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Background: The unprecedented reemergence of Zika virus (ZIKV) has startled the world with reports of increased microcephaly in Brazil. ZIKV can infect human neural progenitors and impair brain growth. However, direct evidence of ZIKV infection in human fetal brain tissues remains elusive., Methods: Investigations were performed with brain cell preparations obtained from 9 donors. Virus infectivity was assessed by detection of virus antigen by flow cytometry together with various hematopoietic cell surface markers. Virus replication was determined by viral RNA quantification. Cytokine levels in supernatant obtained from virus-infected fetal brain cells were measured simultaneously in microbead-based immunoassays., Results: We also show that ZIKV infection was particularly evident in hematopoietic cells with microglia, the brain-resident macrophage population being one of the main targets. Infection induces high levels of proinflammatory immune mediators such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and monocyte chemotactic protein 1 (MCP-1)., Conclusions: Our results highlight an important role for microglia and neuroinflammation during congenital ZIKV pathogenesis., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background:  Dengue virus infection typically causes mild dengue fever, but, in severe cases, life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur. The pathophysiological hallmark of DHF and DSS is plasma leakage that leads to enhanced vascular permeability, likely due to a cytokine storm., Methods:  Ninety patients with dengue during 2010-2012 in Singapore were prospectively recruited and stratified according to their disease phase, primary and secondary infection status, and disease severity, measured by plasma leakage. Clinical parameters were recorded throughout the disease progression. The levels of various immune mediators were quantified using comprehensive multiplex microbead-based immunoassays for 46 immune mediators., Results:  Associations between clinical parameters and immune mediators were analyzed using various statistical methods. Potential immune markers, including interleukin 1 receptor antagonist, interferon γ-inducible protein 10, hepatocyte growth factor, soluble p75 tumor necrosis factor α receptor, vascular cell adhesion molecule 1, and matrix metalloproteinase 2, were significantly associated with significant plasma leakage. Secondary dengue virus infections were also shown to influence disease outcome in terms of disease severity., Conclusions:  This study identified several key markers for exacerbated dengue pathogenesis, notably plasma leakage. This will allow a better understanding of the molecular mechanisms of DHF and DSS in patients with dengue., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: Individuals infected with chikungunya virus (CHIKV) normally exhibit a variety of clinical manifestations during the acute phase of infection. However, studies in different patient cohorts have revealed that disease manifestations vary in frequency., Methods: Disease profiles between patients with acute CHIKV-infection and febrile patients without CHIKV were compared and examined to determine whether any clinical presentations were associated with the clinical outcome of CHIKV infection. Circulatory immune mediators profiles were then characterized and compared with data from 14 independent patient cohort studies. The particular immune mediator signature that defines acute CHIKV infection was determined., Results: Our findings revealed a specific pattern of clinical presentations of joint-specific arthralgia from this CHIKV cohort. More importantly, we identified an immune mediator signature dominated by proinflammatory cytokines, which include interferon α and γ and interleukin 2, 2R, 6, 7, 12, 15, 17, and 18, across different patient cohorts of CHIKV load associated with arthralgia., Conclusions: To our knowledge, this is the first study that associated levels of CHIKV load with arthralgia as an indicator of acute CHIKV infection. Importantly, our findings also revealed specific immune mediator signatures that can be used to better define CHIKV infection., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Background: Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. Although infected individuals clear the virus from the blood, some develop debilitating and prolonged arthralgia., Methods: We investigated specificity and strength of antibody responses in a longitudinal study on CHIKV-infected patients and analyzed their association with viral load, cytokine profile, and severity., Results: We found that CHIKV-specific response is dominated by immunoglobulin G3 (IgG3) antibodies. The antibodies were neutralizing, and patients with high viremia rapidly developed high levels of anti-CHIKV antibodies of this specific isotype. Although these patients endured a more severe disease progression during the acute viremic phase, they cleared the virus faster and did not experience persistent arthralgia. However, significant persistent arthralgia was observed in patients with low viremia who developed IgG3 at a later stage., Conclusions: Absence of early CHIKV-specific IgG3 may therefore serve as a specific marker of patients with increased risk of disease.

The 'Random Mutation Capture' assay allows for the sensitive quantitation of DNA mutations at extremely low mutation frequencies. This method is based on PCR detection of mutations that render the mutated target sequence resistant to restriction enzyme digestion. The original protocol prescribes an end-point dilution to about 0.1 mutant DNA molecules per PCR well, such that the mutation burden can be simply calculated by counting the number of amplified PCR wells. However, the statistical aspects associated with the single molecular nature of this protocol and several other molecular approaches relying on binary (on/off) output can significantly affect the quantification accuracy, and this issue has so far been ignored. The present work proposes a design of experiment (DoE) using statistical modeling and Monte Carlo simulations to obtain a statistically optimal sampling protocol, one that minimizes the coefficient of variance in the measurement estimates. Here, the DoE prescribed a dilution factor at about 1.6 mutant molecules per well. Theoretical results and experimental validation revealed an up to 10-fold improvement in the information obtained per PCR well, i.e. the optimal protocol achieves the same coefficient of variation using one-tenth the number of wells used in the original assay. Additionally, this optimization equally applies to any method that relies on binary detection of a small number of templates.

Background: Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients., Methods: A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated., Results: When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-α and interleukin (IL)-6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor., Conclusions: The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients.