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.

Non-Targeted Metabolomic Analysis of Chicken Kidneys in Response to Coronavirus IBV Infection Under Stress Induced by Dexamethasone.

Stress in poultry can lead to changes in body metabolism and immunity, which can increase susceptibility to infectious diseases. However, knowledge regarding chicken responses to viral infection under stress is limited. Dexamethasone (Dex) is a synthetic glucocorticoid similar to that secreted by animals under stress conditions, and has been widely used to induce stress in chickens. Herein, we established a stress model in 7-day-old chickens injected with Dex to elucidate the effects of stress on IBV replication in the kidneys. The metabolic changes, immune status and growth of the chickens under stress conditions were comprehensively evaluated. Furthermore, the metabolic profile, weight gain, viral load, serum cholesterol levels, cytokines and peripheral blood lymphocyte ratio were compared in chickens treated with Dex and infected with IBV. An LC-MS/MS-based metabolomics method was used to examine differentially enriched metabolites in the kidneys. A total of 113 metabolites whose abundance was altered after Dex treatment were identified, most of which were lipids and lipid-like molecules. The principal metabolic alterations in chicken kidneys caused by IBV infection included fatty acid, valine, leucine and isoleucine metabolism. Dex treatment before and after IBV infection mainly affected the host's tryptophan, phenylalanine, amino sugar and nucleotide sugar metabolism. In addition, Dex led to up-regulation of serum cholesterol levels and renal viral load in chickens, and to the inhibition of weight gain, peripheral blood lymphocytes and IL-6 production. We also confirmed that the exogenous cholesterol in DF-1 cells promoted the replication of IBV. However, whether the increase in viral load in kidney tissue is associated with the up-regulation of cholesterol levels induced by Dex must be demonstrated in future experiments. In conclusion, chick growth and immune function were significantly inhibited by Dex. Host cholesterol metabolism and the response to IBV infection are regulated by Dex. This study provides valuable insights into the molecular regulatory mechanisms in poultry stress, and should support further research on the intrinsic link between cholesterol metabolism and IBV replication under stress conditions.

Interference between avian corona and influenza viruses: The role of the epithelial architecture of the chicken trachea.

Respiratory viral infections are among the major causes of disease in poultry. While viral dual infections are known to occur, viral interference in chicken airways is mechanistically hardly understood. The effects of infectious bronchitis virus (IBV) infection on tissue morphology, sialic acid (sia) expression and susceptibility of the chicken trachea for superinfection with IBV or avian influenza virus (AIV) were studied. In vivo, tracheal epithelium of chickens infected with IBV QX showed marked inflammatory cell infiltration and loss of cilia and goblet cells five days post inoculation. Plant lectin staining indicated that sialic acids redistributed from the apical membrane of the ciliated epithelium and the goblet cell cytoplasm to the basement membrane region of the epithelium. After administration of recombinant viral attachment proteins to slides of infected tissue, retained binding of AIV hemagglutinin, absence of binding of the receptor binding domain (RBD) of IBV M41 and partial reduction of IBV QX RBD were observed. Adult chicken trachea rings were used as ex vivo model to study the effects of IBV QX-induced pathological changes and receptor redistribution on secondary viral infection. AIV H9N2 infection after primary IBV infection was delayed; however, final viral loads reached similar levels as in previously uninfected trachea rings. In contrast, IBV M41 superinfection resulted in 1000-fold lower viral titers over the course of 48 h. In conclusion, epithelial changes in the chicken trachea after viral infection coincide with redistribution and likely specific downregulation of viral receptors, with the extend of subsequent viral interference dependent on viral species.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

Role of Stress Granules in Suppressing Viral Replication by the Infectious Bronchitis Virus Endoribonuclease.

Infectious bronchitis virus (IBV), a γ-coronavirus, causes the economically important poultry disease infectious bronchitis. Cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation. Previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. Here, we aimed to delineate the molecular mechanisms regulating the SG response to pathogenic IBV strain infection. We found that most chicken embryo kidney (CEK) cells formed no SGs during IBV infection and IBV replication inhibited arsenite-induced SG formation. This inhibition was not caused by changes in the integrity or abundance of SG proteins during infection. IBV nonstructural protein 15 (Nsp15) endoribonuclease activity suppressed SG formation. Regardless of whether Nsp15 was expressed alone, with recombinant viral infection with Newcastle disease virus as a vector, or with EndoU-deficient IBV, the Nsp15 endoribonuclease activity was the main factor inhibiting SG formation. Importantly, uridine-specific endoribonuclease (EndoU)-deficient IBV infection induced colocalization of IBV N protein/dsRNA and SG-associated protein TIA1 in infected cells. Additionally, overexpressing TIA1 in CEK cells suppressed IBV replication and may be a potential antiviral factor for impairing viral replication. These data provide a novel foundation for future investigations of the mechanisms by which coronavirus endoribonuclease activity affects viral replication. IMPORTANCE Endoribonuclease is conserved in coronaviruses and affects viral replication and pathogenicity. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal, and reproductive systems, causing millions of dollars in lost revenue to the poultry industry worldwide annually. Mutating the viral endoribonuclease poly(U) resulted in SG formation, and TIA1 protein colocalized with the viral N protein and dsRNA, thus damaging IBV replication. These results suggest a new antiviral target design strategy for coronaviruses.

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.

Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present.

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

Infectious bronchitis virus in Australia: a model of coronavirus evolution - a review.

Infectious bronchitis virus (IBV) was first isolated in Australia in 1962. Ongoing surveillance and characterization of Australian IBVs have shown that they have evolved separately from strains found throughout the rest of the world, resulting in the evolution of a range of unique strains and changes in the dominant wild-type strains, affecting tissue tropism, pathogenicity, antigenicity, and gene arrangement. Between 1961 and 1976 highly nephropathogenic genotype GI-5 and GI-6 strains, causing mortalities of 40% to 100%, predominated, while strains causing mainly respiratory disease, with lower mortality rates, have predominated since then. Since 1988, viruses belonging to two distinct and novel genotypes, GIII and GV, have been detected. The genome organization of the GIII strains has not been seen in any other gammacoronavirus. Mutations that emerged soon after the introduction of vaccination, incursion of strains with a novel lineage from unknown sources, recombination between IBVs from different genetic lineages, and gene translocations and deletions have contributed to an increasingly complex IBV population. These processes and the consequences of this variation for the biology of these viruses provide an insight into the evolution of endemic coronaviruses during their control by vaccination and may provide a better understanding of the potential for evolution of other coronaviruses, including SARS-CoV-2. Furthermore, the continuing capacity of attenuated IBV vaccines developed over 40 years ago to provide protection against viruses in the same genetic lineage provides some assurance that coronavirus vaccines developed to control other coronaviruses may continue to be effective for an extended period.

Transcriptome analysis of primary chicken cells infected with infectious bronchitis virus strain K047-12 isolated in Korea.

Infectious bronchitis virus (IBV), an avian coronavirus, is highly contagious. Chickens with IBV infection develop acute pathogenesis in multiple organs, including the respiratory and urogenital tracts. Frequent recombination in the spike (S) glycoprotein gene has made vaccine strategies ineffective. To understand IBV pathogenesis, we analyzed the genetic distance between Korean IBV isolates and other coronaviruses, including SARS-CoV-2. To obtain comprehensive information about early immune responses such as innate cytokine production and associated immune regulation during IBV infection, we infected primary chicken embryonic kidney cells and performed transcriptome analysis. We observed that the functional pathways of innate immunity are regulated and confirmed expression of genes that coordinate early immune responses. Understanding the immune profile of the host cell may assist in vaccine development.

A multifactorial score including autophagy for prognosis and care of COVID-19 patients.

In less than eleven months, the world was brought to a halt by the COVID-19 outbreak. With hospitals becoming overwhelmed, one of the highest priorities concerned critical care triage to ration the scarce resources of intensive care units. Which patient should be treated first? Based on what clinical and biological criteria? A global joint effort rapidly led to sequencing the genomes of tens of thousands of COVID-19 patients to determine the patients' genetic signature that causes them to be at risk of suddenly developing severe disease. In this commentary, we would like to consider some points concerning the use of a multifactorial risk score for COVID-19 severity. This score includes macroautophagy (hereafter referred to as autophagy), a critical host process that controls all steps harnessed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Abbreviation list: ATG5: autophagy related 5; BECN1: beclin 1; COVID-19: coronavirus infectious disease-2019; EGR1: early growth response 1; ER: endoplasmic reticulum; DMVs: double-membrane vesicles; IBV: infectious bronchitis virus; MAP1LC3: microtubule associated protein 1 light chain 3; LC3-I: proteolytically processed, non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; MEFs: mouse embryonic fibroblasts; MERS-CoV: Middle East respiratory syndrome-coronavirus; MHV: mouse hepatitis virus; NSP: non-structural protein; PEDV: porcine epidemic diarrhea virus; PLP2-TM: membrane-associated papain-like protease 2; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TGEV: transmissible gastroenteritis virus.

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.

Multiple recombination events between field and vaccine strains resulted in the emergence of a novel infectious bronchitis virus with decreased pathogenicity and altered replication capacity.

In this study, we isolated and identified 2 infectious bronchitis virus (IBV) strains from layer chickens soon after vaccination with the Massachusetts-Connecticut bivalent vaccine (Conn) and H120 and 4/91 booster vaccines in China in 2011. The results of cross-virus-neutralization tests and phylogenetic analysis of the S1 subunit of spike gene of these vaccine strains and other reference strains showed that strain LJL/110302 was of GI-19 lineage, whereas LLN/111169 was of the GI-1 lineage of the Conn serotype. Further comparative genomic analysis revealed that LLN/111169, an IBV strain with novel traits, originated from multiple recombination events (at least 3 recombination sites) between GI-19 and the Conn and 4/91 vaccine strains. LLN/111169 was pathogenic to specific pathogen-free (SPF) chickens. This is of prime importance because while IBV prevention measures worldwide are mainly dependent on modified live vaccine strains, our results showed that recombination between field and vaccine strains has produced a novel pathogenic IBV strain. In addition, LLN/111169 showed relatively broad tissue tropism (trachea, lungs, kidneys, and cecal tonsils) in infected SPF chickens. These results emphasize the importance of IBV surveillance in chicken flocks.

Three Amino Acid Changes in Avian Coronavirus Spike Protein Allow Binding to Kidney Tissue.

Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here, we elucidate the determinants for kidney tropism by studying interactions between the receptor-binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the nonnephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by preincubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by enzyme-linked immunosorbent assay (ELISA), demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99 to 159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110 to 112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor-binding site for QX is located at a different location on the spike than that of M41.IMPORTANCE Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic (example, IBV-M41) and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field.

Emerging lethal infectious bronchitis coronavirus variants with multiorgan tropism.

Infectious bronchitis virus (IBV) causes respiratory diseases in chickens and poses an economic threat to the poultry industry worldwide. Despite vaccine use, there have been field outbreaks of IBV in Taiwan. This study aimed to characterize the emerging IBV variants circulating in Taiwan. The analysis of the structural protein genes showed that these variants emerged through frequent recombination events among Taiwan strains, China strains, Japan strains and vaccine strains. Cross-neutralization tests revealed that two of the variants exhibited novel serotypes. Clinicopathological assessment showed that two of the variants caused high fatality rates of 67% and 20% in one-day-old SPF chicks, and all the variants possessed multiorgan tropisms, including trachea, proventriculus and urogenital tissues. Furthermore, the commercial live-attenuated Mass-type vaccine conferred poor protection against these variants. This study identified novel genotypes, serotypes and pathotypes of emerging IBV variants circulating in Taiwan. There is an urgent need for effective countermeasures against these variant strains.

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.

Isolation and Propagation of Coronaviruses in Embryonated Eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.

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.

A highly pathogenic GI-19 lineage infectious bronchitis virus originated from multiple recombination events with broad tissue tropism.

In the present study, an IBV strain I0305/19 was isolated from a diseased commercial broiler flock in 2019 in China with high morbidity and mortality. The isolate I0305/19 was clustered together with viruses in sublineage D of GI-19 lineage on the basis of the complete S1 sequence analysis. Isolate I0305/19 and other GI-19 viruses isolated in China have the amino acid sequence MIA at positions 110-112 in the S protein. Further analysis based on the complete genomic sequence showed that the isolate emerged through at least four recombination events between GI-19 ck/CH/LJS/120848- and GI-13 4/91-like strains, in which the S gene was found to be similar to that of the GI-19 ck/CH/LJS/120848-like strain. Pathological assessment showed the isolate was a nephropathogenic IBV strain that caused high morbidity of 100 % and mortality of 80 % in 1-day-old specific-pathogen-free (SPF) chicks. The isolate I0305/19 exhibited broader tropisms in different tissues, including tracheas, lungs, bursa of Fabricius, spleen, liver, kidneys, proventriculus, small intestines, large intestines, cecum, and cecal tonsils. Furthermore, subpopulations of the virus were found in tissues of infected chickens; this finding is important in understanding how the virulent IBV strains can potentially replicate and evolve to cause disease. This information is also valuable for understanding the mechanisms of replication and evolution of other coronaviruses such as the newly emerged SARS-CoV-2.

Effect of TLR agonist on infections bronchitis virus replication and cytokine expression in embryonated chicken eggs.

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-ß, IFN-γ, IL-1ß, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1ß and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.