Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Type I interferon-dependent CCL4 is induced by a cGAS/STING pathway that bypasses viral inhibition and protects infected tissue, independent of viral burden.

Type I interferons (T1-IFN) are critical in the innate immune response, acting upon infected and uninfected cells to initiate an antiviral state by expressing genes that inhibit multiple stages of the lifecycle of many viruses. T1-IFN triggers the production of Interferon-Stimulated Genes (ISGs), activating an antiviral program that reduces virus replication. The importance of the T1-IFN response is highlighted by the evolution of viral evasion strategies to inhibit the production or action of T1-IFN in virus-infected cells. T1-IFN is produced via activation of pathogen sensors within infected cells, a process that is targeted by virus-encoded immunomodulatory molecules. This is probably best exemplified by the prototypic poxvirus, Vaccinia virus (VACV), which uses at least 6 different mechanisms to completely block the production of T1-IFN within infected cells in vitro. Yet, mice lacking aspects of T1-IFN signaling are often more susceptible to infection with many viruses, including VACV, than wild-type mice. How can these opposing findings be rationalized? The cytosolic DNA sensor cGAS has been implicated in immunity to VACV, but has yet to be linked to the production of T1-IFN in response to VACV infection. Indeed, there are two VACV-encoded proteins that effectively prevent cGAS-mediated activation of T1-IFN. We find that the majority of VACV-infected cells in vivo do not produce T1-IFN, but that a small subset of VACV-infected cells in vivo utilize cGAS to sense VACV and produce T1-IFN to protect infected mice. The protective effect of T1-IFN is not mediated via ISG-mediated control of virus replication. Rather, T1-IFN drives increased expression of CCL4, which recruits inflammatory monocytes that constrain the VACV lesion in a virus replication-independent manner by limiting spread within the tissue. Our findings have broad implications in our understanding of pathogen detection and viral evasion in vivo, and highlight a novel immune strategy to protect infected tissue.

Viral reduction of human blood by ultraviolet A-photosensitized vitamin K5.

Blood product transfusion can transmit viral pathogens. Pathogen reduction methods for blood products have been developed but, so far, are not available for whole blood. We evaluated if vitamin K5 (VK5) and ultraviolet A (UVA) irradiation could be used for virus inactivation in plasma and whole blood. Undiluted human plasma and whole blood diluted to 20% were spiked with high levels of vaccinia or Zika viruses. Infectious titers were measured by standard TCID50 assay before and after VK5/UVA treatments. Up to 3.6 log of vaccinia and 3.2 log of Zika were reduced in plasma by the combination of 500 µM VK5 and 3 J/cm2 UVA, and 3.1 log of vaccinia and 2.9 log of Zika were reduced in diluted human blood (20%) by the combination of 500 µM VK5 and 70 J/cm2 UVA. At end of whole blood treatment, hemolysis increased from 0.18% to 0.41% but remained below 1% hemolysis, which is acceptable to the Food and Drug Administration for red cell transfusion products. No significant alteration of biochemical parameters of red blood cells occurred with treatment. Our results provide proof of the concept that a viral pathogen reduction method based on VK5/UVA may be developed for whole blood.

Cell-to-cell spread of vaccinia virus is promoted by TGF-ß-independent Smad4 signalling.

The induction of Smad signalling by the extracellular ligand TGF-ß promotes tissue plasticity and cell migration in developmental and pathological contexts. Here, we show that vaccinia virus (VACV) stimulates the activity of Smad transcription factors and expression of TGF-ß/Smad-responsive genes at the transcript and protein levels. Accordingly, infected cells share characteristics to those undergoing TGF-ß/Smad-mediated epithelial-to-mesenchymal transition (EMT). Depletion of the Smad4 protein, a common mediator of TGF-ß signalling, results in an attenuation of viral cell-to-cell spread and reduced motility of infected cells. VACV induction of TGF-ß/Smad-responsive gene expression does not require the TGF-ß ligand or type I and type II TGF-ß receptors, suggesting a novel, non-canonical Smad signalling pathway. Additionally, the spread of ectromelia virus, a related orthopoxvirus that does not activate a TGF-ß/Smad response, is enhanced by the addition of exogenous TGF-ß. Together, our results indicate that VACV orchestrates a TGF-ß-like response via a unique activation mechanism to enhance cell migration and promote virus spread.

Design and synthesis of novel Imidazo[2,1-b]thiazole derivatives as potent antiviral and antimycobacterial agents.

A series of novel acyl-hydrazone (4a-d) and spirothiazolidinone (5a-d, 6a-d) derivatives of imidazo[2,1-b]thiazole were synthesized and evaluated for their antiviral and antimycobacterial activity. The antituberculosis activity was evaluated by using the Microplate Alamar Blue Assay and the antiviral activity was evaluated against diverse viruses in mammalian cell cultures. According to the biological activity studies of the compounds, 5a-c displayed hope promising antitubercular activity, 6d was found as potent for Coxsackie B4 virus, 5d was found as effective against Feline corona and Feline herpes viruses. Consequently, the obtained results displayed that, 5a-d and 6d present a leading structure for future drug development due to its straightforward synthesis and relevant bioactivity.

Study of Antiviral Activity of Metabolites of a New Serratia species K-57 Strain.

The results of studies of a newly isolated Serratia species K-57 strain are presented. The strain is characterized by antiviral activity towards human influenza A/Aichi/2/68/H3N2, vaccinia, mouse smallpox, and herpes simplex-2 viruses. The detected characteristics of the strain, including the data on activities on nucleolytic enzymes, recommend it for the development of therapeutic and preventive antiviral drugs.

Preemptive Tecovirimat Use in an Active Duty Service Member Who Presented With Acute Myeloid Leukemia After Smallpox Vaccination.

Smallpox vaccine is contraindicated in immunosuppression due to increased risk for adverse reactions (eg, progressive vaccinia). We describe the first-ever use of tecovirimat as a preemptive vaccinia virus treatment strategy during induction chemotherapy in an active duty service member who presented with acute leukemia and inadvertent autoinoculation after smallpox vaccination.

Screening and evaluation of potential inhibitors against vaccinia virus from 767 approved drugs.

The development of therapies for human smallpox is needed due to the increasing concern over the potential use of smallpox virus as a biological weapon. Here, we report a high-throughput screening for anti-smallpox virus drugs from a 767-small-molecule library, employing two vaccinia virus (VACV) strains containing firefly luciferase (VTT-Fluc and VG9-Fluc) as surrogate viruses. Using an eight-point dose response format assay, 26 compounds of different pharmacological classes were identified with in vitro anti-VACV activities. Mycophenolate mofetil (MMF) and tranilast (TRA) were detected to possess the highest anti-VACV potency (selectivity index values of >334 and >74, respectively); they could inhibit VTT-Fluc replication in nude mice at 5 days post-infection by 99% (10 mg/kg, P < .01) and 59% (45 mg/kg, P = .01), respectively, as indicated by bioluminescent intensity. In conclusion, MMF and TRA are promising anti-smallpox virus candidates for further optimization and repurposing for use in clinical practice.

Tungsten carbide nanoparticles show a broad spectrum virucidal activity against enveloped and nonenveloped model viruses using a guideline-standardized in vitro test.

Five tungsten carbide nanoparticle preparations (denoted WC1-WC5) were investigated for broad spectrum virucidal activity against four recommended model viruses. These are modified vaccinia virus Ankara (MVA), human adenovirus type 5 (HAdV-5), poliovirus type 1 (PV-1) and murine norovirus (MNV). All virucidal tests were performed two to five times using the quantitative suspension test, which is a highly standardized test method to evaluate the virucidal efficacy of disinfectants in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Quantitative detection of viruses was conducted by endpoint titration and quantitative real-time PCR. Results showed that three of the five tested compounds (WC1-WC3) were able to reduce the infectivity of all model viruses by at least four log10 of tissue culture infective dose 50% per ml after 15 min, whereas the other two compounds exhibited only limited efficacy (WC4) or showed cytotoxicity (WC5). Virucidal activity of nanoparticles increased with incubation time and a dose-effect curve showed dependence of virucidal activity with particle concentration. Whereas WC1-WC4 showed little cytotoxicity, WC5 which was doped with copper exhibited a significant cytotoxic effect. These findings propose tungsten carbide nanoparticles to be very promising in terms of new disinfection techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study investigates the virucidal activity of tungsten carbide nanoparticles using the quantitative suspension test in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Due to highly standardized assay conditions, results of this test are considered very reliable for evaluation of the virucidal activity of disinfectants. Broad-spectrum activity and high efficacy of three different tungsten carbide nanoparticles preparations is concluded.

Inhibition of Poxvirus Gene Expression and Genome Replication by Bisbenzimide Derivatives.

Virus infection of humans and livestock can be devastating for individuals and populations, sometimes resulting in large economic and societal impact. Prevention of virus disease by vaccination or antiviral agents is difficult to achieve. A notable exception was the eradication of human smallpox by vaccination over 30 years ago. Today, humans and animals remain susceptible to poxvirus infections, including zoonotic poxvirus transmission. Here we identified a small molecule, bisbenzimide (bisbenzimidazole), and its derivatives as potent agents against prototypic poxvirus infection in cell culture. We show that bisbenzimide derivatives, which preferentially bind the minor groove of double-stranded DNA, inhibit vaccinia virus infection by blocking viral DNA replication and abrogating postreplicative intermediate and late gene transcription. The bisbenzimide derivatives are potent against vaccinia virus and other poxviruses but ineffective against a range of other DNA and RNA viruses. The bisbenzimide derivatives are the first inhibitors of their class, which appear to directly target the viral genome without affecting cell viability.IMPORTANCE Smallpox was one of the most devastating diseases in human history until it was eradicated by a worldwide vaccination campaign. Due to discontinuation of routine vaccination more than 30 years ago, the majority of today's human population remains susceptible to infection with poxviruses. Here we present a family of bisbenzimide (bisbenzimidazole) derivatives, known as Hoechst nuclear stains, with high potency against poxvirus infection. Results from a variety of assays used to dissect the poxvirus life cycle demonstrate that bisbenzimides inhibit viral gene expression and genome replication. These findings can lead to the development of novel antiviral drugs that target viral genomes and block viral replication.

Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome.

Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions.IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones.

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

Tachypleus tridentatus Lectin Enhances Oncolytic Vaccinia Virus Replication to Suppress In Vivo Hepatocellular Carcinoma Growth.

Lectins play diverse roles in physiological processes as biological recognition molecules. In this report, a gene encoding Tachypleus tridentatus Lectin (TTL) was inserted into an oncolytic vaccinia virus (oncoVV) vector to form oncoVV-TTL, which showed significant antitumor activity in a hepatocellular carcinoma mouse model. Furthermore, TTL enhanced oncoVV replication through suppressing antiviral factors expression such as interferon-inducible protein 16 (IFI16), mitochondrial antiviral signaling protein (MAVS) and interferon-beta (IFN-ß). Further investigations revealed that oncoVV-TTL replication was highly dependent on ERK activity. This study might provide insights into a novel way of the utilization of TTL in oncolytic viral therapies.

Discovery of a New Class of Inhibitors of Vaccinia Virus Based on (-)-Borneol from Abies sibirica and (+)-Camphor.

A series of the bornyl ester/amide derivatives with N-containing heterocycles were designed and synthesized as vaccinia virus (VV) inhibitors. Bioassay results showed that among the designed compounds, derivatives 6, 13, 14, 34, 36 and 37 showed the best inhibitory activity against VV with the IC50 values of 12.9, 17.9, 3.4, 2.5, 12.5 and 7.5 µm, respectively, and good cytotoxicity. The primary structure-activity relationship (SAR) study suggested that the combination of a saturated N-heterocycle, such as morpholine or 4-methylpiperidine, and a 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold was favorable for antiviral activity.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity.

CONTEXT: Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. OBJECTIVE: This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). MATERIALS AND METHODS: Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. RESULTS: KPB-100 and KPB-200 inhibited HHV-2 at IC50 values of 2.5 and 2.9 µg/mL, respectively, and VACV at IC50 values of 3.1 and 7.4 µg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC50 values of 1.63 and 13.2 µg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 µg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC50 value of 4.5 µg/mL in CPE inhibition assay and HHV-1 at an IC90 of 3.0 µg/mL in VYR assay. DISCUSSION AND CONCLUSION: Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.

Antiviral Activity of Lady's Mantle (Alchemilla vulgaris L.) Extracts against Orthopoxviruses.

We studied toxicity and antiviral activity of bioactive substances extracted from the roots (ethylacetate extracts) and aerial parts (ethanol extracts) of lady's mantle (Alchemilla vilgaris L.). Plant extracts are characterized by low toxicity for continuous Vero cell culture, but inhibit the reproduction of orthopoxviruses (vaccinia virus and ectromelia virus) in these cells. Of all studied extracts, ethylacetate extract from lady's mantle roots characterized by the highest content of catechins in comparison with other samples demonstrated the highest activity in vitro towards the studied viruses (neutralization index for vaccinia and ectromelia viruses were 4.0 and 3.5 lg, respectively). The antiviral effect of Alchemilla vulgaris L. extracts was shown to be dose dependent.

Destabilization of α-Helical Structure in Solution Improves Bactericidal Activity of Antimicrobial Peptides: Opposite Effects on Bacterial and Viral Targets.

We have previously examined the mechanism of antimicrobial peptides on the outer membrane of vaccinia virus. We show here that the formulation of peptides LL37 and magainin-2B amide in polysorbate 20 (Tween 20) results in greater reductions in virus titer than formulation without detergent, and the effect is replicated by substitution of polysorbate 20 with high-ionic-strength buffer. In contrast, formulation with polysorbate 20 or high-ionic-strength buffer has the opposite effect on bactericidal activity of both peptides, resulting in lesser reductions in titer for both Gram-positive and Gram-negative bacteria. Circular dichroism spectroscopy shows that the differential action of polysorbate 20 and salt on the virucidal and bactericidal activities correlates with the α-helical content of peptide secondary structure in solution, suggesting that the virucidal and bactericidal activities are mediated through distinct mechanisms. The correlation of a defined structural feature with differential activity against a host-derived viral membrane and the membranes of both Gram-positive and Gram-negative bacteria suggests that the overall helical content in solution under physiological conditions is an important feature for consideration in the design and development of candidate peptide-based antimicrobial compounds.

Mutational analysis of vaccinia virus E3 protein: the biological functions do not correlate with its biochemical capacity to bind double-stranded RNA.

UNLABELLED: Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. IMPORTANCE: dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs.

Postchallenge administration of brincidofovir protects healthy and immune-deficient mice reconstituted with limited numbers of T cells from lethal challenge with IHD-J-Luc vaccinia virus.

UNLABELLED: Protection from lethality by postchallenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nude, nu/nu) BALB/c mice infected with vaccinia virus (VACV). Whole-body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 10(5) PFU of recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve (AUCs) were calculated for individual mice to assess viral loads. A three-dose regimen of 20 mg/kg BCV administered every 48 h starting either on day 1 or day 2 postchallenge protected 100% of mice. Initiating BCV treatment earlier was more efficient in reducing viral loads and in providing protection from pox lesion development. All BCV-treated mice that survived challenge were also protected from rechallenge with IHD-J-Luc or WRvFire VACV without additional treatment. In immune-deficient mice, BCV protected animals from lethality and reduced viral loads while animals were on the drug. Viral recrudescence occurred within 4 to 9 days, and mice succumbed ∼10 to 20 days after treatment termination. Nude mice reconstituted with 10(5) T cells prior to challenge with 10(4) PFU of IHD-J-Luc and treated with BCV postchallenge survived the infection, cleared the virus from all organs, and survived rechallenge with 10(5) PFU of IHD-J-Luc VACV without additional BCV treatment. Together, these data suggest that BCV protects immunocompetent and partially T cell-reconstituted immune-deficient mice from lethality, reduces viral dissemination in organs, prevents pox lesion development, and permits generation of VACV-specific memory. IMPORTANCE: Mass vaccination is the primary element of the public health response to a smallpox outbreak. In addition to vaccination, however, antiviral drugs are required for individuals with uncertain exposure status to smallpox or for whom vaccination is contraindicated. Whole-body bioluminescence imaging was used to study the effect of brincidofovir (BCV) in normal and immune-deficient (nu/nu) mice infected with vaccinia virus, a model of smallpox. Postchallenge administration of 20 mg/kg BCV rescued normal and immune-deficient mice partially reconstituted with T cells from lethality and significantly reduced viral loads in organs. All BCV-treated mice that survived infection were protected from rechallenge without additional treatment. In immune-deficient mice, BCV extended survival. The data show that BCV controls viral replication at the site of challenge and reduces viral dissemination to internal organs, thus providing a shield for the developing adaptive immunity that clears the host of virus and builds virus-specific immunological memory.