Emergence of biased hypermutation in a heterologous additional transcription unit in recombinant lentogenic Newcastle disease virus

Recombinant Newcastle Disease virus (rNDV) strains engineered to express foreign genes from an additional transcription unit (ATU) are considered as candidate live-attenuated vector vaccines for human and veterinary use. Early during the COVID-19 pandemic we and others have generated COVID-19 vaccine candidates based on rNDV expressing a partial or complete SARS-CoV-2 spike (S) protein. In our studies, a number of the rNDV constructs did not show high S expression levels in cell culture or seroconversion in immunized hamsters. Sanger sequencing showed the presence of frequent A-to-G transitions characteristic of adenosine deaminase acting on RNA (ADAR). Subsequent whole genome rNDV sequencing revealed that this biased hypermutation was exclusively localized in the ATU expressing the spike gene.