Development And Application Of A High-throughout Antiviral Screening Platform Based On Recombinant Human Coronavirus OC43 Expressing The Luciferase Reporter Gene

Coronaviruses (CoVs) are enveloped ss RNA viruses, representing the largest genome of all the RNA viruses. CoVs are widely distributed among a broad range of animal specials,including humans, mammals and birds, causing respiratory or enteric illness but in some cases hepatic or neurological diseases of varying severity. Even though the first animal CoV (Infectious Bronchitis Virus, IBV) was first isolated in chicken embryos in 1937 and the first human coronaviruses (HCoVs) have been identified in 1960s,HCoVs gained people’s attention in the past decade until the global pandemic of highly pathogenic severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) in 2012, respectively. The two epidemic outbreaks of the infectious diseases indicated that the human medical science and veterinarian should pay more attention about the research of coronaviruses. However, there is lack of effective drug for the treatment of CoVs. Thus, it is necessary to establish an anti-CoVs drug screening platform for the development of new drugs. Human coronavirus OC43 (HCoV-OC43) is one of the six types of HCoVs have been identified. Together with HCoV-HKU1、SARS-CoV and MERS-CoV, are classified into the Betacoronavirus genus. HCoV-OC43 is the most related to SARS-CoV and MERS-CoV, with genomes display high identity with the two viruses,especially in the ORF1b region, which comprises the RdRp and helicase motifs. Hence, it could be used as a model for the study of the biological functions and screen of antivirals of the CoVs without need for Biological safety level-3 (BSL3) laboratory. The availability of the reverse genetics technique provides a new approach for the screening of antiviral drugs. The introduction of a foreign gene into the viral genome using reverse genetics technique provides the opportunity to develop a reporter virus can be used in the study of viral protein functions,viral replication and drug discovery. In this study, we generated a recombinant human coronaviruses OC43 (rOC43) which highly expressing the reporter Renilla luciferase (Rluc)gene named as rOC43-ns2DelRluc, and evaluate it in the host antiviral genes or drugs screening assay. Furthermore, rOC43-ns2DelRluc was successfully used to screen drugs in a 96-well plate, and screen out 38 drugs which could restrain the replication of HCoV-OC43 effectively. The principal results are as follows:1. Generation and screening of the rHCoV-OC43 expressing the Rluc gene.The infectious rHCoVs-OC43 (rOC43-ns2DelRluc, rOC43-ns12.9StopRIuc,rOC43-ns2FusionRluc and rOC43-ns12.9FusionRluc) were obtained using overlapping PCR following replacement of ns 12.9 or ns2 gene with Rluc gene or inserting Rluc gene in frame into ns 12.9 or ns2 gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc showed a robust luciferase activity, had a similar growth characteristic with the parental HCoV-OC43-WT and remained stable both in vivo and in vitro during passages. Moreover,deletion of ns2 had no influence on the pathogenicity of rOC43-ns2DelRluc in BALB/c mice.The experiment proved that the genetic of rOC43-ns2DelRluc is stable and can be reproduced from passages. Moreover, it can be successfully used for animal models.2. Screening of eukaryotic cell Lines for HCoV-OC43 susceptibilit using rOC43-ns2DelRluc,28 different eukaryotic cell lines were tested for the susceptibility to HCoV-OC43 infection based on rOC43-ns2DelRluc and verified by HCoV-OC43-WT. Eighteen cell lines were found could support the replication of recombinant HCoV-OC43, of which five cell lines showed high susceptibility, including BHK-21, 293T, Huh7.0 and RD cells. The replication peak of HCoV-OC43 in these cels lines were appeared at 5 days post-incubation (dpi). Ours findings have provided a reference for the isolation, pathogenesis and antiviral screening assays of HCoV-OC43.3. Screening for host factors that inhibit HCoV-OC43 replication.Eight previously reported potential antiviral host factors were selected in RNAi screening assay using rOC43-ns2DelRluc. Double-stranded-RNA-activated protein kinase(PKR) and DEAD box RNA helicases (DDX3X) were identified that exhibited anti-HCoV-OC43 activities, which were further verified by the use of HCoV-OC43-WT.These data demonstrated the feasibility of using rOC43-ns2DelRluc for identifying antiviral host factors.4. High-throughput antiviral drug screening using rOC43-ns2DelRluc.To verify whether rOC43-ns2DelRluc was suitable for high-throughput antiviral drug screening,rOC43-ns2DelRluc was used to evaluate of the antiviral activities of chloroquine and ribavirin in parallel. The Rluc activity of rOC43-ns2DelRluc was reduced in the presence of increasing levels of chloroquine or ribavirin in a dose-dependent manner, with IC50s similar to those with HCoV-OC43-WT. Next, we successfully screened 38 drugs from the compound library using the rOC43-ns2DelRluc-baesd HTS assays.To our knowledge, this is the first study on development and application of a luciferase-based recombinant HCoV-OC43 for high-throughout quantitative antiviral assays.The platform will pave a way for discovery and investigation of host factors and drugs against CoVs infection.