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Prevalence of Antibodies to Japanese Encephalitis Virus among Inhabitants in Java Island, Indonesia, with a Small Pig Population

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  • 1 Department of International Health, Kobe University Graduate School of Health Sciences, Kobe, Japan; Department of Microbiology and Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan; International Center for Medical Research and Treatment, Kobe University School of Medicine, Kobe, Japan; Indonesia-Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases, Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia

Japanese encephalitis virus (JEV) is maintained through a transmission cycle between amplifier swine and vector mosquitoes in a peridomestic environment. Thus, studies on natural JEV activities in an environment with a small size of pig population have been limited. Here, we surveyed antibodies against JEV in inhabitants of Jakarta and Surabaya located in Java Island (Indonesia), which has a small swine population. Overall, 2.2% of 1,211 sera collected in Jakarta and 1.8% of 1,751 sera collected in Surabaya had neutralizing antibody titers of ≥ 1:160 (90% plaque reduction). All the samples with titers of ≥ 1:160 against JEV were also examined for neutralizing antibodies against each of four dengue viruses to confirm that JEV antibody prevalences obtained in the present survey were not attributable to serologic cross-reactivities among flaviviruses distributed in Java. These results indicated that people in Java Island are exposed to natural JEV infections despite a small swine population.

INTRODUCTION

Japanese encephalitis (JE) is the most important cause of viral encephalitis in Asia.1 Although the majority of humans infected with JE virus are asymptomatic, some develop acute encephalitis at a ratio of subclinical to clinical infections in the range of 25:1 to 1,000:1.2 Mortality is approximately 20% and half of the survivors have severe neuropsychiatric sequelae. Fifty thousand cases are reported annually with one billion people at risk of infection. Japanese encephalitis is a vaccine-preventable disease. The number of patients dramatically reduced after introduction of vaccination in some countries, including Japan, South Korea, and Taiwan.3 Therefore, most of the patients currently occur in tropical and subtropical regions of Asia.

Swine are a major amplifying host of Japanese encephalitis virus (JEV) in a peridomestic environment. Mosquitoes acquire the virus from viremic swine and may transmit it to humans. 4,5 In most swine-raising countries in Asia, antibodies against JEV have been detected in humans and animals, indicating active JEV circulation in nature, for instance as reported in India6 and Nepal.7 This is also the case even in countries where JE is controlled by vaccination in humans: recent surveys in Korea revealed prevalences of 12.1% in goats8 and 51.3% in cattle.9 However, relatively high antibody prevalences in animals were also reported in Singapore, 10,11 where pig farming has been eliminated and human cases have been rare since the early 1990s. 12

Java, one of the main islands of the Indonesian archipelago, constitutes an environment with a small size of pig population: it is difficult to raise pigs in this island because of the religious precepts and laws of the Muslim community. Consistent with the small swine population, laboratory-confirmed JE patients are rare in Java Island, if any. 13 However, studies on natural JEV activities, though limited, reported virus isolation from mosquitoes 1418 and swine 15 in and around Jakarta, West Java. Some of these studies also determined Culex tritaeniorhynchus and Culex gelidus as the predominant vectors. 17,18

Antibody surveys have also been limited in Java Island. In contrast to the small swine population, relatively high prevalence rates have been reported: for humans 8.4% in Madjalengka, West Java 19 and approximately 10% 20 or 2% 21 in Surabaya, East Java; for pigs approximately 90% at a slaughterhouse in Jakarta 22; and for horses 50% near Jakarta. 23 However, the antibody titers in these studies were determined mainly by a hemagglutination-inhibiting (HAI) test that shows high serologic cross-reactions against dengue viruses, other flavivirus members distributed in Indonesia. Although some studies 21,23 used neutralization tests, which are more specific than HAI tests for differentiation from anti-dengue antibodies, the cross-reactivity between JEV and each of four dengue viruses in their neutralization test systems, and neutralizing antibody titers against dengue viruses in samples positive for antibodies to JEV, were not seen. Even using neutralization tests, it is highly probable that significant levels of cross-reactivity against dengue viruses may cause false-positive results for JEV antibodies, particularly in areas where dengue is highly endemic, like Indonesia. 24

The present study was carried out to survey antibodies to JEV in inhabitants of Jakarta and Surabaya. A total of 2,962 sera were examined for JEV antibodies by a neutralization test. To eliminate potential false-positive results in the neutralization test because of serologic cross-reactivities against dengue viruses, all the samples found positive for neutralizing antibodies against JEV were examined for neutralizing antibodies against any of four types of dengue viruses. The results indicated that 2.2% and 1.8% of populations in Jakarta and Surabaya, respectively, had neutralizing antibody titers of ≥ 1:160 in a 90% plaque reduction assay, indicating that people were exposed to natural JEV infections in Java Island, an area with a small swine population.

MATERIALS AND METHODS

Study subjects.

All the sera used in this survey were also used in our earlier surveys of antibodies to a different infectious agent.25,26 A total of 1,211 serum samples were collected from patients at general practitioners and hospitals in Jakarta in 2001, and 1,751 sera at the Emergency Unit of Doctor Soetomo Hospital in Surabaya from 1999 to 2000. The survey populations were mostly the inhabitants of Jakarta or Surabaya City and were randomly selected without any bias relating to JE. The Jakarta City had a population of 7,423,379 people in 661.52 km2 in 2001, 27 whereas the Surabaya City had a population of 2,709,334 people in 326.36 km2 in 2000 28; thus, the present survey populations corresponded to 0.016% and 0.065% of the total populations of Jakarta and Surabaya, respectively. The survey subjects ranged from 20 to 85 years of age in the Jakarta samples and from 0 to 100 years of age in the Surabaya samples. Mean ages (±standard deviation) in the Jakarta samples were 39.9 (±12.3) years in males, 42.3 (±14.1) years in females, and 41.2 (±13.4) years in total, whereas those in the Surabaya samples were 44.7 (±21.2) years in males, 45.1 (±19.6) years in females, and 44.9 (±20.4) years in total: there were no significant differences between genders (P > 0.05 by the Student’s two-sample t test) in both samples (see Tables 3 and 4 for age and gender compositions). The gender (male: female) compositions in the present survey populations were 46%:54% in Jakarta and 52%:48% in Surabaya, whereas those of the general populations were 52%:48% in Jakarta and 50%:50% in Surabaya. Serum samples from babies < 6 months of age, which may contain maternally transferred antibodies, were not used in this survey. For transportation of sera at room temperature from Indonesia to Japan, sodium azide was added to the serum samples at a final concentration of 0.1%. The study protocol was reviewed and approved by the Ethical Committee of Kobe University Graduate School of Medicine (Ethical Committee Approval Number 561).

Antibodies.

Hyperimmune mouse ascitic fluids (HMAFs) against the Nakayama strain of JEV, the Mochizuki strain of dengue type 1 virus (DENV1), the New Guinea C (NGC) strain of dengue type 2 virus (DENV2), the H87 strain of dengue type 3 virus (DENV3), and the H241 strain of dengue type 4 virus (DENV4) have been described previously. 29 Briefly, these were collected from adult ICR mice, which were repeatedly immunized with each of the JEV and DENV1–4 in a form of 10% homogenate of suckling mouse brain, followed by the inoculation with sarcoma 180 cells.

Serology.

Neutralizing antibodies were titrated using plaque reduction assays performed with the Nakayama strain of JEV in the absence of complement, as previously described. 30 Briefly, 2-fold serial dilutions of test specimens starting from 1:10 were mixed with JEV and incubated overnight on ice. The antibody-virus mixture was then titrated on Vero cells. The neutralization titer was expressed as the maximum serum dilution yielding a 90% reduction in plaque number. For testing human sera, the serum dilution started from 1:160, because sodium azide contained in the sera affected most of the Vero cell monolayers at serum dilutions of ≤ 1:80. Antibodies to each of DENV1–4 were titrated by neutralization tests, as described previously for JEV antibody titration, except for the inclusion of rabbit complement (Low-Tox-M Rabbit Complement; Cedarlane, Hornby, Canada) in the virus-antibody mixture at a final concentration of 5% and the use of immunochemical staining to count foci. The viruses used for the tests were DENV1 (Mochizuki), DENV2 (NGC), DENV3 (H87), and DENV4 (H241). 29 The addition of complement usually showed 4- to 8-fold increases in neutralizing antibody titer in our test system. For immunostaining, cells were fixed, blocked with phosphate-buffered saline (PBS) containing normal horse serum at 1%, and then incubated serially with HMAF specific for each dengue virus, biotinylated anti-mouse IgG, the avidin-biotin complex (ABC) reagents, and the VIP substrate (Vector Laboratories, Burlingame, CA).

Statistical analysis.

The significance of differences in antibody prevalence was evaluated by the χ2 test with the Yates’ correction factor. Significance of difference in geometric mean neutralizing antibody titers was evaluated by the Student’s two-sample t test. Probability levels (P) of < 0.05 were considered significant.

RESULTS

Pig populations.

The pig populations in Java Island obtained from statistics from 1990 to 2006, available in literature, 3134 were 126,000–432,000 heads with an average of 232,353 per year; calculated to be 0.99–3.39 with an average of 1.82 heads/ km2 as adjusted by the land area. These numbers were considerably smaller than the corresponding numbers in a pig-raising area in Indonesia (Bali Island, neighboring Java) 3134 and another country (Japan). 35 Specifically, the average pig populations and their densities were, respectively, 965,294 heads and 171.36 heads/km2 in Bali Island and 10,222,062 heads and 27.05 heads/km2 in Japan.

Levels of cross-neutralizing antibodies.

Our neutralization test system showed small levels of cross-reactivity among JEV and DENV1–4, as previously described. 29 Specifically, HMAF against each of DENV1–4 showed neutralizing antibody titers of 1:5,120, or 1:10,240 against the homologous virus, but ≤ 1:10 against JEV, indicating that the cross-neutralization titers were ≤ 1/512 of the specific titer. More specifically, HMAF against DENV1 showed titers of 1:5,120 against DENV1 and < 1:10 against JEV, indicating a difference of > 512-fold. Similarly, 512-, > 1,024-, and 512-fold differences were shown between homologous virus and JEV in titers of HMAFs against DENV2, DENV3, and DENV4, respectively. Although all the neutralization tests for the previous comparisons were performed in the presence of complement to increase the test sensitivity, 29 we included complement only in the test for dengue antibodies but not for JEV antibodies in the present survey; thus, the difference in titers against JEV and dengue viruses seems to be larger than those described previously. Although different assay systems were used for this comparison (focus-forming method for DENV1–4 but plaque-forming method for JEV), previous experiments in our laboratory have shown that equivalent neutralizing antibody titers were obtained with both methods. 36

To further evaluate the effect of cross-reactivity of dengue virus antibodies on neutralizing antibody titers against JEV, we examined a mixture of HMAFs against DENV1–4 for neutralizing antibodies against JEV. For this experiment, the mixture was prepared with an equal volume of HMAF and complement was included in the virus-antibody mixture. Although data are not shown, all 11 different combinations of any of the 4 HMAFs (against DENV1–4) showed undetectable antibody titers (< 1:10), suggesting that antibodies to one type of dengue virus do not synergistically increase their titers against JEV in the presence of antibodies to other type(s) of dengue virus. In addition, to examine if the presence of dengue virus antibodies would increase a titer against JEV, serial 2-fold dilutions of HMAF against JEV were mixed with the mixture of 4 HMAFs (against DENV1–4). The results showed no increase in JEV antibody titers at any dilutions of HMAF against JEV within the range from < 1:10 to 1:160 even in the presence of complement in the neutralization test system (data not shown). Finally, we assessed the effect of HMAFs against DENV1–4 on titers of JEV antibodies included in human sera. A human serum sample that had a titer of 1:1280 against JEV (see Table 2, a male sample aged 39 years from Surabaya) was diluted 128-fold with each of HMAFs against DENV1–4 or the mixture of 4 HMAFs, as well as PBS as a control. The complement was not included in the virus-antibody mixture in this experiment. The result showed that the neutralizing antibody titer against JEV was ≤ 1:10 in all serum-HMAF and serum-PBS mixtures without any increase resulting from the presence of HMAF at titers of 1:5,120–1:10,240 or 1:1,280–1:2,560 (when 4 HMAFs were mixed) against dengue viruses (data not shown). Although these comparisons have limitations because of the use of HMAFs prepared with standard dengue virus strains, these results support the data of the previous experiment showing the cross-neutralization titers within ≤ 1/512 of the specific titer and indicate that the cross-reactivity did not increase even under the condition where antibodies against more than one type of dengue virus were included.

Neutralizing antibody titers against dengue viruses.

Examination of 2,962 sera for neutralizing antibodies against JEV provided titers of ≥ 1:160 in 27 samples collected in Jakarta and 31 samples collected in Surabaya. Tables 1 and 2 show the gender and age of these samples, as well as the neutralizing antibody titers against JEV and DENV1–4. The maximum titers against dengue viruses were 1:5,120 for DENV1 and DENV3, and 1:10,240 for DENV2 and DENV4. On the basis of the previous cross-neutralization experiment using HMAF, these results indicated that all the present samples showing neutralizing titers of ≥ 1:160 against JEV possessed specific JEV antibodies: our system does not seem to show these high JEV antibody titers only because of cross-reactivities of dengue antibodies contained in these samples. Although sodium azide was included in human sera at 0.1%, we confirmed no effect of this chemical on neutralizing antibody titers by demonstrating that titers in 10 selected human sera were not altered after extensive dialysis against PBS to remove sodium azide from the sera (data not shown).

Prevalence of antibodies to JEV.

Serum samples collected in Jakarta and Surabaya were grouped in 10-year increments, except for those over 60 (for Jakarta) and 80 (for Surabaya) years of age, which were grouped in one age group. As shown in Tables 3 and 4, the overall prevalence of antibodies showing titers of ≥ 1:160 against JEV was 2.2% in Jakarta and 1.8% in Surabaya.

No significant differences in JEV antibody prevalence were detected between males and females and between ages in Jakarta populations (P > 0.05). On the other hand, JEV antibody prevalence in Surabaya populations showed significant differences between age groups of ≤ 9 and 50–59 years (4.2% versus 0.4%, P < 0.05) and between age groups of 10–19 and 40–49 years (4.9% versus 0.4%, P < 0.05) or 50–59 years (4.9% versus 0.4%, P < 0.01). The prevalence in males was not significantly different from those in females in each age group and the total age group of the Surabaya population (P > 0.05). Comparisons between Jakarta and Surabaya populations of corresponding ages/genders showed no significant differences, except for total populations in the age group of 40–49 years (3.5% versus 0.4%, P < 0.05).

Quantitative analysis of neutralizing antibody titers against JEV.

Individual neutralizing antibody titers against JEV ranged from < 1:160 to 1:640 and < 1:160 to 1:1,280 in the Jakarta and Surabaya populations, respectively (Tables 1 and 2). Comparisons of geometric mean neutralizing antibody titers using samples that showed titers of ≥ 1:160 indicated no statistically significant differences between Jakarta (1:248) and Surabaya (1:256) populations (P > 0.05). In addition, no differences were detected between genders or between age groups in each of the Jakarta and Surabaya populations that showed titers of ≥ 1:160 (P > 0.05; data not shown).

DISCUSSION

The present study revealed that 2.2% and 1.8% of Jakarta and Surabaya populations, respectively, had neutralizing antibody titers of ≥ 1:160 against JEV. Because there should be populations showing titers of < 1:160, the real prevalence of JEV antibodies is considered to be higher than these percentages. In Indonesia where regular mass vaccination programs against JE are not used, the presence of neutralizing antibodies indicate previous exposure(s) to JEV infection. Because the present study eliminated the possibility to increase JE seropositivity resulting from high serologic cross-reactivity against dengue viruses, the results indicate a relatively high prevalence of antibodies to JEV among inhabitants in Jakarta and Surabaya. On the other hand, there remains the possibility that some of the present subjects possessing neutralizing antibodies against JEV had been infected with JEV outside Java Island where JE might be endemic.

The number and density of pigs were related to JE incidence, when these factors in Java Island were compared with those in Bali Island during and around the period of serum collection in Jakarta (2001) and Surabaya (1999–2000). Specifically, information from the statistics 3134 indicates the pig populations in Java were considerably smaller than those in Bali. Although the numbers of laboratory-confirmed JE patients were not reported from the Indonesian government, an earlier literature reported no or few, if any, JE patients in Java Island. 13 On the other hand, JE cases have been reported in Bali 37,38 and in travelers returning from Bali. 3942 One study revealed that the annual incidence per 100,000 children less than 10 years of age in Bali was 7.1. 38 In Japan, more than a thousand JE cases occurred annually in the past, but following the wide distribution of an inactivated vaccine in 1967, the annual number of cases dramatically reduced, staying below 10 out of approximately 100 million people since 1992, 43 even though pigs have been raised in this country. 35

In contrast, the prevalence of JEV antibodies does not seem to be significantly related to the pig population and the number of patients, when comparison was made in Java, Bali, and Japan. In Japan, the national JE surveillance program reported that 2 of 227 (0.9%) people 40 to 49 years of age had neutralizing antibody titers of ≥ 1:160 in 2004 43: the effect of vaccination carried out before 15 years of age is considered negligible in this age group and thus the neutralizing antibodies possessed by this population would represent natural exposure to JEV infection. This percentage (0.9%) was approximately a half of those obtained in the present study for populations of Jakarta (2.2%) and Surabaya (1.8%) who showed corresponding antibody titers (≥ 1:160). Although seroprevalence data in Balinese were not available, 70% of pigs in Bali possessed HAI antibodies. 38 Because the national JE surveillance program in Japan reported 50–100% seropositivities in pigs in most of the southern and western areas of Japan, 43 the natural JEV activity would be comparable in Bali and Japan. Thus, it should be emphasized that in Java, JEV transmission still continues despite a relatively small number of pigs and no (or few) human JE cases. This situation resembles that in Singapore where pigs are not raised. 1012

Low JE incidence in Japan is considered to be a result of vaccination, whereas that in Java is probably related to a small pig population. One of the potential reasons for low incidence under continued JEV transmission in Java may be the inoculum size per exposed person, which is closely related to the number and density of viremic pigs. It is speculated that the ratio of subclinical to clinical infections decreases with decreased inoculum size. Because the tropical climate and the style of pig farming are similar in neighboring islands of the same country, the difference in the number of JE cases between Java and Bali seems to be mostly attributed to the difference in pig population. Further studies are needed to elucidate other epidemiologic factors involved in natural JEV activities in Indonesia. Although JEV activities in an environment with a small swine population are poorly understood, they would provide important indications and implications for the ecology of JEV and epidemiology of JE.

Comparative statistical analyses of JEV antibody prevalences between genders, age groups, or areas detected significant differences in a few populations. A possible explanation for the difference would be a difference in the opportunities to acquire natural exposure to JEV infections, but factor(s) involved in the opportunities could not be identified. Because the annual JE incidence was almost constant during several years in other endemic countries in Asia without introduction of vaccination,3 and therefore natural JEV activities are considered similar in each year also in Java, we consider at this moment that the significant difference is a random variation and people are equally exposed to infective mosquito bites. Jakarta is more urbanized and has a wider city area than Surabaya. Although no recent reports on seasonal abundance of vector (Culex) mosquitoes are available, exposure to infected mosquito bites seems equally frequent in Jakarta and Surabaya inhabitants, because Culex mosquitoes are generally believed to have the ability to fly for long distances and in this case infected mosquitoes are considered to move from rural area surrounding these cities.

In conclusion, people in Jakarta and Surabaya are still exposed to natural JEV infections, despite a relatively small number of pigs. On the other hand, the JE incidence in Java is very few, if any, to the best of our knowledge. Thus, the introduction of mass vaccination against JE may not be urgently needed in this region. However, considering relatively high antibody prevalence, JEV is circulated in nature and people are exposed to infective mosquito bites: the transmission cycle through vector mosquitoes seems to be established. Therefore, it would be required to continuously monitor the incidence of JE and to be prepared for a potential increase in JE patients.

Table 1

Neutralizing antibody titers against Japanese encephalitis virus (JEV) and DENV1–4 in Jakarta samples*

Table 1
Table 2

Neutralizing antibody titers against Japanese encephalitis virus (JEV) and DENV1–4 in Surabaya samples*

Table 2
Table 3

Prevalence of Japanese encephalitis virus (JEV) antibodies in the Jakarta population

Table 3
Table 4

Prevalence of Japanese encephalitis virus (JEV) antibodies in the Surabaya population

Table 4

*

Address correspondence to Eiji Konishi, Department of International Health, Kobe University Graduate School of Health Sciences, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0412, Japan. E-mail: ekon@kobe-u.ac.jp

Authors’ addresses: Eiji Konishi, Yohei Sakai, and Yoko Kitai, Department of International Health, Kobe University Graduate School of Health Sciences, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0412, Japan, Tel/Fax: 81-78-796-4594, E-mails: ekon@kobe-u.ac.jp, sakatom2002@yahoo.co.jp, and sunbaby_spring5nw9@yahoo.co.jp. Atsushi Yamanaka, Indonesia-Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases, Institute of Tropical Disease, Airlangga University, Kampus C, UNAIR, Jl. Mulyorejo, Surabaya 60115, Indonesia, Tel/Fax: 62-31-599-2445, E-mail: paradios99@yahoo.co.jp.

Acknowledgments: We thank Dr. Pudjiatmoko of the Indonesian Embassy in Tokyo, Japan and Mr. Kris Cahyo Mulyatno of Institute of Tropical Disease, Airlangga University, Indonesia for their assistance in collecting information about sizes of human and pig populations in Indonesia.

Financial support: This work was supported in part by a grant-in-aid through the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and through Research on Emerging and Re-emerging Infectious Diseases, the Ministry of Health and Welfare of Japan.

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