed in an area of suspected transmission and followed for the development of viremia or seroconversion). Identity is confirmed by demonstrating that the isolate is neutralized by serum known to contain antibody specific for Japanese encephalitis virus or by reaction with specific monoclonal antibodies in an enzyme-linked immunosorbent assay. A number of methods of comparing im-
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munologic and genetic properties of different strains of the virus have been used. These methods often suggest groupings of strains based on the property being measured, but, to date, the groupings have revealed limited insight into virus evolution or distribution. There is only one serotype of Japanese encephalitis virus; however, two immunotypes (Nakayama and JaGAr) have been hypothesized based on serologic studies (911). While convenient groupings can be made using genetic analyses, the geographic distribution of the groups does not indicate that the genotypes are distributed in an obvious geographic pattern. Strains from different regions have been compared using RNA oligonucleotide analysis. Twelve strains produced similar patterns while two others produced patterns which indicated considerable genetic differences (12, 13). Analysis of epitopes with monoclonal antibodies suggests that strains can be grouped into the same two groups (based on only four isolates evaluated by both techniques) (14). Sequence data on a 240 nucleotide region in the preM gene of 65 isolates suggests four distinct genotypic groups: 1) those from northern Thailand and Cambodia; 2) those from southern Thailand, Malaysia, and Indonesia; 3) those from Japan, China, Taiwan, the Philippines, Sri Lanka, India, and Nepal; and 4) other isolates from Indonesia (15, 16). Eight strains were studied using both epitope mapping and limited sequencing. Seven strains were classified in the same way, with both methods suggesting that both nucleotide sequencing and epitope mapping identify related characteristics. Nucleotide analysis and RNA oligonucleotide analysis resulted in a similar clarification of four strains, with three isolates in one group and the fourth in a second grouping. Japanese encephalitis virus is thought to gain entry into cells via adsorption of the viral envelope spikes onto the plasma membrane, dissolving the plasma membrane at the adsorption site, and penetrating into the cytoplasm through this plasma-membrane disruption. Virions seem to disintegrate at or near the penetration sites (17-19). RNA synthesis takes place in the perinuclear re-
gion with virion production at the rough endoplasmic reticulum, maturation within the golgi, and release from the cell by exocytosis. Ultrastructural studies reveal that viral particles can be seen in cisternae of the rough endoplasmic reticulum. Cell architecture is not heavily disrupted by infection (20-23). CLINICAL SYNDROMES
Infection with Japanese encephalitis virus is most known for the severe meningomyeloencephalitis which it causes; however, infections may also be manifested by a mild febrile illness or aseptic meningitis. Most infections are asymptomatic. Encephalitis
Clinical aspects. The incubation period is probably 1 -2 weeks. Patients generally give a history of 1-3 days of headache often accompanied by nausea or vomiting. Patients are always febrile and may be stuporous or comatose. Generalized seizures may occur, especially in children. Physical findings often include, in addition to fever, a depressed state of consciousness with impairment of cranial and motor nerves. Stupor progresses to coma which, in nonfatal cases, may resolve in 1-2 weeks. The mortality rate is approximately 25 percent with 50 percent suffering neuropsychiatric sequelae and 25 percent recovering fully (2426). A severely depressed sensorium at the time of presentation is associated with a poor outcome (25, 27, 28). Patients in coma may experience respiratory arrest and require ventilatory support; however, clinicians with extensive experience treating Japanese encephalitis cases indicate that once ventilator dependent, patients with this disease rarely recover, presumably due to irreversible damage to respiratory centers. Long-term disabilities in survivors include weakness, ataxia, tremor, athetoid movements, paralysis, memory loss, and abnormal emotional behavior (26, 29). Examination of cerebrospinal fluid reveals normal or moderately increased pressure,
Epidemiology of Japanese Encephalitis
slightly increased total protein, and a lymphocyte pleocytosis of 10 to 1,000 mononuclear cells per milliliter (mean white blood cell count of 260 with 25 percent polymorphonuclear and 72 percent mononuclear cells). In some areas where diagnostic support is limited and where bacterial meningitis is also frequent, a presumptive diagnosis of Japanese encephalitis is made if the cerebrospinal fluid is clear. Presence of Japanese encephalitis virus-specific immunoglobulin M in the cerebrospinal fluid is thought to be diagnostic of Japanese encephalitis (30, 31). The presence of infectious virus in the cerebrospinal fluid and low levels of virusspecific immunoglobulin G and immunoglobulin M in both cerebrospinal fluid and serum at the time of admission are associated with a poor outcome (27). Most clinical cases are associated with acute, short-lived infection followed by a prolonged convalescence. However, recurrence of Japanese encephalitis has been suggested in three of 40 cases followed prospectively in northern India. Japanese encephalitis virus was isolated from peripheral blood monocytes during co-cultivation with mouse embryo fibroblasts in three of eight children who had remained asymptomatic for 8 months after serologically confirmed Japanese encephalitis virus illness (32). Late cultivation of the virus from T lymphocytes in mice has been reported (33). The virus may infect the fetus transplacentally. Chaturvedi et al. (34) observed five pregnant women with Japanese encephalitis during an epidemic in Uttar Pradesh, India. Two of the women delivered apparently normal babies, two aborted, and the outcome of one case was not known. Japanese encephalitis virus was isolated from the brain, liver, and placental tissues of one of the aborted fetuses. Pathology/pathogenesis. Mechanisms of penetration of the blood brain barrier are not known. The virus may replicate in blood vessels, facilitating early replication in the brain (35). Grossly, the brain appears to have vascular congestion, mild edema, and minima' overlying cellular exudate (36). Virus can be isolated from all areas of the brain,
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with virus and viral antigens most commonly found in the thalamus and brain stem (37). The infection and destruction of neurons in the brain stem explain the profound coma and respiratory failure frequently seen. In patients dying many years after illness from Japanese encephalitis virus, lesions in the brain are generally localized to the thalamus, substantia nigra, and Ammon's horn, with relatively mild lesions in the cerebral cortices (38). Similar lesions can be seen in surviving patients using magnetic resonance imaging (39). Japanese encephalitis appears to be a systemic viral infection rather than a localized infection of the central nervous system. This statement is based on autopsy data from 1924 forward suggesting involvement of several organ systems, including the lungs, kidneys, and reticuloendothelial system (36, 40). Treatment. There is no known effective treatment of Japanese encephalitis beyond supportive care. Dexamethasone has been evaluated and shown to neither reduce mortality nor provide any other benefit (25, 41). Various interferons have shown promise. Interferon-alpha inhibits replication in vitro (42). 6-MFA (Central Drug Research Institute, Lucknow, India), a fungal interferon inducer given intravenously, reduced incidence of illness 75 percent (compared with controls) in bonnet macaques challenged intranasally with an Indian strain of Japanese encephalitis virus (43). Human recombinant interferon-alpha (F. Hoffmann-La Roche and Company Ltd., Basle, Switzerland) has also appeared to be effective in unmasked studies; of 12 human cases of Japanese encephalitis treated, a favorable clinical response was seen in 11. Only one of the 12 treated patients died as compared with four deaths among five control patients (44). In another study, two interferon-alphatreated patients recovered well while two control patients died (45). Larger masked, placebo-controlled studies will be necessary to prove the efficacy of this treatment. The use of interferons at the start of an epidemic or in persons with prodromal symptoms has been suggested (46).
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Vaughn and Hoke
Asymptomatic infection
Most Japanese encephalitis virus infections do not result in obvious illness (47). The data on the ratio of apparent to inapparent infections is limited (table 1) and may be different in different populations. Between 50 and 300 infections occur for each clinical case which is identified. A number of explanations may account for the low ratio of symptomatic to asymptomatic cases, including viral factors, such as route, titer, and neurovirulence of inoculum, or host factors, such as age, genetic makeup, general health, or preexisting immunity. Virus inoculated by the infecting mosquito directly into the lumina of a blood vessel may be transported more rapidly to the central nervous system, while virus inoculated into subcutaneous tissues may proliferate harmlessly for several cycles before entering the circulatory system, allowing time for an immune response (35, 48). Inoculation of larger quantities of virus as a result of higher titers in saliva of certain vector species under optimal climatic conditions may increase the risk of developing encephalitis (49). Inoculation of strains of greater neurovirulence may predispose to symptomatic illness. Variations in host resistance related to age (50), genetic variation (51), other simultaneous infection (52), or previous flavivirus infection (53-57) may modify the susceptibility of the host to serious infection. LABORATORY DIAGNOSIS
Many methods of detecting Japanese encephalitis virus antibodies have been developed. The most reliable method at present
is a carefully standardized enzyme-linked immunosorbent assay (31, 58-60). Tests which were used widely in the past include hemagglutination inhibition (61), compliment fixation (62), virus neutralization (63, 64), indirect hemagglutination (65), indirect immunofluorescence (66), staphylococcal coagglutination (67), and single radial hemolysis in gel (68). The hemagglutination inhibition test can be performed entirely from locally produced or reusable components, whereas enzyme-linked immunosorbent assays may be more difficult to standardize and require expensive or unobtainable reagents and/or equipment, making them impractical in some developing areas. Where they can be afforded, wellstandardized immunoglobulin M antibody capture enzyme-linked immunosorbent assays are the diagnostic procedure of choice. By careful standardization of enzyme-linked immunosorbent assays, Japanese encephalitis and dengue infections can be distinguished, as can primary and secondary flavivirus infections (by comparing the number of units of specific immunoglobulin M versus immunoglobulin G antibody) (69). Antibody test results may be confusing due to considerable cross-reactivity by hemagglutination inhibition serology with other flaviviruses (70). In Southeast Asia, where dengue and Japanese encephalitis virus cocirculate, previous exposure to either virus in an individual patient increases the difficulty of virus-specific serologic diagnosis. For antigen detection, a reverse passive hemagglutination test to detect Japanese encephalitis virus antigens in the cerebrospinal fluid has been developed (71, 72). The poly-
TABLE 1. Reported ratios of apparent to inapparent Japanese encephalitis infections in various studies Country
Year
Population
Ratio
Korea
1958
American military
1:25
Thailand
1970
Chiangmai Valley residents
1:300
Thailand
1972
American military
1:63
India
1980
West Bengal villagers
1:113 to 1:387
China
1982
Beijing residents
1:1,000 (estimate)
Reference
Hallstead and Grosz (207) Grossman et al. (163) Benenson et al. (208) Chakraborty et al. (209) Huang (35)
Epidemiology of Japanese Encephalitis
merase chain reaction is being developed as a tool to detect viral genome (73). Viral isolation from serum in mosquito or other cell lines gives proof of Japanese encephalitis virus as etiology, but unless brain is obtained postmortem, the culture is rarely positive (74-77). ECOLOGY Epidemic cycle
Japanese encephalitis virus is transmitted by zoophilic mosquito vectors; consequently, wild and domesticated animals are the principal hosts. Man is considered to be a dead-end host for this virus due to the short duration and low titer of viremia in man and the relative preference of vector mosquitoes for animals over man (78). Although the virus has been transmitted as an aerosol in laboratory animals (79), such transmission does not occur in nature. Figure 1 illustrates the sequence of events in an epidemic studied on northern Honshu Island, Japan, during the summer of 1964 (80). Sera was collected each week from
10 Pigs
;
previously non-immune pigs in four different areas and tested for the presence of hemagglutination inhibition antibody against Japanese encephalitis virus. Mosquitoes near the pigs were trapped once a week, and the females were pooled for attempted virus isolation. Pigs became viremic starting approximately 4 days after inoculation and developed antibody to the virus approximately 10 days after inoculation. Human clinical cases of Japanese encephalitis were confirmed by a four-fold increase in hemagglutination inhibition antibody. Twenty percent of pigs were infected in early July. Acquiring the virus from viremic pigs, mosquitoes became infectious after a 14-day extrinsic incubation period. These mosquitoes transmit a second wave of Japanese encephalitis virus infections to pigs, leading to development of antibody in 100 percent of the animals. With a larger number of viremic pigs, many more mosquitoes are infected. Following another 4 days for pigs to develop viremia, plus another 14-day extrinsic incubation period, the mosquitoes transmit the virus to the human population, with subsequent appearance of clinical cases of enceph-
100
10 i
r
r -
Second Outbreak '
First Outbreak
201
Third outbreak
Percentage of pigs bearing hemagglutination inhibition antibody
80 60
•
40
i s
20
Number of Virus isolations
Mosquitoes]
14
Cases of Japanese encephalitis
Human beings
27 20 1 3 5 7 9 11 13 15 17 18 21 23 25 27 28 31 2 4
JUNE
i
JULY
I
6 8 10 12 14 16 18 20 22 24 26 28 30 1 3 5 7 8 11 13 15 17 18 21 23 25
AUGUST
I
SEPTEMBER
FIGURE 1. Schematic representation of an outbreak of Japanese encephalitis infections among pig, mosquito, and human populations of northem Honshu Island, Japan, 1964.
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Vaughn and Hoke
alitis. Other studies have shown that epidemics of Japanese encephalitis follow the peak isolation rate of the virus in mosquitoes by 17-20 days (81). Infections in man probably occur later than in amplifying animals (pigs and birds) because man is less frequently bitten by the vector, is not bitten in large numbers until the density of infected vectors is maximal, and because infectious mosquitos are relatively rare until amplification has occurred in pigs (82). Animal reservoirs
Studies of antibody prevalence in animal species provide an indication of the degree of exposure of the species as well as its susceptibility to infection by the virus. High prevalence of antibody to Japanese encephalitis virus has been documented among pigs, horses, and birds, and lower rates in cattle, sheep, dogs, and monkeys. Pigs and ardeid birds are the most important hosts for maintenance, amplification, and spread of the virus (2). Rodents appear to be unimportant hosts (83). Pigs are thought to be an important source of viremic blood for mosquitoes on the basis of 1) a high incidence of natural swine infection, 2) the occurrence of viremia in pigs following natural infection, 3) the demonstration that viremia in pigs lasts 2-4 days and occurs in titers adequate to infect Culex tritaeniorhynchus mosquitoes, 4) transmission of virus from pig to pig by laboratory reared Culex tritaeniorhynchus mosquitoes, 5) large numbers of Culex tritaeniorhynchus mosquitoes found biting pigs in nature, and 6) the presence of a large population of susceptible pigs replenished each year due to commercial slaughtering of the animals at 10-16 months of age (84, 85). Bird-mosquito cycles are thought to be important in maintaining and amplifying Japanese encephalitis virus in the environment (86). Viremia frequently follows infection of both wild and domestic birds with the virus. The amount of virus circulating in the blood of birds following experimental infection by syringe or laboratory-reared in-
fected mosquitoes is adequate to infect other mosquitoes (87). In India, neutralizing antibody to the virus was found in 34.8 percent of 514 birds (pond herons and cattle egrets) indicating frequent exposure of these birds to the virus (88). Japanese encephalitis virus antibody is passively transferred from immune hens, as well as from 25 percent of immune wild herons and egrets, to their progeny and remains detectable until the third to fifth week of life (89). However, even antibody-positive young birds can be infected given an adequate dose of virus. Once birds have been infected, they are immune and no longer able to amplify the virus (90, 91). Animals and mosquitoes infected with Japanese encephalitis virus generally remain asymptomatic, though fatal encephalitis occurs in horses and fetal wastage may occur in infected sows (2). These effects on domestic animals have led to the development and distribution of animal vaccines. A formalin-inactivated vaccine was used to protect horses during an epizootic in Japan during 1947-1949, and since 1972, a live attenuated vaccine has been licensed in Japan for use in pigs to prevent abortions and prevent virus amplification (92, 93). Vectors
Vector competence for transmission is defined by a number of factors. To be an efficient vector, the female of a candidate mosquito species must be infectable by inoculation of infectious materials and become infected by a blood meal containing virus at a titer similar to that found in the blood of animal hosts. The candidate vector must support replication of virus to high titer, have demonstrable virus in the salivary glands, transmit virus following a bite, and be a demonstrable source of virus isolation in the wild. In addition, potential vectors must breed to extremely high numbers and be present where humans might be bitten and, at least on occasion, seek a blood meal from humans. Many mosquitoes have been evaluated as potential vectors for Japanese
Epidemiology of Japanese Encephalitis
encephalitis virus. Only a few species consistently meet requirements to be classified as important vectors, and one emerges as the most important. The Culex tritaeniorhynchus mosquito is the main vector to humans in Asia (94). At least 11 other species have been infected in the laboratory (42). Japanese encephalitis virus has also been found in field collections of many other species of mosquito (table 2) (95). Many ecologic behavioral features of Culex tritaeniorhynchus mosquitoes have been characterized (96-99). In most regions, Culex tritaeniorhynchus mosquitoes are present in enormous numbers for a short time period each year following periods of heavy rain. Although the Culex tritaeniorhynchus mosquito is zoophilic, preferring pigs and
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birds over man, it bites man with enough frequency to account for transmission of the virus. This mosquito breeds in rice fields some distance from human dwellings but flies to peridomestic areas for blood meals. Culex tritaeniorhynchus mosquitoes can fly for a distance of up to 1.5 kilometers and have been found in treetops 43-50 feet above the ground, where virus could be spread among birds (100). Vertical transmission of Japanese encephalitis virus has been demonstrated in three different strains of Culex tritaeniorhynchus mosquitoes, as well as in Culex pipiens, Aedes albopictus, Aedes togoi, Culex annulus, Culex quinquefasciatus, and Armigeres subalbatus mosquitoes (101-103). Vertical transmission probably occurs at oviposition
TABLE 2. Vectors and possible vectors of Japanese encephalitis virus Species
Distribution
Vector efficiency*
Culex vishnui annulus
Taiwan, Hong Kong
Good (limited distribution) Occasional Marginal Marginal Good
Culex bitaeniorhynchus Culex epidesmus Culex fuscocephala Culex gelidus
Oriental region South Asia Oriental region Oriental region
Culex pipiens pallens Culex pseudovishnui
Possible East Asia Pan oriental (especially northeast Good India) Marginal Worldwide
References
160,210-212 213,214 214
95, 173,215-217 2, 151, 173, 181,216, 217
Culex pipiens quinquefasciatus Culex tritaeniorhynchus Oriental region, Middle East, North and East Africa Culex vishnui Culex whitmorei Aedes albopictus
Aedes japonicus Anopheles subpictus Mansonia annulifera Mansonia bonneae/ dives Mansonia uniformis
East Asia, South Asia, Southeast Asia Oriental region, Australia, India East Asia, South Asia, Southeast Asia, Oceania, southern United States Japan, maritime Russia, Korea, Taiwan East Asia, South Asia, Southeast Asia, western Oceania Oriental region Southeast Asia India, Indonesia
35,95 2,95,106, 181,217, 218
2,95,212,217
Excellent
95, 181, 151, 160, 173, 211,212,216,217, 219-221 214,217,220
Marginal Marginal
214,217 35,214
Marginal
95, 104
Excellent
'Good (in localized areas) 217 Occasional Occasional
210 2
Occasional
217,222
* Vector efficiency defined as 1) excellent, widely distributed, consistently trapped in areas of outbreaks, and a predictable source of virus isolates; 2) good, biologically capable of transmission but of limited distribution; 3) occasional, rarely achieves large populations necessary for transmission to man; and 4) marginal, may transmit disease but has limited involvement in human disease and/or zoonotic amplification.
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Vaughn and Hoke
rather than transovarially and might account for the persistence of the virus in nature (103, 104). Seasonally Transmission of Japanese encephalitis virus in the tropics may occur year round. Where seasonal epidemics occur, they generally begin during the rainy seasons when mosquito populations are maximal (105). An epidemiologic survey in Tamil Nadu, India, demonstrated the sequence of increasing rainfall followed by an increase in vectors, seroconversion in sentinel farm animals, and, finally, seroconversion and illness in the human population (106). This sequence of events has been observed elsewhere. In Thailand, non-immune sentinel pigs in the dry hot season do not become infected until several weeks after the first rains of the wet season occur (107). The Karnataka state of India has two epidemics each year, with a severe one from April to July and a milder one from September to December along with the rest of India (108). In contrast, seroconversion of sentinel pigs in peninsular India occurs during 8 months of the year, with increased transmission correlating with increased numbers of clinical cases of encephalitis (109). Overwintering
An important question has been how the virus survives the cool season in temperate and subtropical climates. Several theories have been put forward; one or more of these theories may explain how the virus persists from one epidemic season to the next. The virus may overwinter in hibernating mosquitoes, in mosquito eggs, in reptiles, or it may be reintroduced by migrating birds. Some investigators have proposed that the virus remains in hibernating mosquitoes or is transmitted to their offspring. In Korea, a collection of 50,499 mosquitoes during the winter months over a 6-year period revealed two strains of Japanese encephalitis virus (one in December and one in February) (110). Japanese encephalitis virus has also been isolated from larvae collected in June (one of 382,000 examined) suggesting verti-
cal transmission of the virus in Culex tritaeniorhynchus mosquitoes as a possible interepidemic viral survival mechanism (111). Maintenance of virus in hibernating mosquitoes may be the principal method of overwintering (112). The virus may be transmitted year round in tropical climates and reintroduced into temperate climates by migrating birds or by mosquitoes that are blown by the wind or carried in vehicles (110). The fact that migrating birds moving north are adults and extremely resistant to experimental infection with Japanese encephalitis virus makes this mechanism unlikely (113). Other investigators have suggested that the virus may be latent in birds and be reactivated due to the stress of migration or hormonal changes (114). A recent demonstration of genetic dissimilarity between the northern and southern Japanese encephalitis virus genomes tends to lessen the liklihood of frequent reintroduction by migrating birds (15). A small body of data provides suggestive evidence that snakes may play a role in maintenance of Japanese encephalitis virus in Korea. Reptiles undergo immune suppression during hibernation. Between 1965 and 1970, 40 percent of 2,051 snakes collected in Korea were found to be positive for Japanese encephalitis virus antibody by hemagglutination inhibition. Two isolates were obtained from 747 snakes collected (one isolation each in July and October) (115). Under artificial hibernation conditions, the virus could be recovered from snakes and frogs after 6 months of winter hibernation in Korea (116). EPIDEMIOLOGY
Epidemiologic data is clouded by the fact that most national disease reporting systems report the total number of encephalitis cases. Few cases of Japanese encephalitis in endemic regions ever have a specific etiologic diagnosis. Adding to the confusion is the problem that the serologic tests used, or surveys performed, to detect existing immunoglobulin G antibodies cannot distin-
Epidemiology of Japanese Encephalitis
guish antibodies to Japanese encephalitis virus from those directed against other flaviviruses. Dengue virus is also widespread in several areas where Japanese encephalitis occurs, confounding serologic results. Consequently, descriptions of the epidemiology of Japanese encephalitis are somewhat less precise than is desirable. This lack of diagnostic precision has increased the difficulty of undertaking focused disease control programs for Japanese encephalitis. Age distribution
The age distribution of disease varies between regions. The highest age-specific attack rates usually occur in children aged 36 years. If all children were at equal risk, one would expect the highest rates to be in younger children (aged 1-3 years) following the loss of maternal antibodies. The observation that higher morbidity occurs in children aged 3-6 years is consistent with a higher risk in this group due to behavioral factors; perhaps increased play outside, especially after dusk, allows increased exposure (25, 35). Tapering off of age-specific attack rates after age 14 years is accompanied by increased prevalence of neutralizing antibody in this age group, indicating that the reduced attack rates in heavily affected populations is due to immunity resulting from natural exposure and subclinical infections. In some areas of northern India, Nepal, and Sri Lanka, all age groups are affected, suggesting that the virus was recently introduced into these relatively non-immune populations (105). In addition, adult travelers to areas where Japanese encephalitis virus is transmitted are susceptible to devastating infections. Geographic distribution and incidence
Countries with proven epidemics of Japanese encephalitis are India, Nepal, Sri Lanka, Burma, Laos, Thailand, Kampuchea, Vietnam, Malaysia, Singapore, Philippines, Indonesia, Saipan, China, maritime Siberia, Korea, and Japan (figure 2). Regular seasonal epidemics occur in northern Southeast Asia, China, and Korea (105). Available
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reports on the disease from various countries are summarized below and in table 3. In most instances, few cases have been serologically confirmed. Most cases are reported based on clinical and epidemiologic similarity with confirmed or past cases. Bangladesh. Little is known about the epidemiology of Japanese encephalitis in Bangladesh. The disease was reported for the first time in 1977 based on a clinical diagnosis without serologic confirmation (117). However, ecologic and epidemiologic factors make it likely that the disease occurs in this country. China. Ten thousand or more cases per year of Japanese encephalitis are reported from China (1986 reference) (118). Disease is widespread with clinically and epidemiologically compatible cases reported from all provinces except for the two most western ones, Xinjiang and Xizang. The virus was first isolated in China by Yen in 1940 (35). There was a sharp decrease in the annual morbidity rates of the disease in Beijing in 1982 and 1983. A decrease in the Culex tritaeniorhynchus mosquito population was noted in 1980 and 1981, possibly due to reduced rainfall (118). At present, outbreaks appear to have been controlled by massive immunization programs. Guam. Hammon et al. (119) reported 49 cases of Japanese encephalitis in Guam between December 1947 and March 1948 just after the months of maximum rainfall. India. India appears to have outbreaks in different regions in a rather unpredictable fashion. Cases of Japanese encephalitis were first recognized in India in 1955 (120). A total of 63 cases were reported in south India between 1955 and 1966 (121), with the first reported epidemic of encephalitis due to Japanese encephalitis virus in West Bengal reported in 1973 (763 cases, 325 deaths). Epidemics occurred again in 1976 (307 cases, 126 deaths) and 1978 (1,256 cases, 544 deaths). Serodiagnosis could be made in 64.4 percent and 69.2 percent of paired sera tested in 1976 and 1978, respectively (122126). In 1979, 995 cases of Japanese encephalitis were reported with a 24 percent mortality rate (127). There was an extensive
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Vaughn and Hoke
FIGURE 2.
Distribution of suspected or proven cases of Japanese encephalitis.
epidemic in 1981 (128, 129). A total of 688 cases, with 238 deaths, were reported in the fall of 1983 following 3,516 cases and 1,261 deaths in 1982(130). An outbreak of encephalitis in the Bellary district of Karnataka state (110 patients) and adjoining areas of Andhra Pradesh (109 patients) occurred during October 1986 to January 1987. Sixty-one patients died for a case fatality rate of 27.9 percent. A total of 153 patients were evaluated serologically; 67 cases were clearly due to Japanese encephalitis virus and 48 to an undifferentiated flavivirus (Japanese encephalitis/West Nile/ dengue). There was no serologic evidence of involvement of flaviviruses in 38 patients (131). Efforts to manufacture vaccine in India are well advanced, though targeting of persons at risk remains a problem. Vaccine is manufactured from mouse brains, much as is done in Japan (132-135). Indonesia. Until recently, no outbreaks of Japanese encephalitis have been reported in
Indonesia. Cases of clinical encephalitis range from 1,000 to 2,500 per year. Serologic confirmation of etiology had been lacking (136). Recently a case of Japanese encephalitis in a 10-year-old Australian tourist (137) prompted a hospital-based surveillance of pediatric encephalitis cases in Denpasar on the Island of Bali. Acute and convalescent sera and cerebrospinal fluid collected from patients with encephalitis, tested by enzymelinked immunosorbent assay, confirmed Japanese encephalitis virus as the etiology of the encephalitis in four of 17 children (24 percent) (138). Japanese encephalitis virus has been isolated from man, pigs, and mosquitos. The percentage of persons with neutralizing antibody in areas east of Wallace's line (an imaginary line that runs nearly north and south from the Philippines south between Borneo and Sulawesi and between Java and the Lesser Sunda Islands) is quite low except for Lombok (14 percent), while the seropositivity rate was 52 percent in Bali and approximately 25 percent in Borneo
LE 3. Japanese encephalitis in various countries (see text for references) Country
Number of cases
Antibody prevalence
Bangladesh
Unknown
Unknown
China
10,000/year; cases in all but two most western provinces
Unknown
Guam
49 cases in 1948
India
995-5,200 cases/year
Indonesia
1,000-2,500 cases encephalitis/year; only five confirmed as Japanese encephalitis
Vol. 14,1992 Printed in U.S.A.
The Epidemiology of Japanese Encephalitis: Prospects for Prevention
David W. Vaughn and Charles H. Hoke, Jr.
In Japan, minor epidemics of "summer encephalitis," presumed to be due in large part to Japanese encephalitis, were recorded every year between 1873 and 1968. Major epidemics occurred in 1924, 1935, and 1948 (1). Japanese encephalitis is now recognized throughout much of Asia and is considered the most important mosquito-borne viral encephalitis, with an estimated worldwide incidence of 45,000 cases each year primarily in children. Approximately 25 percent of cases die and 50 percent develop permanent neurologic and psychiatric sequelae (2). The magnitude of the problem is even more impressive when it is considered that the disease often occurs in epidemics that are somewhat predictable and that Japanese encephalitis is a vaccine preventable disease. CLASSIFICATION SYSTEMS/VIROLOGY
Japanese encephalitis virus was first isolated in Japan in 1935 from the brain of a patient dying from encephalitis (3). It was originally called type B encephalitis to differentiate it from the type A encephalitis (von Economo encephalitis or encephalitis lethargica) which occurred during winter months with a different clinical presentation. Encephalitis epidemics in 1933 and 1937 in the United States were initially thought to be caused by the same virus (type From the Department of Virus Diseases, The Walter Reed Army Institute of Research, Washington, D.C. Reprint requests to Dr. Charles H. Hoke, Jr., Department of Virus Diseases, The Waiter Reed Army Institute of Research, Washington, DC 20307-5100. The authors thank Dr. John Gingrich for his assistance in reviewing the known vectors of Japanese encephalitis virus and Dr. Rebecca Bent for her time spent editing this manuscript.
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B); cross-challenge and cross-neutralization studies in mice demonstrated that the virus from North America (St. Louis encephalitis virus) was, in fact, distinct from the virus of Asia, designated the "Japanese" type B encephalitis virus (4). The designation "type B" was later dropped. Although the virus had been classified with other flaviviruses as a group B arbovirus in the Togavirus family (5), it has more recently been classified into a separate family, the family Flaviviridae (6). Yellow fever is the prototype, and all members of the family are antigenically related, having similar genomic organization and structure. Characteristics of this family include virion size of 40-50 nanometers, a lipid envelope, three structural proteins (C, M, and E), and a single-stranded (positive sense) RNA genome of approximately 10 kilobases in length. Sequences of several isolates are available (7). The virus grows in a variety of primary and continuous cell cultures including those of hamster, pig, chicken, monkey, and mosquito origin (8). The virus is most commonly isolated from mosquito pools in areas of transmission, from brain tissue obtained from fatal cases, and from the blood of non-immune sentinel pigs (pigs known to be Japanese encephalitis virus non-immune placed in an area of suspected transmission and followed for the development of viremia or seroconversion). Identity is confirmed by demonstrating that the isolate is neutralized by serum known to contain antibody specific for Japanese encephalitis virus or by reaction with specific monoclonal antibodies in an enzyme-linked immunosorbent assay. A number of methods of comparing im-
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munologic and genetic properties of different strains of the virus have been used. These methods often suggest groupings of strains based on the property being measured, but, to date, the groupings have revealed limited insight into virus evolution or distribution. There is only one serotype of Japanese encephalitis virus; however, two immunotypes (Nakayama and JaGAr) have been hypothesized based on serologic studies (911). While convenient groupings can be made using genetic analyses, the geographic distribution of the groups does not indicate that the genotypes are distributed in an obvious geographic pattern. Strains from different regions have been compared using RNA oligonucleotide analysis. Twelve strains produced similar patterns while two others produced patterns which indicated considerable genetic differences (12, 13). Analysis of epitopes with monoclonal antibodies suggests that strains can be grouped into the same two groups (based on only four isolates evaluated by both techniques) (14). Sequence data on a 240 nucleotide region in the preM gene of 65 isolates suggests four distinct genotypic groups: 1) those from northern Thailand and Cambodia; 2) those from southern Thailand, Malaysia, and Indonesia; 3) those from Japan, China, Taiwan, the Philippines, Sri Lanka, India, and Nepal; and 4) other isolates from Indonesia (15, 16). Eight strains were studied using both epitope mapping and limited sequencing. Seven strains were classified in the same way, with both methods suggesting that both nucleotide sequencing and epitope mapping identify related characteristics. Nucleotide analysis and RNA oligonucleotide analysis resulted in a similar clarification of four strains, with three isolates in one group and the fourth in a second grouping. Japanese encephalitis virus is thought to gain entry into cells via adsorption of the viral envelope spikes onto the plasma membrane, dissolving the plasma membrane at the adsorption site, and penetrating into the cytoplasm through this plasma-membrane disruption. Virions seem to disintegrate at or near the penetration sites (17-19). RNA synthesis takes place in the perinuclear re-
gion with virion production at the rough endoplasmic reticulum, maturation within the golgi, and release from the cell by exocytosis. Ultrastructural studies reveal that viral particles can be seen in cisternae of the rough endoplasmic reticulum. Cell architecture is not heavily disrupted by infection (20-23). CLINICAL SYNDROMES
Infection with Japanese encephalitis virus is most known for the severe meningomyeloencephalitis which it causes; however, infections may also be manifested by a mild febrile illness or aseptic meningitis. Most infections are asymptomatic. Encephalitis
Clinical aspects. The incubation period is probably 1 -2 weeks. Patients generally give a history of 1-3 days of headache often accompanied by nausea or vomiting. Patients are always febrile and may be stuporous or comatose. Generalized seizures may occur, especially in children. Physical findings often include, in addition to fever, a depressed state of consciousness with impairment of cranial and motor nerves. Stupor progresses to coma which, in nonfatal cases, may resolve in 1-2 weeks. The mortality rate is approximately 25 percent with 50 percent suffering neuropsychiatric sequelae and 25 percent recovering fully (2426). A severely depressed sensorium at the time of presentation is associated with a poor outcome (25, 27, 28). Patients in coma may experience respiratory arrest and require ventilatory support; however, clinicians with extensive experience treating Japanese encephalitis cases indicate that once ventilator dependent, patients with this disease rarely recover, presumably due to irreversible damage to respiratory centers. Long-term disabilities in survivors include weakness, ataxia, tremor, athetoid movements, paralysis, memory loss, and abnormal emotional behavior (26, 29). Examination of cerebrospinal fluid reveals normal or moderately increased pressure,
Epidemiology of Japanese Encephalitis
slightly increased total protein, and a lymphocyte pleocytosis of 10 to 1,000 mononuclear cells per milliliter (mean white blood cell count of 260 with 25 percent polymorphonuclear and 72 percent mononuclear cells). In some areas where diagnostic support is limited and where bacterial meningitis is also frequent, a presumptive diagnosis of Japanese encephalitis is made if the cerebrospinal fluid is clear. Presence of Japanese encephalitis virus-specific immunoglobulin M in the cerebrospinal fluid is thought to be diagnostic of Japanese encephalitis (30, 31). The presence of infectious virus in the cerebrospinal fluid and low levels of virusspecific immunoglobulin G and immunoglobulin M in both cerebrospinal fluid and serum at the time of admission are associated with a poor outcome (27). Most clinical cases are associated with acute, short-lived infection followed by a prolonged convalescence. However, recurrence of Japanese encephalitis has been suggested in three of 40 cases followed prospectively in northern India. Japanese encephalitis virus was isolated from peripheral blood monocytes during co-cultivation with mouse embryo fibroblasts in three of eight children who had remained asymptomatic for 8 months after serologically confirmed Japanese encephalitis virus illness (32). Late cultivation of the virus from T lymphocytes in mice has been reported (33). The virus may infect the fetus transplacentally. Chaturvedi et al. (34) observed five pregnant women with Japanese encephalitis during an epidemic in Uttar Pradesh, India. Two of the women delivered apparently normal babies, two aborted, and the outcome of one case was not known. Japanese encephalitis virus was isolated from the brain, liver, and placental tissues of one of the aborted fetuses. Pathology/pathogenesis. Mechanisms of penetration of the blood brain barrier are not known. The virus may replicate in blood vessels, facilitating early replication in the brain (35). Grossly, the brain appears to have vascular congestion, mild edema, and minima' overlying cellular exudate (36). Virus can be isolated from all areas of the brain,
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with virus and viral antigens most commonly found in the thalamus and brain stem (37). The infection and destruction of neurons in the brain stem explain the profound coma and respiratory failure frequently seen. In patients dying many years after illness from Japanese encephalitis virus, lesions in the brain are generally localized to the thalamus, substantia nigra, and Ammon's horn, with relatively mild lesions in the cerebral cortices (38). Similar lesions can be seen in surviving patients using magnetic resonance imaging (39). Japanese encephalitis appears to be a systemic viral infection rather than a localized infection of the central nervous system. This statement is based on autopsy data from 1924 forward suggesting involvement of several organ systems, including the lungs, kidneys, and reticuloendothelial system (36, 40). Treatment. There is no known effective treatment of Japanese encephalitis beyond supportive care. Dexamethasone has been evaluated and shown to neither reduce mortality nor provide any other benefit (25, 41). Various interferons have shown promise. Interferon-alpha inhibits replication in vitro (42). 6-MFA (Central Drug Research Institute, Lucknow, India), a fungal interferon inducer given intravenously, reduced incidence of illness 75 percent (compared with controls) in bonnet macaques challenged intranasally with an Indian strain of Japanese encephalitis virus (43). Human recombinant interferon-alpha (F. Hoffmann-La Roche and Company Ltd., Basle, Switzerland) has also appeared to be effective in unmasked studies; of 12 human cases of Japanese encephalitis treated, a favorable clinical response was seen in 11. Only one of the 12 treated patients died as compared with four deaths among five control patients (44). In another study, two interferon-alphatreated patients recovered well while two control patients died (45). Larger masked, placebo-controlled studies will be necessary to prove the efficacy of this treatment. The use of interferons at the start of an epidemic or in persons with prodromal symptoms has been suggested (46).
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Vaughn and Hoke
Asymptomatic infection
Most Japanese encephalitis virus infections do not result in obvious illness (47). The data on the ratio of apparent to inapparent infections is limited (table 1) and may be different in different populations. Between 50 and 300 infections occur for each clinical case which is identified. A number of explanations may account for the low ratio of symptomatic to asymptomatic cases, including viral factors, such as route, titer, and neurovirulence of inoculum, or host factors, such as age, genetic makeup, general health, or preexisting immunity. Virus inoculated by the infecting mosquito directly into the lumina of a blood vessel may be transported more rapidly to the central nervous system, while virus inoculated into subcutaneous tissues may proliferate harmlessly for several cycles before entering the circulatory system, allowing time for an immune response (35, 48). Inoculation of larger quantities of virus as a result of higher titers in saliva of certain vector species under optimal climatic conditions may increase the risk of developing encephalitis (49). Inoculation of strains of greater neurovirulence may predispose to symptomatic illness. Variations in host resistance related to age (50), genetic variation (51), other simultaneous infection (52), or previous flavivirus infection (53-57) may modify the susceptibility of the host to serious infection. LABORATORY DIAGNOSIS
Many methods of detecting Japanese encephalitis virus antibodies have been developed. The most reliable method at present
is a carefully standardized enzyme-linked immunosorbent assay (31, 58-60). Tests which were used widely in the past include hemagglutination inhibition (61), compliment fixation (62), virus neutralization (63, 64), indirect hemagglutination (65), indirect immunofluorescence (66), staphylococcal coagglutination (67), and single radial hemolysis in gel (68). The hemagglutination inhibition test can be performed entirely from locally produced or reusable components, whereas enzyme-linked immunosorbent assays may be more difficult to standardize and require expensive or unobtainable reagents and/or equipment, making them impractical in some developing areas. Where they can be afforded, wellstandardized immunoglobulin M antibody capture enzyme-linked immunosorbent assays are the diagnostic procedure of choice. By careful standardization of enzyme-linked immunosorbent assays, Japanese encephalitis and dengue infections can be distinguished, as can primary and secondary flavivirus infections (by comparing the number of units of specific immunoglobulin M versus immunoglobulin G antibody) (69). Antibody test results may be confusing due to considerable cross-reactivity by hemagglutination inhibition serology with other flaviviruses (70). In Southeast Asia, where dengue and Japanese encephalitis virus cocirculate, previous exposure to either virus in an individual patient increases the difficulty of virus-specific serologic diagnosis. For antigen detection, a reverse passive hemagglutination test to detect Japanese encephalitis virus antigens in the cerebrospinal fluid has been developed (71, 72). The poly-
TABLE 1. Reported ratios of apparent to inapparent Japanese encephalitis infections in various studies Country
Year
Population
Ratio
Korea
1958
American military
1:25
Thailand
1970
Chiangmai Valley residents
1:300
Thailand
1972
American military
1:63
India
1980
West Bengal villagers
1:113 to 1:387
China
1982
Beijing residents
1:1,000 (estimate)
Reference
Hallstead and Grosz (207) Grossman et al. (163) Benenson et al. (208) Chakraborty et al. (209) Huang (35)
Epidemiology of Japanese Encephalitis
merase chain reaction is being developed as a tool to detect viral genome (73). Viral isolation from serum in mosquito or other cell lines gives proof of Japanese encephalitis virus as etiology, but unless brain is obtained postmortem, the culture is rarely positive (74-77). ECOLOGY Epidemic cycle
Japanese encephalitis virus is transmitted by zoophilic mosquito vectors; consequently, wild and domesticated animals are the principal hosts. Man is considered to be a dead-end host for this virus due to the short duration and low titer of viremia in man and the relative preference of vector mosquitoes for animals over man (78). Although the virus has been transmitted as an aerosol in laboratory animals (79), such transmission does not occur in nature. Figure 1 illustrates the sequence of events in an epidemic studied on northern Honshu Island, Japan, during the summer of 1964 (80). Sera was collected each week from
10 Pigs
;
previously non-immune pigs in four different areas and tested for the presence of hemagglutination inhibition antibody against Japanese encephalitis virus. Mosquitoes near the pigs were trapped once a week, and the females were pooled for attempted virus isolation. Pigs became viremic starting approximately 4 days after inoculation and developed antibody to the virus approximately 10 days after inoculation. Human clinical cases of Japanese encephalitis were confirmed by a four-fold increase in hemagglutination inhibition antibody. Twenty percent of pigs were infected in early July. Acquiring the virus from viremic pigs, mosquitoes became infectious after a 14-day extrinsic incubation period. These mosquitoes transmit a second wave of Japanese encephalitis virus infections to pigs, leading to development of antibody in 100 percent of the animals. With a larger number of viremic pigs, many more mosquitoes are infected. Following another 4 days for pigs to develop viremia, plus another 14-day extrinsic incubation period, the mosquitoes transmit the virus to the human population, with subsequent appearance of clinical cases of enceph-
100
10 i
r
r -
Second Outbreak '
First Outbreak
201
Third outbreak
Percentage of pigs bearing hemagglutination inhibition antibody
80 60
•
40
i s
20
Number of Virus isolations
Mosquitoes]
14
Cases of Japanese encephalitis
Human beings
27 20 1 3 5 7 9 11 13 15 17 18 21 23 25 27 28 31 2 4
JUNE
i
JULY
I
6 8 10 12 14 16 18 20 22 24 26 28 30 1 3 5 7 8 11 13 15 17 18 21 23 25
AUGUST
I
SEPTEMBER
FIGURE 1. Schematic representation of an outbreak of Japanese encephalitis infections among pig, mosquito, and human populations of northem Honshu Island, Japan, 1964.
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Vaughn and Hoke
alitis. Other studies have shown that epidemics of Japanese encephalitis follow the peak isolation rate of the virus in mosquitoes by 17-20 days (81). Infections in man probably occur later than in amplifying animals (pigs and birds) because man is less frequently bitten by the vector, is not bitten in large numbers until the density of infected vectors is maximal, and because infectious mosquitos are relatively rare until amplification has occurred in pigs (82). Animal reservoirs
Studies of antibody prevalence in animal species provide an indication of the degree of exposure of the species as well as its susceptibility to infection by the virus. High prevalence of antibody to Japanese encephalitis virus has been documented among pigs, horses, and birds, and lower rates in cattle, sheep, dogs, and monkeys. Pigs and ardeid birds are the most important hosts for maintenance, amplification, and spread of the virus (2). Rodents appear to be unimportant hosts (83). Pigs are thought to be an important source of viremic blood for mosquitoes on the basis of 1) a high incidence of natural swine infection, 2) the occurrence of viremia in pigs following natural infection, 3) the demonstration that viremia in pigs lasts 2-4 days and occurs in titers adequate to infect Culex tritaeniorhynchus mosquitoes, 4) transmission of virus from pig to pig by laboratory reared Culex tritaeniorhynchus mosquitoes, 5) large numbers of Culex tritaeniorhynchus mosquitoes found biting pigs in nature, and 6) the presence of a large population of susceptible pigs replenished each year due to commercial slaughtering of the animals at 10-16 months of age (84, 85). Bird-mosquito cycles are thought to be important in maintaining and amplifying Japanese encephalitis virus in the environment (86). Viremia frequently follows infection of both wild and domestic birds with the virus. The amount of virus circulating in the blood of birds following experimental infection by syringe or laboratory-reared in-
fected mosquitoes is adequate to infect other mosquitoes (87). In India, neutralizing antibody to the virus was found in 34.8 percent of 514 birds (pond herons and cattle egrets) indicating frequent exposure of these birds to the virus (88). Japanese encephalitis virus antibody is passively transferred from immune hens, as well as from 25 percent of immune wild herons and egrets, to their progeny and remains detectable until the third to fifth week of life (89). However, even antibody-positive young birds can be infected given an adequate dose of virus. Once birds have been infected, they are immune and no longer able to amplify the virus (90, 91). Animals and mosquitoes infected with Japanese encephalitis virus generally remain asymptomatic, though fatal encephalitis occurs in horses and fetal wastage may occur in infected sows (2). These effects on domestic animals have led to the development and distribution of animal vaccines. A formalin-inactivated vaccine was used to protect horses during an epizootic in Japan during 1947-1949, and since 1972, a live attenuated vaccine has been licensed in Japan for use in pigs to prevent abortions and prevent virus amplification (92, 93). Vectors
Vector competence for transmission is defined by a number of factors. To be an efficient vector, the female of a candidate mosquito species must be infectable by inoculation of infectious materials and become infected by a blood meal containing virus at a titer similar to that found in the blood of animal hosts. The candidate vector must support replication of virus to high titer, have demonstrable virus in the salivary glands, transmit virus following a bite, and be a demonstrable source of virus isolation in the wild. In addition, potential vectors must breed to extremely high numbers and be present where humans might be bitten and, at least on occasion, seek a blood meal from humans. Many mosquitoes have been evaluated as potential vectors for Japanese
Epidemiology of Japanese Encephalitis
encephalitis virus. Only a few species consistently meet requirements to be classified as important vectors, and one emerges as the most important. The Culex tritaeniorhynchus mosquito is the main vector to humans in Asia (94). At least 11 other species have been infected in the laboratory (42). Japanese encephalitis virus has also been found in field collections of many other species of mosquito (table 2) (95). Many ecologic behavioral features of Culex tritaeniorhynchus mosquitoes have been characterized (96-99). In most regions, Culex tritaeniorhynchus mosquitoes are present in enormous numbers for a short time period each year following periods of heavy rain. Although the Culex tritaeniorhynchus mosquito is zoophilic, preferring pigs and
203
birds over man, it bites man with enough frequency to account for transmission of the virus. This mosquito breeds in rice fields some distance from human dwellings but flies to peridomestic areas for blood meals. Culex tritaeniorhynchus mosquitoes can fly for a distance of up to 1.5 kilometers and have been found in treetops 43-50 feet above the ground, where virus could be spread among birds (100). Vertical transmission of Japanese encephalitis virus has been demonstrated in three different strains of Culex tritaeniorhynchus mosquitoes, as well as in Culex pipiens, Aedes albopictus, Aedes togoi, Culex annulus, Culex quinquefasciatus, and Armigeres subalbatus mosquitoes (101-103). Vertical transmission probably occurs at oviposition
TABLE 2. Vectors and possible vectors of Japanese encephalitis virus Species
Distribution
Vector efficiency*
Culex vishnui annulus
Taiwan, Hong Kong
Good (limited distribution) Occasional Marginal Marginal Good
Culex bitaeniorhynchus Culex epidesmus Culex fuscocephala Culex gelidus
Oriental region South Asia Oriental region Oriental region
Culex pipiens pallens Culex pseudovishnui
Possible East Asia Pan oriental (especially northeast Good India) Marginal Worldwide
References
160,210-212 213,214 214
95, 173,215-217 2, 151, 173, 181,216, 217
Culex pipiens quinquefasciatus Culex tritaeniorhynchus Oriental region, Middle East, North and East Africa Culex vishnui Culex whitmorei Aedes albopictus
Aedes japonicus Anopheles subpictus Mansonia annulifera Mansonia bonneae/ dives Mansonia uniformis
East Asia, South Asia, Southeast Asia Oriental region, Australia, India East Asia, South Asia, Southeast Asia, Oceania, southern United States Japan, maritime Russia, Korea, Taiwan East Asia, South Asia, Southeast Asia, western Oceania Oriental region Southeast Asia India, Indonesia
35,95 2,95,106, 181,217, 218
2,95,212,217
Excellent
95, 181, 151, 160, 173, 211,212,216,217, 219-221 214,217,220
Marginal Marginal
214,217 35,214
Marginal
95, 104
Excellent
'Good (in localized areas) 217 Occasional Occasional
210 2
Occasional
217,222
* Vector efficiency defined as 1) excellent, widely distributed, consistently trapped in areas of outbreaks, and a predictable source of virus isolates; 2) good, biologically capable of transmission but of limited distribution; 3) occasional, rarely achieves large populations necessary for transmission to man; and 4) marginal, may transmit disease but has limited involvement in human disease and/or zoonotic amplification.
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Vaughn and Hoke
rather than transovarially and might account for the persistence of the virus in nature (103, 104). Seasonally Transmission of Japanese encephalitis virus in the tropics may occur year round. Where seasonal epidemics occur, they generally begin during the rainy seasons when mosquito populations are maximal (105). An epidemiologic survey in Tamil Nadu, India, demonstrated the sequence of increasing rainfall followed by an increase in vectors, seroconversion in sentinel farm animals, and, finally, seroconversion and illness in the human population (106). This sequence of events has been observed elsewhere. In Thailand, non-immune sentinel pigs in the dry hot season do not become infected until several weeks after the first rains of the wet season occur (107). The Karnataka state of India has two epidemics each year, with a severe one from April to July and a milder one from September to December along with the rest of India (108). In contrast, seroconversion of sentinel pigs in peninsular India occurs during 8 months of the year, with increased transmission correlating with increased numbers of clinical cases of encephalitis (109). Overwintering
An important question has been how the virus survives the cool season in temperate and subtropical climates. Several theories have been put forward; one or more of these theories may explain how the virus persists from one epidemic season to the next. The virus may overwinter in hibernating mosquitoes, in mosquito eggs, in reptiles, or it may be reintroduced by migrating birds. Some investigators have proposed that the virus remains in hibernating mosquitoes or is transmitted to their offspring. In Korea, a collection of 50,499 mosquitoes during the winter months over a 6-year period revealed two strains of Japanese encephalitis virus (one in December and one in February) (110). Japanese encephalitis virus has also been isolated from larvae collected in June (one of 382,000 examined) suggesting verti-
cal transmission of the virus in Culex tritaeniorhynchus mosquitoes as a possible interepidemic viral survival mechanism (111). Maintenance of virus in hibernating mosquitoes may be the principal method of overwintering (112). The virus may be transmitted year round in tropical climates and reintroduced into temperate climates by migrating birds or by mosquitoes that are blown by the wind or carried in vehicles (110). The fact that migrating birds moving north are adults and extremely resistant to experimental infection with Japanese encephalitis virus makes this mechanism unlikely (113). Other investigators have suggested that the virus may be latent in birds and be reactivated due to the stress of migration or hormonal changes (114). A recent demonstration of genetic dissimilarity between the northern and southern Japanese encephalitis virus genomes tends to lessen the liklihood of frequent reintroduction by migrating birds (15). A small body of data provides suggestive evidence that snakes may play a role in maintenance of Japanese encephalitis virus in Korea. Reptiles undergo immune suppression during hibernation. Between 1965 and 1970, 40 percent of 2,051 snakes collected in Korea were found to be positive for Japanese encephalitis virus antibody by hemagglutination inhibition. Two isolates were obtained from 747 snakes collected (one isolation each in July and October) (115). Under artificial hibernation conditions, the virus could be recovered from snakes and frogs after 6 months of winter hibernation in Korea (116). EPIDEMIOLOGY
Epidemiologic data is clouded by the fact that most national disease reporting systems report the total number of encephalitis cases. Few cases of Japanese encephalitis in endemic regions ever have a specific etiologic diagnosis. Adding to the confusion is the problem that the serologic tests used, or surveys performed, to detect existing immunoglobulin G antibodies cannot distin-
Epidemiology of Japanese Encephalitis
guish antibodies to Japanese encephalitis virus from those directed against other flaviviruses. Dengue virus is also widespread in several areas where Japanese encephalitis occurs, confounding serologic results. Consequently, descriptions of the epidemiology of Japanese encephalitis are somewhat less precise than is desirable. This lack of diagnostic precision has increased the difficulty of undertaking focused disease control programs for Japanese encephalitis. Age distribution
The age distribution of disease varies between regions. The highest age-specific attack rates usually occur in children aged 36 years. If all children were at equal risk, one would expect the highest rates to be in younger children (aged 1-3 years) following the loss of maternal antibodies. The observation that higher morbidity occurs in children aged 3-6 years is consistent with a higher risk in this group due to behavioral factors; perhaps increased play outside, especially after dusk, allows increased exposure (25, 35). Tapering off of age-specific attack rates after age 14 years is accompanied by increased prevalence of neutralizing antibody in this age group, indicating that the reduced attack rates in heavily affected populations is due to immunity resulting from natural exposure and subclinical infections. In some areas of northern India, Nepal, and Sri Lanka, all age groups are affected, suggesting that the virus was recently introduced into these relatively non-immune populations (105). In addition, adult travelers to areas where Japanese encephalitis virus is transmitted are susceptible to devastating infections. Geographic distribution and incidence
Countries with proven epidemics of Japanese encephalitis are India, Nepal, Sri Lanka, Burma, Laos, Thailand, Kampuchea, Vietnam, Malaysia, Singapore, Philippines, Indonesia, Saipan, China, maritime Siberia, Korea, and Japan (figure 2). Regular seasonal epidemics occur in northern Southeast Asia, China, and Korea (105). Available
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reports on the disease from various countries are summarized below and in table 3. In most instances, few cases have been serologically confirmed. Most cases are reported based on clinical and epidemiologic similarity with confirmed or past cases. Bangladesh. Little is known about the epidemiology of Japanese encephalitis in Bangladesh. The disease was reported for the first time in 1977 based on a clinical diagnosis without serologic confirmation (117). However, ecologic and epidemiologic factors make it likely that the disease occurs in this country. China. Ten thousand or more cases per year of Japanese encephalitis are reported from China (1986 reference) (118). Disease is widespread with clinically and epidemiologically compatible cases reported from all provinces except for the two most western ones, Xinjiang and Xizang. The virus was first isolated in China by Yen in 1940 (35). There was a sharp decrease in the annual morbidity rates of the disease in Beijing in 1982 and 1983. A decrease in the Culex tritaeniorhynchus mosquito population was noted in 1980 and 1981, possibly due to reduced rainfall (118). At present, outbreaks appear to have been controlled by massive immunization programs. Guam. Hammon et al. (119) reported 49 cases of Japanese encephalitis in Guam between December 1947 and March 1948 just after the months of maximum rainfall. India. India appears to have outbreaks in different regions in a rather unpredictable fashion. Cases of Japanese encephalitis were first recognized in India in 1955 (120). A total of 63 cases were reported in south India between 1955 and 1966 (121), with the first reported epidemic of encephalitis due to Japanese encephalitis virus in West Bengal reported in 1973 (763 cases, 325 deaths). Epidemics occurred again in 1976 (307 cases, 126 deaths) and 1978 (1,256 cases, 544 deaths). Serodiagnosis could be made in 64.4 percent and 69.2 percent of paired sera tested in 1976 and 1978, respectively (122126). In 1979, 995 cases of Japanese encephalitis were reported with a 24 percent mortality rate (127). There was an extensive
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Vaughn and Hoke
FIGURE 2.
Distribution of suspected or proven cases of Japanese encephalitis.
epidemic in 1981 (128, 129). A total of 688 cases, with 238 deaths, were reported in the fall of 1983 following 3,516 cases and 1,261 deaths in 1982(130). An outbreak of encephalitis in the Bellary district of Karnataka state (110 patients) and adjoining areas of Andhra Pradesh (109 patients) occurred during October 1986 to January 1987. Sixty-one patients died for a case fatality rate of 27.9 percent. A total of 153 patients were evaluated serologically; 67 cases were clearly due to Japanese encephalitis virus and 48 to an undifferentiated flavivirus (Japanese encephalitis/West Nile/ dengue). There was no serologic evidence of involvement of flaviviruses in 38 patients (131). Efforts to manufacture vaccine in India are well advanced, though targeting of persons at risk remains a problem. Vaccine is manufactured from mouse brains, much as is done in Japan (132-135). Indonesia. Until recently, no outbreaks of Japanese encephalitis have been reported in
Indonesia. Cases of clinical encephalitis range from 1,000 to 2,500 per year. Serologic confirmation of etiology had been lacking (136). Recently a case of Japanese encephalitis in a 10-year-old Australian tourist (137) prompted a hospital-based surveillance of pediatric encephalitis cases in Denpasar on the Island of Bali. Acute and convalescent sera and cerebrospinal fluid collected from patients with encephalitis, tested by enzymelinked immunosorbent assay, confirmed Japanese encephalitis virus as the etiology of the encephalitis in four of 17 children (24 percent) (138). Japanese encephalitis virus has been isolated from man, pigs, and mosquitos. The percentage of persons with neutralizing antibody in areas east of Wallace's line (an imaginary line that runs nearly north and south from the Philippines south between Borneo and Sulawesi and between Java and the Lesser Sunda Islands) is quite low except for Lombok (14 percent), while the seropositivity rate was 52 percent in Bali and approximately 25 percent in Borneo
LE 3. Japanese encephalitis in various countries (see text for references) Country
Number of cases
Antibody prevalence
Bangladesh
Unknown
Unknown
China
10,000/year; cases in all but two most western provinces
Unknown
Guam
49 cases in 1948
India
995-5,200 cases/year
Indonesia
1,000-2,500 cases encephalitis/year; only five confirmed as Japanese encephalitis
