Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

Long-term persistence of Chikungunya virus neutralizing antibodies in human populations of North Eastern Thailand.

BACKGROUND: Chikungunya virus (CHIKV) outbreak recurrences in Thailand are unpredictable and separated by unexplained and often long silent epidemiological periods that can last for several years. These silent periods could be explained in part by the fact that infection with one CHIKV strain confers lasting natural immunity, even against other CHIKV strains. In this study we evaluated the persistence of CHIKV-specific neutralizing antibodies in the population of Chumpae District, Khon Kaen Province, nineteen years after a CHIKV outbreak occurred in the same area in 1991. FINDINGS: Overall 39% (44/111) of 111 former patients had neutralizing antibodies reacting against CHIKV ECSA strain. Consistently high titers of neutralizing antibodies were found in 75% (33/44) of all positively-reacting sera, 70% of which (23/33) were collected from individuals amongst the >60 years old age group. Although the prevalence found in Pong Haeng village (70%) was significantly higher than the prevalence detected in the Nong Thum village (14%), control study villages without known previous Chikungunya epidemics had a high Chikungunya neutralizing antibody prevalence (65%). CONCLUSIONS: More than one-third of the pre-exposed population had persisting natural immunity that was more likely boosted by recent and repetitive exposure to the emerging ECSA CHIKV in Thailand. Also, Chikungunya virus appears to largely circulate in the country with a great variability appears between villages or area probably associated with the vector abundance and efficiency. Altogether these results show a potential for a lifelong immunity against CHIKV. Given the rapid spread of the highly pathogenic ECSA strain in Southern Thailand, the development of CHIK vaccine is strongly recommended.

Dengue, Japanese encephalitis and Chikungunya virus antibody prevalence among captive monkey (Macaca nemestrina) colonies of Northern Thailand.

The potential of macaque Macaca nemestrina leonina in Thailand to be infected by endemic arboviruses was assessed. The prevalence of antibodies of three arboviruses actively circulating in Thailand was determined by Plaque Reduction Neutralization assay procedures using samples from captive colonies in Northern Thailand. Out of 38 macaques, 9 (24%) presented reacting antibodies against dengue virus, 5 (13%) against Japanese encephalitis virus, and 4 (10%) against Chikungunya virus. Our results indicate that the northern pig-tailed macaque in Thailand can be infected by these arboviruses, inferring therefore that their virus specific vectors have bitten them. Given that, northern pig-tailed macaque represents an abundant population, living in close range to human or in peridomestic setting, they could play a role as potential reservoir host for arboviruses circulating in Thailand.

Change in Japanese encephalitis virus distribution, Thailand.

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.

Complete Genome Sequence of a Zika Virus Strain Isolated from the Serum of an Infected Patient in Thailand in 2006.

The complete genome of Zika virus (ZIKV) strain CVD_06-274 was isolated from the serum of an infected patient in Thailand in 2006. Phylogenetic analysis showed that this strain belongs to the Asian lineage and also high titers in Vero cells (RCB 10-87). It has potential for development as an inactivated ZIKV vaccine.

Potential association of dengue hemorrhagic fever incidence and remote senses land surface temperature, Thailand, 1998.

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.

Silent transmission of virus during a Dengue epidemic, Nakhon Pathom Province, Thailand 2001.

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.

Hantavirus infection in Thailand: first clinical case report.

This study involved 115 cases of Fever of Unknown Origin (FUO) in patients who were admitted to the Department of Medicine, Siriraj Hospital from May 1999 to November 2000. Among the patient sera screened by ELISA for IgG Hantavirus, five were positive for IgG Hantavirus-reacting antibodies and eight tested positive for IgM Hantavirus-reacting antibodies. One serum had both IgG and IgM antibodies. The patient exhibited acute encephalitic febrile illness, thrombocytopenia, high AST and ALT levels, and prolonged coagulation time. It appears that a form of the Hantaan virus is circulating in Thailand, which can infect humans and be pathogenic in some instances.

Hantavirus infection in Thailand: first clinical case report.

This study involved 115 cases of fever of unknown origin (FUO) patients who were admitted to the Department of Medicine, Siriraj Hospital from May 1999 to November 2000. Among the patient sera screened by ELISA for IgG Hantavirus, five were positive for IgG Hantavirus-reacting antibodies and eight tested positive for IgM Hantavirus-reacting antibodies. One serum had both IgG and IgM antibodies. The patient exhibited acute encephalitic febrile illness, thrombocytopenia, high AST and ALT levels, and prolonged coagulation time. It appears that a form of the Hantaan virus is circulating in Thailand, which can infect humans and be pathogenic in some instances.

First isolation of Japanese encephalitis from Culex quinquefasciatus in Thailand.

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.

Structure-activity relationships of dengue antiviral polycyclic quinones.

The virucidal and antiviral photoactivities of three compounds, hypericin, tetrabromohypericin and gymnochrome B, were evaluated against dengue viruses. All the three products were active, and both the virucidal and antiviral activities were enhanced by light. Gymnochrome B was more potent than hypericin and tetrabromohypericin. The presence of the side chains on the hypericin core of gymnochromes appears to be beneficial for both virucidal and antiviral activities. This enhanced activity is likely to be linked to a complementary mechanism independent of photoactivation.

Geographic dynamics of viral encephalitis in Thailand.

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.

Seroepidemiological survey among schoolchildren during the 2000-2001 dengue outbreak of Ratchaburi Province, Thailand.

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.

Generation and preclinical evaluation of a DENV-1/2 prM+E chimeric live attenuated vaccine candidate with enhanced prM cleavage.

In the absence of a vaccine or sustainable vector control measures, illnesses caused by dengue virus infection remain an important public health problem in many tropical countries. During the export of dengue virus particles, furin-mediated cleavage of the prM envelope protein is usually incomplete, thus generating a mixture of immature, partially mature and mature extracellular particles. Variations in the arrangement and conformation of the envelope proteins among these particles may be associated with their different roles in shaping the antibody response. In an attempt to improve upon live, attenuated dengue vaccine approaches, a mutant chimeric virus, with enhanced prM cleavage, was generated by introducing a cleavage-enhancing substitution into a chimeric DENV-1/2 virus genome, encoding the prM+E sequence of a recent DENV-1 isolate under an attenuated DENV-2 genetic background. A modest increase in virus specific infectivity observed in the mutant chimeric virus affected neither the attenuation phenotype, when assessed in the suckling mouse neurovirulence model, nor multiplication in mosquitoes. The two chimeric viruses induced similar levels of anti-DENV-1 neutralizing antibody response in mice and rhesus macaques, but more efficient control of viremia during viral challenge was observed in macaques immunized with the mutant chimeric virus. These results indicate that the DENV-1/2 chimeric virus, with enhanced prM cleavage, could be useful as an alternative live, attenuated vaccine candidate for further tests in humans.

Elevated Japanese encephalitis virus activity monitored by domestic sentinel piglets in Thailand.

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.

Detection of host virus-reactive antibodies in blood meals of naturally engorged mosquitoes.

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.

Hantaan virus antibody prevalence in rodent populations of several provinces of northeastern Thailand.

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.