Infectious Bronchitis Virus Infection Research Articles

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.

Mitigation of False Layer Syndrome Through Maternal Antibodies Against Infectious Bronchitis Virus.

The relationship between passive immunity and the development of false layer syndrome (FLS) and its associated lesions was investigated in this study by comparing the long-term reproductive effects of an infectious bronchitis virus (IBV) DMV/1639 wild-type strain and the GA08 vaccine in birds with and without maternal antibodies. There was a clear protective effect provided by maternal antibodies against both the early vaccination and challenge. It was also observed that vaccination at an early age, in the absence of maternal antibodies, can induce reproductive issues, such as reduced egg production and FLS-associated lesions (e.g., cystic oviduct and egg yolk coelomitis). This might indicate that maternal antibodies and the timing of IBV infection are more important in the generation of FLS than the IBV strain type.

Comparative pathogenicity of CA1737/04 and Mass infectious bronchitis virus genotypes in laying chickens.

Infectious bronchitis virus (IBV) is a respiratory virus causing atropism in multiple body systems of chickens. Recently, the California 1737/04 (CA1737/04) IBV strain was identified as one of the circulating IBV variants among poultry operations in North America. Here, the pathogenicity and tissue tropism of CA1737/04 IBV strain in specific-pathogen-free (SPF) hens were characterized in comparison to Massachusetts (Mass) IBV. In 30 weeks-old SPF hens, Mass or CA1737/04 IBV infections were carried out, while the third group was maintained as a control group. Following infection, we evaluated clinical signs, egg production, viral shedding, serology, necropsy examination, and histopathology during a period of 19 days. Also, certain tissue affinity parameters were investigated, which involved the localization of viral antigens and the detection of viral RNA copies in designated tissues. Our findings indicate that infection with CA1737/04 or Mass IBV strain could induce significant clinical signs, reduced egg production, and anti-IBV antibodies locally in oviduct wash and systemically in serum. Both IBV strains showed detectable levels of viral RNA copies and induced pathology in respiratory, renal, enteric, and reproductive tissues. However, the CA1737/04 IBV strain had higher pathogenicity, higher tissue tropism, and higher replication in the kidney, large intestine, and different segments of the oviduct compared to the Mass IBV strain. Both IBV strains shed viral genome from the cloacal route, however, the Mass IBV infected hens shed higher IBV genome loads via the oropharyngeal route compared to CA1737/04 IBV-infected hens. Overall, the current findings could contribute to a better understanding of CA1737/04 IBV pathogenicity in laying hens.

Baicalin Protects Broilers against Avian Coronavirus Infection via Regulating Respiratory Tract Microbiota and Amino Acid Metabolism.

An increasing amount of evidence indicates that Baicalin (Bai, a natural glycosyloxyflavone compound) exhibits an antiviral effect against avian viruses. However, it remains unclear if the antiviral effect of Bai against infectious bronchitis virus (IBV) is exerted indirectly by modulating respiratory tract microbiota and/or their metabolites. In this study, we investigated the protection efficacy of Bai in protecting cell cultures and broilers from IBV infection and assessed modulation of respiratory tract microbiota and metabolites during infection. Bai was administered orally to broilers by being mixed in with drinking water for seven days. Ultimately, broilers were challenged with live IBV. The results showed that Bai treatment reduced respiratory tract symptoms, improved weight gain, slowed histopathological damage, reduced virus loads and decreased pro-inflammation cytokines production. Western blot analysis demonstrated that Bai treatment significantly inhibited Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88) and nuclear factor kappa-B (NF-κB) expression both in cell culture and cells of the trachea. Bai treatment reversed respiratory tract microbiota dysbiosis, as shown by 16S rDNA sequencing in the group of broilers inoculated with IBV. Indeed, we observed a decrease in Proteobacteria abundance and an increase in Firmicutes abundance. Metabolomics results suggest that the pentose phosphate pathway, amino acid and nicotinamide metabolism are linked to the protection conferred by Bai against IBV infection. In conclusion, these results indicated that further assessment of anti-IBV strategies based on Bai would likely result in the development of antiviral molecule(s) which can be administered by being mixed with feed or water.

Antiviral effect of palmatine against infectious bronchitis virus through regulation of NF-κB/IRF7/JAK-STAT signalling pathway and apoptosis

ABSTRACT 1. Infectious bronchitis virus (IBV), a gamma-coronavirus, can infect chickens of all ages and leads to an acute contact respiratory infection. This study evaluated the anti-viral activity of palmatine, a natural non-flavonoid alkaloid, against IBV in chicken embryo kidney (CEK) cells. 2. The half toxic concentration (CC50) of palmatine was 672.92 μM, the half inhibitory concentration (IC50) of palmatine against IBV was 7.76 μM and the selection index (SI) was 86.74. 3. Mode of action assay showed that palmatine was able to directly inactivate IBV and inhibited the adsorption, penetration and intracellular replication of IBV. 4. Palmatine significantly upregulated TRAF6, TAB1 and IKK-β compared with the IBV-infected group, leading to the increased expressions of pro-inflammatory cytokines IL-1β and TNF-α in the downstream NF-κB signalling pathway. 5. Palmatine significantly up-regulated the levels of MDA5, MAVS, IRF7, IFN-α and IFN-β in the IRF7 pathway, inducing type I interferon production. It up-regulated the expression of 2’5’-oligoadenylate synthase (OAS) in the JAK-STAT pathway. 6. IBV infection induced cell apoptosis and palmatine-treatment delayed the process of apoptosis by regulation of the expression of apoptosis-related genes (BAX, BCL-2, CASPASE-3 and CASPASE-8). 7. Palmatine could exert anti-IBV activity through regulation of NF-κB/IRF7/JAK-STAT signalling pathways and apoptosis, providing a theoretical basis for the utilisation of palmatine to treat IBV infection.

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.

Seroprevalence and molecular characterisation of infectious bronchitis virus (IBV) in broiler farms in Sabah, Malaysia.

Infectious bronchitis virus (IBV) is classified as a highly contagious viral agent that causes acute respiratory, reproductive and renal system pathology in affected poultry farms. Molecular and serological investigations are crucial for the accurate diagnosis and management of IBV. The purpose of this study was to determine the seroprevalence of IBV and to characterise the circulating IBV in poultry farms in Sabah Province, Malaysia. To determine IBV antibodies, a total of 138 blood samples and 50 organ samples were collected from 10 commercial broiler flocks in 3 different farms by using the enzyme-linked immunosorbent assay (ELISA) (IDEXX Kit) and reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing. A total of 94.2% (130/138) of the samples were seropositive for IBV in the vaccinated flock, and 38% (52/138) of the birds was the IBV titre for infection. The selected seropositive samples for IBV were confirmed by RT-PCR, with 22% (11/50) being IBV positive amplified and sequenced by targeted highly conserved partial nucleocapsid (N) genes. Subsequently, phylogenetic analysis constructed using amplified sequences again exposed the presence of Connecticut, Massachusetts, and Chinese QX variants circulating in poultry farms in Sabah, Malaysia. The unexpectedly increasing mean titres in serology indicated that post infection of IBV and highly prevalent IBV in selected farms in this study. The sequencing and phylogenetic analysis revealed the presence of multiple IBV variants circulating in Malaysian chicken farms in Sabah, which further monitoring of genetic variation are needed to better understand the genetic diversity.

Synergistic anti-infectious bronchitis virus activity of Phillygenin combined Baicalin by modulating respiratory microbiota and improving metabolic disorders

Phillygenin (PHI) and Baicalin (Bai) are the major chemical ingredients extracted from Forsythia suspensa and Scutellaria baicalensis, respectively. The mixture of Forsythia suspensa and Scutellaria baicalensis according to the theories of Traditional Chinese Veterinary Medicine, compounded formulation can effectively exert heat-clearing and detoxifying effect, but the synergistic anti-IBV activity of PHI combined with Bai was unclear. Here, the protection of PHI combined with Bai on avian infectious bronchitis virus (IBV) M41 infection and the change of respiratory microbiota and metabolomics profiles in broilers that infected with IBV were investigated. According to the experimental findings, the combination of PHI and Bai effectively alleviated broilers' slowing-growth weight and respiratory symptoms. This was accompanied by a reduction in viral copies and histopathological changes, as well as an increase of antiviral protein (G3BP1) level in tracheas and anti-IBV antibody levels in serum. In addition, 16s RNA sequencing revealed that IBV infection significantly changed respiratory microbiota composition at different taxonomic levels and respiratory metabolism composition in broilers. Interestingly, PHI combined with Bai modulated the composition of respiratory microfloras, especially the abundance of Firmicutes and Lactobacillaceae were upregulated, as well as the abundance of Proteobacteria was downregulated. The metabolomics results indicated that PHI combined with Bai involved in glucose, lipids, amino acids and nucleotide metabolism during IBV infection. In summary, PHI combined with Bai exhibited a synergistic effect on preventing infectious bronchitis (IB), with the protection being closely associated with the composition of respiratory microbiota and metabolites. Therefore, adding the mixture of PHI and Bai to the chicken drinking water is recommended to prevent and control IB in clinical.

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

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.

Comparative proteomics analysis of kidney in chicken infected by infectious bronchitis virus

The gamma coronavirus infectious bronchitis virus (IBV) is known to cause an acute and highly contagious infectious disease in poultry. Here, this study aimed to investigate the impact of virulent or avirulent IBV infection on the avian host by conducting proteomics with data-independent acquisition mass spectrometry (DIA-MS) in the kidneys of IBV-infected chickens. The results revealed 267, 489, and 510 differentially expressed proteins (DEPs) in the chicken kidneys at 3, 5, and 7 days postinfection (dpi), respectively, when infected with the GD17/04 strain, which is a highly nephrogenic strain and belongs to the 4/91 genotype. In contrast, the attenuated 4/91 vaccine resulted in the identification of 144, 175, and 258 DEPs at 3, 5, and 7 dpi, respectively. Functional enrichment analyses indicated distinct expression profiles between the 2 IBV strains. Upon GD17/04 infection, metabolic pathways respond initially in the early stage (3 dpi) and immune-related signaling pathways respond in the middle and late stages (5 and 7 dpi). The 4/91 vaccine elicited a completely opposite response compared to the GD17/04 infection. Among all DEPs, 62 immune-related DEPs were focused on and found to be mainly enriched in the type I interferon (IFN-I) signaling pathway and involved in humoral and cellular immunity. Notably, key molecules in the IFN-I signaling pathway including MDA5, LGP2, and TBK1 may serve as regulatory targets of IBV. Overall, this study highlights similarities and discrepancies in the patterns of protein expression at different stages of infection with virulent and avirulent IBV strains, with the IFN-I signaling pathway emerging as a critical response to IBV infection.

SILVER NANOPARTICLES INHIBIT INFECTIOUS BRONCHITIS VIRUS REPLICATION

Objective: Avian infectious bronchitis virus (IBV) threatens the poultry industry and causes global economic losses. The IBV is highly variable. Thus, no effective drugs are available. Objective of the present study was to evaluate silver nanoparticles against it as an antiviral agent. Methods: Silver nanoparticles (AgNPs) have been evaluated as antivirals against IBV. P. betle leaf extract biosynthesizes AgNPs from silver nitrate. UV/vis absorption, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) have been used to characterize AgNPs. TEM indicated particle sizes of 5–30 nm, and XRD demonstrated their characteristic AgNPs structure. The antiviral activity of AgNPs was measured by the log embryo infective dose 50 (logEID50)/ml and the number of IBV genome copies. Results: XRD analysis showed a structure for AgNPs, and transmission electron microscopy showed a size of 5–30 nm for AgNPs. AgNPs at a noncytotoxic concentration inhibit the interaction between the virus and the cell, preventing the virus from entering the cell and reducing the number of IBV genome copies (per µl) in ovo by preventing the formation of the IBV RNA genome, resulting in a significant reduction in the IBV titer. Conclusion: AgNPs possess antiviral properties that inhibit IBV replication in ovo. The findings indicate that AgNPs are a promising drug candidate for treating or preventing IBV infection.

Andrographolide inhibits infectious bronchitis virus-induced apoptosis, pyroptosis, and inflammation.

Avian infectious bronchitis virus (IBV), a coronavirus, causes a huge economic loss to the poultry industry. Andrographolide (APL) is a compound with a variety of pharmacological properties, including antiviral and anti-inflammatory effects. In this study, APL was evaluated for antiviral activity by its anti-apoptotic, anti-pyroptosis, and anti-inflammatory effects. The cytotoxicity of APL was determined by the MTT method. We investigated the therapeutic impact of APL on IBV through a plate assay. We explored that APL inhibited IBV-induced apoptosis, pyroptosis, and inflammation in HD11 cells by RT-qPCR and immunofluorescence. Also, it was verified in the clinical chicken embryo trial. We found that APL down-regulated apoptosis-related genes Caspase-3, Caspase-8, Caspase-9, Bax, Bid, and Bak, down-regulated pyroptosis gene DFNA5, and down-regulated inflammation-related genes (NF-κB, NLRP3, iNOS, TNF-α, and IL-1β). In addition, APL reduced the reactive oxygen species (ROS) production in cells. Finally, clinical trials showed that APL inhibited IBV-induced apoptosis, pyroptosis, and inflammation, as well as reduced the mortality and malformation of chicken embryos. In this study, we delved into the antiviral properties of APL in the context of chicken macrophage (HD11) infection with IBV. Our findings confirm that andrographolide effectively inhibits apoptosis, pyroptosis, and inflammation by IBV infection. Furthermore, this inhibition was verified on chicken embryos in vivo. This inhibition suggests a substantial potential for APL as a therapeutic agent to mitigate the harmful effects of IBV on host cells.

Study of coinfection with local strains of infectious bursal disease virus and infectious bronchitis virus in specific pathogen-free chickens

Immunosuppressive diseases cause great losses in the poultry industry, increasing the susceptibility to infections by other pathogens and promoting a suboptimal response to vaccination. Among them, infectious bursal disease virus (IBDV) arises as one of the most important around the world. IBDV infects immature B lymphocytes, affecting the immune status of birds and facilitating infections by other pathogens such as avian infectious bronchitis virus (IBV). Although it has been reported that the interaction between these viruses increases IBV clinical signs, there are no actual studies about the interaction between regional circulating isolates that validate this statement. In this context, the objective of our work was to evaluate the effect of the interaction between local isolates of IBDV (belonging to genogroup 4) and IBV (lineage GI-16) in chickens. Thus, specific pathogen-free chickens were orally inoculated with IBDV genogroup (G) 4 or with PBS at 5 d of age. At 14-days postinoculation (dpi) the animals were intratracheally inoculated with a GI-16 IBV or with PBS. At multiple time points, groups of birds were euthanized and different parameters such as histological damage, viral load, lymphocyte populations and specific antibodies were evaluated. The success of IBDV infection was confirmed by the severity of bursal atrophy, viral detection, and presence of anti-IBDV antibodies. In IBV-infected animals, the presence of viral genome was detected in both kidney and bursa. The coinfected animals showed higher degree of lymphocyte infiltration in kidney, higher rate of animals with IBV viral genome in bursa at 28 dpi, and a clear decrease in antibody response against IBV at 28, 35, and 40 dpi. The results indicate that the infection with the local isolate of IBDV affects the immune status of the chickens, causing major severe damage, in response to IBV infection, which could consequently severely affect the local poultry industry.

Pathological assessment and tissue tropism of two different Egyptian infectious bronchitis strains.

Avian infectious bronchitis is one of the most common viral infections in chickens affecting all ages. The tropism of infectious bronchitis virus (IBV) strains became broader and more variable posing major implications for the effective control of IBV infection. In this study, two IBV viruses representing classic and variant strains were inoculated intranasally into day-old SPF chicks (105 EID50/0.2ml/bird). Clinical signs were observed for 15 days post-infection (DPI). Five chicks from each group were euthanized at 2, 4, 6, 8, 10, 12, and 15 DPI for histopathology and virus antigen detection by IHC and quantitative rRT-PCR. Results revealed that both classic and variant IBV strains induced mild clinical signs with no mortalities and fewer various histopathological lesions in infected SPF chickens. Although the viruses were detected by rRT-PCR up to 12DPI, the affected tissues showed regeneration after 10DPI with IHC revealing no IBV antigen. In summary, no differences were found in the behaviour of both IBV isolates in chickens. The broad tissue tropism for both IBV strains as indicated by viral antigen detection in various organs with no clinical or gross lesion suggest that the main cause of death in IBV infection under field conditions occurs as a result of complication with secondary infections rather single IBV infection. Due to positive immunostaining in the bursa, it is thought that IBV infection has immunosuppressive consequences, hence further study is required to validate this impact.

Development and Immunoprotection of Bacterium-like Particle Vaccine against Infectious Bronchitis in Chickens.

Infectious bronchitis (IB) is a major threat to the global poultry industry. Despite the availability of commercial vaccines, the IB epidemic has not been effectively controlled. The exploration of novel IBV vaccines may provide a new way to prevent and control IB. In this study, BLP-S1, a bacterium-like particle displaying the S1 subunit of infectious bronchitis virus (IBV), was constructed using the GEM-PA surface display system. The immunoprotective efficacy results showed that BLP-S1 can effectively induce specific IgG and sIgA immune responses, providing a protection rate of 90% against IBV infection in 14-day-old commercial chickens. These results suggest that BLP-S1 has potential for the development of novel vaccines with good immunogenicity and immunoprotection.

The impact of the experimental route of challenge on the host responses and pathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus infection in laying chickens

Infectious bronchitis virus (IBV) infection can be associated with respiratory, renal, and/or reproductive diseases in chickens. Under natural conditions, conjunctiva, mucosa of upper respiratory tract, and cloaca are the main routes of IBV entry. Experimentally, the study of IBV infection involved various routes of inoculation. This study investigated the impact of adding the trachea as a potential route of viral entry to the oculo-nasal infection on the host responses, pathogenicity, and tissue tropism of the Canadian IBV Delmarva (DMV/1639) strain in laying chickens. Specific-pathogen-free laying chickens were divided into three experimental groups: control group (Con group), oculo-nasal challenged group (ON group), and oculo-nasal/intratracheal challenged group (ON/IT group); all groups were observed for 12 days post-infection (dpi). The clinical signs and reduction in egg production in the ON/IT group started slightly earlier compared to the ON group. At 12 dpi, the gross lesions in the ON/IT group were confined to the ovary, while the ON group showed regressed ovary and atrophied oviduct. Only the ON group showed significantly higher microscopic lesion scores in the lung, kidney, magnum, and uterus compared to the control group at 12 dpi. The oviduct tissues of the ON group showed a significant increase in B cells infiltration compared to ON/IT and control groups. The viral shedding (detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR)), tissue tropism (detected either by qRT-PCR or immunohistochemistry (IHC)), T/natural killer cells infiltration in reproductive tract (detected by IHC), and antibody-mediated immune responses (measured by enzyme-linked immunosorbent assay) showed similar patterns in the ON and ON/IT groups.

Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

Effect of oral vitamin A supplementation on host immune response to infectious bronchitis virus infection in specific pathogen-free chicken

Vitamin A is a fat-soluble vitamin that is a crucial mediator of the immune system. In this study, we evaluated the effect of oral vitamin A supplementation on host immune responses to infectious bronchitis virus (IBV) infection in chickens. Forty 1-day-old specific pathogen-free (SPF) chickens were fed a basal diet and randomly divided into 2 groups (n=20 birds per group). Chickens in the experimental group were treated orally with vitamin A (dissolved in 0.1 mL soybean oil, at a dose of 8,000 IU per kg diet) daily. Birds in the control group were orally administered 0.1 mL soybean oil without vitamin A until 21 d of age. On d 21 after birth, all chickens were infected with 0.1 mL of 106.5 50% median embryo infectious dose of a pathogenic IBV strain (CK/CH/LDL/091022) by intraocular and intranasal routes. The results demonstrated that oral vitamin A supplementation did not affect the clinical course of disease and growth performance of SPF chickens. However, vitamin A supplementation increased the IBV-specific IgG serum levels and decreased the viral load in some tissues of IBV-infected chickens. In addition, the results demonstrated that vitamin A supplementation decreased the expression levels of most immune-related molecules in some tissues of IBV-infected chickens. Vitamin A supplementation decreased the mRNA expression levels of some avian β-defensins (AvBD2, 3, 6, 7, 11, and 13) and increased the expression levels of AvBD9 and AvBD12 in some tissues of IBV-infected chickens. Similarly, vitamin A supplementation decreased the mRNA expression levels of some cytokines (interferon-γ, interleukin-1β [IL-1β], and IL-6) and increased the mRNA expression levels of IL-2 in some tissues of IBV-infected chickens. Furthermore, vitamin A supplementation decreased the mRNA expression levels of myeloid differentiation primary response protein 88, nuclear factor-κB p65, toll-like receptor 3, toll-like receptor 7, and CD4. In summary, the present study suggests that vitamin A supplementation enhances the immune function of SPF chickens against IBV infection by inhibiting viral replication, increasing the IBV-specific antibody titer, and suppressing the excessive inflammatory responses to IBV infection.

Isolation and Characterization of Avian Coronavirus from Diagnostic Cases of Selected Bird Species in Malaysia

Infectious bronchitis virus (IBV) in chicken (Gallus gallus) is the most common and well-studied Avian coronavirus (ACoV) in avian species. The study aims to molecularly characterize ACoV isolate of selected bird species other than chicken obtained from the archived samples of field diagnostic cases in the Northern Zone Veterinary Laboratory (MVZU), Malaysia. Twelve archived virus isolates from 2013 to 2019 were amplified using selected primers on the 3’ UTR gene and S1 gene for oligonucleotide sequencing. These sequences were then molecularly characterized and compared with common IBV strains in chicken to determine the genetic diversity of the virus among selected avian species. Subsequent analyses of the nucleotides amplified on 3’ UTR conserved region of 12 selected ACoVs isolates originating from peacocks (Pavo cristatus), turkey (Meleagris), jungle fowl (Gallus gallus spadiceous), guinea fowl (Meleagris gallopavo domesticus), goose (Anser anser domesticus), love bird (Agapornis), macaw (Ara macao), and bird (species unidentified) are classified as belonging to the gammacoronavirus (Gamma-CoV) genus and have a high degree of homology. The S1 complete gene sequence analyses of guinea fowl and jungle fowl showed that both ACoV isolates are Gamma-CoV and under genotype I and GI-13 lineages. Both are identified as having a high similarity of 98% and 99%, respectively, with IBV vaccine strain 4/91 (AF093793). Due attention should be given to ACoVs strains, especially the IBV vaccine strains detected in other bird species, because there is a high probability that other bird species could be the source of pathogenic ACoV infection in general and IBV infection in chickens, as reported in other countries.