In Vitro Inhibition of Coronavirus Replications
A search for new anti-coronaviral drugs to treat coronaviral infections was motivated by an outbreak of severe acute respiratory syndrome (SARS). In order to find drugs that treat coronavirus infections, including SARS, we screened traditional medicinal herbal extracts and evaluated their antiviral activities on coronavirus replication. We employed a plaque assay to evaluate the effect of 29 medicinal herbal extracts of virus replication. We determined the 50% effective concentration (EC50) of each extract that was necessary to inhibit the replication of mouse hepatitis virus A59 (MHV-A59); we also determined 50% cytotoxic concentrations (CC50) for each extract. Northern and Western blot analyzes were performed to investigate antiviral activity in MHV-infected DBT cells, including virus entry, viral RNA and protein expression, and virus release. Coronavirus specific inhibition was also demonstrated using porcine epidemic diarrhea virus (PEDV). Cimicifuga rhizome, Meliae cortex, Coptidis rhizome, Phellodendron cortex and Sophora subprostrata radix decreased the MHV production and the intracellular viral RNA and protein expression with EC50 values ranging from 2.0 to 27.5ug/ml. These extracts also significantly decreased PEDV production and less dramatically vesicular stomatitis virus (VSV) production in vitro. Also, TCM-3, -17, -24, -25, -32 decreased the virus productions and the intracellular viral RNA and protein expressions. The 50% effective concentrations (EC50) of five TCMs, as determined by plaque reduction of MHV-A59, were ranged from 0.67±0.1 to 4.75±3.5 ug/ml, while the 50% cytotoxic concentration (CC50) against DBT cells by MTT assay were ranged from 159.0±1.3 to 820.2±31.7 ug/ml. The selective index (SI) of TCM-25 is 1224.1, the highest among the TCMs tested. To determine the inhibitory effect of TCMs on virus adsorption, MHV-A59 were adsorbed at 4℃ followed by the TCM treatment at 37℃. TCM-3 and -32 additionally inhibit MHV-A59 adsorption, too, although only slightly. The extracts selected strongly inhibited MHV replication and could be potential candidates for new anti-coronavirus- drugs.
Many viruses use microtubules toward the cell interior during the entry process and toward the plasma membrane during the egress period. In addition, viruses often remodel microtubules to facilitate the generation of infectious progeny. Murine coronavirus JHM, the causative agent of demyelination, has previously been shown to have used microtubules in viral protein trafficking and distribution. In this study, we examined whether other cytoskeleton is involved in MHV replication or whether microtubules are involved in early step of MHV replication. To search for the cytoskeletons that affect mouse hepatitis virus replication, we treated pharmacolocial agents specifically target the structure and properties of the above cytoskeletal elements in MHV-infected cells. A microtubule-destabilizing drug (nocodazole), microtubule-stabilizing drug (Paclitaxel), Latrunculin A-disrupting actin and Jasplakinolide-stabilizing actin filament were introduced at various time points during infection of DBT cell with MHV-JHM. We show that nocodazole, but not actin related drug, reduced levels of intracellular viral RNA and virus titer in early treatment. These results suggested that microtubules play an essential role in the infectious life cycle of MHV.
