Development of an in ovo system for evaluation of antivirals against Zika virus

Abstract

Zika virus (ZIKV) infection may be associated with congenital malformations in infected fetuses and severe complications in infected adults. Rapid and reproducible viral culture systems are needed for evaluation of antivirals against ZIKV. Viral culture using chick embryos has been shown to be suitable for antiviral evaluation for other viruses because of the eggs’ large size, low cost, and potentially higher throughput than other ranimal models. We investigated whether chick embryos could support virus replication and facilitate antiviral evaluation for ZIKV. Inoculation of chick embryos with 0.2 ml (3.6 log plaque forming units/ ml) of either epidemic (ZIKV-Puerto Rico: ZIKV-PR, human isolate) or pre-epidemic ZIKV (ZIKV-Uganda: ZIKV-U, primate isolate) strains led to significantly higher mean viral loads in the brain (1.6-2.4 log10 copies/ml), muscle (1.2-2.4 log10 copies/ml), and viscera (0.3-2.1 log10 copies/ml) of the chick embryos (P≤0.018). No significant increase in mean viral load was observed in the egg yolk. Abundant ZIKV NS1 protein expression was seen in the brain, muscle, and viscera of ZIKV-inoculated chick embryos. Embryonic lethality was observed in all the ZIKV-inoculated chick embryos. Compared to untreated controls, addition of azithromycin led to a ≤0.88 log10 copies/ml reduction of mean viral load in ZIKV-inoculated chick embryos. These results corroborated with previous findings that showed the moderate in vitro anti-ZIKV activity of azithromycin in cell lines. Further optimization of this in ovo culture system would facilitate the development of therapeutic measures against the emerging ZIKV.