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.
Highlights
Infectious bronchitis virus (IBV) is a pathogenic gamma coronavirus that causes respiratory symptoms, egg drops, nephritis and proventriculitis in domestic fowls [1]
A nephropathogenic field strain, KM91 that had been passaged 5 times through embryonated eggs was kindly provided by Avian Disease Division, animal and Plant Quarantine Agency in Korea
We found that BP-caKII was classified into of BP-caKII, we determined the complete genomic sequence of BP-caKII (MF924724) and performed the KII genotype (Figure 3), which was clearly different from the NC I genotype of the parent phylogenetic analysis with the S1 gene
Summary
Infectious bronchitis virus (IBV) is a pathogenic gamma coronavirus that causes respiratory symptoms, egg drops, nephritis and proventriculitis in domestic fowls [1]. Due to the persistent infection IBVs are difficult to eradicate and easy to mutate into new recombinant viruses in infected flocks [2,3]. 15 nonstructural proteins (nsp) are generated from the large 1a and 1ab proteins by viral proteases, and these nsp play roles in virus replication and pathogenicity [4,5]. The spike protein is a protective antigen that is essential for virus infection in host cells [1]. Nephropathogenic KM91-like viruses were first reported in 1991 and became prevalent in the field. An inactivated oil emulsion vaccine and an embryo-adapted, attenuated live vaccine (K2 strain) have been used to prevent KM91-like virus infection, but occasionally, KM91-like viruses have been isolated in the field [8]
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