Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus

Abstract

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.

Fig. 1

Phylogenetic relationship of full-length genomes. Phylogenetic tree showing nucleotide sequence relatedness computed using neighbor-joining and the Nei-Gojobori method. The bootstrap consensus subtree was constructed from 1,000 replicates (percentage of replicate trees in which associated strains clustered together are presented at nodes). The nucleotide sequences were aligned with ClustalW (DNASTAR, Inc.), and the nucleotide substitutions (×100) are shown in the scale at the bottom of the figure

Fig. 2

Schematic representation of nsp 3 with the number of amino acid changes (relative positions indicated by tick marks, vertical black, blue and red lines) and deletions (triangles) between pathogenic and attenuated viruses indicated below. Domain positions are based on the IBV Beaudette nsp 3 sequence (Acc# P27920) alignment from Ziebuhr et al. [11] and include the acidic acid domain, the papain-like protease 1 (PL1) domain, the ADP-ribose binding protein (ADRP), the papain-like protease 2 (PL2), and the Y domain. The black line represents an unknown region between the PL2 and Y domains (Color figure online)

Fig. 3

Phylogenetic relationships of the spike protein. Phylogenetic tree showing amino acid sequence relatedness computed using neighbor-joining and the Nei-Gojobori method. The bootstrap consensus subtree was constructed from 1,000 replicates (percentage of replicate trees in which associated strains clustered together are presented at nodes). The amino acid sequences were aligned with ClustalW (DNASTAR, Inc.), and the amino acid substitutions (×100) are shown in the scale at the bottom of the figure

Fig. 4

Virus replication expressed as the log₁₀ of viral genome copies. a Virus replication in 10-day-old embryonated eggs. Eggs were inoculated with 1 × 10⁵ 50% embryo infectious doses of virus. Time 0 represents allantoic fluid collected from eggs immediately after inoculation. Numbers are the average viral genome copies (n = 5 except where indicated) calculated from the standard curve for the real-time RT-PCR test (Callison et al. [27]). In the Ark-attenuated group, all the embryos died prior to the 96 h time point. b Virus replication in 1-day-old chicks. Chicks were inoculated intranasally/intraocularly with 1 × 10⁵ 50% embryo infectious doses of virus. Numbers are the average viral genome copies (n = 5) calculated from the standard curve for the real-time RT-PCR test (Callison et al. [27]). *Indicates that the numbers are statistically different P ≤ 0.1 between Mass/Mass41/41 pathogenic and Mass41-attenuated viruses, and dagger indicates that the numbers are statistically different P ≤ 0.1 between Ark/Ark-DPI/81 pathogenic and Ark-attenuated viruses