Characterization of temperature-sensitive (ts) mutants of coronavirus infectious bronchitis virus (IBV).

Abstract

Coronavirus infectious bronchitis virus is a positive-stranded RNA virus, which synthesises a 3’-coterminal nested set of six subgenomic RNAs. Subgenomic RNA transcription and genome replication are directed by the viral replicase, which is expressed in the form of polyproteins 1a and la/b and subsequently processed into smaller nonstructural proteins by the virusencoded proteinases. Major structural proteins S (spike protein), E (envelope protein), M (membrane protein) and N (nucleoprotein) are translated from subgenomic RNAs 2, 3, 4 and 6 and involved in nucleocapsid and virion assembling (Lai & Cavanagh 1998). Previous studies demonstrated that mutations and deletions on the structural proteins conferred the Coronavirus mouse hepatitis virus ts phenotypes (Masters et al 1994 & Luytjes et al 1997). However, little is known about the mechanisms involved. In this study, ts mutants were generated by growing wild type virus at progressively lower temperatures from 35°C to 28°C on Vero cells. Two ts mutants were isolated from passages grown at 29°C (ts291602) and 28°C (ts282902). Sequence analysis reveals that mutations emerged in the S protein and an insertion occurred in the N protein. Biological and biochemical properties of ts mutants were characterised.