Molecular diversity, evolutionary trends, and mutation rates in avian coronavirus infectious bronchitis virus

Abstract

Live attenuated infectious bronchitis viruses (IBV) are routinely used by the poultry industry to control against outbreaks and to prevent infections. These viruses which are adapted to grow in embryonated chicken eggs contain a mixed viral population known as quasispecies. We sequenced the spike gene of the viruses obtained from Massachusetts (Mass), Connecticut (Conn), Arkansas (Ark-DPI), Georgia (GA98) and Delaware (DE072) vaccine vials and also from chickens infected with the same vaccine virus to study the dynamics of IBV vaccine genetic adaptation and selection in their host. We identified the unique spike gene sequence contained in reisolated vaccine viruses, which showed that modified live IBV vaccines contain subpopulations that undergo selection and mutation in chickens following vaccination. Using this data we were able to distinguish infectious bronchitis vaccine viruses circulating in the field from true field viruses. Coronaviruses have a diverse genetic makeup and are known to evolve very rapidly by accumulating genetic changes from nucleotide mutations, insertions and deletions and also through recombination events between viral genomes. In order to better understand genetic diversity and evolutionary rates in coronaviruses we sequenced the entire genome of eleven IB field viruses of serotypes Mass, Conn and California (Cal), isolated over a 40, 25 and 8 year period respectively. We examined molecular changes to determine how rapidly IBV evolves and if recombinations occur. Our data revealed that the viruses were evolving at evolutionary rates that ranged from 10-2 to 10-6 substitutions/site/year. Molecular evidence also revealed that the Mass and Conn viruses that are routinely used by Poultry producers in vaccination programs did not accumulate changes while the Cal viruses for which no vaccine exists, had the highest evolutionary trends. Phylogenetic analysis also revealed that Mass and Conn field viruses grouped with reisolated vaccine viruses and that multiple recombinations occurred between vaccine virus and an unknown number of field or vaccine viruses. This data is significant because it highlights the risk of examining viral evolution in the face of vaccination. Identifying sequence changes that occur when IBV replicate in a new host and changes in viruses isolated over a long period of time is biologically significant because it will broaden our knowledge of the evolutionary rate of coronavirus evolution.