Jeslin J. L. Tan

Caribbean and La Réunion Chikungunya Virus Isolates Differ in Their Capacity To Induce Proinflammatory Th1 and NK Cell Responses and Acute Joint Pathology

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the Western Hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Réunion LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse tail fibroblasts revealed a weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immunophenotyping, and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2 to 3 days postinfection [dpi]) and late acute (6 to 8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker proinflammatory Th1 response and natural killer (NK) cell activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host. IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. The immunopathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immunophenotyping, and gene expression profiles in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Réunion outbreaks in 2005 and 2006. This is the first study that showed an association between key proinflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.

Loss of TLR3 aggravates CHIKV replication and pathology due to an altered virus‐specific neutralizing antibody response

RNA‐sensing toll‐like receptors (TLRs) mediate innate immunity and regulate anti‐viral response. We show here that TLR3 regulates host immunity and the loss of TLR3 aggravates pathology in Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infection is markedly increased in human and mouse fibroblasts with defective TLR3 signaling. Up to 100‐fold increase in CHIKV load was observed in Tlr3−/− mice, alongside increased virus dissemination and pro‐inflammatory myeloid cells infiltration. Infection in bone marrow chimeric mice showed that TLR3‐expressing hematopoietic cells are required for effective CHIKV clearance. CHIKV‐specific antibodies from Tlr3−/− mice exhibited significantly lower in vitro neutralization capacity, due to altered virus‐neutralizing epitope specificity. Finally, SNP genotyping analysis of CHIKF patients on TLR3 identified SNP rs6552950 to be associated with disease severity and CHIKV‐specific neutralizing antibody response. These results demonstrate a key role for TLR3‐mediated antibody response to CHIKV infection, virus replication and pathology, providing a basis for future development of immunotherapeutics in vaccine development.

Zika Virus Infects Human Fetal Brain Microglia and Induces Inflammation

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.

An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.

Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.

Specific Biomarkers Associated With Neurological Complications and Congenital Central Nervous System Abnormalities From Zika Virus–Infected Patients in Brazil

Summary The first systematic large-scale analysis of immune mediators reported in patients with Zika virus (ZIKV) infection. Several key immune mediators have been identified for the control of ZIKV pathogenesis. This will clarify the molecular mechanisms of ZIKV infection in patients.

Severity of Plasma Leakage Is Associated With High Levels of Interferon γ–Inducible Protein 10, Hepatocyte Growth Factor, Matrix Metalloproteinase 2 (MMP-2), and MMP-9 During Dengue Virus Infection

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 &ggr;–inducible protein 10, hepatocyte growth factor, soluble p75 tumor necrosis factor &agr; 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.

Persistence of Zika virus in conjunctival fluid of convalescence patients

A widespread epidemic of Zika fever, caused by Zika virus (ZIKAV) has spread throughout the Pacific islands, the Americas and Southeast Asia. The increased incidences of ocular anomalies observed in ZIKAV-infected infants and adults may be associated with the rapid spread of ZIKAV. The objective of this study was to check if ZIKAV could be detected in human tears after the first week of infection. Twenty-nine patients with PCR confirmed ZIKAV infection during the Singapore August 2016 ZIKAV outbreak were enrolled for the study. Detection and quantification of ZIKAV RNA was performed on conjunctival swabs collected from both eyes of these patients at the late convalescent phase (30 days post-illness). Efficiency of viral isolation from swab samples was confirmed by the limit of detection (as low as 0.1 PFU/µL, equivalent to copy number of 4.9) in spiked swabs with different concentrations of ZIKAV (PFU/µL). Samples from three patients were found positive by qRT-PCR for ZIKAV and the viral RNA copy numbers detected in conjunctival swabs ranged from 5.2 to 9.3 copies respectively. ZIKAV could persist in the tears of infected patients for up to 30 days post-illness, and may therefore possess a potential public health risk of transmission.

Myeloid Cell Arg1 Inhibits Control of Arthritogenic Alphavirus Infection by Suppressing Antiviral T Cells.

Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections.

Zika Virus Infection Preferentially Counterbalances Human Peripheral Monocyte and/or NK Cell Activity

ZIKV reemerged in recent years, causing outbreaks in many parts of the world. Alarmingly, ZIKV infection has been associated with neurological complications such as Guillain-Barré syndrome (GBS) in adults and congenital fetal growth-associated anomalies in newborns. Host peripheral immune cells are one of the first to interact with the virus upon successful transmission from an infected female Aedes mosquito. However, little is known about the role of these immune cells during infection. In this work, the immune responses of monocytes, known target cells of ZIKV infection, were investigated by high-density transcriptomics. The analysis saw a robust immune response being elicited. Importantly, it also divulged that monocytes prime NK cell activities during virus infection. Removal of monocytes during the infection changed the immune milieu, which in turn reduced NK cell stimulation. This study provides valuable insights into the pathobiology of the virus and allows for the possibility of designing novel targeted therapeutics. ABSTRACT Zika virus (ZIKV) has reemerged in the population and caused unprecedented global outbreaks. Here, the transcriptomic consequences of ZIKV infection were studied systematically first in human peripheral blood CD14+ monocytes and monocyte-derived macrophages with high-density RNA sequencing. Analyses of the ZIKV genome revealed that the virus underwent genetic diversification, and differential mRNA abundance was found in host cells during infection. Notably, there was a significant change in the cellular response, with cross talk between monocytes and natural killer (NK) cells as one of the highly identified pathways. Immunophenotyping of peripheral blood from ZIKV-infected patients further confirmed the activation of NK cells during acute infection. ZIKV infection in peripheral blood cells isolated from healthy donors led to the induction of gamma interferon (IFN-γ) and CD107a—two key markers of NK cell function. Depletion of CD14+ monocytes from peripheral blood resulted in a reduction of these markers and reduced priming of NK cells during infection. This was complemented by the immunoproteomic changes observed. Mechanistically, ZIKV infection preferentially counterbalances monocyte and/or NK cell activity, with implications for targeted cytokine immunotherapies. IMPORTANCE ZIKV reemerged in recent years, causing outbreaks in many parts of the world. Alarmingly, ZIKV infection has been associated with neurological complications such as Guillain-Barré syndrome (GBS) in adults and congenital fetal growth-associated anomalies in newborns. Host peripheral immune cells are one of the first to interact with the virus upon successful transmission from an infected female Aedes mosquito. However, little is known about the role of these immune cells during infection. In this work, the immune responses of monocytes, known target cells of ZIKV infection, were investigated by high-density transcriptomics. The analysis saw a robust immune response being elicited. Importantly, it also divulged that monocytes prime NK cell activities during virus infection. Removal of monocytes during the infection changed the immune milieu, which in turn reduced NK cell stimulation. This study provides valuable insights into the pathobiology of the virus and allows for the possibility of designing novel targeted therapeutics.

Longitudinal Study of Cellular and Systemic Cytokine Signatures to Define the Dynamics of a Balanced Immune Environment During Disease Manifestation in Zika Virus–Infected Patients

Understanding the host immune response during Zika virus (ZIKV) infection will provide valuable insights into ZIKV immunopathogenesis. In this study, characterization of cellular changes and immune mediators of ZIKV-infected patients was performed, allowing for the identification of key infection-associated markers.