Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing.

Infectious bronchitis virus (IBV) is a coronavirus that infects chickens, which exhibits a broad tropism for epithelial cells, infecting the tracheal mucosal epithelium, intestinal mucosal epithelium, and renal tubular epithelial cells. Utilizing single-cell RNA sequencing (scRNA-seq), we systematically examined cells in renal, bursal, and tracheal tissues following IBV infection and identified tissue-specific molecular markers expressed in distinct cell types. We evaluated the expression of viral RNA in diverse cellular populations and subsequently ascertained that distal tubules and collecting ducts within the kidney, bursal mucosal epithelial cells, and follicle-associated epithelial cells exhibit susceptibility to IBV infection through immunofluorescence. Furthermore, our findings revealed an upregulation in the transcription of proinflammatory cytokines IL18 and IL1B in renal macrophages as well as increased expression of apoptosis-related gene STAT in distal tubules and collecting duct cells upon IBV infection leading to renal damage. Cell-to-cell communication unveiled potential interactions between diverse cell types, as well as upregulated signaling pathways and key sender-receiver cell populations after IBV infection. Integrating single-cell data from all tissues, we applied weighted gene co-expression network analysis (WGCNA) to identify gene modules that are specifically expressed in different cell populations. Based on the WGCNA results, we identified seven immune-related gene modules and determined the differential expression pattern of module genes, as well as the hub genes within these modules. Our comprehensive data provides valuable insights into the pathogenesis of IBV as well as avian antiviral immunology.

Host Immune Response Modulation in Avian Coronavirus Infection: Tracheal Transcriptome Profiling In Vitro and In Vivo.

Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host-pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.

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.

Pathogenicity of the Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) on Female Reproductive Tract of Chickens.

Infectious bronchitis virus (IBV) infection causes significant economic losses to various sectors of the poultry industry worldwide. Over the past few years, the incidence of false layer syndrome in Eastern Canadian layer flocks has been associated with the increased prevalence of the IBV Delmarva (DMV)/1639 strain. In this study, 1-day-old specific-pathogen-free (SPF) hens were infected with the Canadian DMV/1639 strain and observed until 16 weeks of age in order to determine if the IBV DMV/1639 strain is causing false layer syndrome. Early after infection, the virus showed a wide tissue distribution with characteristic gross and histopathological lesions in the respiratory tract and kidney. Around 60-70% of the infected hens demonstrated continuous cloacal viral shedding until the end of the experiment (at 16 weeks) which was associated with high IBV genome loads detected in the cecal tonsils. The experiment confirmed the field observations that the Canadian DMV/1639 strain is highly pathogenic to the female reproductive tract causing marked cystic lesions in the oviduct. Moreover, significant histopathological damage was observed in the ovary. Our study provides a detailed description of the pathological consequences of the IBV DMV/1639 strain circulating in an important poultry production sector.

Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Protección incrementada por el virus recombinante de la enfermedad de Newcastle que expresa el ectodominio de la espícula del virus de la bronquitis infecciosa y el factor estimulante de colonias de granulocitos y macrófagos del pollo. Anteriormente se reportó que la cepa LaSota recombinante del virus de la enfermedad de Newcastle (rLS) que expresa el ectodominio de la espícula trimérica (S) de tipo Arkansas (Ark) del virus de la bronquitis infecciosa (IBV) (Se; rLS/ArkSe) proporciona una protección subóptima contra la exposición al virus de la bronquitis infecciosa. Ahora se ha desarrollado hemos desarrollado una cepa LaSota recombinante (rLS) que expresa el factor estimulante de colonias de granulocitos y macrófagos de pollo (GMCSF) y la espícula del virus de bronquitis Arkansas en un intento para mejorar la efectividad de la vacuna. En el estudio actual, primero se comparó la protección conferida por la vacunación con los virus rLS/ArkSe y rLS/ArkSe.GMCSF. Los pollos vacunados se desafiaron con un virus Arkansas virulento y la protección se determinó mediante los signos clínicos, la carga viral y la histomorfometría de la tráquea. Los resultados mostraron que la coexpresión del factor estimulante de colonias de granulocitos y macrófagos de pollo y la espícula de la cepa recombinante LaSota redujo significativamente la carga viral traqueal y las lesiones traqueales en comparación con los pollos vacunados con el virus rLS/ArkSe. En un segundo experimento, se evaluó el incremento en la protección cruzada por una vacuna atenuada de Massachusetts (Mass) mediante la primovacunación o la vacunación de refuerzo con rLS/ArkSe.GMCSF. Los pollos vacunados fueron desafiados con el virus Arkansas y se evaluó la protección. Los resultados mostraron que la primovacunación o la vacunación de refuerzo con el virus recombinante aumentó significativamente la protección cruzada conferida por el virus Massachusetts contra el desafío virulento con el virus Arkansas. Se observaron mayores reducciones de las cargas virales en los fluidos traqueales y lagrimales en pollos primovacunadoss con rLS/ArkSe.GMCSF y con vacunación de refuerzo con Massachusetts. De manera consistente, los niveles de anticuerpos Ark Se medidos con placas de ELISA recubiertas con proteína Ark Se recombinante a los 14 días después del refuerzo fueron significativamente más altos en estos pollos. De manera inesperada, el esquema de vacunación inverso, es decir, la primovacunación con Massachusetts y el refuerzo con la vacuna recombinante, resultó menos efectivo. Se concluye que una estrategia de primovacunación y refuerzo mediante el uso de rLS/ArkSe.GMCSF y la vacuna atenuada con Massachusetts usada en todo el mundo proporciona una protección cruzada aumentada. Por tanto, el virus rLS/GMCSF que coexpresa la proteína de la espícula de los serotipos regionales relevantes de bronquitis infecciosa podría usarse en combinación con una vacuna viva Massachusetts para proteger contra cepas variantes del virus de la bronquitis infecciosa que circulan regionalmente.

Interplay of the ubiquitin proteasome system and the innate immune response is essential for the replication of infectious bronchitis virus.

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNß) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNß. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.

Analysis of chicken macrophage functions and gene expressions following infectious bronchitis virus M41 infection.

Infectious bronchitis virus (IBV) is a pathogenic coronavirus with high morbidity and mortality in chicken breeding. Macrophages with normal biofunctions are essential for host immune responses. In this study, the HD11 chicken macrophage cell line and chicken peripheral blood mononuclear cell-derived macrophages (PBMCs-Mφ) were infected with IBV at multiplicity of infection (MOI) of 10. The dynamic changes of their biofunctions, including cell viability, pathogen elimination function, phagocytic ability, and gene expressions of related proteins/mediators in innate and acquired immunity, inflammation, autophagy and apoptosis were analyzed. Results showed that IBV infection decreased chicken macrophage viability and phagocytic ability, and increased pathogen elimination function. Moreover, IBV augmented the gene expressions of most related proteins in macrophages involved in multiple host bioprocesses, and the dynamic changes of gene expressions had a close relationship with virus replication. Among them, MHCII, Fc receptor, TLR3, IFN-α, CCL4, MIF, IL-1ß, IL-6, and iNOS showed significantly higher expressions in IBV-infected cells. However, TLR7, MyD88, MDA5, IFN-γ, MHCII, Fc receptor, MARCO, CD36, MIF, XCL1, CXCL12, TNF-α, iNOS, and IL-10 showed early decreased expressions. Overall, chicken macrophages play an important role in host innate and acquired immune responses to resist IBV infection, despite early damage or suppression. Moreover, the IBV-induced autophagy and apoptosis might participate in the virus-host cell interaction which is attributed to the biological process.

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus.

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2' cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

Evaluation of the reproductive system development and egg-laying performance of hens infected with TW I-type infectious bronchitis virus.

The prevalence of TW I-type infectious bronchitis virus (IBV) has been increasing rapidly, and it has become the second most common genotype of IBV in China threatening the poultry industry. In this study, 1-day-old specific-pathogen-free (SPF) chickens infected with TW I-type IBV were continuously observed for 200 days. TW I-type IBV affected the respiratory, urinary, and female reproductive systems, resulting in a mortality rate of 10% as well as a decrease in egg quantity and an increase in inferior eggs. During the monitoring period, serious lesions occurred in the female reproductive system, such as yolk peritonitis, a shortened oviduct, and cysts of different sizes with effusion in the degenerated right oviduct. The infective viruses persisted in vivo for a long time, and due to the stress of laying, virus shedding was detected again after the onset of egg production. Our findings suggest that TW I-type IBV is deadly to chickens and could cause permanent damage to the oviduct, resulting in the poor laying performance of female survivors and decreasing the breeding value and welfare of the infected flock.

Molecular and pathogenicity of infectious bronchitis virus (Gammacoronavirus) in Japanese quail (Coturnix japonica).

Infectious bronchitis virus (IBV) infection is highly infectious respiratory disease in poultry industry with significant economic importance. The prevalence of IBV in quail industry in Malaysia was not well documented; therefore, its actual role in the epidemiology of the disease is relatively unknown. This study was to determine the susceptibility of Japanese quail, as one of the species in commercial poultry industry, toward IBV. In addition, it will also give a potential impact on the overall health management in the quail industry even though it had been established that quail are resistant to diseases affecting poultry. Moreover, to the best of our knowledge, it is the first experimental study on IBV inoculation in quail. In this experimental study, 20 quails were divided into 4 groups (n = 5 for group A, B, and C, n = 5 for control group). The quails in group A, B, and C were infected via intraocular and intranasal routes with 0.2 mL of 10 × 5 EID50 of the virus. Clinical signs, gross lesions, positive detection of virus, and trachea histopathological scoring were used to assess the susceptibility of these Japanese quails. The results have indicated mild ruffled feathers and watery feces in these inoculated birds. Trachea, lung, and kidney were subjected to one-step reverse transcription polymerase chain reaction for virus detection. The virus was found from trachea and lung samples, whereas it was absent from all kidney samples. Only 3 quails were found with gross lesions. There was a significant difference of tracheal lesion by 0.009 ± 0.845 (P < 0.05) within the treatment groups. In summary, Japanese quails might be susceptible to IBV.

A Multi-Omics Study of Chicken Infected by Nephropathogenic Infectious Bronchitis Virus.

Chicken gout resulting from nephropathogenic infectious bronchitis virus (NIBV) has become a serious kidney disease problem in chicken worldwide with alterations of the metabolic phenotypes in multiple metabolic pathways. To investigate the mechanisms in chicken responding to NIBV infection, we examined the global transcriptomic and metabolomic profiles of the chicken's kidney using RNA-seq and GC-TOF/MS, respectively. Furthermore, we analyzed the alterations in cecal microorganism composition in chickens using 16S rRNA-seq. Integrated analysis of these three phenotypic datasets further managed to create correlations between the altered kidney transcriptomes and metabolome, and between kidney metabolome and gut microbiome. We found that 2868 genes and 160 metabolites were deferentially expressed or accumulated in the kidney during NIBV infection processes. These genes and metabolites were linked to NIBV-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. In addition, the comprehensive correlations between the kidney metabolome and cecal microbial community showed contributions of gut microbiota in the progression of NIBV-infection. Taken together, our research comprehensively describes the host responses during NIBV infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout.

Microarray analysis of infectious bronchitis virus infection of chicken primary dendritic cells.

BACKGROUND: Avian infectious bronchitis virus (IBV) is a major respiratory disease-causing agent in birds that leads to significant losses. Dendritic cells (DCs) are specialised cells responsible for sampling antigens and presenting them to T cells, which also play an essential role in recognising and neutralising viruses. Recent studies have suggested that non-coding RNAs may regulate the functional program of DCs. Expression of host non-coding RNAs changes markedly during infectious bronchitis virus infection, but their role in regulating host immune function has not been explored. Here, microarrays of mRNAs, miRNAs, and lncRNAs were globally performed to analyse how avian DCs respond to IBV. RESULTS: First, we found that IBV stimulation did not enhance the maturation ability of avian DCs. Interestingly, inactivated IBV was better able than IBV to induce DC maturation and activate lymphocytes. We identified 1093 up-regulated and 845 down-regulated mRNAs in IBV-infected avian DCs. Gene Ontology analysis suggested that cellular macromolecule and protein location (GO-BP) and transcription factor binding (GO-MF) were abundant in IBV-stimulated avian DCs. Meanwhile, pathway analysis indicated that the oxidative phosphorylation and leukocyte transendothelial migration signalling pathways might be activated in the IBV group. Moreover, alteration of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) was detected in IBV-stimulated avian DCs. In total, 19 significantly altered (7 up and 12 down) miRNAs and 101 (75 up and 26 down) lncRNAs were identified in the IBV-treated group. Further analysis showed that the actin cytoskeleton and MAPK signal pathway were related to the target genes of IBV-stimulated miRNAs. Finally, our study identified 2 TF-microRNA and 53 TF-microRNA-mRNA interactions involving 1 TF, 2 miRNAs, and 53 mRNAs in IBV-stimulated avian DCs. CONCLUSIONS: Our research suggests a new mechanism to explain why IBV actively blocks innate responses needed for inducing immune gene expression and also provides insight into the pathogenic mechanisms of avian IBV.

Genes involved in mitochondrial biogenesis and function may not show synchronised responses to mitochondria in shell gland of laying chickens under infectious bronchitis virus challenge.

BACKGROUND: Egg formation takes place in the oviduct of laying hens over a 24 h period. Infectious bronchitis virus (IBV) causes pathological lesions in the chicken oviduct. In the current study, mitochondrial counts were determined in three different segments of the oviduct during egg formation in laying chickens challenged with IBV T strain. Nuclear DNA encoded genes that are involved in mitochondrial biogenesis, fission and function were studied in the shell gland of the oviduct undergoing virus multiplication. RESULTS: In the shell gland, the mitochondrial count was significantly lower (P < 0.05) in the challenged group, compared with the control group. However, it did not vary in response to IBV challenge in the isthmus and magnum regions of the oviduct. The gene succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA) was down-regulated in the shell gland by IBV challenge (P < 0.05), while other genes being studied did not show responses to the challenge (P > 0.05). Differential expression of the genes was observed at different time-points of egg-shell formation. The expression levels of citrate synthase (CS), cytochrome C, somatic (CYC, S) and sodium-potassium adenosine triphosphatase (Na+-K+ATPase) genes were significantly higher, while those of SDHA and dynamin related protein 1 (Drp1) genes were significantly lower, at 15 h compared with 5 h following oviposition of the previous egg. The expression level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) did not show significant change at different time-points. CONCLUSIONS: It was concluded that IBV T strain infection in laying hens reduced mitochondrial counts only in the shell gland region of the oviduct. The genes involved in mitochondrial biogenesis or function may not show synchronised responses to that of mitochondria in the shell gland of chickens under T strain of IBV challenge.

Dietary inclusion of mushroom (Flammulina velutipes) stem waste on growth performance and immune responses in growing layer hens.

BACKGROUND: Medicinal mushrooms contain biologically active substances that can be used as an immune-modulating agent in poultry. The present study aimed to investigate the effects of Flammulina velutipes mushroom waste (FVW) on performance, immune response and serum immunity in growing layer hens. RESULTS: No significant differences (P > 0.05) were observed with respect to average daily feed intake, body weight gain and feed conversion ratio among the experimental groups during the entire study period (1-70 days). Antibody titers against Newcastle disease and infectious bronchitis were higher (P < 0.05) in the FVW fed groups than in the control and antibiotic groups. On day 28, serum immunoglobulin (Ig)A and IgG were higher (P < 0.05) in the 6% FVW group than in the control and antibiotic fed groups. On day 70, serum IgA was higher (P < 0.05) in FVW fed groups than in the control group; IgG was higher (P < 0.05) in the FVW groups than in the control and antibiotic groups. However, IgM was higher (P < 0.05) in both the 4% and 6% FVW groups than in the control and antibiotic groups for both experimental periods. Serum cytokine interleukin (IL)-2 and tumor necrosis factor-α concentrations were significantly higher (P < 0.05) in both the 4% and 6% FVW grousp than in the control and antibiotic groups; IL-4 was significantly higher (P < 0.05) in the FVW groups than in the control group; and IL-6 was significantly higher (P < 0.05) in the 6% FVW group than in the control and antibiotic groups. CONCLUSION: FVW at the 6% level can be used as a potential phytogenic feed stuff in growing layer hen rations with respect to improving the immune response without affecting normal weight gain. © 2018 Society of Chemical Industry.

Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non-translated regions of viral RNA and other viral proteins.

The cleavage products from coronavirus polyproteins, known as the non-structural proteins (nsps), are believed to make up the major components of the viral replication/transcription complex. In this study, several nsps encoded by avian gammacoronavirus infectious bronchitis virus (IBV) were screened for RNA-binding activity and interaction with its RNA-dependent RNA polymerase, nsp12. Nsp2, nsp5, nsp8, nsp9 and nsp10 were found to bind to untranslated regions (UTRs), while nsp8 was confirmed to interact with nsp12. Nsp8 has been reported to interact with nsp7 and functions as a primase synthesizing RNA primers for nsp12. Further characterization revealed that nsp8-nsp12 interaction is independent of the UTRs of viral RNA, and nsp8 interacts with both the N- and C-terminal regions of nsp12. These results have prompted a proposal of how the nsp7-nsp8 complex could possibly function in tandem with nsp12, forming a highly efficient complex that could synthesize both the RNA primer and viral RNA during coronavirus infection.

Activation of the c-Jun NH2-terminal kinase pathway by coronavirus infectious bronchitis virus promotes apoptosis independently of c-Jun.

Mitogen-activated protein kinases (MAPKs) are conserved protein kinases that regulate a variety of important cellular signaling pathways. Among them, c-Jun N-terminal kinases (JNK) are known to be activated by various environmental stresses including virus infections. Previously, activation of the JNK pathway has been detected in cells infected with several coronaviruses. However, detailed characterization of the pathway as well as its implication in host-virus interactions has not been fully investigated. Here we report that the JNK pathway was activated in cells infected with the avian coronavirus infectious bronchitis virus (IBV). Of the two known upstream MAPK kinases (MKK), MKK7, but not MKK4, was shown to be responsible for IBV-induced JNK activation. Moreover, knockdown and overexpression experiments demonstrated that JNK served as a pro-apoptotic protein during IBV infection. Interestingly, pro-apoptotic activity of JNK was not mediated via c-Jun, but involved modulation of the anti-apoptotic protein B-cell lymphoma 2 (Bcl2). Taken together, JNK constitutes an important aspect of coronavirus-host interaction, along with other MAPKs.

Infectious Bronchitis Virus Infection Induces Apoptosis during Replication in Chicken Macrophage HD11 Cells.

Avian infectious bronchitis has caused huge economic losses in the poultry industry. Previous studies have reported that infectious bronchitis virus (IBV) infection can produce cytopathic effects (CPE) and apoptosis in some mammalian cells and primary cells. However, there is little research on IBV-induced immune cell apoptosis. In this study, chicken macrophage HD11 cells were established as a cellular model that is permissive to IBV infection. Then, IBV-induced apoptosis was observed through a cell viability assay, morphological changes, and flow cytometry. The activity of caspases, the inhibitory efficacy of caspase-inhibitors and the expression of apoptotic genes further suggested the activation of apoptosis through both intrinsic and extrinsic pathways in IBV-infected HD11 cells. Additionally, ammonium chloride (NH4Cl) pretreated HD11 cells blocked IBV from entering cells and inhibited IBV-induced apoptosis. UV-inactivated IBV also lost the ability of apoptosis induction. IBV replication was increased by blocking caspase activation. This study presents a chicken macrophage cell line that will enable further analysis of IBV infection and offers novel insights into the mechanisms of IBV-induced apoptosis in immune cells.

The Important Role of Lipid Raft-Mediated Attachment in the Infection of Cultured Cells by Coronavirus Infectious Bronchitis Virus Beaudette Strain.

Lipid raft is an important element for the cellular entry of some viruses, including coronavirus infectious bronchitis virus (IBV). However, the exact role of lipid rafts in the cellular membrane during the entry of IBV into host cells is still unknown. In this study, we biochemically fractionated IBV-infected cells via sucrose density gradient centrifugation after depleting plasma membrane cholesterol with methyl-ß-cyclodextrin or Mevastatin. Our results demonstrated that unlike IBV non-structural proteins, IBV structural proteins co-localized with lipid raft marker caveolin-1. Infectivity assay results of Vero cells illustrated that the drug-induced disruption of lipid rafts significantly suppressed IBV infection. Further studies revealed that lipid rafts were not required for IBV genome replication or virion release at later stages. However, the drug-mediated depletion of lipid rafts in Vero cells before IBV attachment significantly reduced the expression of viral structural proteins, suggesting that drug treatment impaired the attachment of IBV to the cell surface. Our results indicated that lipid rafts serve as attachment factors during the early stages of IBV infection, especially during the attachment stage.

Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus.

It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to in vivo attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein.IMPORTANCE The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.

A Coronavirus Associated with Runting Stunting Syndrome in Broiler Chickens.

Runting stunting syndrome (RSS) is a disease condition that affects broilers and causes impaired growth and poor feed conversion because of enteritis characterized by pale and distended small intestines with watery contents. The etiology of the disease is multifactorial, and a large variety of viral agents have been implicated. Here we describe the detection and isolation of an infectious bronchitis virus (IBV) -like coronavirus from the intestines of a flock of 60,000 14-day-old brown/red broiler chicks. The birds showed typical clinical signs of RSS including stunting and uneven growth. At necropsy, the small intestines were pale and distended with watery contents. Histopathology of the intestines revealed increased cellularity of the lamina propria, blunting of villi, and cystic changes in the crypts. Negative stain electron microscopy of the intestinal contents revealed coronavirus particles. Transmission electron microscopy of the intestine confirmed coronavirus in the cytoplasm of enterocytes. Using immunohistochemistry (IHC), IBV antigen was detected in the intestinal epithelial cells as well as in the proventriculus and pancreas. There were no lesions in the respiratory system, and no IBV antigen was detected in trachea, lung, air sac, conjunctiva, and cecal tonsils. A coronavirus was isolated from the intestine of chicken embryos but not from the allantoic sac inoculated with the intestinal contents of the broiler chicks. Sequencing of the S1 gene showed nucleic acid sequence identities of 93.8% to the corresponding region of IBV California 99 and of 85.7% to IBV Arkansas. Nucleic acid sequence identities to other IBV genotypes were lower. The histopathologic lesions in the intestines were reproduced after experimental infection of specific-pathogen-free chickens inoculated in the conjunctiva and nares. Five days after infection, six of nine investigated birds showed enteritis associated with IBV antigen as detected by IHC. In contrast to the field infection, birds in the experimental group showed clear respiratory signs and lesions in the upper respiratory tract. The results suggest a broader tissue tropism of this isolate, which might be related to the mutations in the S1 gene.