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The Japanese encephalitis virus (JEV), an arthropod-born Flavivirus, is the major cause of viral encephalitis, responsible for 10,000-15,000 deaths each year, yet is a neglected tropical disease. Since the JEV distribution area has been large and continuously extending toward new Asian and Australasian regions, it is considered an emerging and reemerging pathogen. Despite large effective immunization campaigns, Japanese encephalitis remains a disease of global health concern. JEV zoonotic transmission cycles may be either wild or domestic: the first involves wading birds as wild amplifying hosts; the second involves pigs as the main domestic amplifying hosts. Culex mosquito species, especially Cx. tritaeniorhynchus, are the main competent vectors. Although five JEV genotypes circulate, neither clear-cut genotype-phenotype relationship nor clear variations in genotype fitness to hosts or vectors have been identified. Instead, the molecular epidemiology appears highly dependent on vectors, hosts' biology, and on a set of environmental factors. At global scale, climate, land cover, and land use, otherwise strongly dependent on human activities, affect the abundance of JEV vectors, and of wild and domestic hosts. Chiefly, the increase of rice-cultivated surface, intensively used by wading birds, and of pig production in Asia has provided a high availability of resources to mosquito vectors, enhancing the JEV maintenance, amplification, and transmission. At fine scale, the characteristics (density, size, spatial arrangement) of three landscape elements (paddy fields, pig farms, human habitations) facilitate or impede movement of vectors, then determine how the JEV interacts with hosts and vectors and ultimately the infection risk to humans. If the JEV is introduced in a favorable landscape, either by live infected animals or by vectors, then the virus can emerge and become a major threat for human health. Multidisciplinary research is essential to shed light on the biological mechanisms involved in the emergence, spread, reemergence, and genotypic changes of JEV.

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Twenty-nine domestic piglets from pig farms located in three provinces of Thailand between 2003 and 2004 were used as sentinel animals for Japanese encephalitis virus (JEV) circulation. Piglets were used as sentinel to underline, on one hand, the role of domestic pigs as JEV amplifying host and, on another hand, to point out the interest of using sentinel animals for Japanese encephalitis surveillance. JEV activity was demonstrated through i/ antibody detection using a specific ELISA test for the identification of Immunoglobulins of class M and G, ii/ virus isolation on cell culture, after experimental mosquito inoculation for virus amplification. Almost 100% and 83% of the piglets, respectively, had specific IgG and IgM JEV antibodies and 35% yielded a virus isolate. Piglets of the growing farm industry act as virus amplifier increasing the risk of transmission for the human community. Conclusively, since piglets JEV infection appears early in life and is generally clinically unnoticed, it represents an exceptional sentinel model for human health threats, which has to be considered by health authorities.

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Although serosurvey in human or animals is a useful and straightforward strategy routinely used for public health, it often faces different types of impediments: ethics, beliefs, limitation by animal owners, hazard of access to wild animals. To survey virus circulation, we applied the enzyme-linked immunosorbent assay (ELISA) technique to detect Dengue and Japanese encephalitis (JE) virus-reactive antibodies in blood meals collected from mosquitoes without regard to the potential of mosquito species to be a virus vector. ELISA was performed on mosquito colonies and wild specimens collected from farms and urban areas. Blood meals from Aedes aegypti freshly fed on naturally infected volunteers showed the same levels of dengue immunoglobulin (Ig)G and IgM as the sera directly collected from volunteers. A significant clearance of antibodies during the digestion process started from 13 hours after blood meal, and a negative baseline was reached after 30 hours. The ELISA test performed on wild mosquitoes showed that 37% of Culex quinquefasciatus mosquitoes that engorged on humans in a dengue urban endemic area tested positive for dengue IgG, and in a JE virus-endemic area, 88% of Culex tritaeniorhynchus mosquitoes that engorged on pigs from a large pig farm tested positive for JE virus antibodies versus 11% in a small farm. The main limitation of the ELISA method is the antibody cross-reactivity among flaviviruses; also, sampling strategy should be adjusted to take into account that the actual host from which the blood meal was taken may not be determined. Nevertheless, ELISA performed on recently (1-2 days) engorged mosquito, or any other hematophagous arthropod species, could potentially be used as a "wild phlebotomist" to monitor the prevalence or emergence of a variety of pathogens, with less of the practical, ethical, or risk limitations due to direct blood collection from humans and wild or domestic animals.

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Japanese encephalitis virus (JEV) genotypes in Thailand were studied in pigs and mosquitoes collected near houses of confirmed human JEV cases in 2003-2005. Twelve JEV strains isolated belonged to genotype I, which shows a switch from genotype III incidence that started during the 1980s.

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Blood-engorged mosquitoes were collected at poultry farms during an outbreak of highly pathogenic avian influenza in Central Thailand during October 2005. These mosquitoes tested positive for H5N1 virus by reverse transcription-polymerase chain reaction (RT-PCR). Results were confirmed by limited sequencing of the H5 and N1 segments. Infection and replication of this virus in the C6/36 mosquito cell line was confirmed by quantitative real-time PCR. However, transmission by mosquitoes was not evaluated, and further research is needed. Collecting and testing mosquitoes engorged with the blood of domestic or wild animals could be a valuable tool for veterinary and public health authorities who conduct surveillance for H5N1 virus spread.

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A pilot study was designed to analyze a potential association between dengue hemorrhagic fever (DHF) incidence and, temperature computed by satellite. DHF is a mosquito transmitted disease, and water vapor and humidity are known to have a positive effect on mosquito life by increasing survival time and shortening the development cycle. Among other available satellite data, Land Surface Temperature (LST) was chosen as an indicator that combined radiated earth temperature and atmospheric water vapor concentration. Monthly DHF incidence was recorded by province during the 1998 epidemic and obtained as a weekly combined report available from the National Ministry of Public Health. Conversely, LST was calculated using remotely sensed data obtained from thermal infrared sensors of NOAA satellites and computed on a provincial scale. Out of nine selected study provinces, five (58.3%) exhibited an LST with a significant positive correlation with rainfall (p < 0.05). In four out of nineteen surveyed provinces (21.3%), LST showed a significant positive correlation with DHF incidence (p < 0.05). Positive association between LST and DHF incidence was significantly correlated in 75% of the cases during non-epidemic months, while no correlation was found during epidemic months. Non-climatic factors are supposed to be at the origin of this discrepancy between seasonality in climate (LST) and DHF incidence during epidemics.

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In the year 2001 a large dengue fever (DF)/dengue hemorrhagic fever (DHF) outbreak occurred in Nakhon Pathom Province, Thailand. Three thousand one hundred twelve cases of DHF were reported, an attack rate of 393 per 100,000 population. The Nakhon Pathom Provincial Health Office immediately carried out a control action according to WHO recommendations. Active serological surveys and viral RNA isolation were carried out to detect silent transmission of dengue virus in 329 healthy volunteers in Nakhon Pathom Province subdistricts where the dengue epidemic had the highest rate of infection of 2.5 per 1000. Eight point eight percent of these volunteers had a serum sample positive for DF/DHF virus IgM antibody. The highest prevalence occurred in the 15 to 40 year old group. In two instances viral RNA was detected by PCR and dengue serotype 3 was subsequently identified. The data support the hypothesis of subclinical infection with dengue virus. This high frequency of virus circulation combined with a high population density, urbanization and increasing breeding sites for mosquitoes, needs to be taken into account in the evaluation of viral transmission during and after epidemics. This underlines the importance of community-based control in informing people of their involvement in virus transmission and the importance of personal protection.

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Isolation of Japanese encephalitis (JE) virus using C6/36 cell and immunofluorescence virus antigen detection techniques was attempted from female mosquitoes collected with CDC gravid traps in Samut Songkhram Province in the central region and in Phuket Province in southern Thailand, in 2003. One thousand and eighty female mosquitoes including 6 species of the Culicidae family (Culex quinquefasciatus, Cx. gelidus, Cx. tritaeniorhynchus, Cx. whitmorei, Cx. vishnui complex, Cx. s.g. culiciomyia) (pooled by specific specimen), were processed for virus isolation. Two pools of Cx. quinquefasciatus yielded a JE virus isolation. This represents the first report of JE virus isolation from Cx. quinquefasciatus in Thailand.

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Viral encephalitis (VE) continues to be a major disease in Asia, causing serious illness which may result in death or have neurological sequelae. This study involves an ecological analysis of the climatic, geographic and seasonal patterns of clinically reported VE in Thailand from 1993 to 1998 to investigate regional and seasonal differences in disease incidence. Three thousand eight hundred and twenty nine cases of VE were clinically diagnosed nationwide during the study period by the Thai Ministry of Public Health. Spearman rank correlations of temporal, spatial and geographic variables with disease incidence were performed. The monthly incidence of VE correlated significantly with seasonal changes in temperature, relative humidity and rainfall in the north-northeast region of Thailand (P < 0.001), whereas incidence in the south-central region correlated only with relative humidity (P = 0.003). Spatial analysis revealed a positive correlation of disease with elevation (P < 0.001), and negative correlations with rice-field cover (P < 0.001), agricultural land-use (P < 0.001) and temperature (P = 0.004) in the north-northeast region. No significant spatial correlation was identified in the south-central region. The spatial distribution of VE suggests that etiologic variations may be responsible, in part, for the geographic patterns of disease. Active etiologic surveillance is necessary in a variety of geographic settings in order to provide physicians with information necessary for disease prevention and clinical management.

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From August 2000 to 2001, a dengue outbreak occurred in Mueang district, Ratchaburi Province, Thailand. About 800 cases of dengue infection were reported, and among them, 49.5% were clinically diagnosed as dengue hemorrhagic fever according to the WHO criteria. During the outbreak, the incidence rate of dengue infection in Hin Gong subdistrict was 2.9 per 1,000 population. A seroepidemiological survey was conducted among primary schoolchildren from July 2000 to June 2001, to monitor dengue transmission. In a baseline survey, 283 children were surveyed for dengue antibody and 71% were IgG seropositive. In June 2001, the rate of dengue infection showed an increase of 8.8% with 8.0% among immune children and 10.3% among naive schoolchildren. Among 283 schoolchildren, 90 were followed up 3 times, in September and December 2000, and June 2001. An increase in the rate of seroconversion was observed in the period September to December 2000, while the peak dengue outbreaks in the dry season occurred in February 2001. Serosurveys among schoolchildren appear to be early warning system, and can be advantageous in early dengue control actions, in order to break the chain of transmission before an impending epidemic.

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We conducted a serological survey of 632 rodents from the northeast region of Thailand in order to assess the presence of Hantaan-like viruses that may be a risk to the human population. Rodents were collected from rice fields, houses and domestic gardens in five northeastern provinces and tested for IgG reacting sera to Hantaan antigen using enzyme-linked immunoassays. The overall prevalence of Hantavirus infection in rodents was 2.1% (13/632). Species that tested positive included Bandicota indica (4.3% positive within species), Rattus exulans (2.1%), R. losea (1.6%) and R. rattus (0.9%). Species such as R. exulans and R. losea are candidate hosts of unidentified Hantaan-like viruses in Thailand.

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Despite the use of a variety of control strategies, dengue hemorrhagic fever (DHF) control is a major and permanent challenge for public health services in Thailand and in Southeast Asia. In order to improve the efficiency of DHF control in Thailand, these activities have to concentrate on areas and populations at higher risk, which implies early identification of higher incidence periods. A retrospective study of spatial and temporal variations of DHF incidence in all 73 provinces of Thailand (1983-1995) allowed discrimination between seasonal (endemic) transmission dependent on climatic variations and vector density and non-seasonal (epidemic) transmission, mainly due to the occurrence of a new virus serotype in a population with low immunity. To identify epidemic months, which appear significantly clustered, a significant deviation from the monthly average incidence was defined. The occurrence of two consecutive epidemic months in a given area has a high probability (P = 0.66) of being followed by a cluster of 2-18 epidemic months (average: 7.7 months). This observation is proposed as a warning of epidemic outbreak enabling an early launch of control activities. As an example, when this method is retrospectively applied to the studied period, 11,388 province months (73 provinces x 156 months), 579 epidemic outbreaks (5.1% of the total) are identified. Control activities can thus be improved through early management and prevention of the 308,636 supplementary cases occurring during epidemics (37.0% of the total recorded).

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