Antiviral Activity of Chloroquine against Human Coronavirus OC43 Infection in Newborn Mice

Until recently, human coronaviruses (HCoVs), such as HCoV strain OC43 (HCoV-OC43), were mainly known to cause 15 to 30% of mild upper respiratory tract infections. In recent years, the identification of new HCoVs, including severe acute respiratory syndrome coronavirus, revealed that HCoVs can be highly pathogenic and can cause more severe upper and lower respiratory tract infections, including bronchiolitis and pneumonia. To date, no specific antiviral drugs to prevent or treat HCoV infections are available. We demonstrate that chloroquine, a widely used drug with well-known antimalarial effects, inhibits HCoV-OC43 replication in HRT-18 cells, with a 50% effective concentration (± standard deviation) of 0.306 ± 0.0091 μM and a 50% cytotoxic concentration (± standard deviation) of 419 ± 192.5 μM, resulting in a selectivity index of 1,369. Further, we investigated whether chloroquine could prevent HCoV-OC43-induced death in newborn mice. Our results show that a lethal HCoV-OC43 infection in newborn C57BL/6 mice can be treated with chloroquine acquired transplacentally or via maternal milk. The highest survival rate (98.6%) of the pups was found when mother mice were treated daily with a concentration of 15 mg of chloroquine per kg of body weight. Survival rates declined in a dose-dependent manner, with 88% survival when treated with 5 mg/kg chloroquine and 13% survival when treated with 1 mg/kg chloroquine. Our results show that chloroquine can be highly effective against HCoV-OC43 infection in newborn mice and may be considered as a future drug against HCoVs.Coronaviruses are large, enveloped, single-stranded, positive-sense RNA viruses with a genome of approximately 30 kb in length, the largest found in any of the RNA viruses. The genus Coronavirus belongs to the family Coronaviridae in the order Nidovirales. The coronaviruses are classified into three groups based on genetic and serological relationships. Group 1 contains porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, canine coronavirus, feline infectious peritonitis virus, human coronavirus 229E (HCoV-229E), and HCoV-NL63. Group 2 contains murine hepatitis virus, bovine coronavirus (BCoV), HCoV-OC43, rat sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus (PHEV), canine respiratory coronavirus, and equine coronavirus. The severe acute respiratory syndrome coronavirus (SARS-CoV) is considered a distant member of group 2 and is therefore placed in a subgroup, 2b (13). Group 3 thus far contains only avian coronaviruses, such as infectious bronchitis virus and turkey coronavirus. Within group 2, HCoV-OC43 is most closely genetic related to BCoV (33). A comparative analysis of the complete genomes of HCoV-OC43, BCoV, and PHEV demonstrated a high genetic similarity among these three coronaviruses in more than two-thirds of their genomes, except in the hemagglutinin-esterase and spike gene region, in which PHEV was remarkably divergent from HCoV-OC43 and BCoV (30, 31, 33).HCoVs cause respiratory infections, but gastroenteritis and neurological disorders can also occur (3, 20). Until now, five HCoVs have been described. HCoV-OC43 and HCoV-229E are responsible for 10 to 30% of all common colds, and infections occur mainly during the winter and early spring (21). In 2003, a novel HCoV displaying only distant antigenic and genetic similarities to the two previously known HCoVs was identified as the causal agent of SARS, and this causes severe lung disorder, leading in some cases to systemic infection and eventually death in about 10% of cases (19). During the two years after the SARS outbreak, two additional previously unrecognized coronaviruses affecting humans, HCoV-NL63 and HCoV-HKU1, were identified (29, 35). HCoV-NL63 infection is related to acute respiratory dysfunction in infected individuals. Furthermore, HCoV-NL63 was identified as the major pathogen responsible for croup in young children (9, 10, 22). The clinical features of HCoV-NL63 infections appear to be more severe than those commonly attributed to infections by HCoV-OC43 and HCoV-229E (4, 12, 29). Infection with HCoV-HKU1 is mostly associated with bronchiolitis and pneumonia (35, 36).Although coronaviruses have been recognized as human pathogens for about 50 years, no effective treatment strategy has been approved. This shortcoming became evident during the SARS-CoV outbreak and was the start of numerous studies. Nevertheless, 5 years after the outbreak, we are still lacking an effective commercially available drug. Chloroquine is a clinically approved drug effective against malaria, and it is known to elicit antiviral effects against several viruses, including human immunodeficiency virus type 1 (23, 26, 28), hepatitis B virus (18), and herpes simplex virus type 1 (27). Savarino and colleagues hypothesized that chloroquine might be of some use for the clinical management of SARS (25). Moreover, chloroquine was reported to inhibit the replication of HCoV-229E (7) and SARS-CoV (16) in vitro. In a previous study, we showed that the 50% effective concentration (EC₅₀) of chloroquine (8.8 ± 1.2 μM) for the inhibition of SARS-CoV in vitro was significantly lower than its cytostatic activity (261.3 ± 14.5 μM), yielding a selectivity index of 30, and that this EC₅₀ approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. The addition of chloroquine to infected cultures could be delayed for up to 5 h postinfection without a significant drop in antiviral activity (16). The antiviral effects of chloroquine against human immunodeficiency virus type 1 replication are currently being tested in clinical trials (25). Chloroquine is a weak base that increases the pH of acidic vesicles. When added extracellularly, the nonprotonated portion of chloroquine enters the cell, where it becomes protonated and concentrated in acidic, low-pH organelles, such as endosomes, Golgi vesicles, and lysosomes. Chloroquine can affect virus infection in many ways, and the antiviral effect depends in part on the extent to which the virus utilizes endosomes for entry. Besides having a direct antiviral effect, chloroquine is endowed with an immunomodulatory activity, suppressing the production and release of tumor necrosis factor alpha and interleukin 6, which mediate the inflammatory complications of several viral diseases (25).In the present study, we further investigated the anticoronaviral properties of chloroquine by testing the in vitro antiviral activity of chloroquine against HCoV-OC43. In addition, we developed a lethal in vivo challenge model to test the antiviral effect of chloroquine against HCoV-OC43.     

In Vitro Antiviral Activity of V-073 against Polioviruses

V-073, an enterovirus capsid inhibitor, was evaluated for its spectrum of antipoliovirus activity. V-073 inhibited all 45 polioviruses tested in a virus-induced cytopathic effect protection assay, with 50% effective concentration (EC₅₀) values ranging from 0.003 to 0.126 μM. Ninety percent of the polioviruses tested were inhibited at EC₅₀s of ≤0.076 μM (MIC₉₀ = 32 ng/ml). V-073 is a promising antiviral candidate for the posteradication management of poliovirus incidents.     

Antiviral Activity of Tobacco Smoke Condensate on Encephalomyocarditis Infection in Mice

A water-soluble nontumorigenic acidic fraction of tobacco smoke condensate of cigarettes has been found to have antiviral activity against encephalomyocarditis (EMC) virus infection in mice. The portion of lower molecular weight was inhibitory to the growth of EMC virus, vesicular stomatitis virus, reovirus type 2, vaccinia virus, and poliovirus type 2, but not against adenovirus type 12, in KB cell cultures. The cigarette smoke agent did not induce serum interferon although it protected mice from EMC disease by pretreatment.     

Antiviral Activity of a Selective Ribonucleotide Reductase Inhibitor against Acyclovir-Resistant Herpes Simplex Virus Type 1 In Vivo

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAA^r5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAA^r5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAA^r5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAA^r5 infections. The effect of BILD 1633 SE against HSV-1 PAA^r5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 10⁶ and 10⁷ PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1 dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAA^r5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.     

Discovery of Structurally Diverse Small-Molecule Compounds with Broad Antiviral Activity against Enteroviruses

Antiviral drugs do not currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. We conducted high-throughput molecular screening and identified a structurally diverse set of compounds that inhibit the replication of coxsackievirus B3, a commonly encountered enterovirus. These compounds did not interfere with the function of the viral internal ribosome entry site or with the activity of the viral proteases, but they did drastically reduce the synthesis of viral RNA and viral proteins in infected cells. Sequence analysis of compound-resistant mutants suggests that the viral 2C protein is targeted by most of these compounds. These compounds demonstrated antiviral activity against a panel of the most commonly encountered enteroviruses and thus represent potential leads for the development of broad-spectrum anti-enteroviral drugs.     

Antiviral Activity of 3-Methyleneoxindole

3-Methyleneoxindole (MO), an oxidation product of the plant auxin indole-3-acetic acid, can selectively inhibit the replication of herpes-, mengo-, polioviruses, and Sindbis virus. The antiviral action of MO, a sulfhydryl binding compound, is neutralized by 2-mercaptoethanol if the latter is added soon after exposure of infected cells to MO. If addition of 2-mercaptoethanol is delayed, the antiviral action of MO appears to be irreversible. Data are presented which indicate that the antiviral action of MO is not mediated by interferon.     

Antiviral Activity of Broad-Spectrum and Enterovirus-Specific Inhibitors against Clinical Isolates of Enterovirus D68

We investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided.     

Multiple Classes of Antiviral Agents Exhibit In Vitro Activity against Human Rhinovirus Type C

Human rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagated in vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by a Renilla luciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIβ (PI4K-IIIβ) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.     

Engineering T Cells to Functionally Cure HIV-1 Infection

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients'' lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1–resistant cells, redirecting HIV-1–specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1–specific lymphocytes within a patient''s own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy–mediated functional cure.     

Antiviral Activity of (+)-Rutamarin against Kaposi's Sarcoma-Associated Herpesvirus by Inhibition of the Catalytic Activity of Human Topoisomerase II

Kaposi''s sarcoma-associated herpesvirus (KSHV) is an etiological agent of several AIDS-associated malignancies, including Kaposi''s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman''s disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesions, lytic viral replication, production of virion particles, and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in the treatment of KSHV-associated diseases. However, there is no effective anti-KSHV drug currently available. Our previous work showed that human topoisomerase II (Topo II) is indispensable for KSHV lytic replication and is suggested to be an effective target for antiviral drugs. Here, we report the discovery and characterization of a novel catalytic inhibitor of human Topo IIα, namely, (+)-rutamarin. The binding mode of (+)-rutamarin to the ATPase domain of human Topo IIα was established by docking and validated by molecular dynamics (MD) simulations. More importantly, (+)-rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with a half-maximal inhibitory concentration (IC₅₀) of 1.12 μM and blocks virion production with a half-maximal antiviral effective concentration (EC₅₀) of 1.62 μM. It possesses low cytotoxicity, as indicated by the selectivity index (SI) of 84.14. This study demonstrated great potential for (+)-rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.     

Small-Molecule Inhibitors of Lethal Factor Protease Activity Protect against Anthrax Infection

Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.