Cyclooxygenase-2/prostaglandin E2 pathway regulates infectious bronchitis virus replication in avian macrophages.

Infectious bronchitis virus (IBV) is a significant respiratory pathogen that affects chickens worldwide. As an avian coronavirus, IBV leads to productive infection in chicken macrophages. However, the effects of IBV infection in macrophages on cyclooxygenase-2 (COX-2) expression are still to be elucidated. Therefore, we investigated the role of IBV infection on the production of COX-2, an enzyme involved in the synthesis of prostaglandin E2 (PGE2) in chicken macrophages. The chicken macrophage cells were infected with two IBV strains, and the cells and culture supernatants were harvested at predetermined time points to measure intracellular and extracellular IBV infection. IBV infection was quantified as has been the COX-2 and PGE2 productions. We found that IBV infection enhances COX-2 production at both mRNA and protein levels in chicken macrophages. When a selective COX-2 antagonist was used to reduce the COX-2 expression in macrophages, we observed that IBV replication decreased. When IBV-infected macrophages were treated with PGE2 receptor (EP2 and EP4) inhibitors, IBV replication was reduced. Upon utilizing a selective COX-2 antagonist to diminish PGE2 expression in macrophages, a discernible decrease in IBV replication was observed. Treatment of IBV-infected macrophages with a PGE2 receptor (EP2) inhibitor resulted in a reduction in IBV replication, whereas the introduction of exogenous PGE2 heightened viral replication. Additionally, pretreatment with a Janus-kinase two antagonist attenuated the inhibitory effect of recombinant chicken interferon (IFN)-γ on viral replication. The evaluation of immune mediators, such as inducible nitric oxide (NO) synthase (iNOS), NO, and interleukin (IL)-6, revealed enhanced expression following IBV infection of macrophages. In response to the inhibition of COX-2 and PGE2 receptors, we observed a reduction in the expressions of iNOS and IL-6 in macrophages, correlating with reduced IBV infection. Overall, IBV infection increased COX-2 and PGE2 production in addition to iNOS, NO, and IL-6 expression in chicken macrophages in a time-dependent manner. Inhibition of the COX-2/PGE2 pathway may lead to increased macrophage defence mechanisms against IBV infection, resulting in a reduction in viral replication and iNOS and IL-6 expressions. Understanding the molecular mechanisms underlying these processes may shed light on potential antiviral targets for controlling IBV infection.

Recombinant chimpanzee adenovirus expressing spike protein protects chickens against infectious bronchitis virus.

Infectious bronchitis (IB) is an acute and highly contagious disease caused by avian infectious bronchitis virus (IBV), resulting in significant economic losses in the global poultry industry. In this study, we utilized a replication-incompetent adenovirus vector derived from chimpanzees for the first time to express the S gene of IBV. The adenovirus was successfully rescued and demonstrated convenient production, good growth performance, and stability on HEK293 A cells. Morphologically, the recombinant adenovirus (named PAD-S) appeared normal under transmission electron microscopy, and efficient expression of the exogenous gene was confirmed through immunofluorescence analysis and immunoblotting. Administration of PAD-S via ocular and nasal routes induced a strong immune response in the chicken population, as evidenced by specific antibody and cytokine measurements. PAD-S was unable to replicate within chickens and showed low pre-existing immunity, demonstrating high safety and environmental friendliness. The robust immune response triggered by PAD-S immunization effectively suppressed viral replication in various tissues, alleviating clinical symptoms and tissue damage, thus providing complete protection against viral challenges in the chicken population. In conclusion, this study successfully developed an IBV candidate vaccine strain that possesses biosafety, high protective efficacy, and ease of production.

Forsythoside A inhibits apoptosis and autophagy induced by infectious bronchitis virus through regulation of the PI3K/Akt/NF-κB pathway.

IMPORTANCE: Infectious bronchitis virus (IBV) is an acute and highly infectious viral disease that seriously endangered the development of the chicken industry. However, due to the limited effectiveness of commercial vaccines, there is an urgent need to develop safe and effective anti-IBV drugs. Forsythoside A (FTA) is a natural ingredient with wide pharmacological and biological activities, and it has been shown to have antiviral effects against IBV. However, the antiviral mechanism of FTA is still unclear. In this study, we demonstrated that FTA can inhibit cell apoptosis and autophagy induced by IBV infection by regulating the PI3K/AKT/NF-κB signaling pathway. This finding is important for exploring the role and mechanism of FTA in anti-IBV infection, indicating that FTA can be further studied as an anti-IBV drug.

Revealing Novel-Strain-Specific and Shared Epitopes of Infectious Bronchitis Virus Spike Glycoprotein Using Chemical Linkage of Peptides onto Scaffolds Precision Epitope Mapping.

The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.

Baicalin mitigates nephropathogenic infectious bronchitis virus infection-induced spleen injury via modulation of mitophagy and macrophage polarization in Hy-Line chick.

Nephropathogenic infectious bronchitis virus (NIBV) infections continue to pose a significant hazard in the poultry industry. Baicalin is a natural flavonoid that has been reported to have antiviral activity, but its function in NIBV infection largely remains unclear. In this study, the antiviral mechanism of baicalin in the spleen of NIBV-infected chicks was mainly elucidated in mitophagy and macrophage polarization. 28-day-old Hy-Line brown chicks were randomly divided into four groups: the group of chicks was treated intranasally (in) with normal saline (0.2 mL) and subsequently divided into two groups: the Con group (basic diet), the Con+BA group (basic diet+10 mg/kg Baicalin); another group of chicks was intranasally infected with SX9 (10-5/0.2 mL) and subsequently divided into two groups: the Dis group (basic diet), the Dis+BA group (basic diet+10 mg/kg Baicalin). Spleen tissues were collected at 3, 7, and 11 days post infection (dpi). NIBV copy number was strikingly decreased in the spleens under BA treatment with infectious time. Histopathological examination showed enlarged and hemorrhagic white pulp and no clearly defined boundary between white pulp and red pulp in the Dis group, which could be improved by BA treatment. Meanwhile, the loss of cristae structure and vacuolization in mitochondria caused by NIBV infection was repaired in the Dis+BA group by ultrastructure observation. In addition, BA treatment inhibited the induction of mitophagy by NIBV infection. BA treatment also promoted innate immunity by enhancing type I IFN levels. Moreover, BA treatment up-regulated M1-related cytokines (iNOS, TNF-α, IL-1ß, IL-6) and inhibited M2-related cytokines (ARG2, IL-4, IL-10, Pparg) at the mRNA and protein levels. However, the results from the splenic tissues at 11 dpi are opposite results from 3 and 7 dpi. Immunofluorescence analysis for M1 macrophage marker iNOS and M2 macrophage marker CD163 further validated this result. Collectively, BA inhibited mitophagy and triggered IFN activation, and M1 polarization, which contributed to the inhibition of NIBV infection.

[Angiotensin converting enzyme 2 alleviates infectious bronchitis virus-induced cellular inflammation by suppressing IL-6/JAK2/STAT3 signaling pathway].

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.

Efficacy of a plant-produced infectious bronchitis virus-like particle vaccine in specific pathogen-free chickens.

Infectious bronchitis (IB) Gammacoronavirus causes a highly contagious respiratory disease in chickens that is listed by the World Organisation for Animal Health (WOAH). Its high mutation ability has resulted in numerous variants against which the commercially available live or recombinant vaccines singly offer limited protection. Agrobacterium-mediated transient expression in Nicotiana benthamiana (tobacco) plants was used here to produce a virus-like particle (VLP) vaccine expressing a modified full-length IBV spike (S) protein of a QX-like IB variant. In a challenge study with the homologous live IB QX-like virus, VLP-vaccinated birds produced S protein-specific antibodies comparable to those produced by live-vaccinated birds seroconverting with mean geometric titers of 6.8 and 7.2 log2, respectively. The VLP-vaccinated birds had reduced oropharyngeal and cloacal viral shedding compared to an unvaccinated challenged control and were more protected against tracheal ciliostasis than the live-vaccinated birds. While the results appeared similar, plant-produced IB VLPs are safer, more affordable, easier to produce and update to antigenically match any emerging IB variant, making them a more suitable alternative to IBV control than live-attenuated vaccines.

Infectious Bronchitis Virus California Variant CA1737 Isolated from a Commercial Layer Flock with Cystic Oviducts and Poor External Egg Quality.

False layer syndrome is a condition in which the reproductive tract of chicks is infected with infectious bronchitis virus (IBV) strains that cause permanent damage to the oviduct. These chickens subsequently develop cystic oviducts and do not lay eggs, and affected flocks fail to reach expected egg production peaks. The California Animal Health and Food Safety laboratory, Turlock Branch, received four separate case submissions from a 25-to-28-wk-old commercial ISA Brown layer flock. Birds were submitted for diagnostic evaluation due to suboptimal egg production and vent pecking. Submissions totaled 31 birds and consisted of live layers, recent mortality, and a flat of eggs. No clinical signs were observed in the submitted live birds. The most common gross findings included cystic left oviducts, signs of vent pecking, ovarian regression, and yolk coelomitis. The eggs were abnormally shaped with irregular, white, gritty deposits on the surface of the shell. Microscopically, there was atrophy of the oviducts, glandular hypoplasia, and lymphocytic salpingitis. In addition, lymphoplasmacytic tracheitis was observed, and renal tubules were dilated with multifocal areas of mineralization. IBV was identified by reverse transcription quantitative PCR from cecal tonsil tissue pools and tracheal swab pools. Sequencing of the S1 hypervariable region of IBV and whole-genome IBV sequencing were 97% homologous to the California variant CA1737/04. Definitive proof of the CA1737 strain's causing reproductive abnormalities will require challenge studies with fulfillment of Koch's postulates and evaluation of confounding and risk factors.

Reporte de caso- Virus de la bronquitis infecciosa Variante de California CA1737 aislada de una parvada comercial de ponedoras con oviductos quísticos y mala calidad externa del huevo. El síndrome de la falsa capa es una condición en la cual el tracto reproductivo de las gallinas está infectado con cepas del virus de la bronquitis infecciosa (IBV) que causan daño permanente al oviducto. Posteriormente, estas gallinas desarrollan oviductos quísticos y bajas en la postura de huevo, las parvadas afectadas no alcanzan los picos de producción de huevos esperados. El laboratorio de Salud Animal y Seguridad Alimentaria de California, con sede en Turlock, recibió cuatro casos separados de una parvada comercial de ponedoras ISA Brown de 25 a 28 semanas de edad. Las aves se enviaron para evaluación diagnóstica debido a una producción de huevos subóptima y por presencia de picoteo en las cloacas. Se recibieron un total de 31 aves y consistieron en aves de postura vivas, mortalidad reciente y además una charola de huevos. No se observaron signos clínicos en las aves vivas enviadas. Los hallazgos macroscópicos más comunes incluyeron oviductos izquierdos quísticos, signos de picoteo en las cloacas, regresión ovárica y celomitis de la yema. Los huevos tenían una forma anormal con depósitos irregulares, blancos y arenosos en la superficie de la cáscara. Microscópicamente, había atrofia de los oviductos, hipoplasia glandular y salpingitis linfocítica. Además, se observó traqueítis linfoplasmocítica y túbulos renales dilatados con áreas multifocales de mineralización. El virus de la bronquitis infecciosa se identificó mediante PCR cuantitativa de transcripción inversa a partir de grupos de tejidos de tonsilas cecales y muestras agrupadas de hisopos traqueales. La secuenciación de la región hipervariable S1 de IBV y la secuenciación de IBV del genoma completo fueron homólogas en un 97 % a la variante de California CA1737/04. La prueba definitiva de las anomalías reproductivas causantes de la cepa CA1737 requerirá estudios de desafío con el cumplimiento de los postulados de Koch y la evaluación de los factores de riesgo y de confusión.

Optimization of infectious bronchitis virus-like particle expression in Nicotiana benthamiana as potential poultry vaccines.

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease in chickens, with a severe economic impact on poultry production globally. The rapid emergence of regional variants of this Gammacoronavirus warrants new vaccine approaches that are more humane and rapid to produce than the current embryonated chicken egg-based method used for IB variant vaccine propagation (chemically-inactivated whole viruses). The production of virus-like particles (VLPs) expressing the Spike (S) glycoprotein, the major antigen which induces neutralizing antibodies, has not been achieved in planta up until now. In this study, using the Agrobacterium-mediated Nicotiana benthamiana (tobacco plant) transient expression system, the highest levels of VLPs displaying a modified S protein of a QX-like IB variant were obtained when the native transmembrane (TM) domain and cytoplasmic tail were substituted with that of the Newcastle disease virus (NDV) fusion glycoprotein, co-infiltrated with the NDV Matrix protein. In comparison, the native IB modified S co-infiltrated with IB virus membrane, envelope and nucleocapsid proteins, or substituted with the TM and CT of an H6-subtype influenza A virus hemagglutinin glycoprotein yielded lower VLP expression levels. Strong immunogenicity was confirmed in specific pathogen free chickens immunized intramuscularly with VLPs adjuvanted with Emulsigen®-P, where birds that received doses of 5 µg or 20 µg (S protein content) seroconverted after two weeks with mean hemaggluttination inhibition titres of 9.1 and 10 log2, respectively. Plant-produced IB VLP variant vaccines are safer, more rapid and cost effective to produce than VLPs produced in insect cell expression systems or the traditional egg-produced inactivated whole virus oil emulsion vaccines currently in use, with great potential for improved IB disease control in future.

Evaluation of protection and immunity induced by infectious bronchitis vaccines administered by oculonasal, spray or gel routes in commercial broiler chicks.

Broiler chicks' responses following combined IBV live attenuated Massachusetts and 793B strains through gel, spray or oculonasal (ON) vaccination routes were cross-compared. Subsequently, the responses following IBV M41 challenge of the unvaccinated and vaccinated groups were also assessed. Post-vaccination humoral and mucosal immune responses, alongside viral load kinetics in swabs and tissues, were determined using commercial ELISA assays, monoclonal antibody-based IgG and IgA ELISA assays and qRT-PCR respectively. After challenged with IBV-M41 strain, humoral and mucosal immune responses, ciliary protection, viral load kinetics, and immune gene mRNA transcriptions between the three vaccination methods were examined and compared. Findings showed that post-vaccinal humoral and mucosal immune responses were similar in all three vaccination methods. Post vaccinal viral load kinetics is influenced by method of administration. The viral load peaked in the ON group within the tissues and the OP/CL swabs in the first and third weeks respectively. Following M41 challenge, ciliary protection and mucosal immune responses were not influenced by vaccination methods as all three methods offered equal ciliary protection. Immune gene mRNA transcriptions varied by vaccination methods. Significant up-regulation of MDA5, TLR3, IL-6, IFN-α and IFN-ß genes were recorded for ON method. For both spray and gel methods, significant up-regulation of only MDA5 and IL-6 genes were noted. The spray and gel-based vaccination methods gave equivalent levels of ciliary protection and mucosal immunity to M41 virulent challenge comparable to those provided by the ON vaccination. Analysis of viral load and patterns of immune gene transcription of the vaccinated-challenged groups revealed high similarity between turbinate and choanal cleft tissues compared to HG and trachea. With regards to immune gene mRNA transcription, for all the vaccinated-challenged groups, similar results were found except for IFN-α, IFN-ß and TLR3, which were up-regulated only in ON compared to gel and spray vaccination methods.

In Silico Identification of Potential Inhibitors of the SARS-CoV-2 Main Protease among a PubChem Database of Avian Infectious Bronchitis Virus 3CLPro Inhibitors.

Remarkable structural homologies between the main proteases of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the avian infectious bronchitis virus (IBV) were revealed by comparative amino-acid sequence and 3D structural alignment. Assessing whether reported IBV 3CLPro inhibitors could also interact with SARS-CoV-2 has been undertaken in silico using a PubChem BioAssay database of 388 compounds active on the avian infectious bronchitis virus 3C-like protease. Docking studies of this database on the SARS-CoV-2 protease resulted in the identification of four covalent inhibitors targeting the catalytic cysteine residue and five non-covalent inhibitors for which the binding was further investigated by molecular dynamics (MD) simulations. Predictive ADMET calculations on the nine compounds suggest promising pharmacokinetic properties.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

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.

Chicken interferon-induced transmembrane protein 1 promotes replication of coronavirus infectious bronchitis virus in a cell-specific manner.

Interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit numerous virus infections by impeding viral entry into target cells. However, increasing evidence suggests diverse functions of IFITMs in virus infection, especially with the coronavirus. We analyzed the effect of chicken interferon-induced transmembrane proteins (chIFITMs) on coronavirus infectious bronchitis virus (IBV) infection in vitro. We demonstrated that the antiviral effects of IFITMs are dependent on cell and virus types. The overexpression of chIFITM1 dramatically promoted the replication of IBV Beaudette strain in the chicken hepatocellular carcinoma cell line, LMH. Mechanistically, chIFITMs share roughly the same subcellular localization in different host cells, and overexpressed of chIFITM1 have no effect of viral attachment and entry. Further studies revealed that mutations of amino acids at key positions (60KSRD63, 68KDFV71) in the intracellular loop domain (CIL) caused loss of the promoted function. Interaction with downstream proteins in co-response to viral infection could be the primary reason behind variable functions of chIFITM1 in different cells. In all, our study explored the functions of chIFITMs in viral infection from a new perspective.

Direct Interaction of Coronavirus Nonstructural Protein 3 with Melanoma Differentiation-Associated Gene 5 Modulates Type I Interferon Response during Coronavirus Infection.

Coronavirus nonstructural protein 3 (nsp3) is a multi-functional protein, playing a critical role in viral replication and in regulating host antiviral innate immunity. In this study, we demonstrate that nsp3 from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian coronavirus infectious bronchitis virus (IBV) directly interacts with melanoma differentiation-associated gene 5 (MDA5), rendering an inhibitory effect on the MDA5-mediated type I interferon (IFN) response. By the co-expression of MDA5 with wild-type and truncated nsp3 constructs, at least three interacting regions mapped to the papain-like protease (PLpro) domain and two other domains located at the N- and C-terminal regions were identified in SARS-CoV-2 nsp3. Furthermore, by introducing point mutations to the catalytic triad, the deubiquitylation activity of the PLpro domain from both SARS-CoV-2 and IBV nsp3 was shown to be responsible for the suppression of the MDA5-mediated type I IFN response. It was also demonstrated that both MDA5 and nsp3 were able to interact with ubiquitin and ubiquitinated proteins, contributing to the interaction between the two proteins. This study confirms the antagonistic role of nsp3 in the MDA5-mediated type I IFN signaling, highlighting the complex interaction between a multi-functional viral protein and the innate immune response.

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.

Engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes infectious bronchitis, an acute and highly contagious respiratory disease of chickens. IBV evolution under the pressure of comprehensive and widespread vaccination requires surveillance for vaccine resistance, as well as periodic vaccine updates. Reverse genetics systems are very valuable tools in virology, as they facilitate rapid genetic manipulation of viral genomes, thereby advancing basic and applied research. We report here the construction of an infectious clone of IBV strain Beaudette as a bacterial artificial chromosome (BAC). The engineered full-length IBV clone allowed the rescue of an infectious virus that was phenotypically indistinguishable from the parental virus. We used the infectious IBV clone and examined whether an enhanced green fluorescent protein (EGFP) can be produced by the replicase gene ORF1 and autocatalytically released from the replicase polyprotein through cleavage by the main coronavirus protease. We show that IBV tolerates insertion of the EGFP ORF at the 3' end of the replicase gene, between the sequences encoding nsp13 and nsp16 (helicase, RNA exonuclease, RNA endonuclease, and RNA methyltransferase). We further show that EGFP is efficiently cleaved from the replicase polyprotein and can be localized in double-membrane vesicles along with viral RNA polymerase and double-stranded RNA, an intermediate of IBV genome replication. One of the engineered reporter EGFP viruses were genetically stable during passage in cultured cells. We demonstrate that the reporter EGFP viruses can be used to study virus replication in host cells and for antiviral drug discovery and development of diagnostic assays. IMPORTANCE Reverse genetics systems based on bacterial artificial chromosomes (BACs) are the most valuable systems in coronavirus research. Here, we describe the establishment of a reverse genetics system for the avian coronavirus strain Beaudette, the most intensively studied strain. We cloned a copy of the avian coronavirus genome into a BAC vector and recovered infectious virus in permissive cells. We used the new system to construct reporter viruses that produce enhanced green fluorescent protein (EGFP). The EGFP coding sequence was inserted into 11 known cleavage sites of the major coronavirus protease in the replicase gene ORF1. Avian coronavirus tolerated the insertion of the EGFP coding sequence at three sites. The engineered reporter viruses replicated with parental efficiency in cultured cells and were sufficiently genetically stable. The new system facilitates functional genomics of the avian coronavirus genome but can also be used for the development of novel vaccines and anticoronaviral drugs.

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum.

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55-1.65% and 2.58-6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

Infection with IBV DMV/1639 at a Young Age Leads to Increased Incidence of Cystic Oviduct Formation Associated with False Layer Syndrome.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes respiratory disease but can affect the reproductive tract of laying-type chickens. If infection occurs in pullets, false layer syndrome, which is characterized by the development of large, fluid-filled cystic oviducts, can occur. Recently, IBV strain DMV/1639 has been detected in parts of Canada and the U.S., where false layer syndrome has occurred, though it is not clear if IBV is the sole cause or if age at infection is an influencing variable. Our study investigates the role and timing of IBV infection on the development of false layer syndrome, using the IBV types DMV/1639 and Massachusetts (Mass). Six groups of 120 SPF chickens were challenged at either three, seven, or fourteen days of age, using either DMV/1639 or Mass IBV. Cystic oviducts were seen in all the challenged groups, and the pullets challenged at 14 days of age had fewer cystic oviducts than pullets challenged at 3 or 7 days of age. The highest percentage of severe histology lesion scores were seen in the 3-day challenge groups. The data collected in this experiment confirm that IBV DMV/1639 causes cystic oviducts and indicate that age at infection plays a role in the pathogenesis of false layer syndrome.

Pathological effect of different avian infectious bronchitis virus strains on the bursa of Fabricius of chickens.

Infectious bronchitis is an acute and highly contagious disease caused by avian infectious bronchitis virus (IBV). As well as the typical clinical respiratory signs, such as dyspnoea and tracheal rales, QX genotype strains can also cause damage to the urinary system and reproductive system. Our previous studies found that chickens infected with QX-type IBV also displayed damage to the bursa of Fabricius. To investigate the effects of different genotypes of IBV on the bursa of Fabricius, we challenged one-week-old SPF chickens with Mass, QX and TW genotype IBV strains and compared the clinical signs, gross lesions, histopathological damage, viral loads, and expression levels of inflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-α,ß, γ and TNF-α). The results showed that all three strains caused tissue damage, while significant temporal variations in the viral loads of the different infected groups were detected. IBV infection seriously interfered with the natural immune response mediated by inflammatory cytokines (IFN-α, IFN-ß, IL-6 and IFN-γ) in chickens. Our results suggested that IBV has potential immunological implications for chickens that may lead to poor production efficiency. RESEARCH HIGHLIGHTSAvian coronavirus IBV is an important pathogen of chickens.IBV has potential immunological implications in chickens.The bursal viral load of different IBV strains varies significantly.