Equine Polyclonal Antibodies Prevent Acute Chikungunya Virus Infection in Mice.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.

In Vitro Characterization and Antiviral Susceptibility of Ophidian Serpentoviruses.

Ophidian serpentoviruses, positive-sense RNA viruses in the order Nidovirales, are important infectious agents of both captive and free-ranging reptiles. Although the clinical significance of these viruses can be variable, some serpentoviruses are pathogenic and potentially fatal in captive snakes. While serpentoviral diversity and disease potential are well documented, little is known about the fundamental properties of these viruses, including their potential host ranges, kinetics of growth, environmental stability, and susceptibility to common disinfectants and viricides. To address this, three serpentoviruses were isolated in culture from three unique PCR-positive python species: Ball python (Python regius), green tree python (Morelia viridis), and Stimson's python (Antaresia stimsoni). A median tissue culture infectious dose (TCID50) was established to characterize viral stability, growth, and susceptibility. All isolates showed an environmental stability of 10-12 days at room temperature (20 °C). While all three viruses produced variable peak titers on three different cell lines when incubated at 32 °C, none of the viruses detectably replicated at 35 °C. All viruses demonstrated a wide susceptibility to sanitizers, with 10% bleach, 2% chlorhexidine, and 70% ethanol inactivating the virus in one minute and 7% peroxide and a quaternary ammonium solution within three minutes. Of seven tested antiviral agents, remdesivir, ribavirin, and NITD-008, showed potent antiviral activity against the three viruses. Finally, the three isolates successfully infected 32 unique tissue culture cell lines representing different diverse reptile taxa and select mammals and birds as detected by epifluorescent immunostaining. This study represents the first characterization of in vitro properties of growth, stability, host range, and inactivation for a serpentovirus. The reported results provide the basis for procedures to mitigate the spread of serpentoviruses in captive snake colonies as well as identify potential non-pharmacologic and pharmacologic treatment options for ophidian serpentoviral infections.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

An Isomer of Galidesivir That Potently Inhibits Influenza Viruses and Members of the Bunyavirales Order.

We report for the first time the antiviral activities of two iminovirs (antiviral imino-C-nucleosides) 1 and 2, structurally related to galidesivir (Immucillin A, BCX4430). An iminovir containing the 4-aminopyrrolo[2,1-f][1,2,4-triazine] nucleobase found in remdesivir exhibited submicromolar inhibition of multiple strains of influenza A and B viruses, as well as members of the Bunyavirales order. We also report the first syntheses of ProTide prodrugs of iminovir monophosphates, which unexpectedly displayed poorer viral inhibition than their parent nucleosides in vitro. An efficient synthesis of the 4-aminopyrrolo[2,1-f][1,2,4-triazine]-containing iminovir 2 was developed to enable preliminary in vivo studies, wherein it displayed significant toxicity in BALB/c mice and limited protection against influenza. Further modification of this anti-influenza iminovir will therefore be required to improve its therapeutic value.

Therapeutic neutralizing monoclonal antibody administration protects against lethal yellow fever virus infection.

Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks.

Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

Meeting report: 34th international conference on antiviral research.

As a result of the multiple gathering and travels restrictions during the SARS-CoV-2 pandemic, the annual meeting of the International Society for Antiviral Research (ISAR), the International Conference on Antiviral Research (ICAR), could not be held in person in 2021. Nonetheless, ISAR successfully organized a remote conference, retaining the most critical aspects of all ICARs, a collegiate gathering of researchers in academia, industry, government and non-governmental institutions working to develop, identify, and evaluate effective antiviral therapy for the benefit of all human beings. This article highlights the 2021 remote meeting, which presented the advances and objectives of antiviral and vaccine discovery, research, and development. The meeting resulted in a dynamic and effective exchange of ideas and information, positively impacting the prompt progress towards new and effective prophylaxis and therapeutics.

Remdesivir efficacy against yellow fever in a hamster model.

Yellow fever virus (YFV) continues to cause periodic outbreaks of severe disease throughout tropical regions of South America and Africa despite the availability of an effective vaccine. Despite efforts to control this virus for the last century, no antivirals have been approved for the treatment of YFV. The purpose of this study was to evaluate the broadly active antiviral compound remdesivir (RDV) in a hamster model of disease. Yellow fever (YF) disease in hamsters was prevented when treatment with RDV was initiated just prior to virus challenge, which was confirmed in a second study. Disease parameters including viremia, serum ALT and weight loss were significantly improved with RDV treatment in a dose-dependent manner. RDV was also effective when treatment was initiated as late as 4 days post-virus infection (dpi). These results demonstrate therapeutic efficacy of RDV in the treatment of YF in a relevant animal model of disease.

Isolation of a Potently Neutralizing and Protective Human Monoclonal Antibody Targeting Yellow Fever Virus.

Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in areas of endemicity. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed the identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting the YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV-17D vaccine strain in vitro. YFV-136 also potently inhibited infection by multiple wild-type YFV strains, in part, at a postattachment step in the virus replication cycle. YFV-136 showed therapeutic protection in two animal models of YFV challenge, including hamsters and immunocompromised mice engrafted with human hepatocytes. These studies define features of the antigenic landscape of the YFV E protein recognized by the human B cell response and identify a therapeutic antibody candidate that inhibits infection and disease caused by highly virulent strains of YFV. IMPORTANCE Yellow fever virus (YFV) is a mosquito-borne virus that occasionally causes outbreaks of severe infection and disease in South America and sub-Saharan Africa. There are very effective live-attenuated (weakened) yellow fever virus vaccines, but recent problems with their production and distribution have left many people in affected areas vulnerable. Here, we sought to isolate an antibody targeting the surface of the virus for possible use in the future as a biologic drug to prevent or treat YFV infection. We isolated naturally occurring antibodies from individuals who had received a YFV vaccine. We created antibodies and tested them. We found that the antibody with the most powerful antiviral activity was a beneficial treatment in two different small-animal models of human infection. These studies identified features of the virus that are recognized by the human immune system and generated a therapeutic antibody candidate that inhibits infection caused by highly virulent strains of YFV.

Therapeutic and prophylactic treatment with a virus-specific antibody is highly effective in rodent models of Chikungunya infection and disease.

Chikungunya virus (CHIKV) has re-emerged as a significant human pathogen in the 21st century, causing periodic, and sometimes widespread, outbreaks over the past 15 years. Although mortality is very rare, a debilitating arthralgia is very common and may persist for months or years. There are no antivirals that are approved for the treatment of CHIKV infection, and current treatment options consist of supportive care only. Herein, we demonstrate the efficacy of a CHIKV-specific antibody in the prophylactic and therapeutic treatment of CHIKV in mouse models of disease. The fully human anti-CHIKV monoclonal Ab SVIR023 demonstrated broad in vitro activity against representative strains from the three major CHIKV clades. Therapeutic treatment with SVIR023 administered 1- or 3-days post-infection resulted in reduced virus in various tissues in a dose- and time-dependent manner. Prophylactic treatment up to 4 weeks prior to virus challenge was also effective in preventing disease in mice. Mice treated with SVIR023 and infected with CHIKV were resistant to secondary challenge and no evidence of antibody enhancement of disease was observed. Treatment with SVIR023 was effective in mouse models of CHIKV infection and disease and further evaluation towards clinical development is warranted.

AT-752, a double prodrug of a guanosine nucleotide analog, inhibits yellow fever virus in a hamster model.

Yellow fever virus (YFV) is a zoonotic pathogen re-emerging in parts of the world, causing a viral hemorrhagic fever associated with high mortality rates. While an effective vaccine is available, having an effective antiviral against YFV is critical against unexpected outbreaks, or when vaccination is not recommended. We have previously identified AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, as a potent inhibitor of YFV in vitro, with a 50% effective concentration (EC50) of 0.31 µM. In hamsters infected with YFV (Jimenez strain), viremia rose about 4 log10-fold and serum alanine aminotransferase (ALT) 2-fold compared to sham-infected animals. Treatment with 1000 mg/kg AT-752 for 7 days, initiated 4 h prior to viral challenge, reduced viremia to below the limit of detection by day 4 post infection (pi) and returned ALT to normal levels by day 6 pi. When treatment with AT-752 was initiated 2 days pi, the virus titer and ALT dropped >2 log10 and 53% by day 4 and 6 pi, respectively. In addition, at 21 days pi, 70-100% of the infected animals in the treatment groups survived compared to 0% of the untreated group (p<0.001). Moreover, in vivo formation of the active triphosphate metabolite AT-9010 was measured in the animal tissues, with the highest concentrations in liver and kidney, organs that are vulnerable to the virus. The demonstrated in vivo activity of AT-752 suggests that it is a promising compound for clinical development in the treatment of YFV infection.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

Evaluation of AT-752, a Double Prodrug of a Guanosine Nucleotide Analog with In Vitro and In Vivo Activity against Dengue and Other Flaviviruses.

Every year, millions of people worldwide are infected with dengue virus (DENV), with a significant number developing severe life-threatening disease. There are currently no broadly indicated vaccines or therapeutics available for treatment of DENV infection. Here, we show that AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, was a potent inhibitor of DENV serotypes 2 and 3 in vitro, requiring concentrations of 0.48 and 0.77 µM, respectively, to inhibit viral replication by 50% (EC50) in Huh-7 cells. AT-281 was also a potent inhibitor of all other flaviviruses tested, with EC50 values ranging from 0.19 to 1.41 µM. Little to no cytotoxicity was observed for AT-281 at concentrations up to 170 µM. After oral administration of AT-752, substantial levels of the active triphosphate metabolite AT-9010 were formed in vivo in peripheral blood mononuclear cells of mice, rats, and monkeys. Furthermore, AT-9010 competed with GTP in RNA template-primer elongation assays with DENV2 RNA polymerase, which is essential for viral replication, with incorporation of AT-9010 resulting in termination of RNA synthesis. In AG129 mice infected with DENV D2Y98P, treatment with AT-752 significantly reduced viremia and morbidity and increased survival. The demonstrated in vitro and in vivo activity of AT-752 suggests that it is a promising compound for the treatment of dengue virus infection and is currently under evaluation in clinical studies.

Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus.

BACKGROUND: Zika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV. METHODOLOGY/PRINCIPLE FINDINGS: We have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge. CONCLUSIONS/SIGNIFICANCE: These studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.

IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro.

The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus; (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC50, of 7.6 ± 5.8 µM); (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC50, >100 µM); (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells; (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity; (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.

Structure activity relationship of novel antiviral nucleosides against Enterovirus A71.

Various (North)-methanocarba adenosine derivatives, containing rigid bicyclo[3.1.0]hexane ribose substitution, were screened for activity against representative viruses, and inhibition was observed after treatment of Enterovirus A71 with a 2-chloro-N6-1-cyclopropyl-2-methylpropan-1-yl derivative (17). µM activity was also seen when testing 17 against other enteroviruses in the Picornaviridae family. Based on this hit, structural congeners of 17, containing other N6-alkyl groups and 5' modifications, were synthesized and tested. The structure activity relationship is relatively narrow, with most modifications of the adenine or the methanocarba ring reducing or abolishing the inhibitory potency. 4'-Truncated 31 (MRS5474), 4'-fluoromethyl 48 (MRS7704) and 4'-chloromethyl 49 nucleosides displayed EC50 ~3-4 µM, and 31 and 48 achieved SI ≥10. However, methanocarba analogues of ribavirin and N6-benzyladenosine, shown previously to have anti-EV-A71 activity, were inactive. Thus, we identified methanocarba nucleosides as a new scaffold for enterovirus inhibition with a narrow structure activity relationship and no similarity to previously published anti-enteroviral nucleosides.

Strain-dependent disease and response to favipiravir treatment in mice infected with Chikungunya virus.

Antiviral countermeasures are needed to reduce the morbidity associated with Chikungunya virus (CHIKV) infection. This arbovirus reemerged in 2004 and causes periodic outbreaks in various areas throughout the world. While infection is rarely lethal, the majority of people infected with the virus develop a hallmark arthralgia as well as other disease manifestations. The virus is classified within three phylogenetic groups, namely, West African, East/Central/South African (ECSA), and Asian. Six strains of CHIKV covering the three phylogenetic groups were studied for their replication in cell culture, their ability to cause disease in susceptible mouse strains and susceptibility to antiviral treatment. Differential replication kinetics were observed for various CHIKV isolates in cell culture, which coincided with a decreased sensitivity to antiviral treatment as compared with ECSA and Asian clade viruses. This was confirmed in mouse infection studies with severe disease observed in mice infected with West African clade viruses, mild disease phenotype after infection with Asian clade viruses and an intermediate disease severity associated with ECSA virus infection. We also tested a broadly active antiviral, Favipiravir (T-705), which activity was inversely proportional to disease severity. These data suggest that some clades of CHIKV may cause more severe disease and may be more difficult to treat.

In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations.

To curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.

Development, Characterization, and Application of Two Reporter-Expressing Recombinant Zika Viruses.

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals.