Bourbon Virus Transmission, New York, USA.

In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Heartland Virus Transmission, Suffolk County, New York, USA.

During 2018, Heartland virus RNA was detected in an Amblyomma americanum tick removed from a resident of Suffolk County, New York, USA. The person showed seroconversion. Tick surveillance and white-tailed deer (Odocoileus virginianus) serosurveys showed widespread distribution in Suffolk County, emphasizing a need for disease surveillance anywhere A. americanum ticks are established or emerging.

Glycosaminoglycan Compositional Analysis of Relevant Tissues in Zika Virus Pathogenesis and in Vitro Evaluation of Heparin as an Antiviral against Zika Virus Infection.

Zika virus (ZIKV) is an enveloped RNA virus from the flavivirus family that can cause fetal neural abnormalities in pregnant women. Previously, we established that ZIKV-EP (envelope protein) binds to human placental chondroitin sulfate (CS), suggesting that CS may be a potential host cell surface receptor in ZIKV pathogenesis. In this study, we further characterized the GAG disaccharide composition of other biological tissues (i.e., mosquitoes, fetal brain cells, and eye tissues) in ZIKV pathogenesis to investigate the role of tissue specific GAGs. Heparan sulfate (HS) was the major GAG, and levels of HS-6-sulfo, HS 0S (unsulfated HS), and CS 4S disaccharides were the main differences in the GAG composition of Aedes aegypti and Aedes albopictus mosquitoes. In human fetal neural progenitor and differentiated cells, HS 0S and CS 4S were the main disaccharides. A change in disaccharide composition levels was observed between undifferentiated and differentiated cells. In different regions of the bovine eyes, CS was the major GAG, and the amounts of hyaluronic acid or keratan sulfate varied depending on the region of the eye. Next, we examined heparin (HP) of various structures to investigate their potential in vitro antiviral activity against ZIKV and Dengue virus (DENV) infection in Vero cells. All compounds effectively inhibited DENV replication; however, they surprisingly promoted ZIKV replication. HP of longer chain lengths more strongly promoted activity in ZIKV replication. This study further expands our understanding of role of GAGs in ZIKV pathogenesis and carbohydrate-based antivirals against flaviviral infection.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 µM, 11.4 µM, and 4.8 µM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 µM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras.

UNLABELLED: The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE: The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein.

UNLABELLED: The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging. IMPORTANCE: This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.

Characterization of a critical interaction between the coronavirus nucleocapsid protein and nonstructural protein 3 of the viral replicase-transcriptase complex.

The coronavirus nucleocapsid protein (N) plays an essential structural role in virions through a network of interactions with positive-strand viral genomic RNA, the envelope membrane protein (M), and other N molecules. Additionally, N protein participates in at least one stage of the complex mechanism of coronavirus RNA synthesis. We previously uncovered an unanticipated interaction between N and the largest subunit of the viral replicase-transcriptase complex, nonstructural protein 3 (nsp3). This was found through analysis of revertants of a severely defective mutant of murine hepatitis virus (MHV) in which the N gene was replaced with that of its close relative, bovine coronavirus (BCoV). In the work reported here, we constructed BCoV chimeras and other mutants of MHV nsp3 and obtained complementary genetic evidence for its association with N protein. We found that the N-nsp3 interaction maps to the amino-terminal ubiquitin-like domain of nsp3, which is essential for the virus. The interaction does not require the adjacent acidic domain of nsp3, which is dispensable. In addition, we demonstrated a complete correspondence between N-nsp3 genetic interactions and the ability of N protein to enhance the infectivity of transfected coronavirus genomic RNA. The latter function of N was shown to depend on both of the RNA-binding domains of N, as well as on the serine- and arginine-rich central region of N, which binds nsp3. Our results support a model in which the N-nsp3 interaction serves to tether the genome to the newly translated replicase-transcriptase complex at a very early stage of infection.

Identification and characterization of novel lineage 1 Powassan virus strains in New York State.

Powassan virus (POWV, family Flaviviridae) is a reemerging tick-borne virus endemic in North America and Russia. In 1997, a POWV-like agent was isolated from Ixodes scapularis in New England and determined to be genetically distinct from the original POWV isolate. This revealed the existence of two lineages: lineage 1, prototype Powassan virus (POWV-1) and lineage 2, deer tick virus (DTV). POWV-1 is thought to be primarily maintained in a cycle between I. cookei and woodchucks and I. marxi and squirrels, while DTV is primarily maintained in a cycle between I. scapularis and small mammal hosts. Recent tick, mammalian, and human isolates from New York State (NYS) have been identified as DTV, but for the first time in 45 years, we detected four POWV-1 isolates, including the first reported isolation of POWV-1 from I. scapularis. We aimed to investigate genotypic and phenotypic characteristics of recent NYS isolates through sequence analysis and evaluation of replication kinetics in vitro and in vivo. Our sequencing revealed genetic divergence between NYS POWV-1 isolates, with two distinct foci. We found that POWV-1 isolates displayed variable replication kinetics in nymphal ticks but not in cell culture. POWV-1 isolated from I. scapularis displayed increased fitness in experimentally infected I. scapularis as compared to historic and recent POWV-1 isolates from I. cookei. These data suggest the emergence of divergent POWV-1 strains in alternate tick hosts and maintenance of genetically and phenotypically discrete POWV-1 foci.

Surveillance and Genetic Analysis of Jamestown Canyon Virus in New York State: 2001-2022.

Jamestown Canyon virus (JCV) (Peribunyavirdae; Orthobunyavirus) is a mosquito-borne pathogen endemic to North America. The genome is composed of three segmented negative-sense RNA fragments designated as small, medium, and large. Jamestown Canyon virus is an emerging threat to public health, and infection in humans can cause severe neurological diseases, including encephalitis and meningitis. We report JCV mosquito surveillance data from 2001 to 2022 in New York state. Jamestown Canyon virus was detected in 12 mosquito species, with the greatest prevalence in Aedes canadensis and Anopheles punctipennis. Detection fluctuated annually, with the highest levels recorded in 2020. Overall, JCV infection rates were significantly greater from 2012 to 2022 compared with 2001 to 2011. Full-genome sequencing and phylogenetic analysis were also performed with representative JCV isolates collected from 2003 to 2022. These data demonstrated the circulation of numerous genetic variants, broad geographic separation, and the first identification of lineage B JCV in New York state in 2022.

Maintenance of a host-specific minority mutation in the West Nile virus NS3.

West Nile virus (WNV), the most prevalent arthropod-borne virus (arbovirus) in the United States, is maintained in a cycle between Culex spp. mosquitoes and birds. Arboviruses exist within hosts and vectors as a diverse set of closely related genotypes. In theory, this genetic diversity can facilitate adaptation to distinct environments during host cycling, yet host-specific fitness of minority genotypes has not been assessed. Utilizing WNV deep-sequencing data, we previously identified a naturally occurring, mosquito-biased substitution, NS3 P319L. Using both cell culture and experimental infection in natural hosts, we demonstrated that this substitution confers attenuation in vertebrate hosts and increased transmissibility by mosquitoes. Biochemical assays demonstrated temperature-sensitive ATPase activity consistent with host-specific phenotypes. Together these data confirm the maintenance of host-specific minority variants in arbovirus mutant swarms, suggest a unique role for NS3 in viral fitness, and demonstrate that intrahost sequence data can inform mechanisms of host-specific adaptation.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Analysis of a crucial interaction between the coronavirus nucleocapsid protein and the major membrane-bound subunit of the viral replicase-transcriptase complex.

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase.

Aedes Albopictus and Cache Valley virus: a new threat for virus transmission in New York State.

We report surveillance results of Cache Valley virus (CVV; Peribunyaviridae, Orthobunyavirus) from 2017 to 2020 in New York State (NYS). Infection rates were calculated using the maximum likelihood estimation (MLE) method by year, region, and mosquito species. The highest infection rates were identified among Anopheles spp. mosquitoes and we detected the virus in Aedes albopictus for the first time in NYS. Based on our previous Anopheles quadrimaculatus vector competence results for nine CVV strains, we selected among them three stains for further characterization. These include two CVV reassortants (PA and 15041084) and one CVV lineage 2 strain (Hu-2011). We analyzed full genomes, compared in vitro growth kinetics and assessed vector competence of Aedes albopictus. Sequence analysis of the two reassortant strains (PA and 15041084) revealed 0.3%, 0.4%, and 0.3% divergence; and 1, 10, and 6 amino acid differences for the S, M, and L segments, respectively. We additionally found that the PA strain was attenuated in vertebrate (Vero) and mosquito (C6/36) cell culture. Furthemore, Ae. albopictus mosquitoes are competent vectors for CVV Hu-2011 (16.7-62.1% transmission rates) and CVV 15041084 (27.3-48.0% transmission rates), but not for the human reassortant (PA) isolate, which did not disseminate from the mosquito midgut. Together, our results demonstrate significant phenotypic variability among strains and highlight the capacity for Ae. albopictus to act as a vector of CVV.

Adaptive evolution of West Nile virus facilitated increased transmissibility and prevalence in New York State.

West Nile virus (WNV; Flavivirus, Flaviviridae) was introduced to New York State (NYS) in 1999 and rapidly expanded its range through the continental United States (US). Apart from the displacement of the introductory NY99 genotype with the WN02 genotype, there has been little evidence of adaptive evolution of WNV in the US. WNV NY10, characterized by shared amino acid substitutions R1331K and I2513M, emerged in 2010 coincident with increased WNV cases in humans and prevalence in mosquitoes. Previous studies demonstrated an increase in frequency of NY10 strains in NYS and evidence of positive selection. Here, we present updated surveillance and sequencing data for WNV in NYS and investigate if NY10 genotype strains are associated with phenotypic change consistent with an adaptive advantage. Results confirm a significant increase in prevalence in mosquitoes though 2018, and updated sequencing demonstrates a continued dominance of NY10. We evaluated NY10 strains in Culex pipiens mosquitoes to assess vector competence and found that the NY10 genotype is associated with both increased infectivity and transmissibility. Experimental infection of American robins (Turdus migratorius) was additionally completed to assess viremia kinetics of NY10 relative to WN02. Modelling the increased infectivity and transmissibility of the NY10 strains together with strain-specific viremia demonstrates a mechanistic basis for selection that has likely contributed to the increased prevalence of WNV in NYS.

Accessory protein 5a is a major antagonist of the antiviral action of interferon against murine coronavirus.

The type I interferon (IFN) response plays an essential role in the control of in vivo infection by the coronavirus mouse hepatitis virus (MHV). However, in vitro, most strains of MHV are largely resistant to the action of this cytokine, suggesting that MHV encodes one or more functions that antagonize or evade the IFN system. A particular strain of MHV, MHV-S, exhibited orders-of-magnitude higher sensitivity to IFN than prototype strain MHV-A59. Through construction of interstrain chimeric recombinants, the basis for the enhanced IFN sensitivity of MHV-S was found to map entirely to the region downstream of the spike gene, at the 3' end of the genome. Sequence analysis revealed that the major difference between the two strains in this region is the absence of gene 5a from MHV-S. Creation of a gene 5a knockout mutant of MHV-A59 demonstrated that a major component of IFN resistance maps to gene 5a. Conversely, insertion of gene 5a, or its homologs from related group 2 coronaviruses, at an upstream genomic position in an MHV-A59/S chimera restored IFN resistance. This is the first demonstration of a coronavirus gene product that can protect that same virus from the antiviral state induced by IFN. Neither protein kinase R, which phosphorylates eukaryotic initiation factor 2, nor oligoadenylate synthetase, which activates RNase L, was differentially activated in IFN-treated cells infected with MHV-A59 or MHV-S. Thus, the major IFN-induced antiviral activities that are specifically inhibited by MHV, and possibly by other coronaviruses, remain to be identified.

Identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein.

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.

Methylene blue is a potent and broad-spectrum inhibitor against Zika virus in vitro and in vivo.

Many flaviviruses including the Dengue virus (DENV), Zika virus (ZIKV), West Nile virus, Yellow Fever virus, and Japanese encephalitis virus are significant human pathogens, unfortunately without any specific therapy. Here, we demonstrate that methylene blue, an FDA-approved drug, is a broad-spectrum and potent antiviral against Zika virus and Dengue virus both in vitro and in vivo. We found that methylene blue can considerably inhibit the interactions between viral protease NS3 and its NS2B co-factor, inhibit viral protease activity, inhibit viral growth, protect 3D mini-brain organoids from ZIKV infection, and reduce viremia in a mouse model. Mechanistic studies confirmed that methylene blue works in both entry and post entry steps, reduces virus production in replicon cells and inhibited production of processed NS3 protein. Overall, we have shown that methylene blue is a potent antiviral for management of flavivirus infections, particularly for Zika virus. As an FDA-approved drug, methylene blue is well-tolerated for human use. Therefore, methylene blue represents a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.

JMX0207, a Niclosamide Derivative with Improved Pharmacokinetics, Suppresses Zika Virus Infection Both In Vitro and In Vivo.

Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight the need to develop effective therapies for this class of viruses. Previously we identified niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting the interface between viral protease NS3 and its cofactor NS2B. Here, we screened a small library of niclosamide derivatives and identified a new analogue with improved pharmacokinetic properties. Compound JMX0207 showed improved efficacy in inhibition of the molecular interaction between NS3 and NS2B, better inhibition of viral protease function, and enhanced antiviral efficacy in the cell-based antiviral assay. The derivative also significantly reduced Zika virus infection on 3D mini-brain organoids derived from pluripotent neural stem cells. Intriguingly, the compound significantly reduced viremia in a Zika virus (ZIKV) animal model. In summary, a niclosamide derivative, JMX0207, was identified, which shows improved pharmacokinetics and efficacy against Zika virus both in vitro and in vivo.