Abstract
The antigen variability of the infectious bronchitis virus (IBV) has hindered vaccine effectiveness and perpetuated its epidemic. We engineered a rapid attenuation method for IBV variants. The strategy involves creating the rH-CPDF7 backbone by recoding a segment of the H120 nonstructural protein (NSP) genome via codon pair deoptimization (CPD), facilitating S gene integration from IBV variants via transformation-associated recombination (TAR) cloning. These recombinant strains exhibited even lower pathogenicity, indicating the effectiveness of CPDF7 in reducing virulence. Importantly, the rH-CPDF7 backbone demonstrated versatility, being applicable to the development of attenuated strains for IBV variants, including the QX-type, TW-type, and GVI-type strains (different genotypes). In conclusion, our method allows for the rapid development of attenuated strains by integrating the S gene of IBV variants into the rH-CPDF7 backbone. These recombinant strains can elicit a strong immune response and provide effective protection against homologous challenges. This strategy is crucial for developing live-attenuated vaccines against emerging IBV strains.
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Introduction
Infectious bronchitis virus (IBV) is a significant avian pathogen that affects the poultry industry worldwide1,2. The mainstay of disease prevention and control has been vaccination2,3. The high mutation rate and genome recombination of IBV have spawned a variety of novel variant strains. However, there is little cross-protection between different genotypes or serotypes4. These variant strains often escape the immune protection offered by current vaccines, leading to a persistent and widespread disease epidemic5,6. The conventional method for producing live attenuated vaccines involves serial passaging of a virulent strain through embryonated chicken eggs7, a time-consuming and labor-intensive process that carries the risk of reverting to virulence6,7. Consequently, there is an urgent and essential need to innovate and develop new vaccines specifically aimed at combating variant strains of IBV.
IBV is a gamma coronavirus—an enveloped, single-stranded positive-sense RNA virus with a genome of approximately 27.6 kb8,9. The genome's 5' end is dominated by a large replicase gene, which encompasses two open reading frames (ORF1a and ORF1b)10. These ORFs encode two polyproteins (pp1a and pp1ab), which are subsequently cleaved by virus-encoded proteases into 15 nonstructural proteins (NSP2-16)11. These nonstructural proteins play pivotal roles in the processes of viral replication and transcription. The remainder of the genome at the 3' end encodes structural and accessory proteins, including the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, as well as the accessory 3a, 3b, 5a, and 5b proteins12,13. Among these, the S glycoprotein is recognized as the primary inducer of protective immunity in the host14,15. The S protein is proteolytically cleaved into two subunits (S1 and S2)15. The variable S1 subunit determines the virus serotype and is responsible for inducing neutralizing antibodies16,17. The S2 subunit has been implicated in mediating the ability of a virus to infect host cells, thereby influencing the tropism of the virus18,19.
The generation of full-length molecular clones of coronaviruses has long been hindered by the large size of their genomes and the instability of certain viral sequences when they are propagated in E. coli20. Yeast-based transformation-associated recombination (TAR) cloning is an innovative assembly method that can yield stable infectious full-length cDNAs of coronaviruses21. TAR cloning provides a more versatile and rapid workflow for manipulating virus genomes20,21,22,23. This platform is widely applicable for the assembly of full-length molecular clones of coronaviruses24. Consequently, the yeast-based TAR cloning reverse genetic platform provides a powerful tool for the rapid manipulation of IBV genomes at the molecular level. This technology has encouraged and empowered researchers to explore the genetic underpinnings of viral pathogenesis, develop novel vaccines, and devise targeted antiviral strategies against IBV and other coronaviruses. The manipulation of IBV genes, including replicase genes, spike genes, and accessory genes (3ab and 5ab), via reverse genetic systems has emerged as a promising approach for the strategic development of attenuated vaccines25,26,27,28. This strategy has been extensively employed to generate attenuated strains of coronaviruses. Notably, alterations in specific amino acid residues within NSP14, NSP15, and NSP16 have been linked to reduced viral pathogenicity. For example, the 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) within NSP1429,30, the endoribonuclease (EndoU) contained within NSP1531,32, and the conserved 2'O-methyltransferase (MTase, NSP16)33,34. Recent research has highlighted the significance of specific mutations in IBV NSPs. For example, the amino acid changes Pro85Leu in NSP10 and Val393Leu in NSP14 have been shown to result in a noticeable attenuation of IBV11. These findings suggest that targeted modifications of the NSP can provide an effective strategy for coronavirus attenuation, providing a solid foundation for the rational development of live attenuated vaccines.
Synthetic attenuated virus engineering (SAVE) is a virus attenuation strategy that enables rapid and highly efficient attenuation of a wide variety of viruses by codon pair deoptimization (CPD) for the recoding of viral genomes35,36,37,38. This approach increases the proportion of rare codon pairs in the viral genome by rearranging existing synonymous codons to deoptimize codon pairs without altering either the amino acid sequence38. Recoded viruses utilize underrepresented codon pairs, resulting in alterations in viral gene expression and attenuation of the virus37. Importantly, attenuation is not subject to reversion, as it results from hundreds or thousands of nucleotide changes37,38. The application of genome recoding through CPD has accelerated the preparation of attenuated viruses, offering a reliable pathway to develop live attenuated vaccines that are both safe and highly effective39,40. This method not only streamlines the vaccine development process but also enhances the safety profile of the resulting vaccines by minimizing the risk of viral reversion to a virulent form. Therefore, SAVE holds promise for shaping the future of vaccine design and development.
In this study, we successfully applied CPD to recode the NSP14, NSP15, and NSP16 genes of the H120 replicase, culminating in the creation of the rH-CPDF7 backbone. The rH-CPDF7 backbone serves as a versatile platform that can be effectively integrated with S genes from various IBV strains via the TAR cloning technique. This capability enables a rapid response to IBV mutant strains and newly emerging strains, facilitating the development of adaptive vaccines. Moreover, our findings demonstrate that ocular and intranasal immunization with a single dose of the rH-CPDF7 recombinant strain is sufficient to elicit a robust immune response in chickens. This immune response effectively confers protection against homologous challenges. This approach offers a promising pathway for the expedited development of live attenuated vaccines designed to combat the variant strains of IBV that are poised to advance vaccine technology.
Results
The methodology for generating IBV attenuated strains using a versatile backbone applicable to variants
The IBV variant presents a continuous challenge for disease prevention and control. We strategically employed attenuated vaccine strains as a robust backbone. By incorporating SAVE, we successfully mitigated the pathogenicity of these viral variants. Our comprehensive screening process identifies an attenuated backbone that is optimally suited for the variant strains of IBV. The attenuated backbone serves as a versatile platform capable of efficiently integrating S genes from various IBV strains through the TAR cloning technique. This targeted approach ensures the development of vaccines with enhanced safety and efficacy profiles tailored to address the unique challenges posed by the ever-evolving IBV landscape. A flowchart summarizing the selection and testing of the attenuated backbone is shown in Fig. 1.
This chart provides a streamlined visual summary of the steps involved in identifying and evaluating the attenuated backbone. For comprehensive insights, refer to the accompanying text. The CPB (purple) of a coding sequence is scored as the mean of each codon pair score. The codon pair score (CPS; blue) is determined by calculating the natural log of the ratio of the total number of times a given codon pair is observed in an organism's coding genome to the number of times the codon pair is expected to appear. CpG and TpA dinucleotides are highlighted in red and green, respectively.
The attenuation of the rH-QX(S) recombinant strain was incomplete
To manipulate the IBV genome rapidly and effectively, we developed an IBV reverse genetic system based on TAR cloning in yeast (Fig. 2A). The rH-QX(S) recombinant strain was rapidly rescued via TAR cloning (Fig. 2B), following a method described in the literature that can attenuate QX-type virulent strains41,42,43. The growth kinetics demonstrated that rH120 and rH-QX(S) replicated with comparable efficiency in chicken embryos (Fig. 2C).
A Schematic workflow of yeast-based TAR cloning for the IBV reverse genetics system. Step 1: PCR amplification of subgenome fragments from IBV; step 2: co-transformation of subgenome DNA fragments and a linearized pYES1L vector into yeast; step 3: screening of single colonies; step 4: sequencing analysis of the recombinant plasmid; and step 5: virus rescue. B Schematic representation of the IBV H120 genome structure and the construction process for the recombinant strains rH120 and rH-QX(S). C Growth kinetic analysis of the rH120 and rH-QX(S) recombinant strains, with viral RNA copies quantified by RT-qPCR. The data are presented as the means ± SDs (n = 3). Statistical significance was determined by Student's t test. D Survival rate of chickens infected with QX (wild-type), rH120, rH-QX(S), or PBS within 14 dpi. E Viral load assessment in the trachea, lungs, and kidneys of chickens from the QX (WT), rH120, and rH-QX(S) groups at 3, 6, 9, and 14 dpi. The dashed line indicates the PBS control group. The data are presented as means ± SDs (n = 2). F Viral shedding analysis of the throat and cloaca of chickens from the QX (WT), rH120, and rH-QX(S) groups at 6 and 12 dpi. The dashed line represents the PBS control group. The data are presented as means ± SDs (n = 10). One-way ANOVA was applied to assess the significance of differences in viral load and shedding among groups (E, F), where ns indicates not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (G) Gross lesion evaluation, (H) histopathological examination, and (I) IHC of trachea, lungs, and kidneys from chickens in the QX (WT), rH120, and rH-QX(S) groups.
The pathogenicity of the rH-QX(S) strain was subsequently assessed. We closely monitored the patients' clinical symptoms and recorded mortality rates from 1 to 14 d post-infection (dpi). The QX (WT) group presented with symptoms such as tracheal rales and sneezing (data not shown), resulting in a mortality rate of 80%. In contrast, the rH-QX(S) group presented milder symptoms and a mortality rate of 10% (Fig. 2D). RT-qPCR analysis of the viral load in tissues revealed that compared with the QX(WT) group, the rH-QX(S) group presented a significant reduction in the trachea at 14 dpi (P < 0.01) and in the kidneys at 3, 6, and 14 dpi (P < 0.05 or P < 0.01) (Fig. 2E). Furthermore, viral shedding in the throat and cloaca was significantly lower in the rH-QX(S) group than in the QX(WT) group at 6 and 12 dpi (P < 0.01 or P < 0.0001) (Fig. 2F). The rH120 group showed lower viral load and shedding than the QX(WT) and r-HQX(S) groups.
Upon necropsy, the QX(WT) group presented significant tissue damage, characterized by pronounced tracheal and lung hemorrhages, along with urate deposition in the kidneys. In stark contrast, the rH-QX(S) group demonstrated notable alleviation of the severity of these lesions, with only minor tracheal hemorrhages, localized lung congestion, and no discernible kidney lesions (Fig. 2G). The histopathology results revealed notable exfoliation of the ciliated epithelium in the trachea, hemorrhage and inflammatory cell infiltration in the lungs, and glomerular atrophy coupled with necrosis of the renal tubule epithelial cells in the QX(WT) group. In comparison, the rH-QX(S) group presented a reduction in histopathological damage across all tissues, with shedding of mucosal epithelial cells and infiltration of inflammatory cells in tracheal tissues, hemorrhage and inflammatory cell infiltration in the lungs, and no significant kidney lesions (Fig. 2H). However, in the rH120 and PBS groups, no significant lesions or histopathological changes were observed in the trachea, lungs, and kidneys (Fig. 2G, H). Immunohistochemistry revealed a notable decrease in IBV antigen-positive cells within the trachea and lung tissues of the rH-QX(S) group. In contrast, no IBV antigen was detected in the kidney tissues (Fig. 2I). These findings indicated that the rH-QX(S) recombinant strain presented reduced virulence.
In conclusion, although the rH-QX(S) recombinant strains have demonstrated a certain level of attenuation, the attenuation of the rH-QX(S) recombinant strain was incomplete, as evidenced by the observed chicken mortality, tracheal hemorrhage, ciliary loss, and IBV antigen-positive cells within the trachea and lung tissues of the rH-QX(S) group. However, this research underscores the potential of S gene swapping with attenuated strains as a precise and effective strategy for attenuating IBV.
The recoded subgenomic fragments of H120 exhibit lower codon pair bias (CPB)
The subgenomic fragments of the H120 strain were recoded to incorporate lower CPB, a strategy aimed at further attenuating the rH-QX(S) recombinant strain. To achieve this, the HF7 (including NSP14, NSP15, and NSP16) nucleotide sequence within the H120 genome was recoded using codon pairs that are strongly underrepresented in Gallus gallus. This approach was applied to the potential virulence regulatory genes NSP14, NSP15, NSP16, and HF7 in H120, resulting in the creation of CPD14, CPD15, CPD16, and CPDF7. A critical consideration in this process was the preservation of the transcription regulatory sequence (TRS) of the S gene, which is located at the 3' end of ORF1b in the H120 genome. To maintain the S gene's functionality downstream of HF7, we retained approximately 100 base pairs (bp) within the TRS region (specifically, the sequence from 20214 to 20313 bp, containing the CUGAACAA motif) during the coding process. Compared with the original parental sequences, the sequences of the recoded subgenomes presented a notable increase in the frequency of CpG and TpA dinucleotides coupled with a reduced CPB (Table 1). This re-coding is expected to contribute to a further reduction in the virulence of the rH-QX(S) strain.
Assessment of pathogenicity of the recombinant strains rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) in 1-day-old SPF chickens
To further diminish the virulence of the rH-QX(S) recombinant strains, this study employed the CPD strategy to recode specific segments of the H120 genome (Fig. 3A). The growth kinetics revealed that at 48 hours postinoculation (hpi), the number of RNA copies of each recombinant strain was significantly lower than that of the rH120 strain, with the following statistical significance: rH-CPD14-QX(S) (P < 0.001), rH-CPD15-QX(S) (P < 0.001), rH-CPD16-QX(S) (P < 0.01), and rH-CPDF7-QX(S) (P < 0.001). These findings suggested that the CPD encoded subgenomic segments influenced the replication efficiency of the recombinant strains (Fig. 3B).
A Schematic representation of the construction process for the recombinant strains rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S). B Growth kinetics analysis of the recombinant strains rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S). Viral RNA copies were quantified via RT-qPCR. The data are expressed as means ± SDs (n = 3). Statistical significance was determined via Student's t test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. C Survival rates of chickens infected with QX (WT), rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), rH-CPDF7-QX(S), and PBS within 14 dpi. D Viral load assessment in the trachea, lungs, and kidneys of chickens from the QX (WT), rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) groups at 3, 6, 9, and 14 dpi. The dashed line represents the PBS control group. The data are presented as means ± SDs (n = 2). E Viral shedding analysis of the throat and cloaca of chickens from the QX (WT), rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) groups at 6 and 12 dpi. The dashed line indicates the PBS control group. The data are shown as means ± SDs (n = 10). One-way ANOVA was used to evaluate the significance of differences in viral load and shedding among groups (D, E), where ns indicates not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. F Gross lesion assessment and (G) histopathological examination of trachea, lung, and kidney tissues from the QX (WT), rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) groups.
The pathogenicity of these recombinant strains was subsequently evaluated in 1-day-old SPF chickens. Clinical symptoms and mortality rates were closely monitored from 1 to 14 dpi. The QX (WT) group presented symptoms such as tracheal rales and sneezing, resulting in an 80% mortality rate. In contrast, no significant clinical symptoms or mortality were observed in the PBS, rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), or rH-CPDF7-QX(S) groups (Fig. 3C). Viral loads in tracheal, lung, and kidney tissues at 3, 6, 9, and 14 dpi, as well as viral shedding at 6 and 12 dpi, were assessed via RT-qPCR. Compared with those in the QX(WT) group, the viral loads in all tissues of the recombinant groups rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) were significantly lower at 3, 6, 9, and 14 dpi (P < 0.05 or P < 0.0001). However, the viral load in the lung tissue of the rH-CPD15-QX(S) group at 6 dpi and the rH-CPD14-QX(S) group at 9 dpi did not differ significantly from that of the QX(WT) group (Fig. 3D). Furthermore, viral shedding in the throat and cloaca was significantly lower in the recombinant groups than in the QX(WT) group at 6 and 12 dpi (P < 0.05 or P < 0.0001) (Fig. 3E). Among all the groups, the rH-CPDF7-QX(S) group presented the least amount of viral shedding in the throat and cloaca at 6 and 12 dpi (P < 0.0001), with no significant difference compared with the rH120 group (Fig. 3E).
Upon necropsy, the QX(WT) group presented with severe tissue damage, which included pronounced tracheal and lung hemorrhages, along with urate deposition in the kidneys. In stark contrast, the PBS, rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) groups presented no significant tissue lesions. Notably, the rH-CPD14-QX(S) group displayed minor punctate hemorrhages in the trachea (Fig. 3F). Histopathological examination further revealed the severity of the lesions in the QX(WT) group, which included the detachment of mucosal epithelial cells and the infiltration of inflammatory cells in the tracheal tissues, hemorrhage and inflammatory cell infiltration in the lungs, and glomerular atrophy along with necrosis in the epithelial cells of the renal tubules. Conversely, the rH120, PBS, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) groups presented no significant histopathological alterations in the trachea, lungs, or kidneys (Fig. 3G). Immunohistochemistry revealed that IBV antigen was absent in the trachea, lung, or kidney tissues of the PBS, rH120, rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S) groups (Supplementary Fig. 5A).
These results indicate that attenuated QX-type recombinant strains can be rapidly developed using the recoded genomes of H120 as a backbone while integrating the S gene from the QX strain. Among these recoded recombinant strains, the rH-CPDF7-QX(S) strain demonstrated the most significant attenuation, with both the viral load in tissues and viral shedding from the throat and cloaca being significantly reduced, indicating its potential as a promising candidate for further vaccine development.
The CPDF7 subgenome plays a critical role in virus attenuation
To evaluate the impact of the CPDF7 subgenome on virus attenuation, the QF7 fragments within the QX strains were replaced with HF7 from the H120 genome, or the CPDF7 subgenome fragments were recoded via TAR cloning, yielding the recombinant strains rQX, rQX-HF7, and rQX-CPDF7 (Fig. 4A). Growth kinetics indicated that the rQX strain achieved peak replication at 36 hpi. Notably, the rQX-CPDF7 and rQX-HF7 recombinant strains presented significantly fewer RNA copies at 36 hpi than the rQX strain did (P < 0.01), indicating that the insertion of the HF7 and CPDF7 gene fragments significantly influenced the growth characteristics of the rQX-HF7 and rQX-CPDF7 strains (Fig. 4B).
A Schematic representation of the construction process for the recombinant strains rQX, rQX-HF7, and rQX-CPDF7. B Growth kinetics analysis of the recombinant strains rQX, rQX-HF7, and rQX-CPDF7. Viral RNA copies were quantified via RT-qPCR. The data are presented as means ± SDs (n = 3). Statistical significance was determined via Student's t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C Survival rate analysis of chickens infected with rQX, rQX-HF7, or rQX-CPDF7 within 14 dpi. D Viral load assessment in the trachea, lungs, and kidneys of chickens from the rQX, rQX-HF7, and rQX-CPDF7 groups at 3, 6, 9, and 14 dpi. The dashed line indicates the PBS control group. The data are shown as means ± SDs (n = 2). E Viral shedding assessment in the throat and cloaca of chickens from the rQX, rQX-HF7, and rQX-CPDF7 groups at 6 and 12 dpi. The dashed line represents the PBS control group. The data are presented as means ± SDs (n = 10). One-way ANOVA was applied to evaluate the significance of differences in viral load and shedding among groups (D, E), with ns indicating not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. F Gross lesion evaluation, (G) histopathological examination, and (H) IHC of trachea, lung, and kidney tissues from the rQX, rQX-HF7, and rQX-CPDF7 groups.
The pathogenicity of these recombinant strains was subsequently evaluated in 1-day-old SPF chickens, and their clinical symptoms and mortality rates were closely monitored from 1 to 14 dpi. The rQX group displayed symptoms such as tracheal rales and sneezing, with a mortality rate reaching 70%. The rQX-HF7 group presented with signs of dyspnea and tracheal rales, with a mortality rate of 30%. In stark contrast, the rQX-CPDF7 strain induced only mild clinical symptoms, and no mortality was recorded (Fig. 4C). RT-qPCR analysis of the viral load in trachea, lung, and kidney tissues revealed that, compared with those in the rQX and rQX-HF7 groups, the viral load in the rQX-CPDF7 group was significantly lower at 3, 6, 9, and 14 dpi (P < 0.05 or P < 0.001) (Fig. 4D). Furthermore, the rQX-CPDF7 group presented significantly less viral shedding in the throat and cloaca at 6 and 12 dpi than the rQX group did (P < 0.001 or P < 0.0001) (Fig. 4E).
Upon necropsy, the rQX and rQX-HF7 groups presented with severe pathological changes, characterized by disseminated hemorrhage in the trachea, lung congestion and necrosis, and the kidneys displayed a "flower-spotted" appearance due to white urate deposits. In contrast, the rQX-CPDF7 group presented only minor tracheal hemorrhages, with no significant damage observed in the lungs or kidneys (Fig. 4F). Histopathological examination revealed that the rQX and rQX-HF7 groups presented severe tissue damage, including detachment of mucosal epithelial cells in the trachea, hemorrhage, inflammatory cell infiltration in the lungs, atrophy of the glomeruli, and necrosis of the epithelial cells of the renal tubules. The rQX-CPDF7 group, however, presented a marked reduction in histopathological lesions across all the tissues examined, with only minor tracheal epithelial cell shedding, slight lung hemorrhage, and no significant kidney damage (Fig. 4G). Compared with the rQX and rQX-HF7 groups, the rQX-CPDF7 group presented a significant reduction in IBV antigen-positive cells within the trachea and lung tissues, whereas IBV antigen was absent in the kidney tissues (Fig. 4H).
Collectively, these findings suggest that the rQX-CPDF7 group demonstrated reduced pathogenicity and that the CPDF7 subgenome exerted a potent attenuating effect compared with the QF7 and HF7 subgenomes. Notably, the CPDF7 subgenome undergoes many nucleotide alterations, all of which do not result in any amino acid changes. This extensive genetic modification significantly reduces the likelihood of the rQX-CPDF7 strain reverting to a wild-type phenotype, thereby effectively minimizing the risk of reversion to virulence. Furthermore, these results suggest that NSP14, NSP15, and NSP16 are promising candidates for the attenuation of IBV.
The rH-CPDF7 backbone is applicable to the rapid attenuation of IBV variants
To explore the applicability of the rH-CPDF7 backbone in attenuating IBV, the spike (S) genes from both TW-type and GVI-type IBV strains were successfully integrated into the rH-CPDF7 backbone via TAR cloning (Fig. 5A). The growth kinetics indicated that the RNA copy numbers of the engineered rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) strains were notably lower at 36 hpi than those of the rH120 strain, with statistical significance at P < 0.05 and P < 0.01, respectively (Fig. 5B).
A Schematic detailing the assembly process for the recombinant strains rH-CPDF7-TW(S) and rH-CPDF7-GVI(S). B Growth kinetics analysis of the recombinant strains rH-CPDF7-TW(S) and rH-CPDF7-GVI(S). Viral RNA copies were quantified via RT-qPCR. The data are presented as means ± SDs (n = 3). Statistical significance was assessed via Student's t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C Survival rate analysis of chickens infected with rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) within 14 dpi. D Viral load measurements in the trachea, lungs, and kidneys of chickens from the rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) groups at 3, 6, 9, and 14 dpi. The dashed line represents the PBS control group. The data are shown as means ± SDs (n = 2). E Viral shedding assessment in the throat and cloaca of chickens from the rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) groups at 6 and 12 dpi. The dashed line indicates the PBS control group. Data are presented as means ± SDs (n = 10). One-way ANOVA was used to evaluate the significance of differences in viral load and shedding among groups (D, E), with ns indicating not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. F Gross lesion evaluation and (G) histopathological examination of trachea, lung, and kidney tissues from the rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) groups.
The pathogenic potential of the rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) strains was subsequently investigated in 1-day-old specific-pathogen-free (SPF) chickens. Clinical signs and mortality rates were meticulously tracked from 1 to 14 dpi. The TW(WT) group presented with clinical manifestations such as tracheal rales and sneezing, resulting in a 30% mortality rate. The GVI(WT) group presented with signs of dyspnea and tracheal rales, yet without any mortality. In stark contrast, neither clinical symptoms nor mortality was detected in the PBS, rH120, rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) groups (Fig. 5C). The viral loads in the trachea, lung, and kidney tissues, as well as viral shedding at 6 and 12 dpi, were quantitatively analyzed via RT-qPCR. Compared with the TW(WT) group, the rH-CPDF7-TW(S) group presented a marked reduction in the viral load within the trachea at 6 dpi and 9 dpi (P < 0.05) and in the kidneys at 3 dpi (P < 0.05) (Fig. 5D). However, the rH-CPDF7-GVI(S) group did not show a significant difference in viral load within tissues compared with the GVI(WT) group (Fig. 5D). Moreover, the rH-CPDF7-TW(S) group experienced a substantial and highly significant decrease in viral shedding from the throat and cloaca at both 6 dpi and 12 dpi (P < 0.001 or P < 0.0001) relative to the TW(WT) group (Fig. 5E). In alignment with these findings, the rH-CPDF7-GVI(S) group also exhibited notable reductions in viral shedding in the throat and cloaca at 6 and 12 dpi (P < 0.05 or P < 0.0001) compared with the GVI(WT) group (Fig. 5E).
An autopsy revealed that the TW(WT) group presented severe tissue damage, characterized by extensive tracheal hemorrhage and lung congestion, along with a distinctive "flower-spotted kidney" appearance caused by white urate deposits in the kidneys. The GVI(WT) group presented with tracheal hemorrhage and mucous discharge, yet no significant pathological changes were noted in the lungs or kidneys. Notably, the PBS, rH120, rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) groups presented no notable lesions in the trachea, lungs, or kidneys (Fig. 5F). Histopathological examination further revealed significant damage to the trachea, lungs, and kidneys of the QX(WT) group, including the shedding of tracheal mucosal epithelial cells, pulmonary hemorrhage, and renal damage characterized by glomerular atrophy and necrosis of the renal tubular epithelium. The GVI(WT) group also presented tracheal lesions with similar characteristics, but no significant kidney pathology was observed. Conversely, the PBS, rH120, rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) groups were free from significant pathological changes in the trachea, lungs, and kidneys (Fig. 5G).
In conclusion, compared with the homologous wild-type strains, the rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) groups presented significant reductions in mortality, tissue viral load, viral shedding, and tissue lesions. These results indicate that the rH-CPDF7 backbone is applicable to the rapid attenuation of IBV variants, offering a promising approach for live-attenuated vaccines against IBV variants.
Assessment of the immune response and protective effect of vaccine candidate strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S)
Finally, to thoroughly analyze the immune response and protective effects of vaccine candidate strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S), a cohort of 1-day-old SPF chickens was immunized (Fig. 6A). The IBV-specific serum antibody titers of the vaccinated groups increased progressively over the 21 d observation period. Compared with those in the rH120-vaccinated group, the serum antibody levels in the rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) groups did not significantly differ at 7, 14, and 21 d postvaccination (dpv) (Fig. 6B). Additionally, at 21 dpv, the levels of cytokines, including IL-2, IL-4, IL-6, and IFN-γ, in the rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) groups were comparable to those in the rH120-vaccinated group, yet both sets of vaccinated groups presented significantly higher cytokine levels than did the PBS group (P < 0.0001) (Fig. 6C). Importantly, the rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) recombinant strains induced higher levels of neutralizing antibodies than did the rH120 group (P < 0.001 or P < 0.0001) (Fig. 6D).
A Schematic representation of the immunization protocol for vaccine candidate strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S). B Serum-specific antibody titers of the rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) vaccinated groups were assessed via commercial ELISA at 7, 14, and 21 dpv. The dashed line indicates the S/P value of the lowest positive sample (0.2). C Detection of the cytokines IL-2, IL-4, IL-6, and IFN-γ at 21 dpv. D Determination of virus neutralization titers at 21 dpv by serum titration in chicken embryos. The virus neutralization titers are expressed as the log2 of the reciprocal of the highest serum dilution that inhibited 50% of the chicken embryo lesions. The data are presented as means ± SDs (n = 5). E Survival rate analysis of chickens following homologous challenge with the QX, TW, and GVI strains at 21 dpv. F Viral load measurements in the trachea, lungs, and kidneys of chickens from the vaccinated groups at 3, 6, 9, and 14 dpc. The horizontal dashed line represents the PBS group. The data are presented as means ± SDs (n = 2). G Viral shedding assessment in the throat and cloaca of chickens from the vaccinated groups at 6 and 12 dpc. The horizontal dashed lines indicate the PBS group. The data are presented as means ± SDs (n = 10). One-way ANOVA was used to assess the significance of differences in viral load and shedding among the groups (B–D and F–G). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (H) Gross lesion evaluation and (I) histopathological examination of trachea, lung, and kidney tissues from the vaccinated groups.
The vaccinated groups were challenged with 105 EID50 of the homologous virulent strains QX(WT), TW(WT), and GVI(WT) via the ocular and intranasal routes at 21 dpv (recorded as 0 days post-challenge, dpc). Clinical signs were closely monitored, and mortality rates were recorded daily from 1 to 14 dpc (Fig. 6A). Viral loads in the trachea, lung, and kidney tissues were measured at 3, 6, 9, and 14 dpc, and viral shedding was assessed at 6 and 12 dpc via RT-qPCR. The PBS/QX group displayed mild tracheal rales and sneezing, with a 20% mortality rate, whereas the rH120/QX group presented similar symptoms but a lower mortality rate of 10%. The PBS/TW, rH120/TW, PBS/GVI, and rH120/GVI groups presented mild tracheal rales and sneezing, but no mortality. In contrast, no clinical signs or mortality were observed in the rH-CPDF7-QX(S)/QX, rH-CPDF7-TW(S)/TW, rH-CPDF7-GVI(S)/GVI, and negative control (NC, PBS group not challenged) groups (Fig. 6E). Comparative analysis revealed that the rH-CPDF7-QX(S)/QX group had a significantly lower viral load in the trachea and kidneys than the PBS/QX and rH120/QX groups did (P < 0.01 or P < 0.0001) (Fig. 6F). However, no significant differences in viral load were noted among the PBS/TW, rH120/TW, and rH-CPDF7-TW(S)/TW groups or the PBS/GVI, rH120/GVI, and rH-CPDF7-GVI(S)/GVI groups (Fig. 6F). The lung tissue had a lower viral load (data not shown). Compared with those in the PBS/QX and rH120/QX groups, viral shedding in the throat and cloaca of the rH-CPDF7-QX(S)/QX group was significantly reduced (P < 0.01 or 0.0001). While there was no significant difference in the tissue viral load among the PBS/TW, rH120/TW, and rH-CPDF7-TW(S)/TW groups, the rH-CPDF7-TW(S)/TW group presented a significant reduction in viral shedding in the throat and cloaca at 6 and 12 dpc compared with the PBS/TW and rH120/TW groups (P < 0.05 or 0.0001) (Fig. 6G). Similarly, no significant differences in tissue viral load were detected among the PBS/GVI, rH120/GVI, and rH-CPDF7-GVI(S)/GVI groups, but the rH-CPDF7-GVI(S)/GVI group exhibited a significant reduction in viral shedding in the throat and cloaca at 6 dpc and 12 dpc compared with the PBS/GVI and rH120/GVI groups (P < 0.001 or P < 0.0001) (Fig. 6G).
The autopsy findings indicated that the PBS/QX and rH120/QX groups suffered from severe tissue damage, characterized by widespread tracheal hemorrhage, lung congestion, and renal swelling with urate deposition. In the PBS/TW and rH120/TW groups, there was evidence of tracheal larynx hemorrhage accompanied by mucous exudation, whereas the lungs and kidneys showed no significant pathological changes. Similarly, the PBS/GVI and rH120/GVI groups presented tracheal punctate hemorrhages with mucous exudation but no apparent lesions in the lungs or kidneys. In contrast, the rH-CPDF7-QX(S)/QX, rH-CPDF7-TW(S)/TW, rH-CPDF7-GVI(S)/GVI, and negative control (NC) groups presented no significant lesions in the trachea, lungs, or kidneys (Fig. 6H). The histopathological examination further revealed severe tracheal lesions in the PBS/QX and rH120/QX groups, including the loss of tracheal cilia, thickening of mucosal epithelial cells, and inflammatory cell infiltration.
Additionally, there was pulmonary hemorrhage and inflammatory cell infiltration, along with renal damage marked by glomerular atrophy and necrosis of the renal tubular epithelium. In the PBS/TW and rH120/TW groups, similar tracheal lesions were observed, as were pulmonary hemorrhage and inflammation. The PBS/TW group also presented renal damage with glomerular atrophy and necrosis, whereas the kidneys of the rH120/TW group appeared unaffected. In the PBS/GVI and rH120/GVI groups, tracheal cilia shedding, mucosal epithelial cell thickening, and inflammatory cell infiltration were noted, along with mild pulmonary hemorrhage and inflammation, but no significant renal lesions were detected. In stark contrast, the rH-CPDF7-QX(S)/QX, rH-CPDF7-TW(S)/TW, rH-CPDF7-GVI(S)/GVI, and NC groups were free from significant pathological changes in the trachea, lungs, and kidneys (Fig. 6I).
In conclusion, the recombinant strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) effectively triggered robust humoral and cellular immune responses in chickens. Notably, these strains produced higher levels of neutralizing antibodies against homologous virulent strains than did the rH120 strain. Vaccination with the rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) strains effectively mitigated clinical symptoms, reduced mortality, and decreased viral loads and shedding in chickens, thereby protecting against homologous strains. In summary, vaccine candidate strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) elicited robust immune responses and protective effects, suggesting their potential as effective vaccines against IBV variants.
Discussion
The extensive deployment of the IB vaccine has successfully mitigated the impact of IBV infection and significantly reduced the mortality rate in chickens3. As IBV continues to evolve, it presents a diverse array of genotypes or serotypes, which poses a challenge for the development of effective control strategies44,45. Live-attenuated vaccines are preferred because of their ease of administration and superior immune efficacy11,46. However, the traditional method of producing these vaccines involves serial passaging of a virulent field isolate in chicken embryos, often necessitating more than 80 passages47. Therefore, there is an urgent need to investigate innovative attenuation techniques to expedite the development of live attenuated IBV vaccines. Recent studies have demonstrated that integrating the S or S1 gene from M41, 4/91, or QX virulent strains into the corresponding position of IBV Beaudette strains results in attenuated recombinant strains BeauR-M41(S), BeauR-4/91(S), or rIBV-Beau-KC(S1)48,49. Additionally, by replacing the S or S1 gene segment of the attenuated vaccine strain H120 with that from the QX virulent strain, recombinant strains such as rH021202/SCS1, rH120-S1/YZ, and rH120-QX(S) have been successfully generated41,42,43. These recombinant strains have been demonstrated to elicit a robust immune response against homologous challenges. Collectively, these findings underscore the potential of S gene swapping with attenuated strains as a precise and efficacious method for the attenuation of IBV, offering a promising pathway for the development of next-generation live attenuated vaccines.
Yeast-based TAR cloning is a powerful method for generating stable, infectious full-length cDNA clones of coronaviruses21. In this study, TAR cloning was employed to explore the rapid development strategy of live attenuated IBV vaccines applicable to variants. Initially, we utilized H120 as the backbone and swiftly substituted the S gene of H120 with the corresponding sequence from the QX-type virulent strain, resulting in the creation of the rH-QX(S) recombinant strain. Although the rH-QX(S) strain exhibited significantly reduced pathogenicity in chicks, we still observed chicken mortality and minor tissue lesions. Previous studies have indicated that the NSP region is a potential target for attenuating coronaviruses11,29,30,31,32,33,34. To further attenuate the rH-QX(S) strain while preserving the integrity of the S gene, we engineered the rH-CPDF7-QX(S) recombinant strain by recoding the subgenome HF7 (encompassing NSP14, NSP15, and NSP16) of the H120 strain via the CPD method. Compared with their parental strains, recovered viruses are less efficient at producing proteins from the recoded genes40. Consequently, this inefficiency in NSP may contribute to the attenuation of the virus, offering a promising strategy for developing safer and more effective live attenuated IBV vaccines.
On the basis of the large-scale recoding of the viral genome through CPD, there is a notable increase in the frequency of CpG or UpA dinucleotides50,51. The elevated CpG content within the viral genome increases the vulnerability of the virus to the antiviral activity of the zinc-finger antiviral protein (ZAP) present in host cells52. Additionally, the coding process may disrupt unidentified functional RNA elements within the coding sequences of RNA viruses, thereby hindering their replication capabilities53. Thus, CPD has emerged as a rapid and effective technique for developing attenuated vaccine candidates. Importantly, the viral genome undergoes hundreds or thousands of nucleotide changes through CPD (without amino acid changes), making it difficult for the recombinant strain to revert to the wild-type phenotype and significantly reducing the risk of reversion to a more virulent form40. We conducted a thorough analysis of the genomes of the passaged viruses, which were subjected to ten rounds of replication. Using RT-PCR and Sanger sequencing techniques, we did not detect any mutations in the recoded regions (results not shown). In coronaviruses, sequence similarity and secondary structure play a role in directing recombination54. However, the introduction of recoding sequences can disrupt consensus sequences and potentially eliminate unknown functional RNA elements, such as those involved in secondary structure formation. This disruption may be beneficial in mitigating the risk of viral recombination, further enhancing the safety profile of the attenuated vaccine candidates developed through this approach. The impact of CPD-induced recoding on the recombination potential of IBV warrants further investigation.
In this study, we developed a series of recombinant strains—rH-CPD14-QX(S), rH-CPD15-QX(S), rH-CPD16-QX(S), and rH-CPDF7-QX(S)—engineered using the recoded H120 genome as a backbone and integrating the S gene from the QX-type strain. Among these recombinant strains, rH-CPDF7-QX(S) demonstrated the most pronounced attenuation, characterized by the lowest levels of tissue viral load, viral shedding, and tissue lesions. This attenuated effect seems to be the cumulative result of CPD14, CPD15, and CPD16, potentially linked to an increase in the number of CpG or UpA dinucleotides, although definitive evidence supporting this correlation is currently lacking. Our findings indicate that live attenuated IBV strains can be efficiently developed by combining the genome recoding of the H120 strain with the S gene from the QX strain. To further assess the attenuating effect of the CPDF7 subgenome, we constructed additional recombinant strains, namely, rQX, rQX-HF7, and rQX-CPDF7. While both the rQX-infected (with a 70% mortality rate) and the rQX-HF7-infected (with a 30% mortality rate) groups experienced significant mortality and tissue damage, no mortality was observed in the rQX-CPDF7 infected group, although some minor tracheal tissue damage was noted. Moreover, compared with the rQX and rQX-HF7 groups, the rQX-CPDF7 group presented a marked reduction in tissue viral load and shedding. These observations suggest that the CPDF7 subgenome plays a critical role in viral attenuation. Furthermore, these findings also highlight NSP14, NSP15, and NSP16 as potential targets for the development of attenuated IBV strains, offering valuable insights for the design of future live-attenuated vaccines.
The virulence of IBV was effectively reduced through the recoding of the NSP sequence (NSP14, NSP15, and NSP16) via CPD. This approach stands out as a promising strategy for attenuating viruses. However, the factors contributing to IBV virulence remain largely unclear, with only a limited subset being explored in this study. The potential benefits of recoding the entire IBV genome to achieve further attenuation are still unclear. Additionally, it is crucial to consider that the attenuation strategy employing CPD could have implications for the replication capability of the virus40. A delicate balance must be achieved between the desired attenuation and the maintenance of sufficient replication ability to ensure that the virus remains immunogenic yet safe for vaccine development. Therefore, the attenuation strategy for IBV must be carefully calibrated to optimize both the reduced pathogenicity and the necessary replication capacity of the virus, leading to the most effective vaccine candidates.
The S genes from the TW-type and GVI-type strains of IBV were subsequently integrated into the rH-CPDF7 backbone via TAR cloning, yielding the recombinant strains rH-CPDF7-TW(S) and rH-CPDF7-GVI(S), respectively (in addition to the QX-type strain, the TW-type and GVI-type genotype strains are also prevalent in China). Compared with homologous wild-type strains, both rH-CPDF7-TW(S) and rH-CPDF7-GVI(S) presented significant decreases in mortality, tissue viral load, viral shedding, and tissue lesions. These results suggest that the rH-CPDF7 backbone is a versatile platform for the rapid attenuation of various IBV variants. Furthermore, the recombinant strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) were able to successfully elicit effective immune responses and provide robust immune protection against homologous challenges. These findings indicate that the rH-CPDF7-based recombinant strains not only attenuate the virus but also retain the ability to stimulate a protective immune response, making them promising candidates for the development of live-attenuated vaccines against diverse IBV strains.
In this study, we successfully established an rH-CPDF7 backbone by applying CPD to the NSP14, NSP15, and NSP16 components of the H120 strain replicase gene. The rH-CPDF7 backbone serves as a robust foundation for the rapid development of attenuated recombinant strains of various IBV variants. Attenuated recombinant strains were rapidly generated by integrating the S genes from these IBV variants into the rH-CPDF7 backbone via TAR cloning. Furthermore, these recombinant strains were able to elicit effective immune responses successfully and provide robust immune protection against homologous challenges. This approach presents a rapid attenuation strategy for IBV variant strains, facilitating the development of live attenuated vaccines against IBV variants.
Materials and methods
Ethical approval
All of the animal experiments were conducted in accordance with the regulations of the administration of affairs concerning experimental animals and approved by the Nanjing Agricultural University Experimental Animal Welfare Ethics Committee with the approval ID: NJAU.No20230313021 and NJAU.No20231229198. All the chickens involved in this study were euthanized via CO2.
Viruses and cells
The H120 vaccine strain (GenBank No. FJ888351) of IBV was supplied by Lihua Nanjing Industrial Research Institute Co., Ltd. The QX-type (GI-19, CK/CH/JS/CZ211063, GenBank No: PP438800), TW-type (CK/CH/JS/CZ210443, GenBank No: PQ273739), and GVI-type (CK/CH/JS/CZ210673, GenBank No: PQ273740) strains were isolated from intensive chicken farms and subsequently stored in laboratory facilities. Baby Hamster Kidney (BHK-21) cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, CA, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS; Gibco) at 37°C in a CO2 incubator. The GeneArt® High-Order Genetic Assembly System was facilitated by Invitrogen, USA (A13286), which comprises MaV203 competent yeast cells, the pYES1L vector, and one Shot® TOP10 Electrocomp™ E. coli. 9-day-old specific pathogen-free (SPF) chicken embryos were supplied by Jinan SAIS Poultry Co. Ltd., China.
Viral RNA extraction, reverse transcription, and primer design
Viral RNA was extracted via an RNA isolation Total RNA Extraction Reagent Kit (R401-01, Vazyme, China). Subsequently, complementary DNA (cDNA) synthesis was performed through reverse transcription with the HiScript II 1st Strand cDNA Synthesis Kit (R211-01, Vazyme, China), following the manufacturer's instructions. The 5' and 3' end sequences of the virus were determined via a SMARTer RACE 5'/3' Kit (634858, TaKaRa, China)55. BioEdit software was used to compare and analyze the sequences on the basis of the complete gene sequences of H120, CK/CH/JS/CZ211063 (QX-type), CK/CH/JS/CZ210443 (TW-type), and CK/CH/JS/CZ210673 (GVI-type)56. Oligo7 was employed to design the fragment primers, with the corresponding primers listed in Supplementary Tables 1 and 2.
Recoding of H120 subgenomic fragments
Given that the attenuation of the rH-QX(S) strains remains incomplete, we focused on the NSP-encoding genes, which have been identified in prior research as promising targets for attenuating coronaviruses11,29,30,31,32,33,34. To further diminish the virulence of the rH-QX(S) strain while preserving the integrity of the S gene, the HF7 nucleotide sequence (including NSP14, NSP15, and NSP16) from the H120 genome underwent CPD to yield CPD14 (Supplementary Note 1: fragment 1), CPD15 (Supplementary Note 2: fragment 2), CPD16 (Supplementary Note 3: fragment 3), and CPDF7 (CPD14+CPD15+CPD16), a sophisticated method that restructures the viral sequences to attenuate their virulence. CPD meticulously rearranges synonymous codons within these sequences, introducing codon-pair combinations that are less frequently observed in the protein-coding genes of the virus's natural host, Gallus gallus37,40. In line with our objective of developing IBV mutants with reduced pathogenicity for chickens, we tailored the recoding process to align with the CPB specific to Gallus gallus57. By applying this species-specific CPB, we ensured that the recoded IBV sequences would be less optimized for replication in the chicken host, potentially leading to the generation of attenuated mutants. As the TRS of the S gene is located at the 3' end of ORF1b of the H120 genome, approximately 100 bp nucleotide sequences (20214-20313 bp) in the TRS (CUGAACAA) region of the S gene are preserved during the recoding process to ensure that the S gene downstream of HF7 can function properly. The features of the recoded sequence are shown in Table 1.
One-step assembly of IBV infectious cDNA clones via TAR cloning in yeast
DNA fragments from the H120 strain (HF1, HF2, HF3, HF4, HF5, HF6, HF7, HF8, HF9, and HF10) or the QX strain (QF1, QF2, QF3, QF4, QF5, QF6, QF7, QF8, QF9, and QF10) were obtained via PCR amplification, each sharing a 40 bp homologous sequence with its adjacent fragment. This process utilized H120 (or QX) cDNA as the template and relied on the Phanta Max Super-Fidelity DNA Polymerase (P505-d1, Vazyme, China) along with the primers listed in Supplementary Table 1 (or Supplementary Table 2). The human cytomegalovirus (CMV) promoter sequence was added to the 5' end of HF1 (or QF1), while the hepatitis D virus ribozyme sequence (HDVRz) and the bovine growth hormone (BGH) polyadenylation signal sequence (collectively referred to as HB) were added to the 3' end of HF10 (or QF10) by overlap-PCR. TAR cloning was performed following the protocol provided by the GeneArt® High-Order Genetic Assembly System (A13286, Invitrogen, USA)23. In brief, 10 DNA fragments, including CMVHF1, HF2, HF3, HF4, HF5, HF6, HF7, HF8, HF9, and HF10HB (or CMVQF1, QF2, QF3, QF4, QF5, QF6, QF7, QF8, QF9, and QF10HB), each at 200 ng, and 100 ng of the linear plasmid pYES1L were transfected into MaV203 competent cells. This meticulous process yielded the positive plasmids pYES1L-rH120 and pYES1L-rQX after a rigorous screening phase. Further refinements were made to obtain specific recombinant plasmids: pYES1L-rH-QX(S) was constructed by substituting HF8S with the QX(S) DNA fragment through TAR cloning. pYES1L-rH-CPD14-QX(S), pYES1L-rH-CPD15-QX(S), pYES1L-rH-CPD16-QX(S), and pYES1L-rH-CPDF7-QX(S) were generated by replacing HF8S with QX(S) and introducing the CPD-modified genes CPD14+NSP15+NSP16, NSP14+CPD15+NSP16, NSP14+NSP15+CPD16, or CPDF7 in place of the HF7 DNA fragment, respectively, via TAR cloning. For the generation of pYES1L-rQX-CPDF7 and pYES1L-rQX-HF7, QF7 was swapped with CPDF7 or HF7 via TAR cloning. Then, the S1 gene sequences of QX-type, TW-type, and GVI-type were aligned using BioEdit software. An evolutionary tree was constructed using MEGA 7.0 with the neighbor-joining method and bootstrap values based on 1000 replicates to assess similarities among the fragments55. Similarly, pYES1L-rH-CPDF7-TW(S) and pYES1L-rH-CPDF7-GVI(S) were created by replacing HF7 with CPDF7 and substituting HF8S with TW(S) or GVI(S), respectively, through TAR cloning.
Rescue of the recombinant IBV strain
To increase the efficiency of virus rescue, helper plasmids pcDNA3.1-H120-N or pcDNA3.1-QX-N were produced to encode the N protein by integrating the H120-N or QX-N genes under the control of the human cytomegalovirus (CMV) promoter within the pcDNA3.1(+) vector58. Then, 5 μg of recombinant plasmid together with 2 μg of the corresponding helper plasmid was co-transfected into BHK-21 cells via Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, USA) to rescue the recombinant IBV strains. After 48 h of transfection, the cell suspensions were collected and centrifuged at 5000 rpm for 5 min, and 500 μL of the supernatant was inoculated into 9-day-old SPF chicken embryos to amplify the rescued virus. After 2 d of incubation, allantoic fluid was harvested, aliquoted and stored at −80°C for later use. The rescue of the recombinant strains was confirmed by WB analysis and a chicken embryo dwarfing assay43. The recombinant IBV strains were sequenced via Sanger methods and visualized with MegAlign (DNAstar 7.1) software.
Viral growth kinetics analysis
The recombinant strains were inoculated into 9-day-old SPF embryos. After 6 d, symptoms of the embryonic lesions, such as stunting and embryo dwarfing, were observed, and the 50% embryo infectious dose (EID50) was calculated via the formula of Reed and Muench59,60. To evaluate the replication ability of the recombinant strains in vitro, 100 EID50 strains of the IBV recombinant strains were inoculated into 9-day-old SPF embryos. Allantoic fluid (500 μL) was collected from each embryo at 12, 24, 36, and 48 hpi, followed by RNA extraction and real-time fluorescent quantitative PCR (RT-qPCR), as described previously61 with minor modifications. Briefly, the following RT-qPCR primers were designed on the basis of the genome sequence of the isolates: IBV F391 (5'-GCTTTTGAGCCTAGCGTT-3') and IBV R533 (5'-GCCATGTTGTCACTGTCTATTG-3'). The standard curve equation was established as y = -3.315x + 39.704 (R2=0.9991). All experiments were performed in triplicate, and the number of RNA copies of each virus was calculated on the basis of the standard curve.
Pathogenicity analysis of the recombinant strains in 1-day-old SPF chickens
1-day-old SPF chickens were randomly assigned to groups, each containing 20 chickens, and were housed in isolated units equipped with food and water. The chickens in each group were infected with 105 EID50 of the IBV recombinant strains via the ocular and intranasal routes, respectively43, with the infection day being marked as 0 dpi. The negative control group was administered an equivalent volume of phosphate-buffered saline (PBS). Clinical symptoms and mortality were monitored and recorded daily for 14 d. At 3, 6, 9, and 14 dpi, two chickens from each group were selected at random for humane euthanasia and subsequent necropsy. All chickens that died during the study were also subjected to autopsy on the day of death. During necropsy, gross lesions were carefully examined, and tissue samples from the trachea, lungs, and kidneys were harvested for assessment of the viral load. Additionally, throat and cloacal swabs were collected at 6 and 12 dpi to evaluate viral shedding within each group.
Determination of tissue viral load and viral shedding
To quantify viral RNA copies in tissues and assess viral shedding, total RNA was extracted from the collected tissue and swab samples via Total RNA Extraction Reagent. For the synthesis of cDNA, 1 µg of total RNA from each sample was reverse transcribed via a random primer and the HiScript II 1st Strand cDNA Synthesis Kit. RT-qPCR was then conducted via the AceQ qPCR SYBR Green Master Mix to quantify the tissue viral load and degree of viral shedding, as described previously55. Each experimental replicate was performed in triplicate, and the number of RNA copies was calculated on the basis of the standard curve.
Histopathological examination
Tissue samples from the trachea, lungs, and kidneys were collected and fixed in 10% neutral formalin for 48 h at room temperature. The fixed samples were subjected to standard processing, embedded in paraffin wax, and sliced into 5 μm-thick sections. The sections were then stained with hematoxylin and eosin (H&E) for observation via light microscopy (TS100, Nikon, Japan) with 40× objectives62.
Immunohistochemistry (IHC)
Tissue sections were processed as previously described. The sections were subjected to antigen retrieval and blocked with 10% normal goat serum in PBS for 30 min to eliminate nonspecific binding. The samples were then incubated with a rabbit polyclonal antibody against IBV-N proteins at a 1:500 dilution in PBS for 12 h at 4°C. Next, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) for 1 h. The reaction was visualized via 3,3'-diaminobenzidine (DAB; Sigma) for 10 min. Finally, the sections were counterstained with hematoxylin and examined via light microscopy (TS100, Nikon, Japan) with 40× objectives63.
Vaccination and challenge protocols for vaccine candidates
1-day-old SPF chickens, with each experimental group containing 20 chickens, were immunized with 104 EID50 of rH120, rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) via the ocular and intranasal routes. The day of immunization was designated 0 dpv. The negative control group received an equivalent volume of PBS. Blood samples were collected at 7, 14, and 21 dpv, and serum samples were separated for the subsequent analysis of serum antibody and cytokine levels. At 21dpv, the immunized groups were challenged with 105 EID50 strains of the homologous wild-type (WT) strains QX (WT), TW (WT), and GVI (WT) via the same routes of inoculation (recorded as 0 dpc), resulting in the formation of various groups, namely the PBS/QX, rH120/QX, rH-CPDF7-QX(S)/QX, PBS/TW, rH120/TW, rH-CPDF7-TW(S)/TW, PBS/GVI, rH120/GVI, and rH-CPDF7-GVI(S)/GVI groups, and a nonchallenged control (NC) group, with 20 chickens per group. Clinical symptoms were monitored, and mortality rates were recorded daily for 14 dpc. Viral loads in trachea, lung, and kidney tissues at 3, 6, 9, and 14 dpc, as well as viral shedding at 6 and 12 dpi, were assessed by RT-qPCR, as described previously. At 6 dpc, two chickens from each group were randomly selected for humane euthanasia and autopsy; any deceased chickens were also subjected to autopsy on the day of death. Gross lesions were observed, and tissue samples from the trachea, lungs, and kidneys were collected for histopathological examination, following the established protocol. The detailed immunization protocol for vaccine candidate strains rH-CPDF7-QX(S), rH-CPDF7-TW(S), and rH-CPDF7-GVI(S) is presented in Supplementary Table 3.
Detection of serum-specific antibodies via ELISA
Whole blood samples obtained from the chickens at 7, 14, and 21 dpv were subjected to centrifugation at 3000 rpm for 5 min to separate the serum. These serum samples were then thermally inactivated in a water bath maintained at 56°C for 30 min. The IBV-specific IgG antibody levels in the serum were subsequently quantified via an indirect enzyme-linked immunosorbent assay (ELISA). First, the serum samples were diluted 100-fold in PBS. The assessment was subsequently conducted with a commercial IBV antibody test kit (IDEXX, Westbrook, MA, USA), which strictly adhered to the manufacturer's guidelines49. Each sample was analyzed in triplicate, and the sample/positive (S/P) ratio was calculated via the following formula: [(sample mean - negative mean) / (positive mean - negative mean)]. An S/P ratio exceeding 0.2 was established as the threshold for a positive result for IBV antibodies15.
Assessment of serum-neutralizing antibodies
To determine the neutralization titer of the serum samples against the IBV strains, a serum neutralization experiment was performed. Briefly, the serum samples, which were collected at 21 dpv, were first inactivated at 56 °C for 30 min. This was followed by a serial two-fold dilution of the serum with PBS, while the virus was diluted with PBS to 200 EID50. An equal volume of the diluted serum was subsequently mixed with the diluted virus and incubated at 37°C for 1 h to allow adequate interaction between the antibodies and the viral particles. This mixture, 0.2 mL in volume, was then inoculated into the allantoic cavity of 9-day-old SPF chicken embryos. After 6 d, the development of the embryos was observed and counted, and the neutralization titer of each serum sample against the virus was calculated via the Reed and Muench methods59.
Assessment of the cellular immune response
To quantitatively measure the cellular immune response induced by the recombinant IBV strain, a focused analysis of key cytokines was conducted. Specifically, the levels of IL-2, IL-4, IL-6, and IFN-γ were evaluated as indicators of cellular immunity64. These cytokines serve as biomarkers for cellular immunity, with IL-2 and IL-4 being indicative of T helper 1 (Th1) and T helper 2 (Th2) cell activation, respectively; IL-6 acting as a proinflammatory cytokine; and IFN-γ being a hallmark of a robust Th1 response. The detection of these cytokines was performed via an indirect ELISA (Jiangsu Medical Industrial Co., Ltd., China) following the manufacturer's instructions.
Statistical analysis
Statistical analyses were performed via GraphPad Prism version 7 (GraphPad, La Jolla, CA, USA). The data are presented as the means ± standard deviations (SDs). The data in Figs. 2C, 3B, 4B, and 5B were analyzed by Student's t-test. The data in Figs. 2E, F, and 3D, E, and 4D, E, and 5D, E, and 6B–D, F, G were analyzed via one-way ANOVA followed by Dunnett's multiple-comparison test. (NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). The statistical details of the experiments can be found in the figure legends.
Data availability
All data pertaining to this study are available within the main body of this paper or in the Supplementary Materials section. In this study, the IBV strains studied comprise the H120 vaccine strain (GenBank No. FJ888351) and the isolated strains of QX-type (GI-19, CK/CH/JS/CZ211063, GenBank No: PP438800), TW-type (CK/CH/JS/CZ210443, GenBank No: PQ273739), and GVI-type (CK/CH/JS/CZ210673, GenBank No: PQ273740). Information regarding the synthetic fragments can be found in the supplementary file. Additionally, the resources, data, and reagents utilized in this study can be obtained from the corresponding author upon reasonable request.
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Acknowledgements
This study was supported by the Xinjiang Uygur Autonomous Region Major Science and Technology Project, the Xinjiang Animal Disease Prevention and Control System Quality Improvement Project (2023A02007), and the "Tianchi Talent" Introduction Program.
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J.P. and Y.L. conceptualized and designed the study. Y.L., Y.Z., H.L., and J.X. performed the experiments. Y.L., Y.Z., H.L., and H.Z. participated in animal immunizations and infections. Y.L., and J.P. prepared the manuscript. Y.L., J.P., N.C., and L.Z. analyzed the data. All authors have examined and validated the contents of the manuscript.
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Lu, Y., Zeng, Y., Luo, H. et al. Rapid development of attenuated IBV vaccine candidates through a versatile backbone applicable to variants. npj Vaccines 10, 60 (2025). https://doi.org/10.1038/s41541-025-01114-z
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DOI: https://doi.org/10.1038/s41541-025-01114-z
