Establishment and Cross-Protection Efficacy of a Recombinant Avian Gammacoronavirus Infectious Bronchitis Virus Harboring a Chimeric S1 Subunit

Abstract

Infectious bronchitis virus (IBV) is a gammacoronavirus that causes a highly contagious disease in chickens and seriously endangers the poultry industry. A diversity of serotypes and genotypes of IBV have been identified worldwide, and the currently available vaccines do not cross-protect. In the present study, an efficient reverse genetics technology based on Beaudette-p65 has been used to construct a recombinant IBV, rIBV-Beaudette-KC(S1), by replacing the nucleotides 21,704-22,411 with the corresponding sequence from an isolate of QX-like genotype KC strain. Continuous passage of this recombinant virus in chicken embryos resulted in the accumulation of two point mutations (G21556C and C22077T) in the S1 region. Further studies showed that the T248S (G21556C) substitution may be essential for the adaptation of the recombinant virus to cell culture. Immunization of chicks with the recombinant IBV elicited strong antibody responses and showed high cross-protection against challenges with virulent M41 and a QX-like genotype IBV. This study reveals the potential of developing rIBV-Beau-KC(S1) as a cell-based vaccine with a broad protective immunity against two different genotypes of IBV.

Keywords: IBV; broad-spectrum protection; cell adaptability; growth characteristics; immunization; reverse genetics.

Figure 1

Construction and rescue of rIBV-Beau-KC(S1). (A) Diagram showing the genome organization of rIBV-Beau-KC(S1). The regions coding for the replicase polyproteins; the structural proteins S, E, M, and N; the accessory proteins 3a, 3b, 5a, and 5b; and the 5′- and 3′-UTR are shown. Also shown are the regions of the five RT-PCR fragments, the T7 promoter at the 5′-end of fragment A, the 30 As at the 3′ end of fragment E, and the replacement region of S1 at nucleotides 21,704–22,411. (B) The reverse genetics approach for the rescue of rIBV-Beau-KC(S1). (C) Assembly of the full-length cDNA clone and in vitro transcription of the full-length and IBV N transcripts. Equal amounts of the five purified fragments were ligated using T4 DNA ligase and analyzed on a 0.4% agarose gel. The in vitro assembled full-length and PCR-amplified IBV N DNA fragments were used as templates for generating the full-length and IBV N transcripts, which were analyzed on a 0.8% agarose gel. (D) RT-PCR identification of p4 and p5 using specific primers d-rIBV02F and d-rIBV02R. The total RNA was extracted from allantoic fluids of p4 and p5 and reverse transcribed into cDNA for RT-PCR identification. RT-PCR products were analyzed on a 0.8% agarose gel. (E) Western blot identification of p2-p5 using rabbit polyclonal antibody anti-IBV S and mouse monoclonal antibody anti-IBV N.

Figure 2

Effects of G21556C (S248T) point mutation in the S1 region on the cultivability of rIBV-Beau-KC(S1) in Vero cells. (A) Emergence and accumulation of G21556C mutation during the passage of rIBV-Beau-KC(S1) in chicken embryos. The regions covering the G21556C point mutation from p4, p6, p8, p10, and p10V9 were sequenced, showing double peaks of G and C in p6, p8, and p10. Mixed clones containing both G and C at the nucleotide position 21,556 were found in the 30 clones from p10, with the G/C ratio of 4:30, whereas the 32 clones from p4 contain only G and the 24 clones from p10V9 contain only C at the same position. (B) Immunofluorescent staining of Vero cells infected with rIBV-Beau-KC(S1). Vero cells were infected with rIBV-Beau-KC(S1)-p4V9 and rIBV-Beau-KC(S1)-p10V9 at MOI ~0.2, respectively. Vero cells were also mock-treated and infected with Beaudette-p65 as negative and positive controls. Cells were analyzed by IFA staining with an anti-IBV N monoclonal antibody and goat-mouse IgG-Fluor488 secondary antibody at 24 hpi. (C) RT-PCR analysis of Vero cells infected with rIBV-Beau-KC(S1). Vero cells were consecutively infected with rIBV-Beau-KC(S1)-p4 and rIBV-Beau-KC(S1)-p10, total RNAs were extracted, and the viral replication was checked by RT-PCR using specific primers d-rIBV02F. The PCR products were analyzed on a 0.8% agarose gel. (D) Western blot analysis of Vero cells infected with rIBV-Beau-KC(S1). Vero cells were infected with rIBV-Beau-KC(S1)-p10V1-V6, and harvested at 24 hpi. Total cell lysates were prepared and subjected to Western blot analysis with anti-IBV N mouse monoclonal antibody and goat-mouse IgG-HRP secondary antibody. (E) Formation of syncytia in Vero cells infected with rIBV-Beau-KC(S1). Continuous passages of rIBV-Beau-KC(S1)-p4 and p10 were carried out in Vero cells, and the formation of syncytium cells was observed in cells infected with rIBV-Beau-KC(S1)-p10V6 at 36 hpi.

Figure 3

Replication of rIBV-Beau-KC(S1) in Vero, H1299, HeLa, and DF1 cells. (A) IFA of Vero, H1299, HeLa, and DF1 cells infected with rIBV-Beau-KC(S1). Cells were infected with rIBV-Beau-KC(S1)-p10V9 and Beaudette-p65 at an MOI ~0.2 or ~0.05, respectively. IFA was performed with anti-IBV N monoclonal antibody and goat-mouse IgG-Fluor488 secondary antibody. (B) Determination of the maximum EID₅₀ and TCID₅₀ values of rIBV-Beau-KC(S1). Ten-fold serial dilutions of each viral stock were inoculated into 9-day-old SPF ECEs or Vero cells. For each dilution, 0.2 mL of virus suspension was injected into each egg or added to cells in 96-well plates. Five eggs or plates were used for each dilution. The EID₅₀ and TCID₅₀ values were calculated by the method of Reed and Muench. Two-way analysis of variance (ANOVA) was used to analyze significant differences between the indicated samples and the respective control samples. Significance levels are presented by the P-value (ns, non-significant). (C) Growth kinetics of rIBV-Beau-KC(S1) in Vero, H1299, HeLa, and DF1 cells. Cells were infected with rIBV-Beau-KC(S1)-p10V9 and Beaudette-p65 at an indicated MOI, harvested at indicated time post-infection, and total RNAs were extracted. An equal volume of total RNA was reverse transcribed, and the levels of IBV genomic RNA were determined by RT-qPCR.

Figure 4

Effects of G21556C (S248T) point mutation on the absorption, entry, and replication of rIBV-Beau-KC(S1) in Vero cells. For determining the efficiency of viral absorption, entry, and replication, Vero cells were infected with rIBV-Beau-KC(S1)-p4, rIBV-Beau-KC(S1)-p10V9, and Beaudette-p65 at an MOI ~ 0.5, respectively. Cells were then treated and harvested essentially as described in the text, and total RNA was extracted. The levels of IBV genomic RNA were determined by RT-qPCR. Two-way analysis of variance (ANOVA) was used to analyze significant differences between the indicated samples and the respective control samples. Significance levels are presented by the P-value (ns, non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 5

Humoral and cellular immune efficiencies in chickens immunized with rIBV-Beau-KC(S1). (A) Immunization and exsanguination protocol. Prime immunization with PBS, and 105EID50 rIBV-siMutBeau-p5- and 105EID50 rIBV-Beau-KC(S1)-p10-inactivated vaccines, respectively. Boost immunization with PBS, and 105 TCID50 rIBV-siMutBeau-p5V2- and 105 TCID50 rIBV-Beau-KC(S1)-p10V9-activated vaccines, respectively. Blood samples were collected weekly after immunization. (B) Vaccination-induced IBV-specific IgG in SPF chickens. One-day-old chicks were immunized with live or inactivated IBV vaccine with a booster 14 days later. Antibody response was monitored in the serum samples by commercial ELISA kit (IDEXX laboratories, USA) at weekly intervals till 84 dpi (n = 3 or 6/group). Serum neutralization indexes (NI) of rIBV-Beau-KC(S1)-immunized group against KC (QX-like genotype) and M41 were determined by alpha method with certain modifications. KC (10^6.47 EID₅₀/mL) and M41 (10^7.01 EID₅₀/mL) were 10-fold serially diluted, and the titers of each viral dilution were determined after mixing with a fixed dilution (1/5) of serum from the immunized chicks. NI represents the log10 difference between the titer of a viral dilution mixed with the immunized serum and the titer of the same viral dilution treated with the negative serum, and NI > 1.5 indicates a positive neutralization activity of the immunized serum. (C) Serum anti-IBV antibody titers were determined by ELISA (IDEXX laboratories, USA) at the indicated time points. The positive serum samples with 50-fold dilution were 2-fold serially diluted, and endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.2. (D,E) The levels of CD4 and CD8 molecules representing the activation of CD4+ and CD8+ cells in the serum samples were determined by ELISA (CUSABIO, China) at the indicated time points. Two-way analysis of variance (ANOVA) was used to analyze significant differences between the indicated samples and the respective control samples. Significance levels are presented by the P-value (ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.0001). (F) The levels of Bu-1a+, TCRγσ+, CD4+, and CD8+ in peripheral blood samples at 14 dpi were detected by flow cytometry. (G) Statistical analysis for flow cytometric results at 14 dpi and 28 dpi by GraphPad prism 9. Two-way analysis of variance (ANOVA) was used to analyze significant differences between the indicated samples and the respective control samples. Significance levels are presented by the P-value (ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.0001).

Figure 6

Cross-protection against CK/CH/JS/TAHY and M41. (A) Immunization and challenge protocol. One-day-old chicks (n = 15/group) were prime-immunized with a dose of 10⁵ EID₅₀ rIBV-Beau-KC(S1) live vaccine by nasal-ocular route, and were boost-immunized intramuscularly with a dose of 10⁵ TCID₅₀ rIBV-Beau-KC(S1)-inactivated vaccine at 14 days. The same doses of rIBV-siMutBeau and PBS were used as control, and CK/CH/JS/TAHY and M41 challenges were performed at 35 and 84 dpi, respectively. (B,C) At 7 days post-challenge with ~10^5.5 EID₅₀ of CK/CH/JS/TAHY, three chickens were randomly selected from each experimental group, an autopsy was done, and HE stained pathological tissue sections were observed. No obvious macroscopic lesions in the kidneys were observed. HE sections confirmed that the structure of the kidneys of all chickens in the rIBV-Beau-KC(S1)-immunized group was normal, except in one (33.33%) with slight congestion and hemorrhage. However, degeneration and necrosis of renal tubular epithelial cells, inflammatory cell proliferation, and infiltration were seen in kidneys (100%) obtained from all chickens in both control and rIBV-siMutBeau-immunized groups. (D) Within 5 days post-challenge with ~10^7.45 EID₅₀ of M41, the occurrence of tracheal rales in each experimental group was recorded and shown. Chicks in the rIBV-Beau-KC(S1)-immunized group had no clinical symptoms, but all chickens (100%) in the control group had obvious tracheal rales within 5 days post-challenge, and two chickens (22.22%) in the rIBV-siMutBeau-immunized group also had slight tracheal rales at 2 days post-challenge. (E) The necropsy results of the trachea of three randomly selected chickens at 7 days post-challenge with ~10^7.45 EID₅₀ of M41. Tracheas from all the three chicks (100%) in the control group had hemorrhage dots (red arrows) and two (66.67%) had obvious translucent mucus (blue arrows). One chick had hemorrhage dots (33.33%) in the rIBV-siMutBeau-immunized and rIBV-Beau-KC(S1)-immunized groups.