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. 2018 May 2:250:104-113.
doi: 10.1016/j.virusres.2018.04.013. Epub 2018 Apr 21.

Effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus

Dan Shan  1 , Shouguo Fang  2 , Zongxi Han  1 , Hui Ai  1 , Wenjun Zhao  1 , Yuqiu Chen  1 , Lei Jiang  1 , Shengwang Liu  3
Affiliations

Effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus

Dan Shan et al. Virus Res. .

Abstract

To study the roles of hypervariable regions (HVRs) in receptor-binding subunit S1 of the spike protein, we manipulated the genome of the IBV Beaudette strain using a reverse genetics system to construct seven recombinant strains by separately or simultaneously replacing the three HVRs of the Beaudette strain with the corresponding fragments from a QX-like nephropathogenic isolate ck/CH/LDL/091022 from China. We characterized the growth properties of these recombinant IBVs in Vero cells and embryonated eggs, and their pathogenicity, tropism, and serotypes in specific pathogen-free (SPF) chickens. All seven recombinant IBVs proliferated in Vero cells, but the heterogenous HVRs could reduce their capacity for adsorption during in vitro infection. The recombinant IBVs did not significantly increase the pathogenicity compared with the Beaudette strain in SPF chickens, and they still shared the same serotype as the Beaudette strain, but the antigenic relatedness values between the recombinant strain and Beaudette strain generally decreased with the increase in the number of the HVRs exchanged. The results of this study demonstrate the functions of HVRs and they may help to develop a vaccine candidate, as well as providing insights into the prevention and control of IBV.

Keywords: Hypervariable region; Infectious bronchitis virus; Infectious clone; Recombinant IBV; Reverse genetic vaccine.

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Figures

Fig. 1
Fig. 1
Recovery of the recombinant IBVs from cells. (a) Diagram showing the organization of the genomes in the recombinant IBVs. The genome structure of the IBV Beaudette strain is shown in the white rectangle. Regions coding for the hypervariable regions (HVRs) in the IBV strain ck/CH/LDL/091022 are shown in the black rectangle. (b) Immunofluorescent staining of Vero cells infected with the recombinant IBVs. The cells were infected with the recombinant IBVs and the two parental strains. At 24 h post-infection, the cells were fixed and stained with the monoclonal antibody against IBV N protein. (c) Detection of the viral RNA in Vero cells by RT-PCR. Total RNA was extracted from Vero cells electroporated with full-length transcripts at 48 h post-electroporation. RT-PCR was used to amplify the regions corresponding to the 5′-terminal 415 bp and 1010 bp of subgenomic mRNA 4 and 3, respectively.
Fig. 2
Fig. 2
Analysis of the growth properties of the recombinant IBVs in Vero cells. Growth curves for the recombinant IBVs in Vero cells. In order to determine the growth curves for the recombinant IBVs and Beaudette, Vero cells were infected and harvested at 0, 4, 8, 16, 24, and 36 h post-infection. Viral stocks were prepared by freezing/thawing the cells three times and TCID50 was determined by infecting Vero cells in 96-well plates with 10-fold serial dilutions of each viral stock. The experiments were conducted based on three replicates. ANOVA followed Tukey’s multiple comparison tests were performed to compare the virus titers of different strains at the same time point, and differences were considered significant at P < 0.05.
Fig. 3
Fig. 3
Analysis of the growth properties of the recombinant IBVs in embryonated eggs. (a) Detection of the recombinant IBVs in embryonated eggs. A fragment of the N gene was amplified by RT-PCR to detect the replication of the recombinant IBVs, Beaudette, and ck/CH/LDL/091022 in embryonated eggs. (b) Replication kinetics of the recombinant IBVs in embryonated eggs. The rescued IBVs and the parental strains (100 × EID50) were inoculated into the allantoic cavities of three 9-day old embryonated eggs. The allantoic fluids from three eggs in each group were then harvested at 12, 24, 36, 48, and 60 h post-inoculation and pooled to determine the EID50 values in embryonated eggs. The experiments were conducted based on three replicates. ANOVA followed Tukey’s multiple comparison tests were performed to compare the virus titers of different strains at the same time point, and differences were considered significant at P < 0.05.
Fig. 4
Fig. 4
Adsorption and internalization assays. Vero cells were cultured in 96-well plates and infected with the recombinant IBVs and two parental strains (105 × EID50) for 1 h at 4 °C or 1 h at 37 °C. The experiments were conducted based on three replicates. (a) Infected cells were fixed and cELISA was performed as described in the Materials and Methods. Results are shown as viral load adsorption on cell equivalents relative to EID50 compared with that in the Beaudette strain (100%). (b) Vero cells were cultured in 24-well plates and infected with the recombinant IBVs and parental strains (106 × EID50) for 1 h at 4 °C. Total RNA was extracted from the infected cells before quantifying the viral loads using real-time RT-PCR. Data were normalized against the GAPDH expression levels and viral RNA was calculated relative to that in the Beaudette strain (100%). The experiments were conducted based on three replicates. (c) cELISA was performed to evaluate the viral load internalized into cells. (d) Real-time RT-PCR was also used to evaluate the viral load internalized into cells. The experiments were conducted based on three replicates. Differences were considered significant at P < 0.05 using ANOVA followed Tukey’s multiple comparison tests.
Fig. 5
Fig. 5
Pathogenicity test. (a) Mortality was recorded daily for 10 randomly selected infected birds and the survival curve was drawn. (b) At 5 days after challenge, we evaluated the challenged chickens blindly and clinical signs were scored as: 0 = absent, 1 = mild, 2 = moderate, or 3 = severe. Kruskal–Wallis ANOVA followed Dunn’s multiple comparison tests were employed to perform comparisons of the clinical scores using SPSS.
Fig. 6
Fig. 6
Trachea lesion scores. At 5 days after challenge, the other five infected birds in each group were killed humanely using carbon dioxide over inhalation. Tissue samples were collected from the trachea and analyzed by H&E staining. Mucosal thickness (a), deciliation (b), goblet cells (c) and lymphocyte scores (d) for the trachea samples were evaluated blindly and scored from 1 to 5 based on severity. Kruskal–Wallis and Dunn–Bonferroni tests were employed to perform post hoc comparisons of trachea lesion scores using SPSS.
Fig. 7
Fig. 7
Detection of IBV replication in challenged chickens. At 5 days after challenge, the other five infected birds in each group were killed humanely using carbon dioxide over inhalation, and tissue samples of the trachea, lungs, kidneys, and cecum tonsils were collected to determine the presence of IBV by real-time RT-PCR. A Ct value less than 32 was considered to be IBV-positive using real-time RT-PCR. The numbers of tissue samples positive for IBV RNA/the number detected are presented at the bottom. Bars in different colors represent different tissues from chickens, as indicated in the graphical representation. The average copy numbers of IBV RNA in the positive samples are shown. Differences were considered significant at P < 0.05 using ANOVA followed Tukey’s multiple comparison tests.
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