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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. Analyses of antibody dynamics and subclinical infections show that across the population, variability in the infection strength of dengue viruses results in large-scale temporal changes in infection and disease risk that correlate poorly with age.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has emerged as a key upstream regulator of cell death and inflammation. RIPK1-mediated signaling governs the outcome of signaling pathways initiated by tumor necrosis factor receptor 1 (TNFR1), Toll-like receptor 3 (TLR3), TLR4, retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated protein 5 (MDA-5), and Z-binding protein 1 (ZBP1) by signaling for NF-κB activation, mitogen-associated protein kinase (MAPK) and IRF3/7 phosphorylation, and cell death via apoptosis and necroptosis. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has emerged as a key upstream regulator of cell death and inflammation. RIPK1-mediated signaling governs the outcome of signaling pathways initiated by tumor necrosis factor receptor 1 (TNFR1), Toll-like receptor 3 (TLR3), TLR4, retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated protein 5 (MDA-5), and Z-binding protein 1 (ZBP1) by signaling for NF-κB activation, mitogen-associated protein kinase (MAPK) and interferon regulatory factor 3/7 (IRF3/7) phosphorylation, and cell death via apoptosis and necroptosis. Both cell death and inflammatory responses play a major role in controlling virus infections. Therefore, viruses have evolved multifaceted mechanisms to exploit host immune responses by targeting RIPK1. This review focuses on the current understanding of RIPK1-mediated inflammatory and cell death pathways and multiple mechanisms by which viruses manipulate these pathways by targeting RIPK1. We also discuss gaps in our knowledge regarding RIPK1-mediated signaling pathways and highlight potential avenues for future research.

Key Points Dengue is an emerging infection of tropical and subtropical regions caused by a group of four serotypes of dengue virus that are transmitted to humans by Aedes spp. mosquitoes. Infection with one dengue virus serotype provides long-lasting protective immunity to that serotype, but only short-term immunity (lasting less than 6 months) to other serotypes. Sequential infection with different dengue virus serotypes is common in dengue-endemic countries. A small percentage of individuals infected with dengue virus develop a plasma leakage syndrome that can be life-threatening. This syndrome, termed dengue haemorrhagic fever, is associated with high circulating levels of cytokines (a 'cytokine storm') and immune activation. Beyond the first year of life, dengue haemorrhagic fever is significantly more common during an individual's second dengue virus infection than during the primary infection. Antibody responses to dengue virus are principally directed towards two glycoproteins on the surface of the virion envelope, the precursor membrane (pre-M) and envelope (E) proteins, and a secreted viral glycoprotein, non-structural protein 1 (NS1). Depending on the specific epitope targeted and the antibody avidity and concentration, dengue virus-specific antibodies can inhibit viral infection (neutralization) or enhance the uptake of virions into cells bearing immunoglobulin receptors (a process referred to as antibody-dependent enhancement of infection). T cells recognize epitopes that are distributed across the dengue virus genome, and many CD4 + and CD8 + T cells recognize more than one dengue virus serotype. Variant epitope sequences between dengue virus serotypes induce an altered profile of cytokine production and target cell lysis (the altered peptide ligand effect). During a second dengue virus infection, the presence of pre-existing memory B and T cells (which were induced by the prior infection) alters the kinetics and specificity of the immune response, and this is referred to as original antigenic sin. Clinical studies have shown associations between specific immune responses and disease severity. However, no single measure has shown a high correlation with clinical outcome. Further studies are needed to establish reliable correlates of protective or pathological immune responses. Dengue virus vaccines currently in development contain different components of the dengue virus genome. Immune responses to the different vaccines may not be equivalent; incorporation of immunological studies into clinical trials is desirable to establish the necessary knowledge base to guide vaccine development and introduction. Alan Rothman describes the types of immune response that are associated with protection and pathology during dengue virus infection. Although virus-specific antibodies provide protective neutralizing immunity, they can also facilitate infection of other host cells; the author discusses the implications of this for vaccine development. Dengue is a mosquito-borne viral disease of expanding geographical range and incidence. The existence of four viral serotypes and the association of prior dengue virus infection with an increased risk for more severe disease have presented significant obstacles to vaccine development. An increased understanding of the adaptive immune response to natural dengue virus infection and candidate dengue vaccines has helped to define the specific antibody and T cell responses that are associated with either protective or pathological immunity during dengue infection. Further characterization of immunological correlates of disease outcome and the validation of these findings in vaccine trials will be invaluable for developing effective dengue vaccines.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

It is unclear whether dengue serotypes differ in their propensity to cause severe disease. We analyzed differences in serotype-specific disease severity in children presenting for medical attention in Bangkok, Thailand. Prospective studies were conducted from 1994 to 2006. Univariate and multivariate logistic and multinomial logistic regressions were used to determine if dengue hemorrhagic fever (DHF) and signs of severe clinical disease (pleural effusion, ascites, thrombocytopenia, hemoconcentration) were associated with serotype. Crude and adjusted odds ratios were calculated. There were 162 (36%) cases with DENV-1, 102 (23%) with DENV-2, 123 (27%) with DENV-3, and 64 (14%) with DENV-4. There was no significant difference in the rates of DHF by serotype: DENV-2 (43%), DENV-3 (39%), DENV-1 (34%), DENV-4 (31%). DENV-2 was significantly associated with increased odds of DHF grade I compared to DF (OR 2.9 95% CI 1.1, 8.0), when using DENV-1 as the reference. Though not statistically significant, DENV-2 had an increased odds of total DHF and DHF grades II, III, and IV. Secondary serologic response was significantly associated with DHF (OR 6.2) and increased when considering more severe grades of DHF. DENV-2 (9%) and -4 (3%) were significantly less often associated with primary disease than DENV-1 (28%) and -3 (33%). Restricting analysis to secondary cases, we found DENV-2 and DENV-3 to be twice as likely to result in DHF as DEN-4 (p = 0.05). Comparing study years, we found the rate of DHF to be significantly less in 1999, 2000, 2004, and 2005 than in 1994, the study year with the highest percentage of DHF cases, even when controlling for other variables. As in other studies, we find secondary disease to be strongly associated with DHF and with more severe grades of DHF. DENV-2 appears to be marginally associated with more severe dengue disease as evidenced by a significant association with DHF grade I when compared to DENV-1. In addition, we found non-significant trends with other grades of DHF. Restricting the analysis to secondary disease we found DENV-2 and -3 to be twice as likely to result in DHF as DEN-4. Differences in severity by study year may suggest that other factors besides serotype play a role in disease severity.

Dengue viruses are a major cause of morbidity in tropical and subtropical regions of the world. Inapparent dengue is an important component of the overall burden of dengue infection. It provides a source of infection for mosquito transmission during the course of an epidemic, yet by definition is undetected by health care providers. Previous studies of inapparent or subclinical infection have reported varying ratios of symptomatic to inapparent dengue infection. In a prospective study of school children in Northern Thailand, we describe the spatial and temporal variation of the symptomatic to inapparent (S:I) dengue illness ratio. Our findings indicate that there is a wide fluctuation in this ratio between and among schools in a given year and within schools over several dengue seasons. The most important determinants of this S:I ratio for a given school were the incidence of dengue infection in a given year and the incidence of infection in the preceding year. We found no association between the S:I ratio and age in our population. Our findings point to an important aspect of virus-host interactions at either a population or individual level possibly due to an effect of heterotypic cross-reactive immunity to reduce dengue disease severity. These findings have important implications for future dengue vaccines.

Dengue remains one of the most important mosquito-borne diseases worldwide. Infection with one of the serologically related dengue viruses (DENVs) can lead to a wide range of clinical manifestations and severity. Severe dengue is characterized by plasma leakage and abnormal bleeding that can lead to shock and death. There is currently no specific treatment for severe dengue due to gaps in understanding of the underlying mechanisms. The transient period of vascular leakage is usually followed by a rapid recovery and is suggestive of the effects of short-lived biological mediators. Both the innate and the adaptive immune systems are activated in severe dengue and contribute to the cytokine production. We discuss the immunological events elicited during a DENV infection and identify candidate cytokines that may play a key role in the severe manifestations of dengue and possible interventions.

Japanese encephalitis virus (JEV) is a mosquito-borne pathogen that causes severe neurologic disease. Its endemicity in Asia has prompted its inclusion in nationwide immunization programs. However, the Philippines, which is also endemic for related viruses like dengue (DENV), has not yet adopted this practice. Vaccine hesitancy is a major challenge, exacerbated by concerns over cross-reactive antibodies that may enhance viral infection. This study aimed to determine whether IMOJEV vaccination would induce cross-neutralizing or enhancing antibodies against DENV. Pre- and one-month post-vaccination samples from IMOJEV-vaccinated Filipino children (9-24 months old) were analyzed. A reporter virus particle (RVP)-based neutralization assay against JEV showed neutralization in 28/29 subjects post-vaccination. Presence of DENV2-reactive antibodies was measured via DENV2 VLP ELISA, which revealed increased DENV2 binding reactivity post-vaccination. Pre-vaccination DENV2 binding reactivity also had no significant correlation with the JEV vaccine response. RVP-based neutralization and enhancement assays against DENV2 showed that there was no significant change in neutralizing or enhancing antibody activity against DENV2 after JEV vaccination. This study shows that IMOJEV vaccination elicited a JEV neutralizing response in 97% of vaccinees and that the magnitude of JEV neutralizing titers post-vaccination was not associated with pre-existing binding antibodies to DENV2. Further, while live JEV vaccination increases DENV2-binding antibodies, this cross-reactivity does not lead to DENV2 enhancement. These findings contribute to a better understanding of the orthoflavivirus antibody response following immunization and the influence of pre-existing heterologous orthoflavivirus antibodies. This could guide vaccination strategies, especially in orthoflavivirus-endemic regions.

Heterogeneous exposure to mosquitoes determines an individual’s contribution to vector-borne pathogen transmission. Particularly for dengue virus (DENV), there is a major difficulty in quantifying human-vector contacts due to the unknown coupled effect of key heterogeneities. To test the hypothesis that the reduction of human out-of-home mobility due to dengue illness will significantly influence population-level dynamics and the structure of DENV transmission chains, we extended an existing modeling framework to include social structure, disease-driven mobility reductions, and heterogeneous transmissibility from different infectious groups. Compared to a baseline model, naïve to human pre-symptomatic infectiousness and disease-driven mobility changes, a model including both parameters predicted an increase of 37% in the probability of a DENV outbreak occurring; a model including mobility change alone predicted a 15.5% increase compared to the baseline model. At the individual level, models including mobility change led to a reduction of the importance of out-of-home onward transmission ( R , the fraction of secondary cases predicted to be generated by an individual) by symptomatic individuals (up to -62%) at the expense of an increase in the relevance of their home (up to +40%). An individual’s positive contribution to R could be predicted by a GAM including a non-linear interaction between an individual’s biting suitability and the number of mosquitoes in their home (>10 mosquitoes and 0.6 individual attractiveness significantly increased R ). We conclude that the complex fabric of social relationships and differential behavioral response to dengue illness cause the fraction of symptomatic DENV infections to concentrate transmission in specific locations, whereas asymptomatic carriers (including individuals in their pre-symptomatic period) move the virus throughout the landscape. Our findings point to the difficulty of focusing vector control interventions reactively on the home of symptomatic individuals, as this approach will fail to contain virus propagation by visitors to their house and asymptomatic carriers.

Dengue virus (DENV) is an increasingly important human pathogen, with already half of the globe’s population living in environments with transmission potential. Since many cases are missed by direct detection methods (RT-PCR or antigen tests), serological assays play an important role in the diagnostic process. However, individual assays can suffer from low sensitivity and specificity and interpreting results from multiple assays remains challenging, particularly because interpretations from multiple assays may differ, creating uncertainty over how to generate finalized interpretations. We develop a Bayesian mixture model that can jointly model data from multiple paired serological assays, to infer infection events. We first test the performance of our model using simulated data. We then apply our model to 677 pairs of acute and convalescent serum collected as a part of illness and household investigations across two longitudinal cohort studies in Kamphaeng Phet, Thailand, including data from 232 RT-PCR confirmed infections (gold standard). We compare the classification of the new model to prior standard interpretations that independently utilize information from either the hemagglutination inhibition assay (HAI) or the enzyme-linked immunosorbent assay (EIA). We find that additional serological assays improve accuracy of infection detection for both simulated and real world data. Models incorporating paired IgG and IgM data as well as those incorporating IgG, IgM, and HAI data consistently have higher accuracy when using PCR confirmed infections as a gold standard (87–90% F1 scores, a combined metric of sensitivity and specificity) than currently implemented cut-point approaches (82–84% F1 scores). Our results provide a probabilistic framework through which multiple serological assays across different platforms can be leveraged across sequential serum samples to provide insight into whether individuals have recently experienced a DENV infection. These methods are applicable to other pathogen systems where multiple serological assays can be leveraged to quantify infection history.