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.