Identification of Host Proteins Interacting with IBV S1 Based on Tracheal Organ Culture.

Infectious bronchitis virus (IBV) belongs to the gamma-coronavirus genus of Coronaviridae and causes serious infectious diseases in the poultry industry. However, only a few IBV strains can infect avian passage cell lines, seriously hindering the progress of basic research on IBV pathogenesis. Whereas IBV field strains can replicate in tracheal ring organ culture (TOC) without any previous adaptation in chicken embryos or primary cells. In this study, to investigate the potential use of TOC as an in vitro infection model for the study of IBV-host interaction, we first established a chicken embryo TOC culture system and carried out an investigation on the IBV replication kinetics in the system. We found that the selected strains of the IBV GI-1, GI-7, GI-13, GI-19, and GI-22 genotypes could successfully replicate in TOC and bring about damage to the infected trachea. Next, we identified host proteins of the chicken embryo trachea that interact with the IBV S1 protein by immunoprecipitation and protein mass spectrometry. A total of 127 candidate proteins were initially identified with major involvement in cell adhesion pathways and apoptosis- and autophagy-related pathways. The heat shock protein 70 (HSP70) was selected for further investigation in the interaction with IBV viral proteins. Our results showed that HSP70 interacted with IBV S1 in both TOC and CEK cells, whereas HSP70 overexpression inhibited viral replication. This study indicates that TOC is a good system for the elucidation of IBV-host interactions and HSP70 is a potential host antiviral factor.

Characterization of the induction kinetics and antiviral functions of IRF1, ISG15 and ISG20 in cells infected with gammacoronavirus avian infectious bronchitis virus.

Coronavirus infection induces a variety of cellular antiviral responses either dependent on or independent of type I interferons (IFNs). Our previous studies using Affymetrix microarray and transcriptomic analysis revealed the differential induction of three IFN-stimulated genes (ISGs), IRF1, ISG15 and ISG20, by gammacoronavirus infectious bronchitis virus (IBV) infection of IFN-deficient Vero cells and IFN-competent, p53-defcient H1299 cells, respectively. In this report, the induction kinetics and anti-IBV functions of these ISGs as well as mechanisms underlying their differential induction are characterized. The results confirmed that these three ISGs were indeed differentially induced in H1299 and Vero cells infected with IBV, significantly more upregulation of IRF1, ISG15 and ISG20 was elicited in IBV-infected Vero cells than that in H1299 cells. Induction of these ISGs was also detected in cells infected with human coronavirus-OC43 (HCoV-OC43) and porcine epidemic diarrhea virus (PEDV), respectively. Manipulation of their expression by overexpression, knockdown and/or knockout demonstrated that IRF1 played an active role in suppressing IBV replication, mainly through the activation of the IFN pathway. However, a minor, if any, role in inhibiting IBV replication was played by ISG15 and ISG20. Furthermore, p53, but not IRF1, was implicated in regulating the IBV infection-induced upregulation of ISG15 and ISG20. This study provides new information on the mechanisms underlying the induction of these ISGs and their contributions to the host cell antiviral response during IBV infection.

Virulence Properties of GI-23 Infectious Bronchitis Virus Isolated in Poland and Efficacy of Different Vaccination strategies.

Infectious bronchitis virus (IBV) is one of the most important poultry pathogens, leading significant economic losses worldwide. IBV is characterised by highly genetic, serotype, and pathotypic variability. Despite extensive immunoprophylaxis strategies, the emergence of new genetic lineages is frequently observed in the field, causing disease control to be more complicated. In the last decade, the spread of variants assigned to the GI-23 lineage of IBV (formerly known as Var2) started from Middle-Eastern countries and reached Europe in the last few years. Recently, the introduction and fast spread of Var2-like IBVs in Poland was reported. In this study, the virulence properties and efficacy of different vaccination programmes were evaluated against infection with the IBV GI-23 strain gammaCoV/Ck/Poland/G052/2016. The pathogenicity of the Var2 isolate was conducted in one-day-old and three-week-old SPF chickens and showed that the course of the disease is age dependent. Seven vaccination programmes using Mass, 793B, QX alone or in combination, and Var2 live vaccines were tested against the GI-23 infectious bronchitis virus challenge. All groups were scored according to the ciliostasis test at 5 days post challenge. Two immunoprophylaxis strategies generated full protection against gammaCoV/Ck/Poland/G052/2016 infection-Var2 and Mass used in one-day-old chickens boosted by a combination of the QX and 793B vaccine (both with a ciliostasis score of 0 and 100% protection).

Guanylate-binding protein 1 restricts avian coronavirus infectious bronchitis virus-infected HD11 cells.

The Infectious Bronchitis Virus (IBV), a coronavirus, is a key avian pathogen that causes acute and highly infectious viral respiratory diseases. IBV is an enveloped, positive-sense RNA virus, and the host factors that restrict infection and replication of the virus remain poorly understood. Guanylate-binding protein 1 (GBP1), an interferon-gamma (IFN-γ)-inducible guanosine triphosphatase (GTPase), is a major player in host immunity and provides defense against viral replication. However, the role of chicken GBP1 (chGBP1) in the IBV-life cycle is not well understood. Therefore, this study aimed to reveal the potential role of IFN-γ-induced chGBP1 in mediating host anti-IBV infection responses. We identified the host restriction factor, chGBP1, in IBV-infected chicken macrophages HD11 cell lines. We showed that chGBP1 was upregulated by treatment with both IFN-γ and IBV in HD11 cells. chGBP1 inhibited IBV replication in a dose-dependent manner and enhanced IFN-γ anti-IBV activity. Importantly, the GTPase domain of chGBP1 played a pivotal role in its anti-IBV activity. Furthermore, chGBP1 interacts with IBV Nucleocapsids protein to degrade IBV-N protein through the autophagy pathway. Taken together, our results demonstrate a critical role of chGBP1 in anti-IBV in macrophages HD11 cells.

Reverse Genetics of Avian Coronavirus Infectious Bronchitis Virus

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA;that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA dependant RNA polymerase. Copyright © Springer Science+Business Media New York 2016.

Genotyping and In Silico Analysis of Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Spike 1 (S1) Glycoprotein.

Genetic diversity and evolution of infectious bronchitis virus (IBV) are mainly impacted by mutations in the spike 1 (S1) gene. This study focused on whole genome sequencing of an IBV isolate (IBV/Ck/Can/2558004), which represents strains highly prevalent in Canadian commercial poultry, especially concerning features related to its S1 gene and protein sequences. Based on the phylogeny of the S1 gene, IBV/Ck/Can/2558004 belongs to the GI-17 lineage. According to S1 gene and protein pairwise alignment, IBV/Ck/Can/2558004 had 99.44-99.63% and 98.88-99.25% nucleotide (nt) and deduced amino acid (aa) identities, respectively, with five Canadian Delmarva (DMV/1639) IBVs isolated in 2019, and it also shared 96.63-97.69% and 94.78-97.20% nt and aa similarities with US DMV/1639 IBVs isolated in 2011 and 2019, respectively. Further homology analysis of aa sequences showed the existence of some aa substitutions in the hypervariable regions (HVRs) of the S1 protein of IBV/Ck/Can/2558004 compared to US DMV/1639 isolates; most of these variant aa residues have been subjected to positive selection pressure. Predictive analysis of potential N-glycosylation and phosphorylation motifs showed either loss or acquisition in the S1 glycoprotein of IBV/Ck/Can/2558004 compared to S1 of US DMV/1639 IBV. Furthermore, bioinformatic analysis showed some of the aa changes within the S1 protein of IBV/Ck/Can/2558004 have been predicted to impact the function and structure of the S1 protein, potentially leading to a lower binding affinity of the S1 protein to its relevant ligand (sialic acid). In conclusion, these findings revealed that the DMV/1639 IBV isolates are under continuous evolution among Canadian poultry.

Transcriptomic analysis reveals crucial regulatory roles of immediate-early response genes and related signaling pathways in coronavirus infectious bronchitis virus infection.

Coronavirus infection of cells differentially regulates the expression of host genes and their related pathways. In this study, we present the transcriptomic profile of cells infected with gammacoronavirus infectious bronchitis virus (IBV). In IBV-infected human non-small cell lung carcinoma cells (H1299 cells), a total of 1162 differentially expressed genes (DEGs), including 984 upregulated and 178 downregulated genes, was identified. These DEGs were mainly enriched in MAPK and Wnt signaling pathways, and 5 out of the 10 top upregulated genes in all transcripts were immediate-early response genes (IEGs). In addition, the induction of 11 transcripts was validated in IBV-infected H1299 and Vero cells by RT-qPCR. The accuracy, reliability and genericity of the transcriptomic data were demonstrated by functional characterization of these IEGs in cells infected with different coronaviruses in our previous publications. This study provides a reliable transcriptomic profile of host genes and pathways regulated by coronavirus infection.

Infectious Bronchitis Virus Nsp14 Degrades JAK1 to Inhibit the JAK-STAT Signaling Pathway in HD11 Cells.

Coronaviruses (CoVs) are RNA viruses that can infect a wide range of animals, including humans, and cause severe respiratory and gastrointestinal disease. The Gammacoronavirus avian infectious bronchitis virus (IBV) causes acute and contagious diseases in chickens, leading to severe economic losses. Nonstructural protein 14 (Nsp14) is a nonstructural protein encoded by the CoV genome. This protein has a regulatory role in viral virulence and replication. However, the function and mechanism of IBV Nsp14 in regulating the host's innate immune response remain unclear. Here we report that IBV Nsp14 was a JAK-STAT signaling pathway antagonist in chicken macrophage (HD11) cells. In these cells, Nsp14 protein overexpression blocked IBV suppression induced by exogenous chIFN-γ treatment. Meanwhile, Nsp14 remarkably reduced interferon-gamma-activated sequence (GAS) promoter activation and chIFN-γ-induced interferon-stimulated gene expression. Nsp14 impaired the nuclear translocation of chSTAT1. Furthermore, Nsp14 interacted with Janus kinase 1 (JAK1) to degrade JAK1 via the autophagy pathway, thereby preventing the activation of the JAK-STAT signaling pathway and facilitating viral replication. These results indicated a novel mechanism by which IBV inhibits the host antiviral response and provide new insights into the selection of antiviral targets against CoV.

Immunopathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus (IBV): Impact on the reproductive tract in layers.

IBV infection may lead to reduced egg production and poor egg quality in layer flocks. The DMV/1639 strain was recently identified as one of the most dominant IBV variants isolated from Canadian layer flocks with egg production problems. The current study aimed to investigate the immunopathogenesis of the Canadian DMV/1639 strain in laying chickens. Specific-pathogen-free (SPF) layers were infected at the peak of lay (29 weeks; n = 10) with an uninfected control group (n = 10). Egg production in the infected group dropped to 40% by the fifth day post-infection (dpi). Five birds from the infected and the control groups were euthanized at 5 and 10 dpi. Ovarian regression and shortened oviduct with marked histopathological changes were observed in the infected group at 10 dpi. An increase in the IBV viral load in reproductive tissues was accompanied by a significant recruitment (p < 0.05) of KUL01+ macrophages and CD4+ and CD8+ T cell subsets at 10 dpi. Additionally, anti-IBV antibody response was detected in serum and locally in the reproductive tract washes of the infected group. Overall, our findings contribute to the understanding of the pathogenicity of the Canadian DMV/1639 strain and the subsequent host responses in the reproductive tract of chickens.

Human Wharton's Jelly Mesenchymal Stem Cells Secretome Inhibits Human SARS-CoV-2 and Avian Infectious Bronchitis Coronaviruses.

Human SARS-CoV-2 and avian infectious bronchitis virus (IBV) are highly contagious and deadly coronaviruses, causing devastating respiratory diseases in humans and chickens. The lack of effective therapeutics exacerbates the impact of outbreaks associated with SARS-CoV-2 and IBV infections. Thus, novel drugs or therapeutic agents are highly in demand for controlling viral transmission and disease progression. Mesenchymal stem cells (MSC) secreted factors (secretome) are safe and efficient alternatives to stem cells in MSC-based therapies. This study aimed to investigate the antiviral potentials of human Wharton's jelly MSC secretome (hWJ-MSC-S) against SARS-CoV-2 and IBV infections in vitro and in ovo. The half-maximal inhibitory concentrations (IC50), cytotoxic concentration (CC50), and selective index (SI) values of hWJ-MSC-S were determined using Vero-E6 cells. The virucidal, anti-adsorption, and anti-replication antiviral mechanisms of hWJ-MSC-S were evaluated. The hWJ-MSC-S significantly inhibited infection of SARS-CoV-2 and IBV, without affecting the viability of cells and embryos. Interestingly, hWJ-MSC-S reduced viral infection by >90%, in vitro. The IC50 and SI of hWJ-MSC secretome against SARS-CoV-2 were 166.6 and 235.29 µg/mL, respectively, while for IBV, IC50 and SI were 439.9 and 89.11 µg/mL, respectively. The virucidal and anti-replication antiviral effects of hWJ-MSC-S were very prominent compared to the anti-adsorption effect. In the in ovo model, hWJ-MSC-S reduced IBV titer by >99%. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis of hWJ-MSC-S revealed a significant enrichment of immunomodulatory and antiviral proteins. Collectively, our results not only uncovered the antiviral potency of hWJ-MSC-S against SARS-CoV-2 and IBV, but also described the mechanism by which hWJ-MSC-S inhibits viral infection. These findings indicate that hWJ-MSC-S could be utilized in future pre-clinical and clinical studies to develop effective therapeutic approaches against human COVID-19 and avian IB respiratory diseases.

The Characterization of chIFITMs in Avian Coronavirus Infection In Vivo, Ex Vivo and In Vitro.

The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.