Avian Coronavirus Infectious Bronchitis Virus Research Articles

Development of a colloidal gold immunochromatographic strip for rapid detection of avian coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold–mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold–mAb N2D5 conjugation were pH 6.5 and 30 μg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10−1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited areas.

S2 subunit plays a critical role in pathogenesis of TW-like avian coronavirus infectious bronchitis virus

To investigate the critical role of the S gene in determining pathogenesis of TW-like avian infectious bronchitis virus (IBV), we generated two recombinant IBVs (rGDaGD-S1 and rGDaGD-S2) by replacing either the S1 or S2 region of GD strain with the corresponding regions from an attenuated vaccine candidate aGD strain. The virulence and pathogenicity of these recombinant viruses was assessed both in vitro and in vivo. Our results indicated the mutations in the S2 region led to decreased virulence, as evidenced by reduced virus replication in embryonated chicken eggs and chicken embryonic kidney cells as well as observed clinical symptoms, gross lesions, microscopic lesions, tracheal ciliary activity, and viral distribution in SPF chickens challenged with recombinant IBVs. These findings highlight that the S2 subunit is a key determinant of TW-like IBV pathogenicity. Our study established a foundation for future investigations into the molecular mechanisms underlying IBV virulence.

Avian coronavirus infectious bronchitis virus Beaudette strain NSP9 interacts with STAT1 and inhibits its phosphorylation to facilitate viral replication

Avian coronavirus, known as infectious bronchitis virus (IBV), is the causative agent of infectious bronchitis (IB). Viral nonstructural proteins play important roles in viral replication and immune modulation. IBV NSP9 is a component of the RNA replication complex for viral replication. In this study, we uncovered a function of NSP9 in immune regulation. First, the host proteins that interacted with NSP9 were screened. The immune-related protein signal transducer and activator of transcription 1 (STAT1) was identified and the interaction between NSP9 and STAT1 was further confirmed. Furthermore, IBV replication was inhibited in STAT1-overexpressing cells but inversely affected in STAT1 knock-down cells. Importantly, NSP9 inhibited STAT1 phosphorylation. Finally, the expression of JAK/STAT pathway downstream genes IRF7 and ISG20 was significantly decreased in NSP9-overexpressing cells. These results showed the important role of IBV NSP9 in immunosuppression.

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.

Antimicrobial and Antiviral Properties of Triclosan-Containing Polymer Composite: Aging Effects of pH, UV, and Sunlight Exposure

The present study deals with the synthesis and characterization of a polymer composite based on an unsaturated ester loaded with 5 wt.% triclosan, produced by co-mixing on an automated hardware system. The polymer composite's non-porous structure and chemical composition make it an ideal material for surface disinfection and antimicrobial protection. According to the findings, the polymer composite effectively inhibited (100%) the growth of Staphylococcus aureus 6538-P under exposure to physicochemical factors, including pH, UV, and sunlight, over a 2-month period. In addition, the polymer composite demonstrated potent antiviral activity against human influenza virus strain A and the avian coronavirus infectious bronchitis virus (IBV), with infectious activities of 99.99% and 90%, respectively. Thus, the resulting triclosan-loaded polymer composite is revealed to have a high potential as a surface-coating non-porous material with antimicrobial properties.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

A DNA Prime and MVA Boost Strategy Provides a Robust Immunity against Infectious Bronchitis Virus in Chickens.

Infectious bronchitis (IB) is an acute respiratory disease of chickens caused by the avian coronavirus Infectious Bronchitis Virus (IBV). Modified Live Virus (MLV) vaccines used commercially can revert to virulence in the field, recombine with circulating serotypes, and cause tissue damage in vaccinated birds. Previously, we showed that a mucosal adjuvant system, QuilA-loaded Chitosan (QAC) nanoparticles encapsulating plasmid vaccine encoding for IBV nucleocapsid (N), is protective against IBV. Herein, we report a heterologous vaccination strategy against IBV, where QAC-encapsulated plasmid immunization is followed by Modified Vaccinia Ankara (MVA) immunization, both expressing the same IBV-N antigen. This strategy led to the initiation of robust T-cell responses. Birds immunized with the heterologous vaccine strategy had reduced clinical severity and >two-fold reduction in viral burden in lachrymal fluid and tracheal swabs post-challenge compared to priming and boosting with the MVA-vectored vaccine alone. The outcomes of this study indicate that the heterologous vaccine platform is more immunogenic and protective than a homologous MVA prime/boost vaccination strategy.

Synthesis of Novel 1,3,4-Oxadiazole-Derived α-Aminophosphonates/α-Aminophosphonic Acids and Evaluation of Their In Vitro Antiviral Activity against the Avian Coronavirus Infectious Bronchitis Virus.

An efficient and simple approach has been developed for the synthesis of eight dialkyl/aryl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl]phosphonates through the Pudovik-type reaction of dialkyl/arylphosphite with imines, obtained from 5-phenyl-1,3,4-oxadiazol-2-amine and aromatic aldehydes, under microwave irradiation. Five of them were hydrolyzed to lead to the corresponding phosphonic acids. Selected synthesized compounds were screened for their in vitro antiviral activity against the avian bronchitis virus (IBV). In the MTT cytotoxicity assay, the dose-response curve showed that all test compounds were safe in the range concentration of 540-1599 µM. The direct contact of novel synthesized compounds with IBV showed that the diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethoxyphenyl)methyl]phosphonate (5f) (at 33 µM) and the [(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl] phosphonic acid (6a) (at 1.23 µM) strongly inhibited the IBV infectivity, indicating their high virucidal activity. However, virus titers from IBV-infected Vero cells remained unchanged in response to treatment with the lowest non-cytotoxic concentrations of synthesized compounds suggesting their incapacity to inhibit the virus replication inside the host cell. Lack of antiviral activity might presumably be ascribed to their polarity that hampers their diffusion across the lipophilic cytoplasmic membrane. Therefore, the interactions of 5f and 6a were analyzed against the main coronavirus protease, papain-like protease, and nucleocapsid protein by molecular docking methods. Nevertheless, the novel 1,3,4-oxadiazole-based α-aminophosphonic acids and α-amino-phosphonates hold potential for developing new hygienic virucidal products for domestic, chemical, and medical uses.

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.

Genomics and pathogenesis of the avian coronavirus infectious bronchitis virus.

Infectious bronchitis virus (IBV) is a member of the family Coronaviridae, together with viruses such as SARS-CoV, MERS-CoV and SARS-CoV-2 (the causative agent of the COVID-19 global pandemic). In this family of viruses, interspecies transmission has been reported, so understanding their pathobiology could lead to a better understanding of the emergence of new serotypes. IBV possesses a single-stranded, non-segmented RNA genome about 27.6 kb in length that encodes several non-structural and structural proteins. Most functions of these proteins have been confirmed in IBV, but some other proposed functions have been based on research conducted on other members of the family Coronaviridae. IBV has variable tissue tropism depending on the strain, and can affect the respiratory, reproductive, or urinary tracts; however, IBV can also replicate in other organs. Additionally, the pathogenicity of IBV is also variable, with some strains causing only mild clinical signs, while infection with others results in high mortality rates in chickens. This paper extensively and comprehensibly reviews general aspects of coronaviruses and, more specifically, IBV, with emphasis on protein functions and pathogenesis. The pathogenicity of the Australian strains of IBV is also reviewed, describing the variability between the different groups of strains, from the classical to the novel and recombinant strains. Reverse genetic systems, cloning and cell culture growth techniques applicable to IBV are also reviewed.

The Genetic Stability, Replication Kinetics and Cytopathogenicity of Recombinant Avian Coronaviruses with a T16A or an A26F Mutation within the E Protein Is Cell-Type Dependent.

The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.

Exposure to avian coronavirus vaccines is associated with increased levels of SARS-CoV-2-cross-reactive antibodies.

BackgroundAlthough avian coronavirus infectious bronchitis virus (IBV) and SARS‐CoV‐2 belong to different genera of the Coronaviridae family, exposure to IBV may result in the development of cross‐reactive antibodies to SARS‐CoV‐2 due to homologous epitopes. We aimed to investigate whether antibody responses to IBV cross‐react with SARS‐CoV‐2 in poultry farm personnel who are occupationally exposed to aerosolized IBV vaccines.MethodsWe analyzed sera from poultry farm personnel, COVID‐19 patients, and pre‐pandemic controls. IgG levels against the SARS‐CoV‐2 antigens S1, RBD, S2, and N and peptides corresponding to the SARS‐CoV‐2 ORF3a, N, and S proteins as well as whole virus antigens of the four major S1‐genotypes 4/91, IS/1494/06, M41, and D274 of IBV were investigated by in‐house ELISAs. Moreover, live‐virus neutralization test (VNT) was performed.ResultsA subgroup of poultry farm personnel showed elevated levels of specific IgG for all tested SARS‐CoV‐2 antigens compared with pre‐pandemic controls. Moreover, poultry farm personnel, COVID‐19 patients, and pre‐pandemic controls showed specific IgG antibodies against IBV strains. These antibody titers were higher in long‐term vaccine implementers. We observed a strong correlation between IBV‐specific IgG and SARS‐CoV‐2 S1‐, RBD‐, S2‐, and N‐specific IgG in poultry farm personnel compared with pre‐pandemic controls and COVID‐19 patients. However, no neutralization was observed for these cross‐reactive antibodies from poultry farm personnel using the VNT.ConclusionWe report here for the first time the detection of cross‐reactive IgG antibodies against SARS‐CoV‐2 antigens in humans exposed to IBV vaccines. These findings may be useful for further studies on the adaptive immunity against COVID‐19.

Molecular Characterization Analysis of Prevalent Infectious Bronchitis Virus and Pathogenicity Assessment of Recombination Strain in China.

Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, resulting in severe economic losses in the poultry industry. This study aimed to monitor and isolate the molecular identity of IBV in broiler flocks with respiratory symptoms in eight provinces of China. In total, 910 samples (oropharyngeal and cloacal mixed swabs) from broiler flocks showed IBV positive rates of 17.6% (160/910) using PCR assay. Phylogenetic analysis of the complete S1 genes of 160 IBV isolates was performed and revealed that QX-type (GI-19), TW-type (GI-7), 4/91-type (GI-13), HN08-type (GI-22),TC07-2-type (GVI-1), and LDT3-type (GI-28) exhibited IBV positive rates of 58.15, 25, 8.12, 1.86, 5.62, and 1.25%. In addition, recombination analyses revealed that the four newly IBV isolates presented different recombination patterns. The CK/CH/JS/YC10-3 isolate likely originated from recombination events between strain YX10 (QX-type) and strain TW2575-98 (TW-type), the pathogenicity of which was assessed, comparing it with strain GZ14 (TW-type) and strain CK/CH/GD/JR07-7 (QX-type). The complete S1 gene data from these isolates indicate that IBV has consistently evolved through genetic recombination or mutation, more likely changing the viral pathogenicity and leading to larger outbreaks in chick populations, in China.

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.

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

ABSTRACT 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 HIGHLIGHTS Avian 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.

Phylogenetic and Spatiotemporal Analyses of the Complete Genome Sequences of Avian Coronavirus Infectious Bronchitis Virus in China During 1985-2020: Revealing Coexistence of Multiple Transmission Chains and the Origin of LX4-Type Virus.

Infectious bronchitis (IB) virus (IBV) causes considerable economic losses to poultry production. The data on transmission dynamics of IBV in China are limited. The complete genome sequences of 212 IBV isolates in China during 1985–2020 were analyzed as well as the characteristics of the phylogenetic tree, recombination events, dN/dS ratios, temporal dynamics, and phylogeographic relationships. The LX4 type (GI-19) was found to have the highest dN/dS ratios and has been the most dominant genotype since 1999, and the Taiwan-I type (GI-7) and New type (GVI-1) showed an increasing trend. A total of 59 recombinants were identified, multiple recombination events between the field and vaccine strains were found in 24 isolates, and the 4/91-type (GI-13) isolates were found to be more prone to being involved in the recombination. Bayesian phylogeographic analyses indicated that the Chinese IBVs originated from Liaoning province in the early 1900s. The LX4-type viruses were traced back to Liaoning province in the late 1950s and had multiple transmission routes in China and two major transmission routes in the world. Viral phylogeography identified three spread regions for IBVs (including LX4 type) in China: Northeastern China (Heilongjiang, Liaoning, and Jilin), north and central China (Beijing, Hebei, Shanxi, Shandong, and Jiangsu), and Southern China (Guangxi and Guangdong). Shandong has been the epidemiological center of IBVs (including LX4 type) in China. Overall, our study highlighted the reasons why the LX4-type viruses had become the dominant genotype and its origin and transmission routes, providing more targeted strategies for the prevention and control of IB in China.

Identification of Amino Acids within Nonstructural Proteins 10 and 14 of the Avian Coronavirus Infectious Bronchitis Virus That Result in Attenuation In Vivo and In Ovo.

ABSTRACTThe Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens’ eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application.IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens’ eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.

A surveillance for avian coronavirus infectious bronchitis virus, infectious laryngotracheitis virus, avian metapneumovirus, and avian reovirus in poultry flocks with respiratory signs in Türkiye

In this study, avian coronavirus infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (AMPV), and avian reovirus (ARV) were evaluated in broiler and layer flocks. For this purpose, tracheal swabs from 48 broiler and 45 layer flocks with respiratory signs were inoculated SPF embryonated chicken eggs for virus isolation. The viruses were identified by real-time PCR. Results showed that the most common virus in both broiler and layer farms was IBV with incidence rates of 58.33% and 46.67%, respectively. ILTV, AMPV, and ARV incidences in the samples were found to be 22.22%, 13.33%, and 4.44% in layer flocks while 2.08%, 8.33%, and 20.83% in broilers, respectively. The numbers of IBV+AMPV and IBV+ARV coinfections were 5 (11.11%) and 1 (2.22%) in layers, whereas, 1 (2.08%) and 5 (10.42%) broilers, respectively. In addition, 2 broiler flocks (4.17%) had triple infection with IBV, AMPV, and ARV. ILTV was detected always alone from the samples of layer and broiler flocks. Sequencing of S1 gene of selected IBV TR/L45 and TR/B42 isolates showed similarities with IS/1494/06 (HM131453) at the rates of 98.98% and 99.69%, respectively, while TR/L37, TR/L38, and TR/L39 isolates were identical to 4/91 (KF377577) vaccine strain at the rates of 99.01%, 99.01%, and 98.76%, respectively. Sequencing analysis of the ICP4 and TK genes of ILTV isolates revealed that they were all field strains with low virulence. The present data represent actual information on the genotypes of IBV and ILTV circulating in poultry flocks in Türkiye.

Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4+ and CD8+ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

Design and Characterization of a DNA Vaccine Based on Spike with Consensus Nucleotide Sequence against Infectious Bronchitis Virus.

Avian coronavirus infectious bronchitis virus (IBV) causes severe economic losses in the poultry industry, but its control is hampered by the continuous emergence of new genotypes and the lack of cross-protection among different IBV genotypes. We designed a new immunogen based on a spike with the consensus nucleotide sequence (S_con) that may overcome the extraordinary genetic diversity of IBV. S_con was cloned into a pVAX1 vector to form a new IBV DNA vaccine, pV-S_con. pV-S_con could be correctly expressed in HD11 cells with corresponding post-translational modification, and induced a neutralizing antibody response to the Vero-cell-adapted IBV strain Beaudette (p65) in mice. To further evaluate its immunogenicity, specific-pathogen-free (SPF) chickens were immunized with the pV-S_con plasmid and compared with the control pVAX1 vector and the H120 vaccine. Detection of IBV-specific antibodies and cell cytokines (IL-4 and IFN-γ) indicated that vaccination with pV-S_con efficiently induced both humoral and cellular immune responses. After challenge with the heterologous strain M41, virus shedding and virus loading in tissues was significantly reduced both by pV-S_con and its homologous vaccine H120. Thus, pV-S_con is a promising vaccine candidate for IBV, and the consensus approach is an appealing method for vaccine design in viruses with high variability.