Coxsackievirus B3 elicits a sex-specific CD8+ T cell response which protects female mice.

Sex is a significant contributor to the outcome of human infections. Males are frequently more susceptible to viral, bacterial, and fungal infections, often attributed to weaker immune responses. In contrast, a heightened immune response in females enables better pathogen elimination but leaves females more predisposed to autoimmune diseases. Unfortunately, the underlying basis for sex-specific immune responses remains poorly understood. Here, we show a sex difference in the CD8+ T cell response to an enteric virus, Coxsackievirus B3 (CVB3). We found that CVB3 induced expansion of CD8+ T cells in female mice but not in male mice. CVB3 also increased the proportion and number of CD11ahiCD62Llo CD8+ T cells in female mice, indicative of activation. This response was independent of the inoculation route and type I interferon. Using a recombinant CVB3 virus expressing a model CD8+ T cell epitope, we found that the expansion of CD8+ T cells in females is viral-specific and not due to bystander activation. Finally, the depletion of CD8+ T cells, prior to infection, led to enhanced mortality, indicating that CD8+ T cells are protective against CVB3 in female mice. These data demonstrate that CVB3 induces a CD8+ T cell response in female mice and highlight the importance of sex-specific immune responses to viral pathogens.

Redox Regulation and Metabolic Dependency of Zika Virus Replication: Inhibition by Nrf2-Antioxidant Response and NAD(H) Antimetabolites.

Viral infections alter host cell metabolism and homeostasis; however, the mechanisms that regulate these processes have only begun to be elucidated. We report here that Zika virus (ZIKV) infection activates the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2), which precedes oxidative stress. Downregulation of Nrf2 or inhibition of glutathione (GSH) synthesis resulted in significantly increased viral replication. Interestingly, 6-amino-nicotinamide (6-AN), a nicotinamide analog commonly used as an inhibitor of the pentose phosphate pathway (PPP), decreased viral replication by over 1,000-fold. This inhibition was neither recapitulated by the knockdown of PPP enzymes, glucose 6-phosphate dehydrogenase (G6PD), or 6-phosphogluconate dehydrogenase (6PGD), nor prevented by supplementation with ribose 5-phosphate. Instead, our metabolomics and metabolic phenotype studies support a mechanism in which 6-AN depletes cells of NAD(H) and impairs NAD(H)-dependent glycolytic steps resulting in inhibition of viral replication. The inhibitory effect of 6-AN was rescued with precursors of the salvage pathway but not with those of other NAD+ biosynthesis pathways. Inhibition of glycolysis reduced viral protein levels, which were recovered transiently. This transient recovery in viral protein synthesis was prevented when oxidative metabolism was inhibited by blockage of the mitochondrial pyruvate carrier, fatty acid oxidation, or glutaminolysis, demonstrating a compensatory role of mitochondrial metabolism in ZIKV replication. These results establish an antagonistic role for the host cell Nrf2/GSH/NADPH-dependent antioxidant response against ZIKV and demonstrate the dependency of ZIKV replication on NAD(H). Importantly, our work suggests the potential use of NAD(H) antimetabolite therapy against the viral infection. IMPORTANCE Zika virus (ZIKV) is a major public health concern of international proportions. While the incidence of ZIKV infections has declined substantially in recent years, the potential for the reemergence or reintroduction remains high. Although viral infection alters host cell metabolism and homeostasis to promote its replication, deciphering the mechanism(s) involved in these processes is important for identifying therapeutic targets. The present work reveals the complexities of host cell redox regulation and metabolic dependency of ZIKV replication. An antagonistic effect of the Nrf2/GSH/NADP(H)-dependent antioxidant response against ZIKV infection and an essential role of NAD(H) metabolism and glycolysis for viral replication are established for the first time. These findings highlight the potential use of NAD(H) antimetabolites to counter ZIKV infection and pathogenesis.

Mechanistic Target of Rapamycin Signaling Activation Antagonizes Autophagy To Facilitate Zika Virus Replication.

Zika virus (ZIKV), a mosquito-transmitted flavivirus, is linked to microcephaly and other neurological defects in neonates and Guillain-Barré syndrome in adults. The molecular mechanisms regulating ZIKV infection and pathogenic outcomes are incompletely understood. Signaling by the mechanistic (mammalian) target of rapamycin (mTOR) kinase is important for cell survival and proliferation, and viruses are known to hijack this pathway for their replication. Here, we show that in human neuronal precursors and glial cells in culture, ZIKV infection activates both mTOR complex 1 (mTORC1) and mTORC2. Inhibition of mTOR kinase by Torin1 or rapamycin results in reduction in ZIKV protein expression and progeny production. Depletion of Raptor, the defining subunit of mTORC1, by small interfering RNA (siRNA) negatively affects ZIKV protein expression and viral replication. Although depletion of Rictor, the unique subunit of mTORC2, or the mTOR kinase itself also inhibits the viral processes, the extent of inhibition is less pronounced. Autophagy is transiently induced early by ZIKV infection, and impairment of autophagosome elongation by the class III phosphatidylinositol 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA) enhances viral protein accumulation and progeny production. mTOR phosphorylates and inactivates ULK1 (S757) at later stages of ZIKV infection, suggesting a link between autophagy inhibition and mTOR activation by ZIKV. Accordingly, inhibition of ULK1 (by MRT68921) or autophagy (by 3-MA) reversed the effects of mTOR inhibition, leading to increased levels of ZIKV protein expression and progeny production. Our results demonstrate that ZIKV replication requires the activation of both mTORC1 and mTORC2, which negatively regulates autophagy to facilitate ZIKV replication.IMPORTANCE The re-emergence of Zika virus (ZIKV) and its association with neurological complications necessitates studies on the molecular mechanisms that regulate ZIKV pathogenesis. The mTOR signaling cascade is tightly regulated and central to normal neuronal development and survival. Disruption of mTOR signaling can result in neurological abnormalities. In the studies reported here, we demonstrate for the first time that ZIKV infection results in activation of both mTORC1 and mTORC2 to promote virus replication. Although autophagy is activated early in infection to counter virus replication, it is subsequently suppressed by mTOR. These results reveal critical roles of mTOR signaling and autophagy in ZIKV infection and point to a possible mechanism underlying ZIKV-induced pathogenesis. Elucidating the role of mTOR signaling in ZIKV infection will provide insights into the mechanisms of ZIKV-induced neurological complications and potential targets for therapeutic approaches.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

A viral-specific CD4 + T cell response protects female mice from Coxsackievirus B3 infection.

Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4 + T cell response that can protect female mice from mortality. We found that CVB3 can induce expansion of CD62L lo CD4 + T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4 + T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4 + T cells before infection increased mortality in female mice, indicating that CD4 + T cells play a protective role against CVB3 in our model. Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease outcomes.

A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection.

Background: Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4+ T cell response that can protect female mice from mortality. Methods: We inoculated C57BL/6 Ifnar-/- mice with CVB3. We investigated the T cell response in the spleen and mesenteric lymph nodes in male and female mice following infection. Results: We found that CVB3 can induce expansion of CD62Llo CD4+ T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4+ T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4+ T cells before infection increased mortality in female mice, indicating that CD4+ T cells play a protective role against CVB3 in our model. Conclusions: Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease.

A Ferritin Nanoparticle-Based Zika Virus Vaccine Candidate Induces Robust Humoral and Cellular Immune Responses and Protects Mice from Lethal Virus Challenge.

The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain-Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)-γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV.

The cytoplasmic LSm1-7 and nuclear LSm2-8 complexes exert opposite effects on Hepatitis B virus biosynthesis and interferon responses.

Despite many studies on host or viral gene expression, how the cellular proteome responds to internal or external cues during the infection process remains unclear. In this study, we used a Hepatitis B Virus (HBV) replication model and performed proteomic analyses to understand how HBV evades innate immunity as a function of cell cycle progression. Specifically, we performed proteomic analyses of HBV-replicating cells in G1/S and G2/M phases, as a function of IFN-α treatment. We identified that the conserved LSm (Like-Sm1-8) proteins were differentially regulated in HBV replicating cells treated with IFN-α. Specifically, in G2/M phase, IFN-α increased protein level of LSm1, the unique subunit of cytoplasmic LSm1-7 complex involved in mRNA decay. By contrast, IFN-α decreased LSm8, the unique subunit of nuclear LSm2-8 complex, a chaperone of U6 spliceosomal RNA, suggesting the cytoplasmic LSm1-7 complex is antiviral, whereas the nuclear LSm2-8 complex is pro-viral. In HBV replication and infection models, siRNA-mediated knockdown of LSm1 increased all viral RNAs. Conversely, LSm8 knockdown reduced viral RNA levels, dependent on N6-adenosine methylation (m6A) of the epsilon stem-loop at the 5' end of pre-Core/pregenomic (preC/pg) RNA. Methylated RNA immunoprecipitation (MeRIP) assays demonstrated reduced viral RNA methylation by LSm8 knockdown, dependent on the 5' m6A modification, suggesting the LSm2-8 complex has a role in mediating this modification. Interestingly, splicing inhibitor Cp028 acting upstream of the LSm2-8 complex suppressed viral RNA levels without reducing the 5' m6A modification. This observation suggests Cp028 has novel antiviral effects, likely potentiating IFN-α-mediated suppression of HBV biosynthesis.

Dose-Response Effects of 7-Dehydrocholesterol Reductase Inhibitors on Sterol Profiles and Vesicular Stomatitis Virus Replication.

Cholesterol is ubiquitous in cells; it plays a critical role in membrane structure and transport as well as in intracellular trafficking processes. There are suggestions that cholesterol metabolism is linked to innate immunity with inhibitors of DHCR7, the last enzyme in the cholesterol pathway, suggested to have potential as viral therapeutics nearly a decade ago. In fact, there are a number of highly prescribed pharmaceuticals that are off-target inhibitors of DHCR7, causing increased cellular levels of 7-dehydrodesmosterol (7-DHD) and 7-dehydrocholesterol (7-DHC). We report here dose-response studies of six such inhibitors on late-stage cholesterol biosynthesis in Neuro2a cells as well as their effect on infection of vesicular stomatitis virus (VSV). Four of the test compounds are FDA-approved drugs (cariprazine, trazodone, metoprolol, and tamoxifen), one (ifenprodil) has been the object of a recent Phase 2b COVID trial, and one (AY9944) is an experimental compound that has seen extensive use as a DHCR7 inhibitor. The three FDA-approved drugs inhibit replication of a GFP-tagged VSV with efficacies that mirror their effect on DHCR7. Ifenprodil and AY9944 have complex inhibitory profiles, acting on both DHCR7 and DHCR14, while tamoxifen does not inhibit DHCR7 and is toxic to Neuro2a at concentrations where it inhibits the Δ7-Δ8 isomerase of the cholesterol pathway. VSV itself affects the sterol profile in Neuro2a cells, showing a dose-response increase of dehydrolathosterol and lathosterol, the substrates for DHCR7, with a corresponding decrease in desmosterol and cholesterol. 7-DHD and 7-DHC are orders of magnitude more vulnerable to free radical chain oxidation than other sterols as well as polyunsaturated fatty esters, and the effect of these sterols on viral infection is likely a reflection of this fact of Nature.

Inhibition of Zika virus replication by G-quadruplex-binding ligands.

Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control.

Zika virus infection induces endoplasmic reticulum stress and apoptosis in placental trophoblasts.

Zika virus (ZIKV) infection to a pregnant woman can be vertically transmitted to the fetus via the placenta leading to Congenital Zika syndrome. This is characterized by microcephaly, retinal defects, and intrauterine growth retardation. ZIKV induces placental trophoblast apoptosis leading to severe abnormalities in the growth and development of the fetus. However, the molecular mechanism behind ZIKV-induced apoptosis in placental trophoblasts remains unclear. We hypothesize that ZIKV infection induces endoplasmic reticulum (ER) stress in the trophoblasts, and sustained ER stress results in apoptosis. HTR-8 (HTR-8/SVneo), a human normal immortalized trophoblast cell and human choriocarcinoma-derived cell lines (JEG-3 and JAR) were infected with ZIKV. Biochemical and structural markers of apoptosis like caspase 3/7 activity and percent apoptotic nuclear morphological changes, respectively were assessed. ZIKV infection in placental trophoblasts showed an increase in the levels of CHOP mRNA and protein expression, which is an inducer of apoptosis. Next, we also observed increased levels of ER stress markers such as phosphorylated forms of inositol-requiring transmembrane kinase/endoribonuclease 1α (P-IRE1α), and its downstream target, the spliced form of XBP1 mRNA, phosphorylated eukaryotic initiation factor 2α (P-eIF2α), and activation of cJun N-terminal Kinase (JNK) and p38 mitogen activated protein kinase (MAPK) after 16-24 h of ZIKV infection in trophoblasts. Inhibition of JNK or pan-caspases using small molecule inhibitors significantly prevented ZIKV-induced apoptosis in trophoblasts. Further, JNK inhibition also reduced XBP1 mRNA splicing and viral E protein staining in ZIKV infected cells. In conclusion, the mechanism of ZIKV-induced placental trophoblast apoptosis involves the activation of ER stress and JNK activation, and the inhibition of JNK dramatically prevents ZIKV-induced trophoblast apoptosis.

The novel SARS-CoV-2 pandemic: Possible environmental transmission, detection, persistence and fate during wastewater and water treatment.

The contagious SARS-CoV-2 virus, responsible for COVID-19 disease, has infected over 27 million people across the globe within a few months. While literature on SARS-CoV-2 indicates that its transmission may occur predominantly via aerosolization of virus-laden droplets, the possibility of alternate routes of transmission and/or reinfection via the environment requires considerable scientific attention. This review aims to collate information on possible transmission routes of this virus, to ascertain its fate in the environment. Concomitant with the presence of SARS-CoV-2 viral RNA in faeces and saliva of infected patients, studies also indicated its occurrence in raw wastewater, primary sludge and river water. Therefore sewerage system could be a possible route of virus outbreak, a possible tool to assess viral community spread and future surveillance technique. Hence, this review looked into detection, occurrence and fate of SARS-CoV-2 during primary, secondary, and tertiary wastewater and water treatment processes based on published literature on SARS-CoV and other enveloped viruses. The review also highlights the need for focused research on occurrence and fate of SARS-CoV-2 in various environmental matrices. Utilization of this information in environmental transmission models developed for other enveloped and enteric viruses can facilitate risk assessment studies. Preliminary research efforts with SARS-CoV-2 and established scientific reports on other coronaviruses indicate that the threat of virus transmission from the aquatic environment may be currently non-existent. However, the presence of viral RNA in wastewater provides an early warning that highlights the need for effective sewage treatment to prevent a future outbreak of SARS-CoV-2.

Palmitoleate Protects against Zika Virus-Induced Placental Trophoblast Apoptosis.

Zika virus (ZIKV) infection in pregnancy is associated with the development of microcephaly, intrauterine growth restriction, and ocular damage in the fetus. ZIKV infection of the placenta plays a crucial role in the vertical transmission from the maternal circulation to the fetus. Our previous study suggested that ZIKV induces endoplasmic reticulum (ER) stress and apoptosis of placental trophoblasts. Here, we showed that palmitoleate, an omega-7 monounsaturated fatty acid, prevents ZIKV-induced ER stress and apoptosis in placental trophoblasts. Human trophoblast cell lines (JEG-3 and JAR) and normal immortalized trophoblasts (HTR-8) were used. We observed that ZIKV infection of the trophoblasts resulted in apoptosis and treatment of palmitoleate to ZIKV-infected cells significantly prevented apoptosis. However, palmitate (saturated fatty acid) did not offer protection from ZIKV-induced ER stress and apoptosis. We also observed that the Zika viral RNA copies were decreased, and the cell viability improved in ZIKV-infected cells treated with palmitoleate as compared to the infected cells without palmitoleate treatment. Further, palmitoleate was shown to protect against ZIKV-induced upregulation of ER stress markers, C/EBP homologous protein and X-box binding protein-1 splicing in placental trophoblasts. In conclusion, our studies suggest that palmitoleate protects placental trophoblasts against ZIKV-induced ER stress and apoptosis.

Current Status of Zika Virus Vaccines: Successes and Challenges.

The recently emerged Zika virus (ZIKV) spread to the Americas, causing a spectrum of congenital diseases including microcephaly in newborn and Guillain-Barré syndrome (GBS) in adults. The unprecedented nature of the epidemic and serious diseases associated with the viral infections prompted the global research community to understand the immunopathogenic mechanisms of the virus and rapidly develop safe and efficacious vaccines. This has led to a number of ZIKV vaccine candidates that have shown significant promise in human clinical trials. These candidates include nucleic acid vaccines, inactivated vaccines, viral-vectored vaccines, and attenuated vaccines. Additionally, a number of vaccine candidates have been shown to protect animals in preclinical studies. However, as the epidemic has waned in the last three years, further development of the most promising vaccine candidates faces challenges in clinical efficacy trials, which is needed before a vaccine is brought to licensure. It is important that a coalition of government funding agencies and private sector companies is established to move forward with a safe and effective vaccine ready for deployment when the next ZIKV epidemic occurs.

An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model.

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate.

Discovery of a non-nucleoside RNA polymerase inhibitor for blocking Zika virus replication through in silico screening.

Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.