Effects of Naturally Occurring Six- and Twelve-Nucleotide Inserts on Newcastle Disease Virus Replication and Pathogenesis

Newcastle disease virus (NDV) isolates contain genomes of 15,186, 15,192 or 15,198 nucleotides (nt). The length differences reflect a 6-nt insert in the 5′ (downstream) non-translated region (NTR) of the N gene (15,192-nt genome) or a 12-nt insert in the ORF encoding the P and V proteins (causing a 4-amino acid insert; 15,198-nt genome). We evaluated the role of these inserts in the N and P genes on viral replication and pathogenicity by inserting them into genomes of two NDV strains that have natural genome lengths of 15,186 nt and represent two different pathotypes, namely the mesogenic strain Beaudette C (BC) and the velogenic strain GB Texas (GBT). Our results showed that the 6-nt and 12-nt inserts did not detectably affect N gene expression or P protein function. The inserts had no effect on the replication or virulence of the highly virulent GBT strain but showed modest degree of attenuation in mesogenic strain BC. We also deleted a naturally-occurring 6-nt insertion in the N gene from a highly virulent 15,192-nt genome-length virus, strain Banjarmasin. This resulted in reduced replication in vitro and reduced virulence in vivo. Thus, although these inserts had no evident effect on gene expression, protein function, or replication in vivo, they did affect virulence in two of the three tested strains.