Rescue of dual reporter-tagged parainfluenza virus 5 as tool for rapid screening of antivirals in vitro

•Rescue of dual reporter-tagged recombinant parainfluenza virus 5 (PIV5).•Two reporters (eGFP and NLuc) are genetically stable during serial passages.•Antiviral assay using a novel method based on dual reporter-tagged PIV5.•Only ribavirin shows an ideal effect against dual reporter-tagged PIV5. Parainfluenza virus 5 (PIV5) belongs to the genus Orthorubulavirus in the family Paramyxoviridae. PIV5 can infect a range of mammals, but induce mild or even unobservable clinical signs in some animals, except kennel cough in dogs. It is also able to infect a variety of cell lines, but causes minimal or even invisible cytopathic effects on many cells. Sometimes, owing to neither observable cytopathic effects in vitro nor typical clinical signs in vivo, the PIV5 is not easily usable for screening antiviral drugs. To solve this issue, we used reverse genetics to recover a dual reporter-tagged recombinant PIV5 that could simultaneously express enhanced green fluorescence protein (eGFP) and NanoLuc® luciferase (NLuc) in virus-infected cells. Both reporters were genetically stable during twenty serial passages of virus in MDBK cells. The eGFP allowed us to observe virus-infected MDBK cells in real time, and moreover the NLuc made it possible to quantify the degree of viral replication for determining antiviral activity of a given drug. Subsequently, the recombinant PIV5 was used for antiviral assays on five common drugs, i.e., ribavirin, apigenin, 1-adamantylamine hydrochloride, moroxydine hydrochloride and tea polyphenol. The results showed that only the ribavirin had an anti-PIV5 effect in MDBK cells. This study proposed a novel method for rapid screening (or prescreening) of anti-PIV5 drugs.