Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. I. Investigations on Antibody Levels to Japanese Encephalitis Virus in Swine Sera and Vector Mosquito in Okinawa,Miyako and Ishigaki Islands

From 1985 to 1989, serum specimens of swine raised in the northern, central and southern areas in Okinawa island were examined for Japanese encephalitis (JE) virus antibody by ELISA and hemagglutination-inhibition test. The antibody positive rate was found to be higher in the north and central than in the south. The 2-mercaptoethanol sensitive antibody to JE was detected mostly in June and July, and occasionally in other months except February and March. There was no month when all specimens from three areas turned antibody-negative simultaneously, indicating that JE virus transmission to swine lasted longer in Okinawa island than in other temperate areas in Japan. From 1986 to 1991, the vector mosquitoes (Culex tritaeniorhynchus) were collected in a pig farm in the south of Okinawa island. A total of 153 strains of JE virus was isolated from the vector mosquitoes mainly in June. In Miyako and Ishigaki islands, the antibody positive rate in swine sera was found to be extremely low, compared with that in Okinawa island. In Miyako island, where no paddy rice field is cultivated, a few adults as well as larvae of the vector mosquito were collected, while in Ishigaki island, where there are many watered rice fields, a lot of adults as well as larvae were collected. Although the environmental situation is quite different between the two islands, JE virus transmission appeared to be very low in both islands.     

Detection of an invasive gall-inducing pest, Quadrastichus erythrinae (Hymenoptera: Eulophidae), causing damage to Erythrina variegata L. (Fabaceae) in Okinawa Prefecture, Japan

In 2005, Quadrastichus erythrinae Kim, 2004 (Hymenoptera: Eulophidae), which induces stem, petiole, and leaf galls on Erythrina variegata L. (Fabaceae), was found on the following six islands in Okinawa Prefecture, Japan: Okinawa, Kume, Miyako, Ishigaki, Iriomote, and Hateruma. Galls were also found in Vietnam. In Japan, no further infestation records have been reported from any of Japan's other south‐western prefectures where Erythrina species grow. Because no Erythrina galls were observed in Okinawa Prefecture before 2005, Q. erythrinae seems to have invaded quite recently.     

Molecular Epidemiology of Japanese Encephalitis Virus in Okinawa

In order to elucidate the molecular characteristics of Japanese encephalitis (JE) virus in Okinawa, 23 strains of JE virus isolated in a 25-year span were sequenced for the 240 nucleotides of the C-preM junction region and 111 nucleotides of the E gene region and compared with those of reference strains isolated in mainland Japan. The results of phylogenic analysis showed that although all the Okinawan isolates showed more than 96% homology in the nucleotide sequence in each region, they were chronologically divided into two groups: the old group (nine strains) and a new group (14 strains). On the other hand, in a comparison with reference strains in mainland Japan, the Okinawan isolates showed more than 94% nucleotide sequence homology in both regions, indicating that the Okinawan strains belong to the same genotype as that of JE strains in mainland Japan. The nucleotide homology of the old group was relatively higher than that of the new group. Among the 14 strains in the new group, 13 strains were isolated from mosquitoes collected from a pig farm from 1986 through 1992. These strains showed higher nucleotide divergence than the old group strains, isolated from mosquitoes and swine sera collected at several sites, in both regions. A nucleotide substitution at the position 1920 in the E gene was identified in three isolates. This substitution generated an asparagine-proline-threonine sequence capable of serving as an attachment site of carbohydrate.     

Distribution of clubroot disease of a cruciferous weed, Cardamine flexuosa, in major isolated islands, Hokkaido and Okinawa in Japan

To investigate the distribution of clubroot of a cruciferous weed, Cardamine flexuosa, caused by Plasmodiophora brassicae, field surveys were conducted in Hokkaido, Aomori, and Okinawa, and major isolated islands in Japan during 1993–2004. The disease was newly recorded in Aomori and nine islands in five different prefectures, including Sado (Niigata), Oki (Shimane), Mishima (Yamaguchi), Tsushima, Iki and Goto (Nagasaki), and Koshiki, Yakushima, and Tanegashima (Kagoshima). The diseased plants were not found in Hokkaido and Okinawa (islands of Okinawa, Kumejima, Ishigaki, Iriomote, and Kohama). However, inoculation tests showed that most C. flexuosa collected from Hokkaido and Okinawa included many susceptible plants. The result suggests that resistance of the plants is not the reason that the disease was not found in these areas. An erratum to this article is available at .     

Neutralizing antibody responses to Japanese encephalitis vaccine in children

Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of JE virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees had no N antibody to JE virus before the first vaccination and were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithms) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The seroconversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vacciness were also bled before the second vaccination. Most of them showed a considerably high N antibody response against the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization two shots, at least, of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of JE virus.     

A genetic analysis of the Sakishima islanders reveals no relationship with Taiwan aborigines but shared ancestry with Ainu and main‐island Japanese

The Sakishima islands are members of the Ryukyu island chain, stretching from the southwestern tip of the Japanese archipelago to Taiwan in the East China Sea. Archaeological data indicate cultural similarities between inhabitants of prehistoric Sakishima and Neolithic Taiwan. Recent studies based on tooth crown traits show remarkably high inter‐island diversity among Ryukyu islanders, suggesting that the Sakishima islanders might have genetic backgrounds distinct from main‐island Okinawa people. To investigate the genetic diversity of the Ryukyu islanders, we analyzed mtDNA, Y chromosome, and autosomal short tandem repeat loci in a sample of main‐island Okinawa people and Sakishima (Miyako and Ishigaki) islanders whose participated in a previous study of tooth crown morphology. Our phylogenetic analysis of maternal (mtDNA) and paternal (Y chromosome) lineages shows that the Sakishima islanders are more closely related to people from the Japanese archipelago than to Taiwan aborigines. Miyako islanders and the Hokkaido Ainu have the first and second highest frequencies (respectively) of the Y‐chromosomal Alu‐insertion polymorphism, which is a presumable Jomon marker. Genetic diversity statistics show no evidence of demographic reduction or of extreme isolation in each island's population. Thus, we conclude that 1) Neolithic expansion from Taiwan did not contribute to the gene pool of modern Sakishima islanders, 2) male‐lineage of the Ryukyu islanders likely shares a common ancestor with the Hokkaido Ainu who are presumably direct descendants of the Jomon people, and 3) frequent reciprocal gene flow among islands has masked the trace of common ancestry in the Ryukyu island chain. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang,an Eastern Province of China

BackgroundSporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains.Conclusion/SignificancesJE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.     

A comparative study on sweating pattern and heat tolerance. A field study on residents of Okinawa in summer

We proposed a numerical index for evaluating human heat tolerance which is practically useful. The adaptive changes in heat tolerance of unacclimatized subjects during short-term heat acclimatization could be evaluated by this index. The present investigation was performed to examine whether or not our index can reflect effects on heat tolerance of a long-term acclimatization as seen in subtropical natives. Nineteen young male residents born and raised in Okinawa (subtropical zone), 18 male residents born in the Japan Main Islands (temperate zone) but moved to Okinawa and 25 male students in Kyoto (temperate zone) were chosen to be subjects. Their age were from 19 to 29 years. Sweating reaction was examined for 60 min in summer, by immersing legs in stirring water of 42°C by using a mobile climatic chamber of 30°C with 70 RH. Local sweat samples from the chest and back were collected at 15 min interval by the filter paper method. Sweating reaction of the residents born and raised in Okinawa was characterized by smaller volume of sweating and lower salt concentration in sweat, while the rise in rectal temperature and increase in heart rate differ less between the three groups. It is concluded that residents born and raised in Okinawa showed higher heat tolerance than the other two groups when assessed by our factorial index method.     

Sex pheromone and related compounds in the Ishigaki and Okinawa strains of the tussock moth Orgyia postica (Walker) (Lepidoptera: Lymantriidae)

Two distinct electroantennographycally active (EAG-active) components, A and B, and a weakly active component C were found in a solvent extract from virgin females of the Ishigaki strain of the tussock moth, Orgyia postica (Walker). Components A, B, and C were found in the extract of the females at 4.0, 0.5, and 4.0 ng/female respectively. Components A, B, and C were identified as (6Z,9Z,11S,12S)-11,12-epoxyhenicosa-6,9-diene [(11S,12S)-1: posticlure], (6Z)-henicos-6-en-11-one (2), and (6Z,9Z)-henicosa-6,9-diene (3), respectively. Component B was absent in the extract from the Okinawa strain, in which components A and C were present at 2.0 and 1.5 ng/female respectively. (11S,12S)-1 and the racemic mixture showed attractiveness for both the Okinawa and Ishigaki strains, whereas (11R,12R)-1 did not. The addition of 2 significantly reduced the trap catches with (11S,12S)-1 on the Okinawa strain which lacked 2, while there was no significant inhibitory effect on the Ishigaki strain. The addition of 3 to (11S,12S)-1 did not significantly affect trap catches at Ishigaki or Okinawa. This confirmed that the attractant pheromone of O. postica of the Ishigaki strain is also (11S,12S)-1.     

Epidemiology of Japanese Encephalitis in the Philippines: A Systematic Review

BackgroundJapanese encephalitis virus (JEV) is an important cause of encephalitis in most of Asia, with high case fatality rates and often significant neurologic sequelae among survivors. The epidemiology of JE in the Philippines is not well defined. To support consideration of JE vaccine for introduction into the national schedule in the Philippines, we conducted a systematic literature review and summarized JE surveillance data from 2011 to 2014.MethodsWe conducted searches on Japanese encephalitis and the Philippines in four databases and one library. Data from acute encephalitis syndrome (AES) and JE surveillance and from the national reference laboratory from January 2011 to March 2014 were tabulated and mapped.ResultsWe identified 29 published reports and presentations on JE in the Philippines, including 5 serologic surveys, 18 reports of clinical cases, and 8 animal studies (including two with both clinical cases and animal data). The 18 clinical studies reported 257 cases of laboratory-confirmed JE from 1972 to 2013. JE virus (JEV) was the causative agent in 7% to 18% of cases of clinical meningitis and encephalitis combined, and 16% to 40% of clinical encephalitis cases. JE predominantly affected children under 15 years of age and 6% to 7% of cases resulted in death. Surveillance data from January 2011 to March 2014 identified 73 (15%) laboratory-confirmed JE cases out of 497 cases tested.SummaryThis comprehensive review demonstrates the endemicity and extensive geographic range of JE in the Philippines, and supports the use of JE vaccine in the country. Continued and improved surveillance with laboratory confirmation is needed to systematically quantify the burden of JE, to provide information that can guide prioritization of high risk areas in the country and determination of appropriate age and schedule of vaccine introduction, and to measure the impact of preventive measures including immunization against this important public health threat.     

Geographic distribution of phytoseiid mite species (Acari: Phytoseiidae) on crops in Okinawa,a subtropical area of Japan

We extensively identified phytoseiid mites co‐occurring with spider mites on crops throughout the islands of Okinawa, southwestern Japan to obtain fundamental information for controlling spider mites in the area using natural enemies. Of the 19 species found, three were new to Japan and eight were new to Okinawa. Neoseiulus womersleyi was the most common species with respect to the distribution range. Following N. womersleyi, six species including Amblyseius eharai were common, whereas N. californicus was not, suggesting that the recent change in dominant species from N. womersleyi to N. californicus observed on the main islands of Japan had not happened in Okinawa. On one small island (Tarama Island) located in the southwestern part of Okinawa, N. womersleyi was not found, and instead N. longispinosus, which is morphologically similar to N. womersleyi and has not been found in Japan, occurred. This suggests interisland differences in the relative frequencies of N. womersleyi and N. longispinosus in Okinawa.     

Comparative Spatial Dynamics of Japanese Encephalitis and Acute Encephalitis Syndrome in Nepal

Japanese Encephalitis (JE) is a vector-borne disease of major importance in Asia. Recent increases in cases have spawned the development of more stringent JE surveillance. Due to the difficulty of making a clinical diagnosis, increased tracking of common symptoms associated with JE—generally classified as the umbrella term, acute encephalitis syndrome (AES) has been developed in many countries. In Nepal, there is some debate as to what AES cases are, and how JE risk factors relate to AES risk. Three parts of this analysis included investigating the temporal pattern of cases, examining the age and vaccination status patterns among AES surveillance data, and then focusing on spatial patterns of risk factors. AES and JE cases from 2007–2011 reported at a district level (n = 75) were examined in relation to landscape risk factors. Landscape pattern indices were used to quantify landscape patterns associated with JE risk. The relative spatial distribution of landscape risk factors were compared using geographically weighted regression. Pattern indices describing the amount of irrigated land edge density and the degree of landscape mixing for irrigated areas were positively associated with JE and AES, while fragmented forest measured by the number of forest patches were negatively associated with AES and JE. For both JE and AES, the local GWR models outperformed global models, indicating spatial heterogeneity in risks. Temporally, the patterns of JE and AES risk were almost identical; suggesting the relative higher caseload of AES compared to JE could provide a valuable early-warning signal for JE surveillance and reduce diagnostic testing costs. Overall, the landscape variables associated with a high degree of landscape mixing and small scale irrigated agriculture were positively linked to JE and AES risk, highlighting the importance of integrating land management policies, disease prevention strategies and promoting healthy sustainable livelihoods in both rural and urban-fringe developing areas.     

Distribution of marine birnavirus (MABV) in marine organisms from Okinawa,Japan, and a unique sequence variation of the VP2/NS region

Distribution of marine type of Aquabirnavirus (MABV) was examined in shellfish and fish from Okinawa and Ishigaki Islands, Japan, where water temperature is higher than 25°C through the year. Genome detection and virus isolation were performed for shellfish and fish samples, and the results revealed the prevalent distribution of MABV in diverse species in the area, although isolation was not frequently. Detection rate of MABV genome in bivalves was higher than gastropods, which was similar result to former report in mainland of Japan. Furthermore, the unique five-nucleotide deletion was found with a high rate of occurrence in the MABV genome from shellfish and fish. This study showed distribution status of MABV in organisms in subtropical waters by wide monitoring, and discovered new genome variation in VP2/NS region of this virus.     

Thermostability of Japanese encephalitis vaccine produced in India.

Different batches of bulk vaccine, final bulk at in-process level, finished freeze-dried and reconstituted Japanese encephalitis vaccine were assayed for their stability at temperatures of 22, 37 and 40 degrees C. After ultrazonal purification of 50 times concentrated brain suspension, JE Bulk vaccine was found to be stable for up to 2 years at 4 degrees C, however, the percentage loss in potency (log 10 N antibody titre) after 2.5 years was 24%. Three-times concentrated final bulk showed rapid deterioration by the fourth week at 37 and 40 degrees C. Freeze-dried JE vaccine maintained at 22 degrees C for 28 weeks did not show perceptible deterioration. At 37 degrees C, the same vaccine started showing deterioration (14%) after 8 weeks whereas at 40 degrees C the loss of potency was 24% after 8 weeks. The freeze-dried vaccine was found to be stable for up to 2 weeks duration at 40 degrees C.     

Study of seroconversion of antibody to human T-cell lymphotropic virus type-I in children of Okinawa, Japan

From 1983 to 1986, 1,813 children in nursery schools in Ishigaki Island and 1,228 children under 15 years old in the rural area in the Yaeyama District of Okinawa, Japan, were tested for anti-HTLV-I; 18 children (1.0%) in Ishigaki Island and 39 children (3.2%) in the rural area were positive. In order to survey when anti-HTLV-I developed in these children, their older serum samples were investigated retrospectively for 1 to 5 years. Two cases of seroconversion from anti-HTLV-I negative to positive were found between the age of 2 and 4, and there were no cases of seroconversion over 4 years old. Among the children who suffered maternal transmission of HTLV-I and developed anti-HTLV-I before the age of 15, about 80% of the children were considered to have anti-HTLV-I before the age of 2, and the remainder developed anti-HTLV-I before the age of 4.     

Prevalence of Neutralizing Antibodies to Japanese Encephalitis Virus among High-Risk Age Groups in South Korea, 2010

After an extensive vaccination policy, Japanese encephalitis (JE) was nearly eliminated since the mid-1980s in South Korea. Vaccination in children shifted the affected age of JE patients from children to adults. However, an abrupt increase in JE cases occurred in 2010, and this trend has continued. The present study aimed to investigate the prevalence of neutralizing antibodies to the JE virus (JEV) among high-risk age groups (≥40 years) in South Korea. A plaque reduction neutralization test was conducted to evaluate the prevalence of neutralizing antibodies to JEV in 945 subjects within four age groups (30–39, 40–49, 50–59, and 60–69 years) in 10 provinces. Of the 945 enrolled subjects, 927 (98.1%) exhibited antibodies against JEV. No significant differences were found in the prevalence of neutralizing antibodies according to sex, age, or occupation. However, there were significant differences in the plaque reduction rate according to age and occupation; oldest age group had a higher reduction rate, and subjects who were employed in agriculture or forestry also had a higher value than the other occupations. We also found that three provinces (Gangwon, Jeonnam, and Gyeongnam) had a relatively lower plaque reduction rate than the other locations. In addition, enzyme-linked immunosorbent assays were conducted to determine recent viral infections and 12 (2.2%) subjects were found to have been recently infected by the virus. In conclusion, the present study clearly indicated that the prevalence of neutralizing antibodies has been maintained at very high levels among adult age groups owing to vaccination or natural infections, or both. In the future, serosurveillance should be conducted periodically using more representative samples to better understand the population-level immunity to JE in South Korea.     

The Effect of Vaccination Coverage and Climate on Japanese Encephalitis in Sarawak,Malaysia

Background

Japanese encephalitis (JE) is the leading cause of viral encephalitis across Asia with approximately 70,000 cases a year and 10,000 to 15,000 deaths. Because JE incidence varies widely over time, partly due to inter-annual climate variability effects on mosquito vector abundance, it becomes more complex to assess the effects of a vaccination programme since more or less climatically favourable years could also contribute to a change in incidence post-vaccination. Therefore, the objective of this study was to quantify vaccination effect on confirmed Japanese encephalitis (JE) cases in Sarawak, Malaysia after controlling for climate variability to better understand temporal dynamics of JE virus transmission and control.

Methodology/principal findings

Monthly data on serologically confirmed JE cases were acquired from Sibu Hospital in Sarawak from 1997 to 2006. JE vaccine coverage (non-vaccine years vs. vaccine years) and meteorological predictor variables, including temperature, rainfall and the Southern Oscillation index (SOI) were tested for their association with JE cases using Poisson time series analysis and controlling for seasonality and long-term trend. Over the 10-years surveillance period, 133 confirmed JE cases were identified. There was an estimated 61% reduction in JE risk after the introduction of vaccination, when no account is taken of the effects of climate. This reduction is only approximately 45% when the effects of inter-annual variability in climate are controlled for in the model. The Poisson model indicated that rainfall (lag 1-month), minimum temperature (lag 6-months) and SOI (lag 6-months) were positively associated with JE cases.

Conclusions/significance

This study provides the first improved estimate of JE reduction through vaccination by taking account of climate inter-annual variability. Our analysis confirms that vaccination has substantially reduced JE risk in Sarawak but this benefit may be overestimated if climate effects are ignored.     

Effectiveness of the Viet Nam Produced,Mouse Brain-Derived,Inactivated Japanese Encephalitis Vaccine in Northern Viet Nam

Background

Japanese encephalitis (JE) is a flaviviral disease of public health concern in many parts of Asia. JE often occurs in large epidemics, has a high case-fatality ratio and, among survivors, frequently causes persistent neurological sequelae and mental disabilities. In 1997, the Vietnamese government initiated immunization campaigns targeting all children aged 1–5 years. Three doses of a locally-produced, mouse brain-derived, inactivated JE vaccine (MBV) were given. This study aims at evaluating the effectiveness of Viet Nam''s MBV.

Methodology

A matched case-control study was conducted in Northern Viet Nam. Cases were identified through an ongoing hospital-based surveillance. Each case was matched to four healthy controls for age, gender, and neighborhood. The vaccination history was ascertained through JE immunization logbooks maintained at local health centers.

Principal Findings

Thirty cases and 120 controls were enrolled. The effectiveness of the JE vaccine was 92.9% [95% CI: 66.6–98.5]. Confounding effects of other risk variables were not observed.

Conclusions

Our results strongly suggest that the locally-produced JE-MBV given to 1–5 years old Vietnamese children was efficacious.     

Enzyme-linked immunosorbent assay for detection of serum antibody in children vaccinated with Japanese encephalitis vaccine

Serum antibodies in children who had been vaccinated with Japanese encephalitis (JE) vaccine were measured by enzyme-linked immunosorbent assay (ELISA) and neutralization (N) and hemagglutination-inhibition (HI) tests. Of 20 serum samples obtained after two shots of JE vaccine in the first year, all but one showed positive titers in the ELISA and N test, but five showed negative titers in the HI test. All 12 serum samples obtained after booster immunization with JE vaccine in the second year showed positive and considerably higher titers in all three tests. Moreover, a high correlation was found between the ELISA, N and HI titers. These results indicate that the ELISA is useful for detecting antibodies in subjects immunized with JE vaccine.     

Karyological and taxonomic studies of Dugesia japonica from the Southwest Islands of Japan-II

The karyotypes of Dugesia japonica from the southern part of the Southwest Islands of Japan (the Nansei Shotô) include diploidy, triploidy, mixoploidy, and mixoaneuploidy. Animals having karyotypes based on n = 8 were found on Okinawa Island, Ishigaki Island, Iriomote Island, and Yonaguni Island, while animals having karyotypes based on n = 7 were found on Okinawa Island, Miyako Island, and Ishigaki Island. Toward the northern end of this island chain, at Tanegashima Island, Yakushima Island, and Amami-Ôshima Island, karyotypes based on n = 8 occurred; and on one of these, Tanegashima Island, n = 7 also occurred. Our data provide further support of the karyotypical distinction between the two subspecies of D. japonica: D. j. japonica has karyotypes based on n = 8 and D. j. ryukyuensis has karyotypes based on n = 7. Taking into consideration the geological history of the Southwest Islands and the ties of their faunas to those of adjacent areas, we can explain the current geographical distribution of the two subspecies in these islands as the result of two separate invasions by D. japonica, one in the Miocene and one after the early Quaternary.