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1.  Characteristics of Mild Dengue Virus Infection in Thai Children 
A four-year longitudinal cohort and geographic cluster study in rural Thailand was conducted to characterize the clinical spectrum of dengue virus (DENV) infection. Symptomatic DENV infections in the cohort were detected by active school absence–based surveillance that triggered cluster investigations around ill cohort children. Data from 189 cohort children with symptomatic DENV infection and 126 contact children in the clusters with DENV infection were analyzed. Of infected contacts, only 19% were asymptomatic; 81% were symptomatic, but only 65.9% reported fever. Symptom-based case definitions were unreliable for diagnosis. Symptomatic infections in contacts were milder with lower DENV RNA levels than the cohort. Infections in contacts with fever history were more likely to have detectable DENV RNA than infections without fever history. Mild infections identified by cluster investigations account for a major proportion of all DENV infections. These findings are relevant for disease burden assessments, transmission modeling, and determination of vaccine impact.
PMCID: PMC3854884  PMID: 24127167
2.  The Spatial Dynamics of Dengue Virus in Kamphaeng Phet, Thailand 
Dengue is endemic to the rural province of Kamphaeng Phet, Northern Thailand. A decade of prospective cohort studies has provided important insights into the dengue viruses and their generated disease. However, as elsewhere, spatial dynamics of the pathogen remain poorly understood. In particular, the spatial scale of transmission and the scale of clustering are poorly characterized. This information is critical for effective deployment of spatially targeted interventions and for understanding the mechanisms that drive the dispersal of the virus.
Methodology/Principal Findings
We geocoded the home locations of 4,768 confirmed dengue cases admitted to the main hospital in Kamphaeng Phet province between 1994 and 2008. We used the phi clustering statistic to characterize short-term spatial dependence between cases. Further, to see if clustering of cases led to similar temporal patterns of disease across villages, we calculated the correlation in the long-term epidemic curves between communities. We found that cases were 2.9 times (95% confidence interval 2.7–3.2) more likely to live in the same village and be infected within the same month than expected given the underlying spatial and temporal distribution of cases. This fell to 1.4 times (1.2–1.7) for individuals living in villages 1 km apart. Significant clustering was observed up to 5 km. We found a steadily decreasing trend in the correlation in epidemics curves by distance: communities separated by up to 5 km had a mean correlation of 0.28 falling to 0.16 for communities separated between 20 km and 25 km. A potential explanation for these patterns is a role for human movement in spreading the pathogen between communities. Gravity style models, which attempt to capture population movement, outperformed competing models in describing the observed correlations.
There exists significant short-term clustering of cases within individual villages. Effective spatially and temporally targeted interventions deployed within villages may target ongoing transmission and reduce infection risk.
Author Summary
Transmission of dengue virus has long been studied in Kamphaeng Phet, Northern Thailand, but how cases are related in time and space is still unclear, as is the role of human movement in generating these patterns. Because of these knowledge gaps, public health officials cannot make educated decisions on how to target vector control interventions and mechanisms of virus dispersal are not known. We mapped the homes of dengue cases admitted to the main hospital in the province capital from 1994–2008 and quantified the spatial correlation between them. We found an almost three times greater chance that cases from the same month came from the same village than expected, given the overall distribution of cases. Some clustering was also observed between cases in neighboring villages with the overall epidemics experienced by neighboring communities also more correlated than epidemics in villages farther apart. The short-term clustering observed within individual villages implies that effective spatially targeted interventions deployed within villages may reduce infection risk. As the distance between neighboring communities exceeds the typical flight range of the dengue vector, these findings also suggest a potential role for human movement in driving the wider spread of the virus.
PMCID: PMC4161352  PMID: 25211127
3.  High Rate of A(H1N1)pdm09 Infections among Rural Thai Villagers, 2009–2010 
PLoS ONE  2014;9(9):e106751.
Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.
A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.
The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003.
Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.
PMCID: PMC4154756  PMID: 25188434
4.  Neuropathogenesis of Japanese Encephalitis in a Primate Model 
Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.
Methodology/Principal Findings
Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.
The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.
Author Summary
Japanese encephalitis (JE) is one of the most important causes of viral encephalitis worldwide, with no specific antiviral treatment available. Despite some recent successes with widespread vaccination, JE will likely remain an important public health problem; because the virus is mosquito-borne and has natural animal hosts, it will never be eradicated. We have little understanding of what determines the severity and outcome of infection. Data from human post mortem studies is very limited because of cultural constraints on autopsies in areas where JE occurs. Circumstantial evidence suggests that in addition to cytopathology caused directly by infection of neurons, there may be bystander cell death of non-infected neurons, caused by an excessive inflammatory response. Our study used archived brain samples from a prior challenge study in a validated macaque model of JE. We stained for the presence of JEV antigen, apoptosis, and pro-inflammatory markers in affected areas, such as the thalamus and brainstem. We show that bystander neuronal cell death is important, and elucidate the inflammatory and apoptotic mechanisms underlying it. Currently there is no proven efficacious therapy for most viral infections of the central nervous system, including JE. Novel strategies for treating such infections are urgently needed. Our findings suggest new anti-inflammatory and anti-apoptotic therapeutic approaches may be useful in treating this debilitating disease.
PMCID: PMC4125110  PMID: 25102067
5.  Preliminary evaluation of near infrared spectroscopy as a method to detect plasma leakage in children with dengue hemorrhagic fever 
BMC Infectious Diseases  2014;14:396.
Dengue viral infections are prevalent in the tropical and sub-tropical regions of the world, resulting in substantial morbidity and mortality. Clinical manifestations range from a self-limited fever to a potential life-threatening plasma leakage syndrome (dengue hemorrhagic fever). The objective of this study was to assess the utility of near infrared spectroscopy (NIRS) measurements of muscle oxygen saturation (SmO2) as a possible continuous measure to detect plasma leakage in children with dengue.
Children ages 6 months to 15 years of age admitted with suspected dengue were enrolled from the dengue ward at Queen Sirikit National Institute for Child Health. Children were monitored daily until discharge. NIRS data were collected continuously using a prototype CareGuide Oximeter 1100 with sensors placed on the deltoid or thigh. Daily ultrasound of the chest and a right lateral decubitus chest x-ray the day after defervescence were performed to detect and quantitate plasma leakage in the pleural cavity.
NIRS data were obtained from 19 children with laboratory-confirmed dengue. Average minimum SmO2 decreased for all subjects prior to defervescence. Average minimum SmO2 subsequently increased in children with no ultrasound evidence of pleural effusion but remained low in children with pleural effusion following defervescence. Average minimum SmO2 was inversely correlated with pleural space fluid volume. ROC analysis revealed a cut-off value for SmO2 which yielded high specificity and sensitivity.
SmO2 measured using NIRS may be a useful guide for real-time and non-invasive identification of plasma leakage in children with dengue. Further investigation of the utility of NIRS measurements for prediction and management of severe dengue syndromes is warranted.
PMCID: PMC4223418  PMID: 25033831
6.  Space-time analysis of hospitalized dengue patients in rural Thailand reveals important temporal intervals in the pattern of dengue virus transmission 
This study uses space-time analysis to determine the temporal intervals at which spatial clustering of dengue hospitalizations occurs.
Analysis of 262 people hospitalized and serologically confirmed with dengue virus infections in Kamphaeng Phet, Thailand was performed. The cases were observed between January 1, 2009 and May 6, 2011. Spatial coordinates of each patient’s home were captured using the Global Positioning System. A novel methodology based on the Knox test was used to determine the temporal intervals between cases at which spatial clustering occured. These intervals are indicative of the length of time between successive illnesses in the chain of dengue virus transmission.
The strongest spatial clustering occurred at the 15–17 day interval. Therewas also significant spatial clustering over short time intervals (2–5 days). The highest excess risk was observed within 200m of a previous hospitalized case and significantly elevated risk persisted within this distance for as long as 32–34 days.
The analyses indicate that 15–17 days is the most likely serial interval between successive dengue illnesses. This novel methodology relies only on passively-detected, hospitalized case data with household locations and provides a useful tool for understanding region-specific and outbreak-specific dengue virus transmission dynamics.
PMCID: PMC4099473  PMID: 22808917
dengue; transmission; serial interval; space-time; clustering; Thailand
7.  Safety and Immunogenicity of a Rederived, Live-Attenuated Dengue Virus Vaccine in Healthy Adults Living in Thailand: A Randomized Trial 
Safety and immunogenicity of two formulations of a live-attenuated tetravalent dengue virus (TDEN) vaccine produced using rederived master seeds from a precursor vaccine were tested against a placebo control in a phase II, randomized, double blind trial (NCT00370682). Two doses were administered 6 months apart to 120 healthy, predominantly flavivirus-primed adults (87.5% and 97.5% in the two vaccine groups and 92.5% in the placebo group). Symptoms and signs reported after vaccination were mild to moderate and transient. There were no vaccine-related serious adverse events or dengue cases reported. Asymptomatic, low-level viremia (dengue virus type 2 [DENV-2], DENV-3, or DENV-4) was detected in 5 of 80 vaccine recipients. One placebo recipient developed a subclinical natural DENV-1 infection. All flavivirus-unprimed subjects and at least 97.1% of flavivirus-primed subjects were seropositive to antibodies against all four DENV types 1 and 3 months post-TDEN dose 2. The TDEN vaccine was immunogenic with an acceptable safety profile in flavivirus-primed adults.
PMCID: PMC4080550  PMID: 24865677
8.  Dengue Virus and Japanese Encephalitis Virus Epidemiological Shifts in Nepal: A Case of Opposing Trends 
We report on the changing epidemiology of two important flaviviruses in Nepal: Japanese encephalitis (JE) and dengue viruses. Morbidity and mortality in Nepal is in the thousands since JE was introduced in 1978. Nepal launched an extensive laboratory-based JE surveillance in 2004. Nepal experienced a remarkable reduction in disease burden after mass immunizations from 2005 to 2010, when 2,040 JE infections and 205 JE-related deaths were confirmed. With its emergence in 2006, dengue has become a significant challenge in the country, highlighted by a sudden outbreak in 2010 that resulted in 359 confirmed dengue infections. Currently, both viruses cocirculate in Nepal. Here, we document the remarkable expansion of dengue in Nepal, which urgently requires national surveillance to refine the burden and make recommendations regarding control and prevention programs. We believe that the use of existing JE surveillance network for integrated dengue surveillance may represent the most appropriate alternative.
PMCID: PMC3617851  PMID: 23419366
9.  Estimation of the Impact of a Japanese Encephalitis Immunization Program with Live, Attenuated SA 14-14-2 Vaccine in Nepal 
Wider availability of the live, attenuated SA 14-14-2 Japanese encephalitis (JE) vaccine has facilitated introduction or expansion of immunization programs in many countries. However, information on their impact is limited. In 2006, Nepal launched a JE immunization program, and by 2009, mass campaigns had been implemented in 23 districts. To describe the impact, we analyzed surveillance data from 2004 to 2009 on laboratory-confirmed JE and clinical acute encephalitis syndrome (AES) cases. The post-campaign JE incidence rate of 1.3 per 100,000 population was 72% lower than expected if no campaigns had occurred, and an estimated 891 JE cases were prevented. In addition, AES incidence was 58% lower, with an estimated 2,787 AES cases prevented, suggesting that three times as many disease cases may have been prevented than indicated by the laboratory-confirmed JE cases alone. These results provide useful information on preventable JE disease burden and the potential value of JE immunization programs.
PMCID: PMC3592526  PMID: 23358643
10.  Kinetics of Chikungunya Infections during an Outbreak in Southern Thailand, 2008–2009 
The Indian Ocean chikungunya epidemic re-emerged in Thailand in August 2008. Forty-five adults with laboratory-confirmed chikungunya in Songkhla province, Thailand were clinically assessed and serially bled throughout the acute and convalescent phase of the disease. Patient symptoms, antibody responses, and viral kinetics were evaluated using observational assessments, polymerase chain reaction (PCR), and serological assays. All subjects experienced joint pain with 42 (93%) involving multiple joints; the interphalangeal most commonly affected in 91% of the subjects. The mean duration of joint pain was 5.8 days, 11 (25%) experiencing discomfort through the duration of the study. Rash was observed in 37 (82%) subjects a mean 3.5 days post onset of symptoms. Patents were positive by PCR for a mean of 5.9 days with sustained peak viral load through Day 5. The IgM antibodies appeared on Day 4 and peaked at Day 7 and IgG antibodies first appeared at Day 5 and rose steadily through Day 24.
PMCID: PMC3945684  PMID: 24493674
11.  Dengue and US Military Operations from the Spanish–American War through Today 
Emerging Infectious Diseases  2012;18(4):623-630.
Dengue may remain problematic for military personnel until an effective vaccine is licensed.
Dengue is a major cause of illness among travelers and a threat to military troops operating in areas to which it is endemic. Before and during World War II, dengue frequently occurred in US military personnel in Asia and the South Pacific. From the 1960s into the 1990s, dengue often occurred in US troops in Vietnam, the Philippines, Somalia, and Haiti. We found attack rates as high as 80% and periods of convalescence up to 3-1/2 weeks beyond the acute illness. The increase in dengue throughout the world suggests that it will remain a problem for military personnel until an effective vaccine is licensed.
PMCID: PMC3309667  PMID: 22469290
dengue; military personnel; history; fever; viruses; vector-borne infections; United States; Spanish–American War; military operations
12.  Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance 
Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.
Author Summary
Reports from sub-tropical regions of the world suggest a growing risk of introduction and establishment of dengue viruses (DENV) in new locales. Recent dengue epidemics in northern Viet Nam present an opportunity to study how DENV invades and spreads in these environments. The proximity of this region to tropical areas experiencing year-round endemic DENV transmission makes it an ideal site for studying the effects of human population movement and climate on DENV emergence. We performed a phylogenetic analysis using DENV-1 envelope gene sequences from Southeast Asia. We show that DENV are regularly imported into northern and central Viet Nam from southern Vietnam, and that increasingly large seasonal epidemics in the north are caused by newly introduced viruses each year. While tropical Vietnam maintains localized virus populations for multiple years, cool winter temperatures in sub-tropical northern Viet Nam may reduce mosquito populations and virus replication to levels that are not conducive to year-round DENV transmission. Finally, we found that the dispersal of DENV across the region is well-described using human movement and immunity data, and believe that increased epidemiological, entomological, and virological surveillance are needed to understand the processes by which endemic DENV transmission becomes established in new populations.
PMCID: PMC3854975  PMID: 24340118
13.  Interactions between serotypes of dengue highlight epidemiological impact of cross-immunity 
Dengue, a mosquito-borne virus of humans, infects over 50 million people annually. Infection with any of the four dengue serotypes induces protective immunity to that serotype, but does not confer long-term protection against infection by other serotypes. The immunological interactions between serotypes are of central importance in understanding epidemiological dynamics and anticipating the impact of dengue vaccines. We analysed a 38-year time series with 12 197 serotyped dengue infections from a hospital in Bangkok, Thailand. Using novel mechanistic models to represent different hypothesized immune interactions between serotypes, we found strong evidence that infection with dengue provides substantial short-term cross-protection against other serotypes (approx. 1–3 years). This is the first quantitative evidence that short-term cross-protection exists since human experimental infection studies performed in the 1950s. These findings will impact strategies for designing dengue vaccine studies, future multi-strain modelling efforts, and our understanding of evolutionary pressures in multi-strain disease systems.
PMCID: PMC3730691  PMID: 23825116
dengue; infectious disease modelling; cross-protection; time-series models
14.  Prospective Study of Avian Influenza Virus Infections among Rural Thai Villagers 
PLoS ONE  2013;8(8):e72196.
In 2008, 800 rural Thai adults living within Kamphaeng Phet Province were enrolled in a prospective cohort study of zoonotic influenza transmission. Serological analyses of enrollment sera suggested this cohort had experienced subclinical avian influenza virus (AIV) infections with H9N2 and H5N1 viruses.
After enrollment, participants were contacted weekly for 24mos for acute influenza-like illnesses (ILI). Cohort members confirmed to have influenza A infections were enrolled with their household contacts in a family transmission study involving paired sera and respiratory swab collections. Cohort members also provided sera at 12 and 24 months after enrollment. Serologic and real-time RT-PCR assays were performed against avian, swine, and human influenza viruses.
Over the 2 yrs of follow-up, 81 ILI investigations in the cohort were conducted; 31 (38%) were identified as influenza A infections by qRT-PCR. Eighty-three household contacts were enrolled; 12 (14%) reported ILIs, and 11 (92%) of those were identified as influenza infections. A number of subjects were found to have slightly elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2) virus: 21 subjects (2.7%) at 12-months and 40 subjects (5.1%) at 24-months. Among these, two largely asymptomatic acute infections with H9N2 virus were detected by >4-fold increases in annual serologic titers (final titers 1∶80). While controlling for age and influenza vaccine receipt, moderate poultry exposure was significantly associated with elevated H9N2 titers (adjusted OR = 2.3; 95% CI, 1.04–5.2) at the 24-month encounter. One subject had an elevated titer (1∶20) against H5N1 during follow-up.
From 2008–10, evidence for AIV infections was sparse among this rural population. Subclinical H9N2 AIV infections likely occurred, but serological results were confounded by antibody cross-reactions. There is a critical need for improved serological diagnostics to more accurately detect subclinical AIV infections in humans.
PMCID: PMC3745375  PMID: 23977250
15.  Underrecognized Mildly Symptomatic Viremic Dengue Virus Infections in Rural Thai Schools and Villages 
The Journal of Infectious Diseases  2012;206(3):389-398.
Background. The understanding of dengue virus (DENV) transmission dynamics and the clinical spectrum of infection are critical to informing surveillance and control measures. Geographic cluster studies can elucidate these features in greater detail than cohort studies alone.
Methods. A 4-year longitudinal cohort and geographic cluster study was undertaken in rural Thailand. Cohort children underwent pre-/postseason serology and active school absence–based surveillance to detect inapparent and symptomatic dengue. Cluster investigations were triggered by cohort dengue and non-dengue febrile illnesses (positive and negative clusters, respectively).
Results. The annual cohort incidence of symptomatic dengue ranged from 1.3% to 4.4%. DENV-4 predominated in the first 2 years, DENV-1 in the second 2 years. The inapparent-to-symptomatic infection ratio ranged from 1.1:1 to 2.9:1. Positive clusters had a 16.0% infection rate, negative clusters 1.1%. Of 119 infections in positive clusters, 59.7% were febrile, 20.2% were afebrile with other symptoms, and 20.2% were asymptomatic. Of 16 febrile children detected during cluster investigations who continued to attend school, 9 had detectable viremia.
Conclusions. Dengue transmission risk was high near viremic children in both high- and low-incidence years. Inapparent infections in the cohort overestimated the rate of asymptomatic infections. Ambulatory children with mild febrile viremic infections could represent an important component of dengue transmission.
PMCID: PMC3490697  PMID: 22615312
16.  Identification of All Dengue Serotypes in Nepal 
Emerging Infectious Diseases  2008;14(10):1669-1670.
PMCID: PMC2609890  PMID: 18826846
dengue; Nepal; Aedes; serotype; PCR; ELISA; letter
17.  Frequent In-Migration and Highly Focal Transmission of Dengue Viruses among Children in Kamphaeng Phet, Thailand 
Revealing the patterns and determinants of the spread of dengue virus (DENV) at local scales is central to understanding the epidemiology and evolution of this major human pathogen. We performed a phylogenetic analysis of the envelope (E) genes of DENV-1, -2, -3, and -4 isolates (involving 97, 23, 5, and 74 newly collected sequences, respectively) sampled from school-based cohort and village-based cluster studies in Kamphaeng Phet, Thailand, between 2004 and 2007. With these data, we sought to describe the spatial and temporal patterns of DENV spread within a rural population where a future vaccine efficacy trial is planned. Our analysis revealed considerable genetic diversity within the study population, with multiple lineages within each serotype circulating for various lengths of time during the study period. These results suggest that DENV is frequently introduced into both semi-urban and rural areas in Kamphaeng Phet from other populations. In contrast, the persistence of viral lineages across sampling years was observed less frequently. Analysis of phylogenetic clustering indicated that DENV transmission was highly spatially and temporally focal, and that it occurred in homes rather than at school. Overall, the strength of temporal clustering suggests that seasonal bottlenecks in local DENV populations facilitate the invasion and establishment of viruses from outside of the study area, in turn reducing the extent of lineage persistence.
Author Summary
Long-term cohort studies of dengue virus (DENV), the most common vector-borne viral disease of humans, are essential to understand the epidemiology and evolution of this important human pathogen, and may assist in predicting the evolutionary response to vaccination. We utilized DENV gene sequences and information on the locations and timing of infected children within a primary school-based cohort in Kamphaeng Phet, Thailand to investigate the spatial and temporal relationships among viruses isolated from 2004 to 2007. We found that all four DENV serotypes circulated in the region during the study period, with the presence of multiple viral lineages within each serotype. Viruses sampled closely in time and space were generally very closely related. More genetic variation was observed across districts in a given year and within the same district across different years. The high genetic similarity among viruses during each season and the rare persistence of these lineages through multiple seasons suggest that seasonal reductions in the force of infection through changes in mosquito transmission and fluctuations in human population immunity are key factors shaping the genetic diversity of dengue virus diversity in this region. The importation of DENV by human movement from other populations is therefore an important generator of DENV diversity even in hyperendemic areas.
PMCID: PMC3547850  PMID: 23350000
18.  A Phase II, Randomized, Safety and Immunogenicity Study of a Re-Derived, Live-Attenuated Dengue Virus Vaccine in Healthy Adults 
Two formulations of a new live tetravalent dengue virus (DENV) vaccine produced using re-derived master seeds from a precursor vaccine and that same precursor vaccine as a control were compared in a placebo-controlled, randomized, observer-blind, phase II trial of 86 healthy adults. Two vaccine doses were administered 6 months apart; a third dose was offered to a subset. Symptoms and signs of dengue-like illness reported after vaccination were mild to moderate, transient, and occurred with similar frequency among recipients of the new DENV vaccine and placebo, except for rash. Neither dengue nor vaccine-related serious adverse events were reported. The first DENV vaccine dose was moderately immunogenic; the second dose increased the potency and breadth of the neutralizing antibody response. Tetravalent response rates to the new formulations were 60% and 66.7% in unprimed subjects. A third dose did not increase tetravalent antibody rates. The new DENV vaccine candidates merit additional evaluation.
PMCID: PMC3541749  PMID: 23208878
19.  Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells Are Associated with Disease Severity and Preexisting Dengue Immune Status 
PLoS ONE  2012;7(12):e51335.
Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.
The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.
Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.
B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.
PMCID: PMC3526575  PMID: 23284680
20.  B-Cell Responses During Primary and Secondary Dengue Virus Infections in Humans 
The Journal of Infectious Diseases  2011;204(10):1514-1522.
Low-avidity serotype–cross-reactive antibodies are hypothesized to play a key role in triggering severe disease in patients with secondary dengue virus (DENV) infection. However, there is little systematic information about the frequency, avidity, and cross-reactivity of DENV-specific B cells in individuals experiencing primary instead of secondary infection. We compared DENV-specific B-cell responses in a cohort of Thai children with primary or secondary DENV infection. B cells specific for DENV precursor membrane protein, envelope (E) protein, and nonstructural protein 1 were detectable in immune peripheral blood mononuclear cells with the highest frequencies of DENV E-specific B cells detected in patients experiencing primary DENV-1 infections. DENV E-specific B cells were highly serotype-specific after primary DENV infections, whereas most E-specific B cells in patients with secondary infection were serotype–cross-reactive and secreted antibodies with higher avidity to heterologous DENV serotypes. Our data suggest that the minor populations of serotype–cross-reactive B cells generated by primary DENV infection are preferentially expanded during secondary DENV infection.
PMCID: PMC3222107  PMID: 21930609
21.  Evidence for Subclinical Avian Influenza Virus Infections Among Rural Thai Villagers 
Adults with poultry exposure living in rural central Thailand are prospectively studied for evidence of avian influenza virus infections. Report details enrollment methods, findings of enrollment questionnaire data, and the serological investigation of enrollment sera.
Background. Regions of Thailand reported sporadic outbreaks of A/H5N1 highly pathogenic avian influenza (HPAI) among poultry between 2004 and 2008. Kamphaeng Phet Province, in north-central Thailand had over 50 HPAI poultry outbreaks in 2004 alone, and 1 confirmed and 2 likely other human HPAI infections between 2004 and 2006.
Methods. In 2008, we enrolled a cohort of 800 rural Thai adults living in 8 sites within Kamphaeng Phet Province in a prospective study of zoonotic influenza transmission. We studied participants’ sera with serologic assays against 16 avian, 2 swine, and 8 human influenza viruses.
Results. Among participants (mean age 49.6 years and 58% female) 65% reported lifetime poultry exposure of at least 30 consecutive minutes. Enrollees had elevated antibodies by microneutralization assay against 3 avian viruses: A/Hong Kong/1073/1999(H9N2), A/Thailand/676/2005(H5N1), and A/Thailand/384/2006(H5N1). Bivariate risk factor modeling demonstrated that male gender, lack of an indoor water source, and tobacco use were associated with elevated titers against avian H9N2 virus. Multivariate modeling suggested that increasing age, lack of an indoor water source, and chronic breathing problems were associated with infection with 1 or both HPAI H5N1 strains. Poultry exposure was not associated with positive serologic findings.
Conclusions. These data suggest that people in rural central Thailand may have experienced subclinical avian influenza infections as a result of yet unidentified environmental exposures. Lack of an indoor water source may play a role in transmission.
PMCID: PMC3174099  PMID: 21921216
22.  Dengue—How Best to Classify It 
Since the 1970s, dengue has been classified as dengue fever and dengue hemorrhagic fever. In 2009, the World Health Organization issued a new, severity-based clinical classification which differs greatly from the previous classification.
Dengue has emerged as a major public health problem worldwide. Dengue virus infection causes a wide range of clinical manifestations. Since the 1970s, clinical dengue has been classified according to the World Health Organization guideline as dengue fever and dengue hemorrhagic fever. The classification has been criticized with regard to its usefulness and its applicability. In 2009, the World Health Organization issued a new guideline that classifies clinical dengue as dengue and severe dengue. The 2009 classification differs significantly from the previous classification in both conceptual and practical levels. The impacts of the new classification on clinical practice, dengue research, and public health policy are discussed.
PMCID: PMC3202316  PMID: 21832264
23.  Genetic Diversity of Thottapalayam Virus, a Hantavirus Harbored by the Asian House Shrew (Suncus murinus) in Nepal 
Despite the recent discovery of genetically divergent hantaviruses in shrews of multiple species in widely separated geographic regions, data are unavailable about the genetic diversity and phylogeography of Thottapalayam virus (TPMV), a hantavirus originally isolated from an Asian house shrew (Suncus murinus) captured in southern India more than four decades ago. To bridge this knowledge gap, the S, M, and L segments of hantavirus RNA were amplified by reverse transcription polymerase chain reaction from archival lung tissues of Asian house shrews captured in Nepal from January to September 1996. Pair-wise alignment and comparison revealed approximately 80% nucleotide and > 94% amino acid sequence similarity to prototype TPMV. Phylogenetic analyses, generated by maximum likelihood and Bayesian methods, showed geographic-specific clustering of TPMV, similar to that observed for rodent- and soricid-borne hantaviruses. These findings confirm that the Asian house shrew is the natural reservoir of TPMV and suggest a long-standing virus–host relationship.
PMCID: PMC3163881  PMID: 21896819
24.  Safety and Immunogenicity of a Tetravalent Live-Attenuated Dengue Vaccine in Flavivirus-Naive Infants 
A Phase I/II observer-blind, randomized, controlled trial evaluated the safety and immunogenicity of a dengue virus (DENV) vaccine candidate in healthy Thai infants (aged 12–15 months) without measurable pre-vaccination neutralizing antibodies to DENV and Japanese encephalitis virus. Fifty-one subjects received two doses of either DENV (N = 34; four received 1/10th dose) or control vaccine (N = 17; dose 1, live varicella; dose 2, Haemophilus influenzae type b). After each vaccine dose, adverse events (AEs) were solicited for 21 days, and non-serious AEs were solicited for 30 days; serious AEs (SAEs) were recorded throughout the study. Laboratory safety assessments were performed at 10 and 30 days; neutralizing antibodies were measured at 30 days. The DENV vaccine was well-tolerated without any related SAEs. After the second dose, 85.7% of full-dose DENV vaccinees developed at least trivalent and 53.6% developed tetravalent neutralizing antibodies ≥ 1:10 to DENV (control group = 0%). This vaccine candidate, therefore, warrants continued development in this age group (NCT00322049;
PMCID: PMC3144835  PMID: 21813857
25.  Fine Scale Spatiotemporal Clustering of Dengue Virus Transmission in Children and Aedes aegypti in Rural Thai Villages 
Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages.
Methodology/Principal Findings
Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes.
Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).
Author Summary
Dengue is the leading cause of mosquito-borne viral infections globally. An improved understanding of the spatial and temporal distribution of dengue virus (DENV) transmission between humans and the principal vector, Aedes aegypti, can enhance prevention programs. Human DENV infection is known to occur at very fine spatiotemporal scales. We sought to link and quantify human DENV infections with infectious mosquitoes at these fine scales by conducting geographic cluster investigations around febrile children with and without DENV infection. We found that DENV infection in children was positively associated with houses in which infectious mosquitoes were captured. These houses also had more Ae. aegypti pupae and adult female mosquitoes than neighboring houses. However, the neighboring houses still had elevated rates of human DENV infection. Our results indicate that certain houses with high risk of DENV transmission contribute disproportionately to DENV amplification and spread to surrounding houses. At a given point in time, people and mosquitoes from a small portion of houses are responsible for the majority of DENV transmission.
PMCID: PMC3398976  PMID: 22816001

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