Beneath the surface: Amino acid variation underlying two decades of dengue virus antigenic dynamics in Bangkok, Thailand.

Neutralizing antibodies are important correlates of protection against dengue. Yet, determinants of variation in neutralization across strains within the four dengue virus serotypes (DENV1-4) is imperfectly understood. Studies focus on structural DENV proteins, especially the envelope (E), the primary target of anti-DENV antibodies. Although changes in immune recognition (antigenicity) are often attributed to variation in epitope residues, viral processes influencing conformation and epitope accessibility also affect neutralizability, suggesting possible modulating roles of nonstructural proteins. We estimated effects of residue changes in all 10 DENV proteins on antigenic distances between 348 DENV collected from individuals living in Bangkok, Thailand (1994-2014). Antigenic distances were derived from response of each virus to a panel of twenty non-human primate antisera. Across 100 estimations, excluding 10% of virus pairs each time, 77 of 295 positions with residue variability in E consistently conferred antigenic effects; 52 were within ±3 sites of known binding sites of neutralizing human monoclonal antibodies, exceeding expectations from random assignments of effects to sites (p = 0.037). Effects were also identified for 16 sites on the stem/anchor of E which were only recently shown to become exposed under physiological conditions. For all proteins, except nonstructural protein 2A (NS2A), root-mean-squared-error (RMSE) in predicting distances between pairs held out in each estimation did not outperform sequences of equal length derived from all proteins or E, suggesting that antigenic signals present were likely through linkage with E. Adjusted for E, we identified 62/219 sites embedding the excess signals in NS2A. Concatenating these sites to E additionally explained 3.4% to 4.0% of observed variance in antigenic distances compared to E alone (50.5% to 50.8%); RMSE outperformed concatenating E with sites from any protein of the virus (ΔRMSE, 95%IQR: 0.01, 0.05). Our results support examining antigenic determinants beyond the DENV surface.

Reconstruction of antibody dynamics and infection histories to evaluate dengue risk.

As with many pathogens, most dengue infections are subclinical and therefore unobserved 1 . Coupled with limited understanding of the dynamic behaviour of potential serological markers of infection, this observational problem has wide-ranging implications, including hampering our understanding of individual- and population-level correlates of infection and disease risk and how these change over time, between assay interpretations and with cohort design. Here we develop a framework that simultaneously characterizes antibody dynamics and identifies subclinical infections via Bayesian augmentation from detailed cohort data (3,451 individuals with blood draws every 91 days, 143,548 haemagglutination inhibition assay titre measurements)2,3. We identify 1,149 infections (95% confidence interval, 1,135-1,163) that were not detected by active surveillance and estimate that 65% of infections are subclinical. After infection, individuals develop a stable set point antibody load after one year that places them within or outside a risk window. Individuals with pre-existing titres of ≤1:40 develop haemorrhagic fever 7.4 (95% confidence interval, 2.5-8.2) times more often than naive individuals compared to 0.0 times for individuals with titres >1:40 (95% confidence interval: 0.0-1.3). Plaque reduction neutralization test titres ≤1:100 were similarly associated with severe disease. Across the population, variability in the size of epidemics results in large-scale temporal changes in infection and disease risk that correlate poorly with age.

Precision Tracing of Household Dengue Spread Using Inter- and Intra-Host Viral Variation Data, Kamphaeng Phet, Thailand.

Dengue control approaches are best informed by granular spatial epidemiology of these viruses, yet reconstruction of inter- and intra-household transmissions is limited when analyzing case count, serologic, or genomic consensus sequence data. To determine viral spread on a finer spatial scale, we extended phylogenomic discrete trait analyses to reconstructions of house-to-house transmissions within a prospective cluster study in Kamphaeng Phet, Thailand. For additional resolution and transmission confirmation, we mapped dengue intra-host single nucleotide variants on the taxa of these time-scaled phylogenies. This approach confirmed 19 household transmissions and revealed that dengue disperses an average of 70 m per day between households in these communities. We describe an evolutionary biology framework for the resolution of dengue transmissions that cannot be differentiated based on epidemiologic and consensus genome data alone. This framework can be used as a public health tool to inform control approaches and enable precise tracing of dengue transmissions.

An Innovative, Prospective, Hybrid Cohort-Cluster Study Design to Characterize Dengue Virus Transmission in Multigenerational Households in Kamphaeng Phet, Thailand.

Difficulties inherent in the identification of immune correlates of protection or severe disease have challenged the development and evaluation of dengue vaccines. There persist substantial gaps in knowledge about the complex effects of age and sequential dengue virus (DENV) exposures on these correlations. To address these gaps, we were conducting a novel family-based cohort-cluster study for DENV transmission in Kamphaeng Phet, Thailand. The study began in 2015 and is funded until at least 2023. As of May 2019, 2,870 individuals in 485 families were actively enrolled. The families comprise at least 1 child born into the study as a newborn, 1 other child, a parent, and a grandparent. The median age of enrolled participants is 21 years (range 0-93 years). Active surveillance is performed to detect acute dengue illnesses, and annual blood testing identifies subclinical seroconversions. Extended follow-up of this cohort will detect sequential infections and correlate antibody kinetics and sequence of infections with disease outcomes. The central goal of this prospective study is to characterize how different DENV exposure histories within multigenerational family units, from DENV-naive infants to grandparents with multiple prior DENV exposures, affect transmission, disease, and protection at the level of the individual, household, and community.

Dengue Virus (DENV) Neutralizing Antibody Kinetics in Children After Symptomatic Primary and Postprimary DENV Infection.

The immune response to dengue virus (DENV) infection is complex and not fully understood. Using longitudinal data from 181 children with dengue in Thailand who were followed for up to 3 years, we describe neutralizing antibody kinetics following symptomatic DENV infection. We observed that antibody titers varied by serotype, homotypic vs heterotypic responses, and primary versus postprimary infections. The rates of change in antibody titers over time varied between primary and postprimary responses. For primary infections, titers increased from convalescence to 6 months. By comparing homotypic and heterotypic antibody titers, we saw an increase in type specificity from convalescence to 6 months for primary DENV3 infections but not primary DENV1 infections. In postprimary cases, there was a decrease in titers from convalescence up until 6 months after infection. Beginning 1 year after both primary and postprimary infections, there was evidence of increasing antibody titers, with greater increases in children with lower titers, suggesting that antibody titers were boosted due to infection and that higher levels of neutralizing antibody may be more likely to confer a sterilizing immune response. These findings may help to model virus transmission dynamics and provide baseline data to support the development of vaccines and therapeutics.

Reconstruction of 60 Years of Chikungunya Epidemiology in the Philippines Demonstrates Episodic and Focal Transmission.

Proper understanding of the long-term epidemiology of chikungunya has been hampered by poor surveillance. Outbreak years are unpredictable and cases often misdiagnosed. Here we analyzed age-specific data from 2 serological studies (from 1973 and 2012) in Cebu, Philippines, to reconstruct both the annual probability of infection and population-level immunity over a 60-year period (1952-2012). We also explored whether seroconversions during 2012-2013 were spatially clustered. Our models identified 4 discrete outbreaks separated by an average delay of 17 years. On average, 23% (95% confidence interval [CI], 16%-37%) of the susceptible population was infected per outbreak, with >50% of the entire population remaining susceptible at any point. Participants who seroconverted during 2012-2013 were clustered at distances of <230 m, suggesting focal transmission. Large-scale outbreaks of chikungunya did not result in sustained multiyear transmission. Nevertheless, we estimate that >350 000 infections were missed by surveillance systems. Serological studies could supplement surveillance to provide important insights on pathogen circulation.

Sequential dengue virus infections detected in active and passive surveillance programs in Thailand, 1994-2010.

BACKGROUND: The effect of prior dengue virus (DENV) exposure on subsequent heterologous infection can be beneficial or detrimental depending on many factors including timing of infection. We sought to evaluate this effect by examining a large database of DENV infections captured by both active and passive surveillance encompassing a wide clinical spectrum of disease. METHODS: We evaluated datasets from 17 years of hospital-based passive surveillance and nine years of cohort studies, including clinical and subclinical DENV infections, to assess the outcomes of sequential heterologous infections. Chi square or Fisher's exact test was used to compare proportions of infection outcomes such as disease severity; ANOVA was used for continuous variables. Multivariate logistic regression was used to assess risk factors for infection outcomes. RESULTS: Of 38,740 DENV infections, two or more infections were detected in 502 individuals; 14 had three infections. The mean ages at the time of the first and second detected infections were 7.6 ± 3.0 and 11.2 ± 3.0 years. The shortest time between sequential infections was 66 days. A longer time interval between sequential infections was associated with dengue hemorrhagic fever (DHF) in the second detected infection (OR 1.3, 95% CI 1.2-1.4). All possible sequential serotype pairs were observed among 201 subjects with DHF at the second detected infection, except DENV-4 followed by DENV-3. Among DENV infections detected in cohort subjects by active study surveillance and subsequent non-study hospital-based passive surveillance, hospitalization at the first detected infection increased the likelihood of hospitalization at the second detected infection. CONCLUSIONS: Increasing time between sequential DENV infections was associated with greater severity of the second detected infection, supporting the role of heterotypic immunity in both protection and enhancement. Hospitalization was positively associated between the first and second detected infections, suggesting a possible predisposition in some individuals to more severe dengue disease.

Revealing the microscale spatial signature of dengue transmission and immunity in an urban population.

It is well-known that the distribution of immunity in a population dictates the future incidence of infectious disease, but this process is generally understood at individual or macroscales. For example, herd immunity to multiple pathogens has been observed at national and city levels. However, the effects of population immunity have not previously been shown at scales smaller than the city (e.g., neighborhoods). In particular, no study has shown long-term effects of population immunity at scales consistent with the spatial scale of person-to-person transmission. Here, we use the location of dengue patients' homes in Bangkok with the serotype of the infecting pathogen to investigate the spatiotemporal distribution of disease risk at small spatial scales over a 5-y period. We find evidence for localized transmission at distances of under 1 km. We also observe patterns of spatiotemporal dependence consistent with the expected impacts of homotypic immunity, heterotypic immunity, and immune enhancement of disease at these distances. Our observations indicate that immunological memory of dengue serotypes occurs at the neighborhood level in this large urban setting. These methods have broad applications to studying the spatiotemporal structure of disease risk where pathogen serotype or genetic information is known.

A model immunization programme to control Japanese encephalitis in Viet Nam.

In Viet Nam, an inactivated, mouse brain-derived vaccine for Japanese encephalitis (JE) has been given exclusively to ≤ 5 years old children in 3 paediatric doses since 1997. However, JE incidence remained high, especially among children aged 5-9 years. We conducted a model JE immunization programme to assess the feasibility and impact of JE vaccine administered to 1-9 year(s) children in 3 standard-dose regimen: paediatric doses for children aged <3 years and adult doses for those aged ≥ 3 years. Of the targeted children, 96.2% were immunized with ≥ 2 doses of the vaccine. Compared to the national immunization programme, JE incidence rate declined sharply in districts with the model programme (11.32 to 0.87 per 100,000 in pre-versus post-vaccination period). The rate of reduction was most significant in the 5-9 years age-group. We recommend a policy change to include 5-9 years old children in the catch-up immunization campaign and administer a 4th dose to those aged 5-9 years, who had received 3 doses of the vaccine during the first 2-3 years of life.

LABORATORY DIAGNOSIS OF DENGUE VIRUS INFECTIONS.

Dengue diagnosis was one of the topics discussed at "the adult dengue" presentations. In this paper, a review is presented focusing on the main challenges of dengue laboratory diagnosis. Accurate and efficient diagnosis of dengue is important for clinical care, surveillance support, pathogenesis studies, and vaccine research. Laboratory diagnosis is also important for case confirmation. Laboratory dengue diagnosis can be performed through virus isolation, genome and antigen detection and serological studies. For virus detection, dengue viremia is short, usually observed two or three days before onset of fever and lasts four to five days later. Therefore, samples for virus detection must be taken in the first four to five days of the disease during febrile phase. In recent years, PCR (polymerase chain reaction) has become an important tool as a quick method for diagnosis of dengue, another is detection of NS1 antigen, using commercial ELISA kit. Serological studies, for primary infection, the dominant immunoglobulin isotype is IgM, anti-IgM may appear during febrile phase (50% of cases), the other half, it appears within 2-3 days of defervescence. Once detectable, IgM levels rise quickly and appears to peak about 2 weeks after the onset of symptoms, then they decline to undetectable level over 2-3 months. Anti-IgG appears shortly afterwards with very low level. The physiological definition of a primary infection is therefore characterized by a high molar fraction of anti-dengue IgM and low molar fraction of IgG. Secondary dengue infections are characterized by a rapid increase in IgG antibodies, anti-dengue IgM appears in most instances, the level are dramatically lower.

A shorter time interval between first and second dengue infections is associated with protection from clinical illness in a school-based cohort in Thailand.

BACKGROUND: Despite the strong association between secondary dengue virus (DENV) infections and dengue hemorrhagic fever (DHF), the majority of secondary infections are subclinical or mild. The determinants of clinical severity remain unclear, though studies indicate a titer-dependent and time-dependent role of cross-protective anti-DENV antibodies. METHODS: Data from 2 sequential prospective cohort studies were analyzed for subclinical and symptomatic DENV infections in schoolchildren in Kamphaeng Phet, Thailand (1998-2002 and 2004-2007). Children experiencing ≥ 1 DENV infection were selected as the population for analysis (contributing 2169 person-years of follow-up). RESULTS: In total, 1696 children had ≥ 1 DENV infection detected during their enrollment; 268 experienced 2 or more infections. A shorter time interval between infections was associated with subclinical infection in children seronegative for DENV at enrollment, for whom a second-detected DENV infection is more likely to reflect a true second infection (average of 2.6 years between infections for DHF, 1.9 for DF, and 1.6 for subclinical infections). CONCLUSIONS: These findings support a pathogenesis model where cross-reactive antibodies wane from higher-titer, protective levels to lower-titer, detrimental levels. This is one of the first studies of human subjects to suggest a window of cross-protection following DENV infection since Sabin's challenge studies in the 1940s.

Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation.

Revisiting Rayong: shifting seroprofiles of dengue in Thailand and their implications for transmission and control.

Dengue virus has traditionally caused substantial morbidity and mortality among children less than 15 years of age in Southeast Asia. Over the last 2 decades, a significant increase in the mean age of cases has been reported, and a once pediatric disease now causes substantial burden among the adult population. An age-stratified serological study (n = 1,736) was conducted in 2010 among schoolchildren in the Mueang Rayong district of Thailand, where a similar study had been conducted in 1980/1981. Serotype-specific forces of infection (λ(t)) and basic reproductive numbers (R0) of dengue were estimated for the periods 1969-1980 and 1993-2010. Despite a significant increase in the age at exposure and a decrease in λ(t) from 0.038/year to 0.019/year, R0 changed only from 3.3 to 3.2. Significant heterogeneity was observed across subdistricts and schools, with R0 ranging between 1.7 and 6.8. These findings are consistent with the idea that the observed age shift might be a consequence of the demographic transition in Thailand. Changes in critical vaccination fractions, estimated by using R0, have not accompanied the increase in age at exposure. These results have implications for dengue control interventions because multiple countries in Southeast Asia are undergoing similar demographic transitions. It is likely that dengue will never again be a disease exclusively of children.

Hospital-based surveillance of Japanese encephalitis at a tertiary hospital in Manila.

Japanese encephalitis virus (JEV) is endemic in the Philippines but the incidence and burden of disease are not well established. We conducted a prospective hospital-based study at San Lazaro Hospital, a tertiary level hospital in Manila, from September 2005 to December 2006. Cases were determined using an in-house dengue and Japanese encephalitis (JE) enzyme-linked immunosorbent assay in order to detect the proportion of JE cases among the acute encephalitis syndrome (AES) cases admitted to our hospital. Fifteen patients were found to have AES, of whom 6 (40%) had confirmed JE. Of the JE cases, 4 were females and 2 were males with an age range of 3-14 years. Three of the 6 JE cases occurred during July. The most common signs and symptoms on admission among JE cases were: fever, headache, loss of appetite, neck rigidity and altered sensorium. JE likely comprises a significant proportion of hospitalized AES cases among children from Manila and nearby provinces. Further studies on the nation-wide prevalence and distribution of JE in the Philippines are needed to guide health authorities in disease control and prevention strategies.

Underrecognized mildly symptomatic viremic dengue virus infections in rural Thai schools and villages.

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.

Recombination and positive selection identified in complete genome sequences of Japanese encephalitis virus.

The mosquito-borne Japanese encephalitis virus (JEV) causes encephalitis in man but not in pigs. Complete genomes of a human, mosquito and pig isolate from outbreaks in 1982 and 1985 in Thailand were sequenced with the aim of identifying determinants of virulence that may explain the differences in outcomes of JEV infection between pigs and man. Phylogenetic analysis revealed that five of these isolates belonged to genotype I, but the 1982 mosquito isolate belonged to genotype III. There was no evidence of recombination among the Thai isolates, but there were phylogenetic signals suggestive of recombination in a 1994 Korean isolate (K94P05). Two sites of the genome under positive selection were identified: codons 996 and 2296 (amino acids 175 of the non-structural protein NS1 and 24 of NS4B, respectively). A structurally significant substitution was seen at NS4B position 24 of the human isolate compared with the mosquito and pig isolates from the 1985 outbreak in Thailand. The potential importance of the two sites in the evolution and ecology of JEV merits further investigation.

Inferring the serotype associated with dengue virus infections on the basis of pre- and postinfection neutralizing antibody titers.

BACKGROUND: Currently, the only tests capable of determining the serotype associated with dengue virus (DENV) infection require sampling during the period of acute viremia. No test can accurately detect the serotype associated with past DENV infections. The standard assay for determination of serotype-specific antibody against DENV is the plaque reduction neutralization test (PRNT), although performance of this test continues to be evaluated. METHODS: From a cohort study among schoolchildren in Thailand, PRNT values were determined in serum samples collected before and after infection. A multinomial logistic regression model was used to infer the serotype associated with intercurrent DENV infections. Models were validated based on polymerase chain reaction identification of DENV serotypes. RESULTS: The serotype associated with DENV infection inferred by the model corresponded with polymerase chain reaction in 67.6% of cases, and the kappa statistic was 0.479. A model for 35 cases with primary seroconversion correctly identified the DENV serotypes causing infection in 77.1% of cases, compared with 66.9%, using a model for 169 cases with secondary seroconversion. The best model using only postinfection PRNT values correctly inferred the DENV serotype causing infection in 60.3% of cases. CONCLUSIONS: A statistical model based on both pre- and postinfection PRNT values can be used to infer the serotype associated with DENV infections in prospective studies and vaccine trials.

Dengue hemorrhagic fever: the sensitivity and specificity of the world health organization definition for identification of severe cases of dengue in Thailand, 1994-2005.

BACKGROUND: Dengue virus infection causes a spectrum of clinical manifestations, usually classified according to the World Health Organization (WHO) guidelines into dengue fever (DF) and dengue hemorrhagic fever (DHF). The ability of these guidelines to categorize severe dengue illness has recently been questioned. METHODS: We evaluated dengue case definitions in a prospective study at a pediatric hospital in Bangkok, Thailand, during 1994-2005. One thousand thirteen children were enrolled within the first 3 days after onset of fever and observed with standardized data collection. Cases were classified on the basis of application of the strict WHO criteria. All dengue virus infections were laboratory confirmed. We retrospectively grouped patients on the basis of whether they received significant intervention based on fluid replacement and/or requirements for blood transfusion. RESULTS: Eighty-five (58%) of 150 persons with DHF, 40 (15%) of 264 with DF, and 73 (12%) of 599 with other febrile illnesses (OFIs) received significant intervention. Sixty-eight percent of dengue cases requiring intervention met strict WHO criteria for DHF. In contrast, only 1% of OFI cases met WHO criteria for DHF. Plasma leakage and thrombocytopenia were the 2 components contributing to the specificity of the WHO case definition and identified dengue cases that required intervention. Hemorrhagic tendency did not reliably differentiate DF and DHF. In DF cases, thrombocytopenia and bleeding were associated with severity. CONCLUSIONS: Dengue illness is heterogeneous in severity, and severe clinical features occurred in patients whose cases were not characterized as DHF. The WHO case definition of DHF demonstrated sensitivity of 62% and specificity of 92% for identification of dengue illness requiring intervention, without the need for laboratory confirmation of dengue virus infection, in an area of endemicity.

Travelling waves in the occurrence of dengue haemorrhagic fever in Thailand.

Dengue fever is a mosquito-borne virus that infects 50-100 million people each year. Of these infections, 200,000-500,000 occur as the severe, life-threatening form of the disease, dengue haemorrhagic fever (DHF). Large, unanticipated epidemics of DHF often overwhelm health systems. An understanding of the spatial-temporal pattern of DHF incidence would aid the allocation of resources to combat these epidemics. Here we examine the spatial-temporal dynamics of DHF incidence in a data set describing 850,000 infections occurring in 72 provinces of Thailand during the period 1983 to 1997. We use the method of empirical mode decomposition to show the existence of a spatial-temporal travelling wave in the incidence of DHF. We observe this wave in a three-year periodic component of variance, which is thought to reflect host-pathogen population dynamics. The wave emanates from Bangkok, the largest city in Thailand, moving radially at a speed of 148 km per month. This finding provides an important starting point for detecting and characterizing the key processes that contribute to the spatial-temporal dynamics of DHF in Thailand.