A system for mass screening of antiviral agents by using chlorella virus as a surrogate DNA virus

Abstract

Nucleo-cytoplasmic large DNA viruses (NCLDVs), which infect a wide range of eukaryotes, consist of large double-strand DNA (dsDNA) viruses, including Phycodnaviridae, Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, and Mimiviridae. As the incidence of various infectious diseases caused by new viruses such as African swine fever (ASF) and coronavirus disease 2019 (COVID-19), has increased, the need for the development of antiviral drugs has become the center of research efforts. The chlorella virus infecting Chlorella variabilis NC64A, one of the Chlorovirus genera in the Phycodnaviridae family, is genetically and morphologically similar to ASFV, and is easy to handle and harmless to human. Therefore, it can be used as a surrogate for ASFV, one of the dsDNA viruses. In this study, a screening system for antiviral substances against dsDNA virus was developed by using a chlorella-infecting virus called the PKV2 virus isolated from the domestic freshwater environment as a surrogate virus. A total of 19,133 substances (15,635 chemical compounds and 3,498 extracts from plants and marine organisms) were screened for antiviral activity. Twenty out of 15,635 chemical compounds and 31 out of 3,498 extracts were selected from the first-step screening process using a microscope. The antiviral activity of these selected materials was further tested at lower concentrations and two chemical compounds (bergapten, psoralen) and two plant extracts (Calystegia soldanella, Distylium racemosum) were selected as the final candidates. The selected substances were additionally tested by plaque reduction assay for antiviral activity and cytotoxicity by using a water-soluble tetrazolium salts (WST) assay. Both bergapten and psoralen showed increased antiviral activity when they were treated with UVA in the presence of the virus. In the antiviral test with extracts from different parts of the two selected plants, the extract from the flower of C. soldanella and from a branch of D. racemosum showed the highest antiviral activity. In conclusion, the successful screening of substances with antiviral activity suggests that our system can be used to develop antiviral substances against dsDNA viruses.