Avian infectious bronchitis virus (IBV), a gammacoronavirus with substantial agricultural impact, offers a tractable model for dissecting coronavirus evolution. Here, we integrated 20 years of epidemiological surveillance with whole-genome sequence analysis of 624 IBV strains, including 136 newly isolated field samples, to investigate the evolutionary and structural dynamics of N-linked glycosylation at the spike protein. We identified three dominant glycosylation haplotypes defined by residues 51 and 77 of spike protein, which correlate with receptor-binding interfaces, clinical phenotypes, and spatiotemporal transmission patterns. Molecular modeling and docking analyses provided insights into potential mechanistic links between glycan positioning and Neu5Ac alpha 2-3Gal beta 1-3GlcNAc receptor engagement. Complementing these findings, we developed a proof-of-concept machine learning model that shows potential for predicting clinical serotypes directly from the spike protein sequence, achieving high accuracy on a preliminary independent validation set. These findings support the use of glycosylation motifs as structural-genomic markers and highlight the potential of sequence-based serotype prediction. Our work establishes a scalable genomic-structural framework that leverages glycosylation motifs and sequence features as evolutionary markers, providing a powerful approach for forecasting coronavirus adaptation and informing vaccine design and outbreak preparedness.

In the present study, we report the detection of a novel infectious bronchitis virus lineage within genotypes GVII (GVII-2) in commercial flocks in China since 2008. Virus strains within this lineage have been isolated from different geographic regions in China. S1 genes of these viruses have an intralineage divergence of 0.2%-4.4% nucleotides and 0.1%-7.5% amino acids. The pairwise distances of nucleotide and amino acid sequences based on the S1 subunit between GVII-2 and the closest GVII-1 were 28.3% -28.8% and 28.1%-28.9%, respectively. Complete genomic sequence analysis of the first detected strain, ck/CH/LAH/08I (LAH/08I), suggested that it is derived from recombination events with a genomic backbone of the GI-19 ck/CH/LGS/08I-like strain and distinct S, 3a, and 5a genes. The GVII-2 strain represents a unique serotype that differs from the GVII-1 I0636/16 strain in the same genotype and its deduced parental virus GI-19 I0305/19. Strain LAH/08I not only showed strong tissue tropism in the kidneys of SPF chickens but also exhibited high replication capacities in the trachea, kidney, digestive tract, and immune system of infected chickens, attributed to its pathogenicity in SPF chickens. Strain LAH/08I also showed tropism in the oviducts and the ability to induce cystic oviduct formation in adult chickens after infection in baby chicks.

Due to the scarcity of cell-adapted strains and limitations of current reverse genetics techniques, the factors determining cell tropism of infectious bronchitis virus (IBV), a prototype gammacoronavirus, remain unclear. Here, we demonstrated that the expanded cell tropism of CEA, a chicken embryo fibroblast-adapted strain derived from tl/CH/LDT3/03, was related to its enhanced attachment capacity. Then, a reverse genetics platform for IBV based on circular polymerase extension reaction (CPER) was established, enabling the successful recovery of rCEA and rLDT, which retained the biological properties of their respective parental viruses. Using this platform, we constructed a series of chimeric viruses based on the CEA strain by incorporating the entire S gene, the S1 and S2 subunits, and substitutions at differential sites derived from the tl/CH/LDT3/03 strain. Our findings indicated that subunit S1, rather than S2, was associated with viral adaptation in DF-1 cells, with the amino acid residue at position 413 in the C-terminal domain of the S1 protein as a key determinant. Evaluation with recombinant S1 proteins and chimeric viruses revealed that the histidine residue at position 413 (His413) promoted enhanced attachment of both S1 proteins and viruses to cells, while exerting no direct effect on viral entry. Mechanistically, His413 facilitated efficient viral binding to α-2,3-linked sialic acids presented on gangliosides rather than glycoproteins. This interaction activated Src kinase and triggered caveolae-mediated endocytosis, initiating viral entry and subsequent replication. Collectively, the CPER-based reverse genetics platform established in this study represents a significant technical advancement for IBV, and our findings provide novel insights into the cell adaptation and entry mechanisms of IBV.IMPORTANCEAlthough infectious bronchitis virus (IBV) was the first discovered coronavirus, the cellular receptors and cofactors that mediate successful infection, as well as the specific pathways and mechanisms of viral entry into host cells, remain to be elucidated. In this study, we established a reverse genetics platform for IBV using circular polymerase extension reaction for the first time. A series of chimeric viruses were subsequently recovered, and a specific amino acid substitution at position 413 in the C-terminal domain of the S1 subunit was identified as a key determinant for the expanded DF-1 cell tropism of IBV. The histidine residue at this site facilitated viral binding to α-2,3-linked sialic acids on gangliosides, thereby activating the caveola-mediated endocytosis and enabling viral entry into DF-1 cells. This study provides novel insights into the receptor-binding function of the IBV S protein and the strategies employed by the virus for cell adaptation. Additionally, our findings offer new perspectives for developing cell-culture-based vaccines.

Infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus 1, represents a prominent alphaherpesvirus that poses a significant threat to the global poultry industry. Current routine vaccination strategies fail to eliminate latent infection. Host–pathogen interaction networks have become a key focus in antiviral research. Our previous study demonstrated that activation of the MEK/ERK signaling pathway upon ILTV infection restricts host cellular metabolic activity to mount protective host antiviral responses, yet the underlying molecular mechanism remains unclear. The present work systematically dissects the contribution of MEK/ERK signaling to intrinsic host defense against ILTV. The results show that ILTV infection activates the MEK/ERK pathway, which in turn promotes the expression, activation, and nuclear translocation of the transcription factor Fos. As a core transcriptional regulator, Fos represses host metabolic gene expression, thereby restricting viral replication and proliferation. Integrated multi-omics analyses further demonstrate that the MEK/ERK-Fos-metabolic regulatory axis operates uniformly during infection with avian, human, and porcine alphaherpesviruses, suggesting a broadly conserved host antiviral mechanism. This consistent cross species signaling pattern points toward an evolutionarily conserved host antiviral strategy and provides potential molecular targets for the development of broad-spectrum antiviral strategies against alphaherpesviruses.

Ongoing mutations of coronaviruses make control extremely difficult, urgently demanding broad-spectrum antivirals to complement vaccines. In this study, we screened and identified antiviral drugs targeting the coronavirus 3CL protease (3CLpro) and further investigated their broad-spectrum antiviral activity. The results indicated that Isoliquiritigenin (ISL) interacts with the conserved His41 site of 3CLpro through a non-covalent pan-binding mode and exhibits broad-spectrum antiviral activity against six coronaviruses from four genera. ISL exhibits high affinity for the coronavirus 3CLpro (K D = 10 − 6 –10 − 7 M), which is abolished upon the His41 mutation (K D > 10 − 3 M). Its enzyme-inhibitory activity stems from anchoring to 3CLpro His41 via a π-π stacking network. By rationally fortifying this π-π stacking, we developed the optimized derivative ISL-221, achieving an order-of-magnitude leap in pan-3CLpro affinity (K D = 10 − 7 –10 − 8 M). In vivo , ISL-221 demonstrated 83.33%, 100%, and 100% protection against coronavirus PEDV, TGEV, and PDCoV, respectively; ISL-221 markedly reduced lung viral loads to 1.65 ± 0.68 Log 10 copies/g against SARS-CoV-2 infection in mice, exhibiting efficacy comparable to Paxlovid. This study establishes the highly conserved 3CLpro His41 site as a tractable broad-spectrum design site against four genera of coronaviruses, reveals the unique non-covalent π-π stacking mechanism, and validates mechanism-guided optimization for developing potent pan-coronavirus therapeutics.

Background/Objectives: Despite decades of extensive vaccinations against avian infectious bronchitis virus (IBV) infection, outbreaks caused by constantly emerging variants due to genome recombination between different viral strains, including vaccine strains, occur annually worldwide. The development of novel vaccines with favorable safety and effectiveness is required but is hindered by a limited understanding of vaccination against IBV. Methods: Here, we performed a comprehensive analysis of the in vivo dynamics of peripheral blood mononuclear cells (PBMCs) in specific pathogen-free chickens inoculated with the widely used live attenuated IBV vaccine strain H120 at single-cell level, using high-throughput single-cell transcriptome sequencing (scRNA-seq). Results: High-quality sequencing dataset for four scRNA-seq data containing the transcriptomes of 29,846 individual chicken PBMCs were obtained, defining 22 populations and 7 cell types based on distinct molecular signatures and known markers. Further integrative analysis constructed the time series dynamic cell transition and immune response landscapes within the two weeks post-prime vaccination against IBV. Enhanced crosstalk between antigen-presenting cells and T lymphocytes was revealed as early as four days post-vaccination. The specific immune cell populations and their comprehensive cellular and molecular networks involved in the initiation phase of antiviral adaptive immune responses were elucidated in details. Conclusions: Our study provides a comprehensive view of the dynamic initiation of immune responses in chickens against IBV infection at the cellular and molecular levels, which provides theoretical support and potential solutions for the future rational design of safe and effective vaccines, the augmentation of the efficacy of current vaccines, and the optimization of immune programs.

In addition to its nutritional role in regulating calcium and phosphorus metabolism, vitamin D also possesses immunoregulatory effects. Herein, we used chicken embryo fibroblast cells (CEFs) to study the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the immune response induced by infectious bronchitis virus (IBV) infection. We observed the inhibition to virus proliferation in the cells by adding 10 nM 1,25(OH)2D3. In addition, treatment with 1,25(OH)2D3 has significantly increased the mRNA expressions of toll-like receptor 15 (TLR15) (2.75-fold), avian beta-defensins 9 (AvBD9) (25.37-fold), and AvBD14 (2.28-fold) in the IBV-infected CEFs comparing with those of the control group. In contrast, the mRNA expression levels of TLR3 (0.98-fold), TLR4 (0.66-fold), TLR5 (0.44-fold), TLR7 (0.62-fold), and AvBD1 (0.57-fold), AvBD3 (0.54-fold), AvBD8 (0.60-fold), and AvBD12 (0.54-fold) were found to be decreased. In parallel with this results, the mRNA expression levels of the myeloid differentiation primary response protein 88 (MyD88) (0.69-fold), interleukin-8 (IL-8) (0.99-fold), IL-6 (0.98-fold), and IL-1β (0.93-fold) in 1,25(OH)2D3 group were also significantly downregulated comparing with those of control group. Thus, the results suggest that adding of 1,25(OH)2D3 is capable of inducing TLRs and AvBDs gene expressions, suppressing the inflammatory response and enhancing an antiviral defense, which might have a beneficial effect in controlling viral infections in poultry.

As a unique antigenic variant of Newcastle disease virus (NDV) in pigeons, genotype VI NDVs cause serious disease in pigeons and pose a potential threat to domestic poultry. In this study, analyses of genetic variation and evolution of global genotype VI NDVs revealed the worldwide distribution and continuous evolution of genotype VI NDVs. Of importance, VI.2.1.1.2.1 and VI.2.1.1.2.2 are the two prevalent sub-genotypes worldwide. The virulence, replication capacity, and pathogenesis in chickens of representative strains from VI.2.1.1.2.1 and different clusters in VI.2.1.1.2.2 were further evaluated. Compared with the VI.2.1.1.2.1 strain, increased replication capacity and virulence in chickens were observed in strains of VI.2.1.1.2.2. VI.2.1.1.2.2 strains showed preferential binding for α-2,3-linked sialic acids and superior performance in viral entry, cell-cell fusion, and release of progeny virions. Evaluation with recombinant viruses demonstrated that residues at positions 365 and 497 in the HN protein contributed to the differences in biological characteristics of VI.2.1.1.2.2 strains, and residue 365 was a key determinant. Moreover, our results showed that the activity of the viral replication complex contributed to the differences in replication capacity among these viruses, with the P protein being the major individual contributor. The optimal effect was achieved when the NP and L proteins were homologous. Moreover, the 5' terminal trailer region was also found to be involved in the replication capacity of genotype VI NDVs; however, the viral V protein was not related to the replication and virulence of these viruses. Our findings highlight the potential risk of VI.2.1.1.2.2 NDVs due to their persistent circulation and evolution. IMPORTANCE:Genotype VI is the most diverse group of Newcastle disease viruses (NDVs). In addition to infectious disease in pigeons, the potential threat to chicken flocks and the public health implications associated with genotype VI NDVs also need to be addressed. Herein, comprehensive genetic evolution analysis revealed a global distribution pattern and continuous evolution of genotype VI NDVs worldwide. The biological characteristics of genotype VI NDVs belonging to different sub-genotypes were also evaluated. In particular, the widespread transmission, circulation, and constant evolution of currently prevalent sub-genotype VI.2.1.1.2.2 have led to the alteration of receptor binding preference, an increase in replication capacity, and a resultant increase in virulence in chickens. These findings expand our current understanding of the evolution and pathogenesis of genotype VI NDVs.

Cover crops can suppress the following crop diseases and alter soil microbial communities, but the mechanisms of such disease suppressive effects remain uncertain. Here, we studied the effects of brassica and cereal cover crops, along with decomposition solutions from these crop residues, on tomato growth and bacterial wilt. Moreover, tomato rhizosphere microorganisms were analyzed by qPCR and high-throughput sequencing. Rhizosphere transplant experiment was conducted to validate the disease suppressive potential of rhizosphere microorganisms mediated by decomposition solutions from these crop residues. Our findings revealed that brassica and cereal cover crops especially wheat, pakchoi and rape significantly enhanced tomato growth and inhibited bacterial wilt disease. Decomposition solutions from brassica and cereal residues had inhibitory effects on Ralstonia solanacearum and this disease. Moreover, such decomposition solutions can differently alter the abundances, compositions and diversities of tomato rhizosphere bacterial and fungal communities. Notably, decomposition solutions from wheat, pakchoi and rape residues increased the inverse Simpson diversity and the abundances of Bacillus spp. community. In addition, decomposition solutions from wheat and pakchoi residues significantly increased bacterial beta diversity, and decomposition solutions from rape residue significantly increased fungal beta diversity. Rhizosphere transplant experiment confirmed that the rhizosphere microbial changes induced by decomposition solutions contributed to the suppressiveness of tomato bacterial wilt disease. These suppressive effects were stronger in decomposition solutions from wheat, pakchoi and rape residues than those from oilseed rape, wild rocket and Indian mustard residues. Overall, our results demonstrated that decomposition solutions from brassica and cereal residues enhance disease suppression by shaping a beneficial rhizosphere microbiota, providing a promising strategy for sustainable management of bacterial wilt in tomato cultivation.

Entry of Newcastle disease virus (NDV) into target cells via caveolae-mediated endocytosis has been presented. However, the viral factors that mediate the endocytic entry and fusion in intracellular endosomes remain poorly understood. Herein, the potential contributions of HN stalk region and second receptor biding site (site II) located at the globular head region on the endocytosis of NDV and the viral-cell membrane fusion in endosomes were investigated. HN protein mutants with substitutions at positions 89 and 94 in the stalk region and at position 516 in site II were firstly constructed. We demonstrated that mutations in HN stalk region restricted its interaction with F protein and fusion promotion activity. While mutation in HN site II resulted in slightly decreased fusion promotion activity without affected the HN-F interactions. Further evaluation of receptor binding and entry capacities with HN mutant NDVs obtained by reverse genetics demonstrated that both mutations at stalk region and site II reduced the binding abilities of NDV to receptors. Interestingly, site II in HN was not responsible for the endocytic entry of NDV and subsequent viral-cell membrane fusion in endosomes. While the residues at 89 and 94 in HN stalk region were involved in the fusion of NDV in endosomes. Of particular note, the acidic condition was indispensable for efficiently fusion triggering of NDV within endosomes. Besides, residues at 89 and 94 in HN were conserved in different genotypes of NDVs and the endocytic entry of NDV into cells was not determined by viral virulence and genotype.

To adapt the infectious bronchitis virus (IBV) for cell culture, the tl/CH/LDT3/03 strain was subjected to serial passaging in chicken embryo fibroblasts (CEFs) and Vero cells, respectively. Cytopathic effects (CPEs) first became apparent at the 7th passage in CEFs and the 11th passage in Vero cells, respectively. The tl/CH/LDT3/03 strain achieved stable replication and adaptation after 20 passages in CEFs (CEA P20) and 25 passages in Vero cells (VEA P25). Analysis of the genomic sequences of the two adapted viruses identified amino acid substitutions, insertions, and deletions in some of the viral proteins. To evaluate the replication capacity of the cell-adapted viruses, 1-day-old SPF chicks were inoculated with either CEA P20 or VEA P25. Both CEA P20 and VEA P25 exhibited reduced replication capacity in chickens, as determined by viral titration in 11 selected tissues collected at 5 days post-inoculation (dpi). The pathogenicity of the two viruses was also decreased for 1-day-old chicks. Furthermore, VEA P25 elicited significantly reduced neutralizing antibody responses in infected birds, nearly 3-fold lower than that induced by CEA P20. To evaluate protective efficacy for chickens, in ovo vaccination of SPF eggs with either CEA P20 or VEA P25 was carried out. Both cell-adapted viruses provided complete protection against tl/CH/LDT3/03 challenge, therefore represent promising attenuated live vaccine candidates for in ovo vaccination. In conclusion, serial propagation of IBV tl/CH/LDT3/03 in CEFs and Vero cells resulted in successful viral adaptation, which was associated with decreased replication capacity and a consequent attenuation of virulence in chickens, showing the potential of live vaccine candidates against tl/CH/LDT3/03.

Ten infectious bronchitis viruses (IBVs) were isolated from diseased chicken flocks in China between 2018 and 2024. Phylogenetic, homology, and pairwise comparisons of the complete S1 gene revealed that the 10 isolates shared a close genetic relationship with the European L1148 vaccine strain. Complete genomic sequence of isolate I0317/22 confirmed this result. Inoculation of 1-day-old specific pathogen free (SPF) chickens with I0317/22 induced mild clinical signs. The virus replicated at low levels in most of the tissues, except in the respiratory and upper digestive (preventriculus) tracts. We speculate that the 10 isolates derived from the L1148 vaccine strain and spread with low pathogenicity within chicken flocks. In 1-day-old SPF chickens, inoculation with I0317/22 and vaccination with the Mycoplasma gallisepticum (MG) F vaccine strain demonstrated that the MG F vaccine did not promote I0317/22 replication. Similarly, challenge of the chickens inoculated with MG F after 28 days with strain I0305/19 did not enhance I0305/19 replication compared to the control group. However, inoculation with MG F suppressed the neutralizing antibody responses against I0317/22 and I0305/19. We hypothesize that MG F strain induced suppression of neutralizing antibodies by affecting the host immune response to IBV infection, thereby increasing susceptibility to other pathogens, such as Escherichia coli. This likely led to mixed infections where synergistic interactions between pathogens exacerbated disease severity beyond what would be expected under experimental conditions alone.

Biosafe and effective vaccines are urgently needed for the prevention and control of avian infectious bronchitis virus (IBV), the first coronavirus to be discovered, despite extensive vaccination for decades. However, their development has been hindered by our limited understanding of prime vaccination, which is crucial for rational vaccine design. Here, we constructed in vivo dynamic single-cell resolution blood immune landscapes of chickens immunized with live-attenuated or inactivated IBV. Bioinformatic analysis together with in vivo examination revealed that live-attenuated and inactivated vaccines reshaped lymphocytes and led to identical compositions through different mechanisms. Inactivated vaccines activate T lymphocytes through dendritic cells with subsequent T lymphocyte-dependent B lymphocyte expansion upon prime vaccination but induce pathogen-specific antibodies only after boost vaccination. Prime vaccination with a live-attenuated vaccine led to an initial preference for monocytes/macrophages as antigen-presenting cells (APCs), followed by extensive activation of the main APCs, which facilitated rapid T lymphocyte expansion and elicited satisfactory humoral immunity. Along with the disparate utilization of APCs, live-attenuated and inactivated vaccines yielded distinct TCR repertoires and triggered different B lymphocyte dynamics despite their similar final BCR repertoires. Furthermore, APC preference correlated with vaccine effectiveness rather than modality, as prime avian influenza vaccination triggered effective adaptive immune responses with the same APC preference as live-attenuated IBV did. This study comprehensively characterized avian coronavirus prime vaccination and highlighted the key role of APC preference.

Newcastle disease virus (NDV) genotype VI from pigeon origin is an important causative agent for serious disease in pigeons. Although the biological characteristics of genotype VI NDV have been extensively studied, the understanding of the thermostability of this genotype is still incomplete. In this study, an NDV strain, designated P0506, was isolated from a diseased pigeon in China and classified as genotype VI. Phylogenetic analysis on the basis of the Fusion gene coding sequence indicated that P0506 belonged to sub-genotype VI.2.1.1.2.2 of class II. The thermostability may be a universal characteristic of genotype VI NDV. Thus, the thermostability of two strains, including P0506 identified in this study and P0713 identified previously, belonging to VI.2.1.1.2.2, and another previously isolated strain, P0813, in VI.2.1.1.2.1, was investigated. It was indicated that all three viruses presented resistance to heat treatment, but P0713 was more robust than P0813 and P0506. By constructing a series of HN protein mutants, amino acid residues at both residues 365 and 497 in HN protein were found to be involved in the heat resistance. Furthermore, the effects of residues 365 and 497 in HN protein on the thermostability of the virus were further evaluated by using recombinant viruses generated by the reverse genetic system. Our results showed that residue at position 365 in HN protein was the key thermostable determinant of sub-genotype VI.2.1.1.2.2 NDV. These findings will help us better understand the thermostable mechanism of NDV and serve as a foundation for the further development of novel thermostable vaccines.

Therapies targeting virus-host interactions are seen as promising strategies for treating gallid alphaherpesvirus 1 (ILTV) infection. Our study revealed a biphasic activation of two MAPK cascade pathways, MEK/ERK and p38 MAPK, as a notably activated host molecular event in response to ILTV infection. It exhibits antiviral functions at different stages of infection. Initially, the MEK/ERK pathway is activated upon viral invasion, leading to a broad suppression of metabolic pathways crucial for ILTV replication, thereby inhibiting viral replication from the early stage of ILTV infection. As the viral replication progresses, the p38 MAPK pathway activates its downstream transcription factor, STAT1, further hindering viral replication. Interestingly, ILTV overcomes this biphasic antiviral barrier by hijacking host p38-AKT axis, which protects infected cells from the apoptosis induced by infection and establishes an intracellular equilibrium conducive to extensive ILTV replication. These insights could provide potential therapeutic targets for ILTV infection.

ABSTRACT As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors. IMPORTANCE In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

ABSTRACT The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53’s antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53’s genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens. IMPORTANCE The current study conducted a comprehensive analysis, comparing molecular networks underlying chp53’s antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.

Infectious bronchitis virus (IBV) does not only cause disease in millions of chickens worldwide, but IBV-like viruses have also been detected or isolated from other domestic birds. We propose that the pheasant coronavirus (PhCoV) originates from IBV. Indeed, the IBV strains H120 and M41 can replicate but do not cause disease in pheasants. In this study, we found that three chicken nephropathogenic IBV strains, including ck/CH/LDL/091,021, ck/CH/LDL/140,520, and I0305/19, and the viruses recovered from the tissues of pheasants challenged with each IBV strain could replicate in some challenged pheasants with different capacities but could not cause disease. Overall, these viruses showed different capacities of replication and adaptation in pheasants, and the neutralizing antibody against each IBV strain could be detected in different numbers of pheasants challenged with each of the viruses, although the titers were generally low with large variation. Comparatively, ck/CH/LDL/140,520 and 20/P1-D5/Tr1 showed higher adaptation capacities in pheasants. Furthermore, the three IBV strains gained an increased capacity for adaptation when they passed in pheasants once, especially strain ck/CH/LDL/140,520, which gained an increased capacity for adaptation and extended tissue tropism when it was passaged in pheasants. Similar to IBV in chicken, the subpopulations within the virus were selected when the virus replicated and was passaged in pheasants, and the accumulation of mutations and deletions in the genome of each virus subpopulation accounted for the independent evolution of the virus in different tissues of pheasants. Taken together, we suggest that the phCoVs might originate from IBV through interspecies transmission from chickens to pheasants, before gaining increased tissue tropism, adaptation capacities, and disease-causing behaviors in pheasants during intraspecies transmission.

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53–Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it.