Semi-automated diagnostic RT-PCR as a screening assay for antiviral compounds in a 96-well format against highly pathogenic RNA viruses.

Abstract

In response to outbreaks of (re)emerging highly pathogenic RNA viruses, simple and scalable antiviral screening methods are urgently needed. Using established and validated diagnostic methods like RT-PCR for antiviral screening offers a rapid readout of viral replication. This becomes particular important when other traditional viral replication readouts, such as TCID₅₀ or plaque assays cannot be used due to the absence of cytopathic effects, lack of reporter gene-containing recombinant viruses or unavailability of appropriate antibodies - the latter two common challenges when so far unknown viruses emerge. This study evaluated semi-automated diagnostic RT-PCR in a 96-well approach for antiviral compound screening using Marburg virus serving as a case study. Remdesivir, a prodrug that exhibits antiviral activities against multiple RNA viruses, was used as positive control inhibiting replication of filoviruses. Applicability of the protocol to other members of the filovirus family was feasible using the same settings, while for other viruses like Middle East respiratory syndrome coronavirus (MERS-CoV) or Crimean-Congo hemorrhagic fever virus (CCHFV) adaptations to optimal infection settings were necessary. Our results demonstrate a high reproducibility and highlight the rapid adaptability of semi-automated RT-PCR assays as an accelerated antiviral screening assay with high scalability against a wide range of newly or (re)emerging RNA viruses. This is critical especially during outbreak situations where timely antiviral assessments are urgently needed.