Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII)

Abstract

We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 °C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.

Keywords: cold adaptation; comparative genomics; infectious bronchitis virus; persistent infection; premature reproductive tract pathogenicity model.

Figure 1

The growth increment of BP-caKII and K2 in embryonated eggs at 32 °C and 37 °C, over time (hour, h). The time point and the mean fold change of RNA copy (log2) were represented on the x- and the y-axis, respectively. Fold changes were calculated by multiplying the ddCt value and real fold-change of each virus calculated from regression analysis. * Significant difference of K2-37 °C from BP-caKII-37 °C and K2-32 °C; ** significant difference of BP-caKII-32 °C from others (p < 0.05).

Figure 2

Differences in pathological changes between IBV strains according to sexual precocity after IBV infection. The severe aplasia of the ovarian follicles and atrophy of the oviduct was compared across treated (+) and untreated (−) chicks. The yellow arrow indicates a lesion, such as a cyst, in reproductive organs.

Figure 3

Phylogenetic analysis of the S1 genes of infectious bronchitis viruses. Phylogenetic trees are based on the S1 amino acid sequence of IBVs, where the BP-caKII strain is marked with a red filled circle. Phylogenetic trees were constructed with the neighbor-joining method using MEGA 6.0. The bootstrap values were determined from 500 replicates of the original data. The branch number represents the percentage of times that the branch appeared in the tree. Bootstrap values greater than 50% are shown. The p-distance is indicated by the bar at the bottom of the figure.