Selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells

Abstract

An interesting question posed by the current evidence that severe acute respiratory syndrome coronavirus may be originated from an animal coronavirus is how such an animal coronavirus breaks the host species barrier and becomes zoonotic. In this report, we study the chronological order of genotypic changes in the spike protein of avian coronavirus infectious bronchitis virus (IBV) during its adaptation to a primate cell line. Adaptation of the Beaudette strain of IBV from chicken embryo to Vero cells showed the accumulation of 49 amino acid mutations. Among them, 26 (53.06%) substitutions were located in the S protein. Sequencing analysis and comparison of the S gene demonstrated that the majority of the mutations were accumulated and fixed at passage 7 on Vero cells and minor variants were isolated in several passages. Evidence present suggests that the dominant Vero cell-adapted IBV strain may be derived from the chicken embryo passages by selection of and potential recombination between the minor variants. This may explain why adaptation is a rapid process and the dominant strain, once adapted to a new host cell, becomes relatively stable.

Fig. 1

(A) Immunofluorescent staining of cells infected with Vero cell passages p1, p4, p6, p15, p36, and p65. Cells were infected with different passages of IBV, stained with rabbit anti-S antibodies at 24 h postinfection, and detected by FITC-labelled anti-rabbit antibodies. (B) Cell–cell fusion induced by the S protein derived from different passages of IBV. Vero cells expressing S protein derived from chicken embryo passage 3 (EP3), Vero cell passage 15 (p15), 36 (p36), and 65 (p65) were observed by phase-contrast microscopy at 24 h posttransfection.

Fig. 2

(A) Morphological changes of chicken fibroblast cell line, UMNSAH/DF1, infected with chicken embryo passage 3 (EP3) and Vero cell passage 65 (p65). Cells were infected with different passages of IBV and observed by phase-contrast microscopy at 24 h postinfection. (B) Western blot analysis of the nucleocapsid protein in UMNSAH/DF1 cells infected with chicken embryo passage 3 (EP3) and Vero cell passage 65 (p65). Cells were infected with different passages of IBV and harvested at indicated times. Polypeptides were separated by SDS–10% polyacrylamide gel, transferred to nitrocellulose membrane, and analysed by Western blotting with rabbit anti-N antibodies. Numbers on the left indicate molecular masses in kilodaltons.