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Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.

Background Plaque size is a common feature of viral characterization. Small plaque size is used as a marker of attenuation for live-attenuated vaccine development. Objective To investigate whether the naturally occurring plaque size variation reflects virulence of the variants of chikungunya virus (CHIKV). Methods We selected and purified a variant with small plaque size from the primary isolate. The viral variant was tested for the plaque morphology, in vitro growth kinetics and mouse neurovirulence in comparison with the parental wild type. Results The small plaque size variant showed stable homogenous small plaques after 4 plaque purifications. The small plaque virus grew slower and to the lower titer when compared with wild type virus. After 21 days of infection, mice that received small plaque virus showed 98% survival rate while 74% of mice survived after infected with wild type virus. Conclusion The small plaque size variant of CHIKV can be obtained by plaque purification and the virus displays decreased virulence.

Dengue virus (DENV) infection is considered one of the most important mosquito‐borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV‐infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56(hi) CD16(−) and CD56(lo) CD16(+) NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56(hi) CD16(−) subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56(lo) CD16(+) subset compared with those in the CD56(hi) CD16(−) subset. Moreover, although the CD56(lo) CD16(+) subset contained a high frequency of cells expressing skin‐homing markers, the CD56(hi) CD16(−) subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is the etiological agent of chikungunya fever. CHIKV re-emerged from 2004 onwards, and subsequently caused major outbreaks in many parts of the world including the Indian Ocean islands, Asia, and the Americas. In this study, a large plaque variant of CHIKV isolated from patient in Thailand was subjected to repeated cycles of plaque-purification in Vero cells. The resulting virus produced homogenous large plaques and showed a more pathogenic phenotype than the parental wild-type CHIKV. Whole genome analysis of the large plaque virus in comparison to parental isolate revealed a number of mutations, leading to the following amino acid changes: nsP2 (P

Real-time PCR validation of siRNA mediated gene silencing of fatty acid synthase (FASN) gene. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 5 post transfection the level of FASN transcript was determined by real time PCR. Normalization expression data relative to actin is shown. Bars show mean +/−SD. (a) 1 day post transfection, (b) 2 days post transfection, (c) 3 days post transfection, (d) 4 days post transfection and (e) 5 days post transfection. Bars show mean +/−SD (*; p value

List of gene specific primers (PDF 56 kb)

Dengue is the most significant arthropod borne viral disease worldwide, and infection with the dengue virus causes a wide range of symptoms in humans, including bone marrow suppression. While the target cells of the virus remain poorly characterized, cells of the myeloid lineage have been shown to be important mediators of the disease. This study sought to determine whether erythroid precursor cells were susceptible to dengue virus infection, and whether erythroid cells from thalassemia trait carriers showed any protection against infection. Infection with a laboratory adapted high passage DENV-2 resulted in high levels of infection during certain stages of differentiation, and cells derived from thalassemia trait carriers showed significantly reduced susceptibility to dengue virus infection. Infection with low passage isolates resulted in only scattered cells showing evidence of infection, but high bystander apoptosis that was reduced by both a caspase 8 inhibitor and anti-tumor necrosis factor 1 receptor antibodies.

Summary Background An estimated 100 million people have symptomatic dengue infection every year. This is the first report of a phase 3 vaccine efficacy trial of a candidate dengue vaccine. We aimed to assess the efficacy of the CYD dengue vaccine against symptomatic, virologically confirmed dengue in children. Methods We did an observer-masked, randomised controlled, multicentre, phase 3 trial in five countries in the Asia-Pacific region. Between June 3, and Dec 1, 2011, healthy children aged 2–14 years were randomly assigned (2:1), by computer-generated permuted blocks of six with an interactive voice or web response system, to receive three injections of a recombinant, live, attenuated, tetravalent dengue vaccine (CYD-TDV), or placebo, at months 0, 6, and 12. Randomisation was stratified by age and site. Participants were followed up until month 25. Trial staff responsible for the preparation and administration of injections were unmasked to group allocation, but were not included in the follow-up of the participants; allocation was concealed from the study sponsor, investigators, and parents and guardians. Our primary objective was to assess protective efficacy against symptomatic, virologically confirmed dengue, irrespective of disease severity or serotype, that took place more than 28 days after the third injection. The primary endpoint was for the lower bound of the 95% CI of vaccine efficacy to be greater than 25%. Analysis was by intention to treat and per procotol. This trial is registered with ClinicalTrials.gov, number NCT01373281. Findings We randomly assigned 10 275 children to receive either vaccine (n=6851) or placebo (n=3424), of whom 6710 (98%) and 3350 (98%), respectively, were included in the primary analysis. 250 cases of virologically confirmed dengue took place more than 28 days after the third injection (117 [47%] in the vaccine group and 133 [53%] in the control group). The primary endpoint was achieved with 56·5% (95% CI 43·8–66·4) efficacy. We recorded 647 serious adverse events (402 [62%] in the vaccine group and 245 [38%] in the control group). 54 (1%) children in the vaccine group and 33 (1%) of those in the control group had serious adverse events that happened within 28 days of vaccination. Serious adverse events were consistent with medical disorders in this age group and were mainly infections and injuries. Interpretation Our findings show that dengue vaccine is efficacious when given as three injections at months 0, 6, and 12 to children aged 2–14 years in endemic areas in Asia, and has a good safety profile. Vaccination could reduce the incidence of symptomatic infection and hospital admission and has the potential to provide an important public health benefit. Funding Sanofi Pasteur.

The independent, scientific and educational The Association of South East Asian Nations (ASEAN) Member States Dengue Vaccination Advocacy Steering Committee (ADVASC) was established in 2011 to address the practical challenges faced by ASEAN countries as they prepare for the eventual introduction of a dengue vaccine. ADVASC convened a workshop in September 2012 that drew together public health representatives and dengue experts from seven ASEAN countries in order to make practical recommendations to improve current surveillance and diagnostics for dengue to enable countries to assess consistently, and accurately communicate, the impact of a dengue vaccine. The workshop compared surveillance and diagnostic capacity in these ASEAN countries and made recommendations to streamline and harmonize key elements of these systems. In particular, attendees recommended the need for reconciliation and harmonization of the different World Health Organization guidelines, in use in ASEAN countries for case definition and surveillance of dengue.

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5'UTR, prM, E, NS1, NS2A, NS3, and 3'UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5'UTR of V2 was different from P and V1. The secondary structure of 3'UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5'UTR, 3'UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.

A single nucleotide polymorphism located at the promoter -308A of tumour necrosis factor-alpha (TNF-α) gene may affect transcription and increase cytokine production, leading to the severe manifestation of dengue virus infection.To study the association of the TNF-α -308A allele and the severity of patients with dengue infection.112 patients suspected of having dengue infection and 106 normal controls were enrolled in the study. Mean (SD) age was 10.4 (3.6) years. In all, 19 and 82 patients were diagnosed with dengue fever (DF) and dengue haemorrhagic fever (DHF), respectively, while 11 were diagnosed with other febrile illnesses (OFIs). They were tested for the polymorphisms at the promoter -308 position of the TNF-α gene and their TNF-α levels were measured.In the patients with dengue infection (14/202, 6.9%) with OFIs (1/22, 4.5%) and in normal controls (17/212, 8.0%), the frequency of the TNF-α -308A allele was not significantly different. Moreover, no statistically significant difference was found in patients with various clinical manifestations of dengue infection involving DF (5.3%, 2/38), DHF grade I (5.0%, 2/40), DHF grade II (9.5%, 4/42), DHF grade III (2.5%, 1/40) and DHF grade IV (11.9%, 5/42). However, patients with dengue infection and significant bleeding manifestations, apart from petechiae and ecchymosis, tended to have a higher frequency of the TNF-α -308A allele (11.8%, 9/76) than those without significant bleeding manifestations (5/126, 4.0%) (P = 0.056). The levels of TNF-α were additionally measured in 67 patients but the results failed to demonstrate a functional effect in the transcriptional rate of the TNF-α -308A allele.In patients with dengue infection there is an association between the TNF-α -308A allele and the risk of bleeding. The test may be used as one of the predictors of the severity of dengue infection.

Liver is a site of viral replication and liver dysfunction is a characteristic of severe dengue infection. To understand these mechanisms, we analyzed the response of a hepatic cell linage, HepG2 to infection with dengue 3 virus (strain 16562). Steady state levels of mRNA accumulation were assessed for 14 genes involved in modulation of the host immune responses, at 6, 24 and 48 hpi, by quantitative reverse transcription real-time PCR (qRT-PCR). Fourteen genes showed altered expression upon infection with D3V including; cytokines/chemokines (IL-1β, IL-6, IL-8, RANTES, MCP-2, IL-2Rα and TGF-βIIIR), type I interferon (IFN-α and IFN-β), and pattern-recognition receptors (TLR3, TLR8, RIG-1, MDA5 and MyD88). Although these genes are associated with mechanism of innate immune response and anti-viral activity, their altered expression does not inhibit D3V (strain 16562) growth kinetics and virus yield in HepG2 cells. Gene expression in liver may explain pathological changes associated with dengue virus infection.

Background Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Methodology/Principal Findings The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). Conclusions/Significance Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity., Author Summary Dengue is the most important mosquito-borne viral disease of humans and an enormous public health burden in affected countries. Early, sensitive and specific diagnosis of dengue is needed for appropriate patient management as well as for early epidemic detection. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offer the possibility of early and rapid diagnosis. Here we evaluated two commercially available ELISA kits for NS1 detection (Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag). Results were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

The evolution of dengue viruses has resulted in four antigenically similar yet distinct serotypes. Infection with one serotype likely elicits lifelong immunity to that serotype, but generally not against the other three. Secondary or sequential infections are common, as multiple viral serotypes frequently cocirculate. Dengue infection, although frequently mild, can lead to dengue hemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary dengue infections, implying a role for the adaptive immune response in the disease. There is currently much effort toward the design and implementation of a dengue vaccine but these efforts are made more difficult by the challenge of inducing durable neutralizing immunity to all four viruses. Domain 3 of the dengue virus envelope protein (ED3) has been suggested as one such candidate because it contains neutralizing epitopes and it was originally thought that relatively few cross-reactive antibodies are directed to this domain. In this study, we performed a detailed analysis of the anti-ED3 response in a cohort of patients suffering either primary or secondary dengue infections. The results show dramatic evidence of original antigenic sin in secondary infections both in terms of binding and enhancement activity. This has important implications for dengue vaccine design because heterologous boosting is likely to maintain the immunological footprint of the first vaccination. On the basis of these findings, we propose a simple in vitro enzyme-linked immunosorbent assay (ELISA) to diagnose the original dengue infection in secondary dengue cases. Dengue virus is an insect-borne flavivirus transmitted to humans by the bite of an infected mosquito, usually Aedes aegypti (20). There are four circulating serotypes of dengue (dengue serotype 1 [Den1] to Den4) that show up to 70% sequence homology across their genomes (4, 17), and it is common for multiple viral serotypes to cocirculate in countries where dengue is endemic. Most dengue infections are either asymptomatic or lead to uncomplicated dengue fever (DF). However, in 1 to 5% of cases, symptoms can be more severe with the development of plasma leakage and hemorrhage. Such dengue hemorrhagic fever (DHF) can lead to circulatory collapse, resulting in a mortality rate of around 20% if left untreated. The more frequent occurrence of DHF in secondary dengue infections in children and adults suggests a role for the acquired immune system in disease pathogenesis, and there has been considerable research into both the B- and T-cell responses. Antibody-dependent enhancement (ADE) of infection, proposed by Halstead in 1977 (24, 25), is one hypothesis for this increase in severity in secondary infections (23, 36). During a primary infection, antibodies that cross-react with the remaining 3 serotypes are induced. After a few months, when heterologous protection is no longer observed (54), it is hypothesized that these cross-reactive antibodies decline to subneutralizing levels, meaning that a heterologous infecting serotype is not controlled. Antibody made against the primary infecting virus may not be of sufficient avidity to neutralize a second serotype. Instead, these poorly neutralizing, low-avidity cross-reactive antibodies bind the secondary virus and target it to Fcγ receptor-bearing cells, such as macrophage/monocytes (24, 25), leading to internalization and increased virus replication. In vivo, ADE has been shown to induce lethal disease in mice (2) and to drive high virus loads in primates (16, 22). ADE has also been invoked to explain a peak in disease severity in primary cases during the first year of life, as the titers of passively transferred maternal antibody fall (23, 35, 41, 56). The incidence of dengue increased sharply in the middle of the last century and is still increasing at an alarming rate (20). There are estimated to be around 3.6 billion people living in the tropics and subtropics who are at risk from dengue, with up to 50 million predicted infections per annum. To date, however, there are no specific treatments for dengue barring careful attention to fluid replacement. The scale of the problem posed by dengue has spawned much interest in the development of a dengue vaccine, with some candidates in phase II trials (67, 69), and also in anti-dengue drugs that have not yet reached clinical trials. A number of vaccine candidates, ranging from live attenuated viruses to subunit vaccines, are currently being pursued. The envelope (E) protein of dengue is a major target of neutralizing (48, 53, 62) and protective antibodies (32) and, as such, should be a key component for any subunit vaccines. The envelope protein consists of three domains: ED1, ED2, and ED3 (38, 47, 48). ED3 is proposed to be the binding domain for the virus (7, 9, 28), attaching to as yet poorly characterized cellular receptor(s); although heparan sulfate has been implicated in the interaction (29). Indeed, in mice, anti-ED3 monoclonal antibodies are potent neutralizers of dengue virus (5, 18, 19, 27, 43, 52, 55, 60, 61); often neutralizing to greater levels than those targeting ED1 or ED2 (60). ED3 is a target of both serotype-specific (5, 18, 42, 45, 53, 55, 60, 61) and cross-reactive (19, 43, 46, 52, 55, 60, 61) neutralizing antibodies. Of the anti-ED3 antibodies that are strongly neutralizing, however, the majority are usually serotype specific (5, 55, 60), and cross-reactive antibodies are generally weaker neutralizers (19, 61). There have been a number of vaccination studies investigating ED3 as a potential immunogen (3, 30, 57-59). In this study, we have performed a detailed analysis of the anti-ED3 antibody responses in humans, using a cohort of patients experiencing either primary or secondary dengue infections. We demonstrate that low-level dengue cross-reactive anti-ED3 responses are induced upon primary infection and boosted dramatically during a secondary heterotypic infection. We have developed a competition enzyme-linked immunosorbent assay (ELISA) to measure the relative avidities of these cross-reactive antibodies and show that the response is dominated by original antigenic sin. We propose this ELISA as a simple diagnostic test for determining the serotype of the original dengue infection in secondary dengue cases. Furthermore, we go on to show an inverse relationship between the avidity of the antibody response and enhancing activity.

Background Japanese Encephalitis chimeric virus vaccine (JE-CV) and SA14-14-2 vaccine are live-attenuated JE vaccines produced from the same virus strain. Data on interchangeability is limited. Objectives To evaluate the immunogenicity and safety of JE-CV booster after primary vaccination with SA14-14-2 vaccine. Methods This study was an open-label clinical trial in Thai children who had received a primary SA14-14-2 vaccination at 12–24 months before enrollment (ClinicalTrials.gov NCT02602652). JE-CV was administered. A 50% plaque reduction neutralization test (PRNT50) against three virus strains; JE-CV, SA-14-14-2 and wild-type JE virus was measured before and 28-days post vaccination. The laboratory was performed at PRNT50 titers ⩾10 (1/dil) were considered seroprotective against JE. Geometric mean titer (GMT) of PRNT50 was calculated. Adverse events were observed for 28 days. Results From March 2014 to June 2015, 50 children (64% male) were enrolled. Mean age and duration after primary vaccination was 26.9 (SD 4.6) and 12.8 (SD 2.7) months, respectively. The proportion of participants who had PRNT50pre and post-booster vaccination were 92% and 96% against JE-CV virus, 56% and 98% against SA-14-14-2 strain and 70% and 98% against wild-type JE virus, respectively. Solicited injection site reactions including erythema, pain and swelling occurred in 18%, 10% and 4% of subjects, respectively. Four children (8%) had fever (⩾37.7 Celsius). Eight children (16%) had adverse events, which were not related to the vaccine. Conclusions AJE-CV booster dose is highly immunogenic and safe among children who previously received SA14-14-2 vaccine.

Healthy donors exposed to Japanese encephalitis (JE) virus show a CD8+ T cell response that cross reacts with other flaviviruses. Patients that recovered from JE show a CD4+ T cell response that targets structural proteins of JE virus., Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8+ and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4+ and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4+ T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4+ and CD8+ T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus.

The leakage of plasma during febrile episodes in dengue-infected patients is a severe condition leading to dengue shock syndrome. Alteration of cytokines/chemokines is suspected to be a major cause of endothelial cell damage in these patients. The study was designed to demonstrate the alteration of cytokines and chemokines in dengue-infected patients during febrile episodes.The blood samples from 164 patients with dengue fever, dengue hemorrhagic fever and other febrile illnesses were collected daily from the day of hospitalization until discharge and also in the convalescent stage. The levels of cytokines/chemokines were determined using a sandwich chemiluminescent immunoassay, and the hematological parameters were examined by the ADVIA hematological analyzer.Two patterns of cytokines/chemokines alteration were detected at different time points during the febrile episode. The increased factors included interleukin (IL)-4, IL-6, IL-8, IL-10, tumor necrosis factor-α, interferon-γ and monocyte chemoattractant protein-1 whereas IL-1β, IL-2, vascular endothelial growth factor and epidermal growth factor were decreased. Several cytokines were correlated with disease severity especially in dengue hemorrhagic fever/dengue shock syndrome patients.The alteration in the cytokine/chemokine kinetics during a febrile episode can be used as a predictor for severe dengue infection. The increased and decreased levels at different time points can indicate the disease progression related to vascular leakage in dengue hemorrhagic fever/dengue shock syndrome patients.

We herein have reported a case of severe nonfebrile dengue infection complicated with refractory pancytopenia and a large perinephric hematoma with shock in a 16-year-old adolescent during the early postoperative period after kidney transplantation. After the diagnosis of end-stage renal disease she underwent living-related kidney transplantation. Thirteen days after successful transplantation, she exhibited a notable amount of ascites, bilateral pleural effusions, thrombocytopenia, and increased hemoglobin without pre-existent fever. Further investigation revealed positive dengue nonstructural protein 1 antigen (dengue NS1 Ag) and dengue virus serotype 1 by a reverse transcriptase-polymerase chain reaction (RT-PCR) in the patient's serum. She exhibited hemophagocytic syndrome, manifested by refractory pancytopenia and refractory anemia resulting in hypovolemic shock and acute graft failure on day 28 posttransplantation. The anemia was attributed to a large hematoma around the transplanted kidney requiring surgical evacuation of clotted blood. Postoperatively, she gradually recovered with resolution of thrombocytopenia and excellent graft function. Persistent dengue antigenemia and viremia was shown by dengue NS1 Ag and RT-PCR of dengue serotype-1. The viremia was present longer than the dengue antigenemia. Dengue-specific immunoglobulin M (IgM) and IgG by enzyme-linked immunosorbent assay confirmed the primary dengue infection and evidence of a recent donor dengue infection.

There have been no recommendations for revaccination with the Japanese encephalitis (JE) vaccine in post-hematopoietic stem cell transplantation (HSCT) patients. This study aimed to measure the immunogenic response to a live-attenuated JE vaccine (SA 14-14-2) in post-HSCT patients. JE-specific neutralizing Ab titers were measured before and after the JE vaccination. The patients with Ab titers

We report a prospective study of mouse brain derived inactivated Japanese encephalitis (JE) vaccine, given in 3-dose EPI program to human immune deficiency virus (HIV)-exposed Thai infants. 18 HIV-infected receiving antiretroviral therapy with median baseline CD4 of 33.1%, and 92 HIV-uninfected children were studied. All but one HIV-infected child seroconverted after the second dose. The geometric mean titers (GMTs) 3 months after the second and third doses in HIV-infected vs HIV-uninfected children were 247 vs 938 (p = 0.022), and 2273 vs 24069 (p = 0.009), respectively. Urticaria or angioedema found in 4% and 6% in HIV-infected and -uninfected children, respectively (p = 1.0). The vaccine was safe and immunogenic but antibody response in HIV-infected children was not as high as in uninfected children.

Summary Japanese encephalitis (JE) virus is a major cause of disease, disability, and death in Asia. An effective, live, attenuated JE vaccine (LJEV) is available; however, its use in routine immunization schedules is hampered by lack of data on concomitant administration with measles vaccine (MV). This study evaluated the immunogenicity and reactogenicity of LJEV and MV when administered at the same or separate study visits in infants younger than 1 year of age. Three groups of healthy infants were randomized to receive LJEV at age of 8 months and MV at 9 months (Group 1; n = 100); MV and LJEV together at 9 months (Group 2; n = 236); or MV and LJEV at 9 and 10 months, respectively (Group 3; n = 235). Blood was obtained 4 weeks after each vaccine administration to determine antibody levels for measles and JE. Reactogenicity was assessed by parental diaries and clinic visits. Four weeks after immunization, measles seroprotection rates (defined as ≥340 mIU/ml) were high and comparable in all three groups and specifically, rates in the combined MV–LJEV (Group 2) were not statistically inferior to those in Group 3 receiving MV separately (96% versus 100%, respectively). Likewise, the LJEV seroprotection rates were high and similar between the three groups. The reactogenicity profiles of the three vaccine schedules were also analogous. LJEV and MV administered together are well tolerated and immunogenic in infants younger than 1 year. These results should facilitate incorporation of LJEV into routine immunization schedules with MV.

In July 1999, a single dose of live-attenuated SA 14-14-2 Japanese encephalitis (JE) vaccine was administered to children living in the Bardiya, Banke and Kailali districts of Nepal. In 2004, the original vaccinated population experienced a fifth seasonal exposure to JE. We performed a case-control study comparing the prevalence of the administration of vaccine in patients with JE hospitalized in the Bardiya and Bheri Zonal hospitals and in age-sex matched controls resident in the Bardiya district. Among the 219 village controls, 114 had been vaccinated (52.1%) while only one of 20 JE cases had received live-attenuated JE vaccine. Five years after administration of a single dose, SA 14-14-2 provided a protective efficacy of 96.2% (CI 73.1-99.9%).

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.

Susceptibility baselines and diagnostic doses of the technical grade insecticides deltamethrin, permethrin, fenitrothion, and propoxur were established based on Aedes aegypti (L.), Bora (French Polynesia), a reference susceptible strain. Field-collected Aedes mosquitoes from each part of Thailand were subjected to bioassay for their susceptibility to the diagnostic doses of each insecticide. Almost all Ae. aegypti collected were incipient resistant or resistant to deltamethrin and permethrin, except those from some areas of Songkhla (southern) and Phan district of Chiang Rai (northern) province. Susceptibility to fenitrothion was found in mosquitoes from Bangkok (central), Chonburi (eastern), Chiang Rai, Kanchanaburi (western), and Songkhla, whereas they were resistant in almost all areas of Nakhon Sawan (north central) and Nakhon Ratchasima (northeastern) provinces. Most of Ae. aegypti were susceptible to propoxur except those from Mae Wong, Nakhon Sawan province. Various levels of insecticide resistance and susceptibility in adjacent areas revealed a focal susceptible/resistance profile in the country. It could be noted that almost all of Ae. albopictus were susceptible to the insecticides tested at the same diagnostic doses. In conclusion, resistance to pyrethroids (permethrin and deltamethrin) has developed in Ae. aegypti in most of the collected areas, suggesting that an alternative choice of insecticide or other control measures should be applied.

Dengue virus (DENV) is the most important mosquito transmitted human viral pathogen. There are four different dengue viruses (DENV 1 to DENV 4) with multiple genotypes and strains. Whether there are significant differences in how these DENVs interact with and modulate the host cell proteome remains unclear. Using a panel of 12 DENVs representative of one isolate for each DENV from three different origins (lab adapted, low passage isolates from dengue fever patients, low passage isolates from dengue hemorrhagic fever patients) LLC-MK2 cells were equally infected and proteomic alterations compared by MALDI-TOF and principal component analysis and a sub-10 kDa peptidome analysis. There was no clear segregation of data with respect to either virus origin or serotype in either the MALDI-TOF or the peptidome analysis. The two isolates with the greatest variation from the other isolates in the MALDI-TOF analysis were a low passage DENV 3 dengue fever isolate and a low passage DENV 4 dengue hemorrhagic fever isolate. Analysis of the sub-10 kda protein fraction by LC-MS/MS identified 128 proteins of which only 28 (20%) were constantly expressed in all infections, while 80% showed variable expression, with no clear relationship with either serotype or virus origin. These results suggest that the interaction between DENV and the host cell is characterized by a degree of plasticity, whereby the end biological processes are not rigorously determined by specific proteome alterations, and that virus strain plays a role in determining the specific proteome changes.

Background The concept of antibody dependent enhancement (ADE) of dengue virus (DENV) infection is a cornerstone of our current understanding of dengue pathogenesis, although some questions as to the mechanism remain, particularly in regards to the behavior of low and high passage virus isolates. This study utilized two low passage DENV 4 isolates and a laboratory adapted DENV 4 isolate to investigate the potential of low passage isolates to undergo ADE. Results Little or no ADE of infection was observed on day 2 post infection with low passage isolates, while high enhancement of infection was seen with the laboratory adapted virus. However, both of the low passage isolates showed high levels of infection (60–100 %) by day 5 post infection. Conclusions These results show that low passage DENV 4 viruses undergo ADE mediated infection, but that the process is significantly temporally delayed as compared to laboratory adapted DENV 4. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1381-8) contains supplementary material, which is available to authorized users.

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.

Understanding dengue virus infection in children and the kinetics of maternal dengue neutralizing antibodies is essential for effective dengue immunization of children in endemic areas.Serum samples from 219 mother-child pairs and 140 children at 3, 6, 9, 12, 18, and 24 months of age from Bangkok, Thailand, were tested for serotype-specific dengue antibodies. Febrile episodes in the children were recorded.Antibodies were found in 97% of cord serum samples and disappeared in 27%, 80%, and 95% of the children by the age of 6, 9, and 12 months, respectively. Geometric mean titers (GMTs) of the antibodies to 4 dengue serotypes decreased to 5.4-15.5 in 6-month-old infants. Eleven of 12 children acquired dengue virus infection at 6 months of age and beyond; 1 had the infection at 3 months of age. Two exhibited undifferentiated febrile illnesses, and 10 had subclinical infections.Evidence of dengue virus infection and very low GMTs against all dengue serotypes in children at 6 months of age and beyond was demonstrated. There was no evidence that maternal antibodies were harmful to infants. Dengue virus infection rates increase from 12 months of age onward. These data provide information for supporting the optimal age at vaccination.

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax-DEN2) in comparison to that of YF vaccine (YF-VAX). Forty-two healthy YF naive adults randomly received a single dose of either ChimeriVax-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAX by the subcutaneous route (SC). To determine the effect of YF preimmunity on the ChimeriVax-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naive subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVax-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX seroconverted to YF 17D virus but none of YF naive subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3 and 4 were observed in YF naive subjects inoculated with either ChimeriVax-DEN2 or YF-VAX. In contrast, 100% of YF immune subjects inoculated with ChimeriVax-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that (1) the safety and immunogenicity profile of the ChimeriVax-DEN2 vaccine is consistent with that of YF-VAX, and (2) preimmunity to YF virus does not interfere with ChimeriVax-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.

We conducted a Phase 1b study to evaluate the immunogenicity and safety of two live attenuated tetravalent dengue vaccines in healthy adult volunteers. After one injection, all subjects reported systemic reactions consistent with a mild dengue-like syndrome. Seven volunteers developed dengue 3 viraemia after vaccination. All subjects developed a neutralizing antibody response against serotype 3 with partial response against other serotypes. The trial was stopped early (after 10 subjects enrolled) due to formulation issues, which were related to the dengue 3 vaccine component. Managing viral interference and balancing attenuation to produce acceptable tetravalent immunogenicity with minimal reactogenicity may be a recurring problem for future multivalent live vaccines.

Dengue fever and dengue hemorrhagic fever are mosquito-borne viral diseases. Dendritic cell–specific ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), an attachment receptor of dengue virus, is essential for productive infection of dendritic cells1,2. Here, we report strong association between a promoter variant of CD209, DCSIGN1-336, and risk of dengue fever compared with dengue hemorrhagic fever or population controls. The G allele of the variant DCSIGN1-336 was associated with strong protection against dengue fever in three independent cohorts from Thailand, with a carrier frequency of 4.7% in individuals with dengue fever compared with 22.4% in individuals with dengue hemorrhagic fever (odds ratio for risk of dengue hemorrhagic fever versus dengue fever: 5.84, P = 1.4 × 10−7) and 19.5% in controls (odds ratio for protection: 4.90, P = 2 × 10−6). This variant affects an Sp1-like binding site and transcriptional activity in vitro. These results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever. This may have consequences for therapeutic and preventive strategies. Supplementary information The online version of this article (doi:10.1038/ng1550) contains supplementary material, which is available to authorized users.

SUMMARY We did a seroepidemiological study to determine the circulating dengue virus serotypes and the extent to which the Singapore population has been exposed to multiple dengue virus serotypes, using the plaque reduction neutralization assay (PRNT). Of 164 enrolled subjects aged between 18-30 years, 49 subjects (29-8 %) were PRNT positive for at least one dengue serotype. The seroprevalence was 39 (238 %) for dengue virus serotype 1, 37 (22-6%) for type 2, 43 (26-2 %) for type 3, and 30 (18-3 %) for type 4. Of the 49 subjects with PRNT-positive dengue virus results, 28 (57-1 %) were positive to all four virus serotypes, seven (14-3 %) to three serotypes, two (4 %) to two serotypes, and 12 (24-5 %) to a single serotype. All four dengue virus serotypes circulate in Singapore, and a substantial proportion of the adult population in Singapore had exposure to more than one dengue virus serotype. In spite of multiple circulating types, the rate of dengue haemorrhagic fever is low in Singapore.

A comparison of the sequence of the dengue-2 16681 virus with that of the candidate vaccine strain (16681-PDK53) derived from it identified 53 of the 10,723 nucleotides which differed between the strains. Nucleotide changes occurred in genes coding for all virion and nonvirion proteins, and in the 5′ and 3′ untranslated regions. Twenty-seven of the nucleotide changes resulted in amino acid alterations. The greatest amino acid sequence differences in the virion proteins occurred in prM (2.20%; 2 91 amino acids) followed by the M protein (1.33%; 1 75 amino acids), the C protein (0.88%; 1 114 amino acid), and the E protein (0.61%; 3 495 amino acids). Differences in the amino acid sequence of nonvirion proteins ranged from 1.51% ( 6 398 amino acids) in NS4 to 0.33% ( 3 900 amino acids) in NS5. The encoded protein sequences of 16681-PDK53 were also compared with the published sequences of other flaviviruses to obtain a detailed classification of 17 flaviviruses using the neighbor-joining tree method. The analyses of the sequence data produced dendrograms which supported the traditional groupings based on serological evidence, and they suggested that the flaviviruses have evolved by divergent mutational change and there was no evidence of genetic recombination between members of the group. Comparisons of the sequences of the flavivirus polymerase and helicase-like proteins (NS5 and NS3, respectively) with those from other viruses yielded a classification of the flaviviruses indicating that the primary division of the flaviviruses was between those transmitted by mosquitoes and those transmitted by ticks.

The safety and immunogenicity of tetravalent live-attenuated dengue vaccines after a three dose vaccination series were evaluated in Thai children. One hundred three healthy flavivirusseronegative schoolchildren ages 5 to 12 years were randomized to receive either dengue vaccine containing 3 2 1 and 2 log/10 of the 50% cell culture infective dose respectively of the live attenuated dengue vaccine serotypes 1 2 3 and 4 per dose (F3212; n = 40) or 3 3 1 and 3 log/10 of the 50% cell culture infective dose (F3313; n = 42) or purified Vero cell rabies vaccine (control group; n = 21) given in a two dose schedule (3 to 5 months apart). A third dose was administered 8 to 12 months after the second dose to 90 subjects. Safety and immunogenicity were evaluated within 28 days after each injection. No serious adverse event related to the vaccines occurred. Most children experienced mild to moderate fever rash headache and myalgia occurring within 12 days after Dose 1 and generally lasting 3 days or less. One subject in Group F3212 had a 1-week dengue-like fever. Reactogenicity was minimal after Doses 2 and 3. Transient mild variations in liver enzymes and hematologic indices were noted mainly after Dose 1. After the third dose 89% of the subjects in Group F3212 seroconverted (neutralizing antibody response >10) to all four serotypes and all children in Group F3313 seroconverted. This study demonstrates a moderate although improvable reactogenicity and high seroconversion rates against the four serotypes of dengue after a three dose schedule of tetravalent live-attenuated dengue vaccine in children. (authors)

Volume 53, no. 2, p. [382–388][1], 2015. Page 384, Fig. 1: Incorrect images were mistakenly placed in the second (Thai), fourth (S27), and sixth (SV) columns in the row labeled “Alphavirus Antibody.” The figure should appear as shown below. ![Figure][2] Page 385, Table 2: Several

In a dengue-endemic area, sera from 42 mother-infant pairs and sera collected from the infants at follow-up at the ages of 3 months (n =27), 6 months (n =34), 9 months (n =23) and 12 months (n =8) were tested for antibodies to four dengue serotypes using a plaque reduction neutralisation test (PRNT(50)), IgG ELISA and haemagglutination inhibition assay (HAI). The IgG ELISA and HAI tests were less sensitive than PRNT(50) in detecting low levels of antibodies. Levels of maternally transferred dengue neutralising antibodies in the cord sera were very high and identical to those in the mothers. Neutralising antibody prevalences in the newborns were 95% to dengue serotype 1 (DEN-1), 93% to DEN-2 and DEN-3 and 91% to DEN-4. The antibodies decreased with increasing age. At least one dengue serotype neutralising antibody persisted in the infants at ages 3, 6 and 9 months in 92%, 69% and 13%, respectively. No maternally transferred antibody was observed in 12-month-old infants. In endemic areas therefore, where most infants have maternally transferred dengue antibodies, interference with dengue vaccine is likely to be less after 12 months of age than before then.

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

Dengue fever, caused by four serotypes of a mosquito-borne virus, is a growing problem in tropical countries. Currently, there is no treatment or vaccine. We evaluated safety and immunogenicity of two doses, given six months apart, of seven formulations of dengue tetravalent live-attenuated vaccine (containing different concentrations of the component viruses) versus placebo in 59 flavivirus-seronegative Thai adults. The first dose was the more reactogenic. Most volunteers experienced clinically moderate fever, headache, myalgia, eye pain or rash 7-11 days after injection, generally lasting three days or less. Modest decreases in platelets and neutrophils were observed. After one dose, 58% of dengue recipients seroconverted (neutralizing antibody level > or = 1:10) against > or = 3 serotypes; 35% seroconverted against all four. After the second dose, seroconversion was 76% and 71%, respectively. All subjects seroconverted to serotype 3 after one dose. Serotype 4 elicited the lowest primary response but the highest increase in seroconversion after the second dose.

Global Disease Detection Regional Center. Centers for Disease Control. Nairobi, Kenya. Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil. Pasteur Institute of Ho Chi Minh City. Arbovirus Laboratory, Ho Chi Minh City, Vietnam. Mahidol University. Center for Vaccine Development. Bangkok, Thailand. Instituto Pasteur, Paris, France. This paper conclud that assess the overall benefits of vaccination in high burden countries for dengue, long-term surveillance will require a robust, affordable, and user-friendly diagnostic algorithm to detect suspected dengue cases. After that Since a large variability in the results of the diagnostics may occur between different kits and laboratories worldwide, future phase IV studies of CYD-TDV vaccine effectiveness should use the proposed combined diagnostic testing with the same validated and standardized kits/ protocols and international guidelines when designing sentinel sites to ensure quality assurance and to enhance data sharing and comparability across regions.

The vascular leakage was shown by the increment of hematocrit (Hct), dengue viral infected monocyte, monocyte status, and cytokines production in patients infected with dengue virus. Dengue viral antigens were demonstrated in monocytes (CD14+) from peripheral blood mononuclear cells. The increased levels of Hct, interleukin- (IL-) 10, and tumor necrosis factor-alpha (TNF-α) were detected in dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) patients as compared with other febrile illnesses (OFIs). The highest levels of Hct and IL-10 were detected in DSS patients as compared with other groups (P<0.05) especially on one day before and after defervescence. The unstimulated and lipopolysaccharide- (LPS-) stimulated monocytes from DSS patients showed the significantly decreased of intracellular IL-1βand TNF-α. In addition, the lowest level of mean fluorescence intensity (MFI) of CD11b expression on monocytes surface in DSS patients was also demonstrated. Furthermore, the negative correlations between IL-10 levels and intracellular IL-1βand MFI of CD11b expression in unstimulated and LPS-stimulated monocytes were also detected. Nevertheless, not only were the relationships between the prominent IL-10 and the suppression of intracellular monocyte secretion, namely, IL-1β, TNF-α, demonstrated but also the effect of vascular leakage was observed.

International audience; Background: Chikungunya virus (CHIKV) outbreak recurrences in Thailand are unpredictable and separated byunexplained and often long silent epidemiological periods that can last for several years. These silent periods could beexplained in part by the fact that infection with one CHIKV strain confers lasting natural immunity, even against otherCHIKV strains. In this study we evaluated the persistence of CHIKV-specific neutralizing antibodies in the population ofChumpae 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 ECSAstrain. 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 theprevalence found in Pong Haeng village (70%) was significantly higher than the prevalence detected in the NongThum village (14%), control study villages without known previous Chikungunya epidemics had a high Chikungunyaneutralizing antibody prevalence (65%).Conclusions: More than one-third of the pre-exposed population had persisting natural immunity that was morelikely boosted by recent and repetitive exposure to the emerging ECSA CHIKV in Thailand. Also, Chikungunyavirus appears to largely circulate in the country with a great variability appears between villages or area probablyassociated with the vector abundance and efficiency. Altogether these results show a potential for a lifelongimmunity against CHIKV. Given the rapid spread of the highly pathogenic ECSA strain in Southern Thailand, thedevelopment of CHIK vaccine is strongly recommended.

Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%., Author Summary Dengue virus (DENV) infection occurs throughout tropical and sub-tropical regions of the world where dengue is a major public health problem. Laboratory diagnosis of dengue with a single serum specimen obtained during the acute phase of the illness requires tests to detect IgM antibodies to DENV or the virus genome. A previous evaluation of available tests for IgM anti-DENV showed wide variability. The present study examined newly available commercial tests that detect the virus protein NS1, as well as new tests for IgM anti-DENV in microplate or rapid diagnostic test formats. This analytic study used specimens from laboratory confirmed dengue patients worldwide, which makes the results widely generalizable. The study found variability among the microplate ELISAs for both analytes but some tests performed with sensitivity and specificity acceptable for routine dengue diagnostics. The RDT's for both analytes had variable sensitivity that could be considered acceptable for routine clinical diagnostics. There is the need to maintain a network of dengue reference laboratories to conduct similar evaluations as additional dengue diagnostic tests become commercially available in order to guide the use for surveillance, clinical diagnosis and research.

Introduction To facilitate introduction of live attenuated SA 14-14-2 Japanese encephalitis vaccine (LJEV) into the National Immunization Programme of Sri Lanka, we evaluated the safety and immunogenicity of co-administration of LJEV and measles vaccine at 9 months of age. Serum immune responses were evaluated post-vaccination on days 28, 180, and 365 using JE neutralization test and anti-measles IgG ELISA. Results 278 infants received one dose of LJEV and measles vaccine. Of these, 257 were eligible for the per-protocol analysis. On Day 0, 14 infants (5.5%) were seropositive for JE, but none were seropositive for measles. At Day 28, seropositivity rates were 90.7% (95% CI, 86.4–93.9%) for JE and 84.8% (95% CI, 79.8–89.0%) for measles. The geometric mean titer for JE neutralizing antibodies was 111 (95% CI, 90–135), and the geometric mean concentration (GMC) for anti-measles IgG was 375 mIU/mL (95% CI, 351–400 mIU/mL). Over the next year, JE neutralizing antibody responses declined only slightly, with seropositivity at 87.4% (95% CI, 82.6–91.2%) at Day 365. In contrast, measles antibody levels continued to increase over time. Seropositivity for anti-measles IgG reached 97.2% (95% CI, 94.4–98.9%) at Day 365, and the GMC rose to 1202 mIU/mL (95% CI, 1077–1341 mIU/mL). Co-administration of LJEV and measles vaccine was also safe. Most adverse reactions were mild, and no serious adverse events were related to study vaccinations. Conclusion The safety and immunogenicity of LJEV co-administered with measles vaccine in Sri Lankan infants is similar to that seen in other populations, and our results support use of LJEV at 9 months of age. Live SA 14-14-2 vaccine is now prequalified by the WHO for use in infants in Asia, and other countries may wish to introduce LJEV to combat this devastating disease.

The objective of this study was to investigate the possibility of dengue virus infection causing an abnormal neurologic presentation. Between 1996 and 1998, all pediatric patients with clinical manifestations of encephalitis-like illness who were admitted to the Department of Pediatrics, Siriraj Hospital were prospectively studied for any evidence of dengue virus infection. The diagnosis of dengue virus infection was based on mosquito viral isolation and serologic and polymerase chain reaction (PCR) evidence. Of 44 patients with the preliminary diagnosis of acute viral encephalitis, 8 were diagnosed with dengue infection. All of these 8 patients were diagnosed by serology. In addition to the serologic diagnosis, four also had positive PCR, one had positive viral isolation, and one had both positive PCR and viral isolation. Only two patients were diagnosed by serologic evidence alone. All except one had clinical courses and laboratory findings compatible with typical dengue infection. All had obvious encephalitic clinical manifestations with normal cerebrospinal fluid findings except one patient, who had mildly increased cerebrospinal fluid protein. All of these patients recovered completely and had benign clinical courses except one patient, who developed leakage symptoms. None had liver failure. Dengue virus can cause acute encephalopathy with fever. It can masquerade as other types of acute viral encephalitis. However, its clinical course and prognosis are usually favorable ( J Child Neurol 2000; 15:544-547).

Laboratory-based epidemiological studies have been initiated in a cohort of school-aged children in Ratchaburi province, Thailand in preparation for imminent dengue vaccine field trials. In these studies, levels of neutralising antibodies were determined by the plaque reduction neutralisation test (PRNT) while virological confirmation was performed using RT-PCR or mosquito inoculation. Serological confirmation was performed using IgM and IgG ELISA. The incidence rate of dengue in the Ratchaburi cohort was 1635 per 100,000 during 2005-2006. Among 3547 patients in this cohort, 331 were classified with febrile disease and 58 with dengue. All four dengue serotypes were observed in Ratchaburi province. Efforts to identify the infecting serotype in symptomatic patients, based solely upon the neutralising antibody response, were complicated by cross- neutralising antibodies. In addition, 20 symptomatic and 49 asymptomatic cases were identified among 897 subjects investigated during 2005-2006, a ratio of 1:2.5. Serological analysis of asymptomatic dengue infections demonstrated boosting of immune responses due to subclinical infections with dengue or Japanese encephalitis. These results demonstrate that laboratory-confirmed dengue disease in areas of high transmission can be established. Epidemiological data of this kind are critical to dengue vaccine efficacy trials.

Dengue-1 virus PDK13 and isolates from vaccinees (dengue-1 Ib1 and dengue-1 Ib 10), dengue-3 PGMK30F3, and dengue-4 PDK48 were studied for their abilities to infect, disseminate, and replicate in Aedes aegypti mosquitoes by the oral route. In general, infection and dissemination rates were poorer for the vaccine compared with the parent viruses. The transmissibility was also lower for dengue-1 PDK13 than the parent virus, whereas it was not detected for isolates from vaccinees and dengue-3 PGMK30F3. Transmissibility of dengue-4 PDK48 was not determined because no dissemination occurred. Replication rates of vaccine strains were also found to be less efficient than the parent viruses. These imply that vaccination with the candidate vaccine is safe. Moreover, vector attenuation of vaccine viruses was consistent with its phenotypic markers of attenuation, which remained stable after a mosquito passage or after human and mosquito passage.

Seroprevalence of dengue antibodies were determined in healthy children in Bangkok. At 9, 12, and 18 months of age 23%, 9%, and 17% of children respectively had neutralizing antibodies against one or more serotypes. DEN1 was the most prevalent, followed by DEN3, DEN2, and DEN4. Twelve children had serologic evidence of dengue infection. The nadir of dengue antibodies in children was at 12 months of age. In highly endemic areas, dengue vaccination could be needed at an early age.