Article Figures & Data
Figures
- FIG 1
Schematic diagram of the IBV S glycoprotein and identification of Beaudette-specific amino acids in the S2 subunit. (A) The S1 domain contains the receptor binding domain and the cleaved signal sequence. The S2 domain contains two heptad repeats (HR1 and HR2), a transmembrane domain, and a cytoplasmic domain. Beau-R also contains a Beaudette-specific sequence not found in M41. (B) The sequences of the ectodomains of the S2 subunit of IBV strains Beau-CK (GenBank accession number AJ311317), M41 (GenBank accession number X04722), 4/91 (UK) (GenBank accession number JN192154), and QX L1148 (GenBank accession number KY933090) are shown. There are 19 amino acid differences between the S2 subunits of Beau-R and M41-CK. The Beaudette-specific motif is labeled with a horizontal line. A black box surrounds each amino acid change made in the spike glycoproteins of the recombinant viruses generated in this study, and the amino acid positions are shown above.
- FIG 2
Growth characteristics of rIBVs BeauR-M41(S1) and BeauR-M41(S2) P3-CK on CK and Vero cells. (A and B) Chick kidney cells (A) and Vero cells (B) in 6-well plates were infected with Beau-R, M41-CK, BeauR-M41(S), BeauR-M41(S1) P3-CK, and BeauR-M41(S2) P3-CK at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean. (C to H) Vero cells were mock infected (C) or were infected with Beau-R (D), M41-CK (E), BeauR-M41(S) (F), BeauR-M41(S1) P3-CK (G), or BeauR-M41(S2) P3-CK (H). Infected cells were fixed at 24 h postinfection and immunolabeled with mouse anti-dsRNA and secondary antibody Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (green; Invitrogen). Nuclei were labeled with DAPI (blue). Bars, 50 μm.
- FIG 3
Adaptation of rIBV BeauR-M41(S1) to Vero cells. Vero cells in 6-well plates were infected with Beau-R and BeauR-M41(S1) P7-Vero at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean; some error bars are too small to be observed.
- FIG 4
Growth characteristics of rIBVs BeauR-S-MM and BeauR-M41-S-BSM P3-CK on CK and Vero cells. (A and D) Chick kidney cells (A) and Vero cells (D) in 6-well plates were infected with Beau-R, M41-CK, BeauR-M41(S), BeauR-S-MM P3-CK, and BeauR-M41-S-BSM P3-CK at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean. (B and C) Vero cells were infected with BeauR-M41-S-BSM P3-CK (B) and BeauR-S-MM P3-CK (C). Infected cells were fixed at 24 h postinfection and immunolabeled with mouse anti-dsRNA and secondary antibody Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (green; Invitrogen). Nuclei were labeled with DAPI (blue). Bars, 50 μm.
- FIG 5
Adaptation of rIBV BeauR-M41-S-BSM to Vero cells. Vero cells in 6-well plates were infected with Beau-R and BeauR-M41-S-BSM P7-Vero at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, 72, and 96 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean; some error bars are too small to be observed.
- FIG 6
Growth characteristics of rIBVs with modified S2 subunits on CK and Vero cells. (A and B) Chick kidney cells (A) and Vero cells (B) in 6-well plates were infected with Beau-R, BeauR-M41(S), BeauR-M41-S-BSM P3-CK, BeauR-M41-S-BSM-N617S P3-CK, BeauR-M41-S-BSM-L578F-N617S P3-CK, BeauR-M41-S-BSM-I1000V P3-CK, BeauR-M41-S-BSM-L857F-I1000V P3-CK, BeauR-M41-S-BSM-N826S-L857F-I1000V P3-CK, and BeauR-M41-S-BSM-L578F-N617S-N826S-L857F-I1000V P3-CK at an MOI of 0.05. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean. (C to H) Vero cells were infected with BeauR-M41-S-BSM-N617S P3-CK (C), BeauR-M41-S-BSM-L578F-N617S P3-CK (D), BeauR-M41-S-BSM-I1000V P3-CK (E), BeauR-M41-S-BSM-N826S-L857F-I1000V P3-CK (F), BeauR-M41-S-BSM-N826S-L857F-I1000V P3-CK (G), and BeauR-M41-S-BSM-L578F-N617S-N826S-L857F-I1000V P3-CK (H). Infected cells were fixed at 24 h postinfection and immunolabeled with mouse anti-dsRNA and secondary antibody Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (green; Invitrogen). Nuclei were labeled with DAPI (blue). Bars, 50 μm.
- FIG 7
Adaptation of rIBVs with modified S2 subunits to Vero cells. Vero cells in 6-well plates were infected with Beau-R, BeauR-M41-S-BSM P7-Vero, BeauR-M41-S-BSM-N617S P7-Vero, BeauR-M41-S-BSM-L578F-N617S P7-Vero, BeauR-M41-S-BSM-I1000V P7-Vero, BeauR-M41-S-BSM-L857F-I1000V P7-Vero, BeauR-M41-S-BSM-N826S-L857F-I1000V P7-Vero, and BeauR-M41-S-BSM-L578F-N617S-N826S-L857F-I1000V P7-Vero at an MOI of 0.005. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean.
- FIG 8
Proteolytic cleavage of rIBVs at the S2′ site is dependent on the Beaudette-specific motif. Chick kidney cells in 6-well plates were mock infected (lane 1) or infected with Beau-R (lane 2), M41-CK (lane 3), BeauR-M41(S) (lane 4), BeauR-S-MM P3-CK (lanes 5 and 6), or BeauR-M41-S-BSM P3-CK (lanes 7 and 8). Cells were lysed at 24 h postinfection, and spike glycoproteins were analyzed by Western blotting with anti-S2 antibody. Actin was detected with anti-beta-actin antibody. Two replicates of BeauR-S-MM P3-CK and BeauR-M41-S-BSM P3-CK were used. Bands corresponding to uncleaved spike, the S2 subunit, and the S2′ cleavage product are labeled. The S2′ cleavage product was detected in lanes 2, 7, and 8 only.
Tables
- TABLE 1
Mutations identified in the S genes of rIBVs
Virus Passage no.-cell type Mutationa nt postion nt change aa position aa change BeauR-M41(S1) P3-CK 22239 GTT → AAC 625 LF → LT P7-Vero 22239 GTT → AAC 625 LF → LT P7-Vero 23378 C → T 1004 T → I BeauR-M41(S2) P3-CK 21721 G → A 452 A → T BeauR-S-MM BeauR-M41-S-BSM P3-CK 22226 A → C 620 D → A P7-Vero 22226 A → C 620 D → A BeauR-M41-S-BSM-N617S P7-Vero 22962 A → C 866 Q → H BeauR-M41-S-BSM-L578F-N617S P3-CK 21843 A → C 493 E → D P7-Vero 21843 A → C 493 E → D P7-Vero 22962 A → C 866 Q → H BeauR-M41-S-BSM-I1000V P7-Vero 22099 C → T 578 L → F BeauR-M41-S-BSM-L857F-I1000V P7-Vero 22255 C → T 630 P → S BeauR-M41-S-BSM-N826S-L857F-I1000V P3-CK 20494 C → mixed T/C 43 H → Y P7-Vero 21709 G → A 448 V → I P7-Vero 22099 C → T 578 L → F P7-Vero 22342 C → mixed A/C 659 P → T P7-Vero 22936 C → mixed T/C 857 L → F P7-Vero 22962 A → mixed T/A 866 Q → H BeauR-M41-S-BSM-L578F-N617S-N826S-L857F-I1000V P7-Vero 20584 G → A 73 G → S ↵a nt, nucleotide; aa, amino acid.
