Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis.

Mouse models expressing human ACE2 for coronavirus disease 2019 have been frequently used to understand its pathogenesis and develop therapeutic strategies against SARS-CoV-2. Given that human TMPRSS2 supports viral entry, replication, and pathogenesis, we established a double-transgenic mouse model expressing both human ACE2 and TMPRSS2 for SARS-CoV-2 infection. Co-overexpression of both genes increased viral infectivity in vitro and in vivo. Double-transgenic mice showed significant body weight loss, clinical disease symptoms, acute lung injury, lung inflammation, and lethality in response to viral infection, indicating that they were highly susceptible to SARS-CoV-2. Pretreatment with the TMPRSS2 inhibitor, nafamostat, effectively reduced virus-induced weight loss, viral replication, and mortality in the double-transgenic mice. Moreover, the susceptibility and differential pathogenesis of SARS-CoV-2 variants were demonstrated in this animal model. Together, our results demonstrate that double-transgenic mice could provide a highly susceptible mouse model for viral infection to understand SARS-CoV-2 pathogenesis and evaluate antiviral therapeutics against coronavirus disease 2019.

Zika Virus Infection Induces Interleukin-1ß-Mediated Inflammatory Responses by Macrophages in the Brain of an Adult Mouse Model.

During the 2015-2016 Zika virus (ZIKV) epidemic, ZIKV-associated neurological diseases were reported in adults, including microcephaly, Guillain-Barre syndrome, myelitis, meningoencephalitis, and fatal encephalitis. However, the mechanisms underlying the neuropathogenesis of ZIKV infection are not yet fully understood. In this study, we used an adult ZIKV infection mouse model (Ifnar1-/-) to investigate the mechanisms underlying neuroinflammation and neuropathogenesis. ZIKV infection induced the expression of proinflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, gamma interferon, and tumor necrosis factor alpha, in the brains of Ifnar1-/- mice. RNA-seq analysis of the infected mouse brain also revealed that genes involved in innate immune responses and cytokine-mediated signaling pathways were significantly upregulated at 6 days postinfection. Furthermore, ZIKV infection induced macrophage infiltration and activation and augmented IL-1ß expression, whereas microgliosis was not observed in the brain. Using human monocyte THP-1 cells, we confirmed that ZIKV infection promotes inflammatory cell death and increases IL-1ß secretion. In addition, expression of the complement component C3, which is associated with neurodegenerative diseases and known to be upregulated by proinflammatory cytokines, was induced by ZIKV infection through the IL-1ß-mediated pathway. An increase in C5a produced by complement activation in the brains of ZIKV-infected mice was also verified. Taken together, our results suggest that ZIKV infection in the brain of this animal model augments IL-1ß expression in infiltrating macrophages and elicits IL-1ß-mediated inflammation, which can lead to the destructive consequences of neuroinflammation. IMPORTANCE Zika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection in the mouse brain can induce IL-1ß-mediated inflammation and complement activation, thereby contributing to the development of neurological disorders. Thus, our findings reveal a mechanism by which ZIKV induces neuroinflammation in the mouse brain. Although we used adult type I interferon receptor IFNAR knockout (Ifnar1-/-) mice owing to the limited mouse models of ZIKV pathogenesis, our conclusions contributed to the understanding ZIKV-associated neurological diseases to develop treatment strategies for patients with ZIKV infection based on these findings.

Ocular tropism of SARS-CoV-2 in animal models with retinal inflammation via neuronal invasion following intranasal inoculation.

Although ocular manifestations are reported in patients with COVID-19, consensus on ocular tropism of SARS-CoV-2 is lacking. Here, we infect K18-hACE2 transgenic mice with SARS-CoV-2 using various routes. We observe ocular manifestation and retinal inflammation with production of pro-inflammatory cytokines in the eyes of intranasally (IN)-infected mice. Intratracheal (IT) infection results in dissemination of the virus from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasions are confirmed using intracerebral (IC) infection. Notably, the eye-dropped (ED) virus does not cause lung infection and becomes undetectable with time. Ocular and neurotropic distribution of the virus in vivo is evident in fluorescence imaging with an infectious clone of SARS-CoV-2-mCherry. The ocular tropic and neuroinvasive characteristics of SARS-CoV-2 are confirmed in wild-type Syrian hamsters. Our data can improve the understanding regarding viral transmission and clinical characteristics of SARS-CoV-2 and help in improving COVID-19 control procedures.

Dynamics of Viral and Host 3D Genome Structure upon Infection.

Eukaryotic chromatin is highly organized in the 3D nuclear space and dynamically regulated in response to environmental stimuli. This genomic organization is arranged in a hierarchical fashion to support various cellular functions, including transcriptional regulation of gene expression. Like other host cellular mechanisms, viral pathogens utilize and modulate host chromatin architecture and its regulatory machinery to control features of their life cycle, such as lytic versus latent status. Combined with previous research focusing on individual loci, recent global genomic studies employing conformational assays coupled with high-throughput sequencing technology have informed models for host and, in some cases, viral 3D chromosomal structure re-organization during infection and the contribution of these alterations to virus-mediated diseases. Here, we review recent discoveries and progress in host and viral chromatin structural dynamics during infection, focusing on a subset of DNA (human herpesviruses and HPV) as well as RNA (HIV, influenza virus and SARS-CoV-2) viruses. An understanding of how host and viral genomic structure affect gene expression in both contexts and ultimately viral pathogenesis can facilitate the development of novel therapeutic strategies.

Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant.

Diverse severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged since the beginning of the COVID-19 pandemic. We investigated the immunological and pathological peculiarity of the SARS-CoV-2 beta variant of concern (VoC) compared to the ancestral strain. Comparative analysis of phenotype and pathology revealed that the beta VoC induces slower disease progression and a prolonged presymptomatic period in the early stages of SARS-CoV-2 infection but ultimately causes sudden death in the late stages of infection in the K18-hACE2 mouse model. The beta VoC induced enhanced activation of CXCL1/2-CXCR2-NLRP3-IL-1ß signal cascade accelerating neutrophil recruitment and lung pathology in beta variant-infected mice, as evidenced by multiple analyses of SARS-CoV-2-induced inflammatory cytokines and transcriptomes. CCL2 was one of the most highly secreted cytokines in the early stages of infection. Its blockade reduced virus-induced weight loss and delayed mortality. Our study provides a better understanding of the variant characteristics and need for treatment. IMPORTANCE Since the outbreak of COVID-19, diverse SARS-CoV-2 variants have been identified. These variants have different infectivity and transmissibility from the ancestral strains. However, underlying molecular mechanisms have not yet been fully elucidated. In our study, the beta variant showed distinct pathological conditions and cytokine release kinetics from an ancestral strain in a mouse model. It was associated with higher neutrophil recruitment by increased levels of CXCL1/2, CXCR2, and interleukin 1ß (IL-1ß) at a later stage of viral infection. Our study will provide a better understanding of SARS-CoV-2 pathogenesis.

SARS-CoV-2 Infection of Microglia Elicits Proinflammatory Activation and Apoptotic Cell Death.

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in patients with coronavirus disease 2019 (COVID-19). The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we reported that SARS-CoV-2 can directly infect human microglia, eliciting M1-like proinflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA sequencing (RNA-seq) analysis also revealed that endoplasmic reticulum (ER) stress and immune responses were induced in the early, and apoptotic processes in the late phases of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α), but not the anti-inflammatory cytokine IL-10. After this proinflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of proinflammatory microglial IL-6 and TNF-α and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in patients with COVID-19. IMPORTANCE Recent studies reported neurological and cognitive sequelae in patients with COVID-19 months after the viral infection with several symptoms, including ageusia, anosmia, asthenia, headache, and brain fog. Our conclusions raise awareness of COVID-19-related microglia-mediated neurological disorders to develop treatment strategies for the affected patients. We also indicated that HMC3 was a novel human cell line susceptible to SARS-CoV-2 infection that exhibited cytopathic effects, which could be further used to investigate cellular and molecular mechanisms of neurological manifestations of patients with COVID-19.

Receptor-binding domain of SARS-CoV-2 spike protein efficiently inhibits SARS-CoV-2 infection and attachment to mouse lung.

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.

A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses.

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (-1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro -1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential -1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.

Structure-Based Virtual Screening: Identification of a Novel NS2B-NS3 Protease Inhibitor with Potent Antiviral Activity against Zika and Dengue Viruses.

Zika virus (ZIKV), which is associated with severe diseases in humans, has spread rapidly and globally since its emergence. ZIKV and dengue virus (DENV) are closely related, and antibody-dependent enhancement (ADE) of infection between cocirculating ZIKV and DENV may exacerbate disease. Despite these serious threats, there are currently no approved antiviral drugs against ZIKV and DENV. The NS2B-NS3 viral protease is an attractive antiviral target because it plays a pivotal role in polyprotein cleavage, which is required for viral replication. Thus, we sought to identify novel inhibitors of the NS2B-NS3 protease. To that aim, we performed structure-based virtual screening using 467,000 structurally diverse chemical compounds. Then, a fluorescence-based protease inhibition assay was used to test whether the selected candidates inhibited ZIKV protease activity. Among the 123 candidate inhibitors selected from virtual screening, compound 1 significantly inhibited ZIKV NS2B-NS3 protease activity in vitro. In addition, compound 1 effectively inhibited ZIKV and DENV infection of human cells. Molecular docking analysis suggested that compound 1 binds to the NS2B-NS3 protease of ZIKV and DENV. Thus, compound 1 could be used as a new therapeutic option for the development of more potent antiviral drugs against both ZIKV and DENV, reducing the risks of ADE.

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern.

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha's small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

Inhibition of p70 isoforms of S6K1 induces anoikis to prevent transformed human hepatocyte growth.

AIMS: The mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation. MAIN METHODS: THH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA. KEY FINDINGS: p70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-ß1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth. SIGNIFICANCE: Our results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.

Therapeutic Strategies Against COVID-19 and Structural Characterization of SARS-CoV-2: A Review.

The novel coronavirus, SARS-CoV-2, or 2019-nCoV, which originated in Wuhan, Hubei province, China in December 2019, is a grave threat to public health worldwide. A total of 3,672,238 confirmed cases of coronavirus disease 2019 (COVID-19) and 254,045 deaths were reported globally up to May 7, 2020. However, approved antiviral agents for the treatment of patients with COVID-19 remain unavailable. Drug repurposing of approved antivirals against other viruses such as HIV or Ebola virus is one of the most practical strategies to develop effective antiviral agents against SARS-CoV-2. A combination of repurposed drugs can improve the efficacy of treatment, and structure-based drug design can be employed to specifically target SARS-CoV-2. This review discusses therapeutic strategies using promising antiviral agents against SARS-CoV-2. In addition, structural characterization of potentially therapeutic viral or host cellular targets associated with COVID-19 have been discussed to refine structure-based drug design strategies.

Changes in Gait Parameters and Gait Variability in Young Adults during a Cognitive Task while Slope and Flat Walking.

This study investigates the effects of a cognitive task while walking on a slope or a flat surface on gait parameters and gait variability in young adults. The participants consisted of thirty healthy young subjects. They were instructed to walk on a slope or on a flat surface while performing or not performing a cognitive task, which involved speaking a four-syllable word in reverse. A wearable inertia measurement unit (IMU) system was used to measure spatiotemporal parameters and gait variability. Flat gait (FG) while performing the cognitive task (FGC) and uphill gait (UG) while performing the cognitive task (UGC) significantly altered stride times, gait speeds, and cadence as compared with FG and UG, respectively. Downhill gait (DG) while performing the cognitive task (DGC) caused no significant difference as compared with DG. Gait variability comparisons showed no significant difference between UGC and UG or between FGC and FG, respectively. On the other hand, variabilities of stride times and gait speeds were significantly greater for DGC than DG. FGC and UGC induce natural changes in spatiotemporal gait parameters that enable the cognitive task to be performed safely. DGC should be regarded as high complexity tasks involving greater gait variability to reduce fall risk.

Zika Virus-Immune Plasmas from Symptomatic and Asymptomatic Individuals Enhance Zika Pathogenesis in Adult and Pregnant Mice.

Preexisting immunity against dengue virus or West Nile virus was previously reported to mediate antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection in a mouse model. We show here that ZIKV-immune plasma samples from both symptomatic and asymptomatic individuals mediated ZIKV ADE of infection in vitro and in mice. In a lethal infection model with a viral inoculum 10 times higher, both ADE and protection were observed, depending on the amount of infused immune plasma. In a vertical-transmission model, ZIKV-immune plasma infused to timed pregnant mice increased fetal demise and decreased the body weight of surviving fetuses. Depletion of IgG from an immune plasma abolished ADE of infection, and the presence of purified IgG alone mediated ADE of infection. Higher viral loads and proinflammatory cytokines were detected in mice treated with ZIKV-immune plasma samples compared to those receiving control plasma. Together, these data show that passive immunization with homotypic ZIKV antibodies, depending on the concentration, could either worsen or limit a subsequent ZIKV infection.IMPORTANCE Antibody-dependent enhancement (ADE) of virus infection is common to many viruses and is problematic when plasma antibody levels decline to subneutralizing concentrations. ADE of infection is especially important among flaviviruses, many of which are the cause of global health problems. Recently, human plasma samples immune to heterologous flaviviruses were shown to promote Zika virus (ZIKV) infection. Here we showed in immunocompromised mouse models that homologous immune plasma samples protect mice from subsequent infection at high antibody concentrations but that they mediate ADE of infection and increase ZIKV pathogenesis in adult mice and fetal demise during pregnancy at low concentrations.

Multi-Cue-Based Circle Detection and Its Application to Robust Extrinsic Calibration of RGB-D Cameras.

RGB-Depth (RGB-D) cameras are widely used in computer vision and robotics applications such as 3D modeling and human⁻computer interaction. To capture 3D information of an object from different viewpoints simultaneously, we need to use multiple RGB-D cameras. To minimize costs, the cameras are often sparsely distributed without shared scene features. Due to the advantage of being visible from different viewpoints, spherical objects have been used for extrinsic calibration of widely-separated cameras. Assuming that the projected shape of the spherical object is circular, this paper presents a multi-cue-based method for detecting circular regions in a single color image. Experimental comparisons with existing methods show that our proposed method accurately detects spherical objects with cluttered backgrounds under different illumination conditions. The circle detection method is then applied to extrinsic calibration of multiple RGB-D cameras, for which we propose to use robust cost functions to reduce errors due to misdetected sphere centers. Through experiments, we show that the proposed method provides accurate calibration results in the presence of outliers and performs better than a least-squares-based method.

Hepatitis C Virus E2 Envelope Glycoprotein Induces an Immunoregulatory Phenotype in Macrophages.

A comprehensive strategy to control hepatitis C virus (HCV) infection needs a vaccine. Our phase I study with recombinant HCV E1/E2 envelope glycoprotein (EnvGPs) as a candidate vaccine did not induce a strong immune response in volunteers. We analyzed the interactions of HCV EnvGPs with human monocyte-derived macrophages as antigen-presenting cells. HCV E2 induced immune regulatory cytokine interleukin (IL)-10 and soluble CD163 (sCD163) protein expression in macrophages from 7 of 9 blood donors tested. Furthermore, HCV E2 enhanced Stat3 and suppressed Stat1 activation, reflecting macrophage polarization toward M2 phenotype. E2-associated macrophage polarization appeared to be dependent of its interaction with CD81 leading endothelial growth factor receptor (EGFR) activation. Additionally, E2 suppressed the expression of C3 complement, similar to HCV-exposed dendritic cells (DCs), implying potential impairment of immune cell priming. Conclusion: Our results suggest that E2 EnvGP may not be an ideal candidate for HCV vaccine development, and discrete domains within E2 may prove to be more capable of elliciting a protective immune response. (Hepatology 2018).

Complement Regulation and Immune Evasion by Hepatitis C Virus.

A prominent role for complement has been identified in the linkage of innate and adaptive immunity. The liver is the main source of complement and hepatocytes are the primary sites for synthesis of complement components in vivo. We have discovered that hepatitis C virus (HCV) impairs C4 and C3 synthesis. Liver damage may diminish capacity of complement synthesis in patients. However, we observed that the changes in measured complement components in chronically HCV infected patients do not correlate with liver fibrosis or rheumatoid factor present in the blood, serum albumin, or alkaline phosphatase levels. Complement component C3 is of critical importance in B cell activation and T cell-dependent antibody responses. C3 activity is required for optimal expansion of CD8+T cells during a systemic viral infection. Deficiencies in complement may predispose patients to infections via ineffective opsonization, and defects in lytic activity via membrane attack complex. Interestingly, C9 is significantly reduced at the mRNA level in chronically HCV infected liver biopsy specimens, while many hepatocyte derived complement components (C6, C8, Factor B, MASP1, and MBL) and unrelated genes remain mostly unaffected. This implies an HCV specific effect, not a global effect from liver disease.

Author Correction: N-terminal gelsolin fragment potentiates TRAIL mediated death in resistant hepatoma cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Ontogeny of the B- and T-cell response in a primary Zika virus infection of a dengue-naïve individual during the 2016 outbreak in Miami, FL.

Zika virus (ZIKV) is a mosquito-borne flavivirus of significant public health concern. In the summer of 2016, ZIKV was first detected in the contiguous United States. Here we present one of the first cases of a locally acquired ZIKV infection in a dengue-naïve individual. We collected blood from a female with a maculopapular rash at day (D) 5 and D7 post onset of symptoms (POS) and we continued weekly blood draws out to D148 POS. To establish the ontogeny of the immune response against ZIKV, lymphocytes and plasma were analyzed in a longitudinal fashion. The plasmablast response peaked at D7 POS (19.6% of CD19+ B-cells) and was undetectable by D15 POS. ZIKV-specific IgM was present at D5 POS, peaked between D15 and D21 POS, and subsequently decreased. The ZIKV-specific IgG response, however, was not detected until D15 POS and continued to increase after that. Interestingly, even though the patient had never been infected with dengue virus (DENV), cross-reactive IgM and IgG binding against each of the four DENV serotypes could be detected. The highest plasma neutralization activity against ZIKV peaked between D15 and D21 POS, and even though DENV binding antibodies were present in the plasma of the patient, there was neither neutralization nor antibody dependent enhancement (ADE) of DENV. Interestingly, ADE against ZIKV arose at D48 POS and continued until the end of the study. CD4+ and CD8+ T-cells recognized ZIKV-NS2A and ZIKV-E, respectively. The tetramer positive CD8+ T-cell response peaked at D21 POS with elevated levels persisting for months. In summary, this is the first study to establish the timing of the ontogeny of the immune response against ZIKV.