Genotyping of infectious bronchitis virus in Canada.

From 2014-2023, infectious bronchitis virus (IBV) was detected in 6,589 samples from Canada, and partial nucleotide (nt) sequences of the IBV spike protein (S) gene were determined for 1,678 samples. Based on their S gene nt sequence identities and origin, Canadian IBVs could be classified into 4 groups: 1) 50.3% were variant viruses related to strains described in the United States; 2) 45.6% were vaccine-like viruses; 3) 2.1% were Eurasian viruses; 4) 2.0% were Canadian variants. Outbreaks with IBVs related to strains CAL1734/04, 4/91, and DMV/1639/11 were often associated with more severe disease in all chicken commodity groups. With the emergence of numerous IBV strains, the severity of infection and number of affected flocks increased. Outbreaks with various IBV strains overlapped in their emergence, peaked, and regressed, but the introduction of DMV/1639/11 has resulted in a continuous field challenge since its first detection in 2015.

Evaluation of different heterologous-homologous vaccine regimens against challenge with GI-23 lineage infectious bronchitis virus.

This study assesses different IBV vaccination regimens in broiler chickens using commercially available live attenuated GI-23 (Egyptian-VAR2) and GI-1 (H120) vaccines. Vaccines were administered at 1, 14 days of age, or both. The ciliostasis test, following wild-type VAR2 challenge at 28 days of age, indicated that classic H120+VAR2 at one day old followed by the VAR2 vaccine at 14 days of age provided the highest level of protection (89.58%). Similarly, administering VAR2 at 1 day of age and classic H120 at 14 days of age demonstrated substantial protection (85.42%). Conversely, administering only classic H120 and VAR2 at one day old resulted in the lowest protection level (54.17%). Tracheal virus shedding quantification and assessment of trachea and kidney degenerative changes were significantly lower in vaccinated groups compared to the unvaccinated-challenged group. In conclusion, a carefully planned vaccination regimen based on homologous vaccination offers the most effective clinical protection in broiler chickens.

Pathogenicity and molecular characterization of a GI-19 infectious bronchitis virus isolated from East China.

Infectious bronchitis virus (IBV) is responsible for avian infectious bronchitis, a disease prevalent in countries with intensive poultry farming practices. Given the presence of multiple genotypic strains in China, identifying the regionally dominant genotypes is crucial for the implementation of effective prevention and control measures. This study focuses on the IBV strain CK/CH/WJ/215, isolated from a diseased commercial chicken flock in China in 2021. The CK/CH/WJ/215 isolate was genetically characterized through complete S1 sequence analysis. Phylogenetic comparisons were made with prevalent vaccine strains (H120, LDT3-A, and 4/91). Glycosylation patterns in the S1 protein were also analyzed. Pathogenicity was assessed in 7-day-old specific-pathogen-free chicks, monitoring morbidity, mortality, and tissue tropisms. Phylogenetic analysis clustered the CK/CH/WJ/215 isolate within the GI-19 lineage. Identity with the vaccination strains H120, LDT3-A, and 4/91 was low (75.7%, 78.6%, and 77.5% respectively). Novel glycosylation sites at positions 138 and 530 were identified compared to H120 and LDT-A. The isolate demonstrated nephropathogenic characteristics, causing 100% morbidity and 73.3% mortality in SPF chicks, with broader tropisms in tissues including trachea, lungs, kidneys, and bursa of Fabricius. Comprehensive genetic and pathological investigations revealed significant differences between the CK/CH/WJ/215 isolate and common vaccine strains, including novel glycosylation sites and a strong multiorgan infective capability. These findings are crucial for understanding the evolutionary dynamics of IBV and developing more effective prevention and control strategies.

Lessons Learnt on Infectious Bronchitis Virus Lineage GI-23.

ABSTRACTInfectious bronchitis virus (IBV) is the first Coronavirus discovered in the world in the early 1930s and despite decades of extensive immunoprophylaxis efforts, it remains a major health concern to poultry producers worldwide. Rapid evolution due to large poultry population sizes coupled with high mutation and recombination events and the reliance of the antiviral immune response on specific antibodies against the epitopes of the S1 glycoprotein, render the control of IBV extremely challenging. The numerous and rapidly evolving genetic and antigenic IBV types are currently classified based on the whole S1 gene sequence, into 36 lineages clustered in 8 genotypes. Most lineages (29) are grouped in Genotype I (GI). "Variant 2" (Israel/Variant 2/1998) is the prototype strain of lineage GI-23 and since this lineage emergence during the mid-1990s in the Middle East, it has evolved into numerous genetically related strains and disseminated to 5 continents. The hallmarks of IBV Variant 2-like strain infections are high virulence and remarkable nephrotropism and nephropathogenicity, however the molecular mechanisms of these traits remain to be elucidated. Limited protection from previously utilized vaccine strains and accumulated losses to poultry producers have urged the development and implementation of homologous Variant 2-like vaccine strains. The latest Avian Coronavirus biology with specific emphasis on the cumulative knowledge about IBV "Variant 2" and related strains' emergence, characteristics and control are reviewed.

Safety evaluation of recombinant Newcastle disease virus expressing IBV multi-epitope chimeric live vaccine.

Newcastle Disease (ND) and Infectious Bronchitis (IB) are two significant diseases that pose threats to the poultry industry, caused by Newcastle disease virus (NDV) and Infectious bronchitis virus (IBV), respectively. Currently, the control and prevention of these diseases primarily rely on vaccination. However, commercial ND and IB vaccines face challenges such as poor cross-protection of inactivated IBV strains and interference from live vaccines when used together, leading to immunization failures. Previously, we reported the successful rescue of a recombinant NDV expressing multiple epitopes of IBV, named rNDV-IBV-T/B, which showed promising immunoprotective efficacy against both NDV and IBV. This study focuses on the biosafety of the genetically modified recombinant vaccine candidate rNDV-IBV-T/B. Immunization was performed on day-old chicks, ducklings, goslings, and ICR mice. Observations were recorded on clinical symptoms, body weight changes, and post-mortem examination of organs, as well as histopathological preparations of tissue samples. The results indicated that the rNDV-IBV-T/B vaccine candidate had no adverse effects on the growth of targeted animals (chickens) and non-target species (ducks, geese) as well as in mammals (mice). Additionally, histopathological slides confirmed that the vaccine is safe for all tested species. Further studies evaluated the potential of rNDV-IBV-T/B to spread horizontally and vertically post-immunization, and its environmental safety. The findings revealed that the vaccine candidate lacks the capability for both horizontal and vertical transmission and does not survive in the environment. In conclusion, the rNDV-IBV-T/B strain is safe and holds potential as a new chimeric live vaccine for ND and IB.

Wogonin Inhibits Apoptosis and Necroptosis Induced by Nephropathogenic Infectious Bronchitis Virus in Chicken Renal Tubular Epithelial Cells.

NIBV is an acute and highly contagious virus that has a major impact on the poultry industry. Wogonin, as a flavonoid drug, has antiviral effects, but there have been no reports indicating its role in renal injury caused by NIBV infection. The aim of this study is to investigate the antiviral effect of wogonin against NIBV. Renal tubular epithelial cells were isolated and cultured, and divided into four groups: Con, Con+Wog, NIBV and NIBV+Wog. We found that wogonin significantly inhibited the copy number of NIBV and significantly alleviated NIBV-induced cell apoptosis and necrosis. Moreover, wogonin inhibited the reduction in mitochondrial membrane potential and the aberrant opening of mPTP caused by NIBV. In conclusion, wogonin can protect renal tubular epithelial cells from damage by inhibiting the replication of NIBV and preventing mitochondrial apoptosis and necroptosis induced by NIBV.

The N545S and K717N substitution at the N-glycosylation sites of the S2 subunit of avian infectious bronchitis virus can significantly enhance viral pathogenicity.

The S2 subunit of infectious bronchitis virus (IBV) is a heavily glycosylated protein that can impact various characteristics of the virus. It is currently known that N-glycosylation modifications are predominantly located on the S2 subunit. However, the exact role of their N-glycosylation modification remains undisclosed. To elucidate the function of these N-glycosylation sites, we identified 14 common sites distributed on the S2 subunit of the 5 genotypes of IBV in present study. Subsequently, we selected 7 sites to generate mutants and assessed their impact on viral virulence, replication ability, and antigenicity. Our finding revealed that only 2 substitutions, N545S and K717N, increased the viral replication titer and antigenicity, and ultimately the pathogenicity in chicks. To delve into the mechanisms underlying this increased pathogenicity, we discovered that K717N can change the structure of antigenic epitopes. The N545S substitution not only influenced antigenic epitope structure, but also enhanced the ability of the virus to enter CEKs during the early stages of viral replication. These results suggest that the enhanced viral pathogenicity associated with N545S and K717N substitutions is multifaceted, with acceleration of the viral membrane fusion process and alterations in epitope structure representing crucial factors in the capability of N-glycosylation modifications to boost viral virulence. These insights provide valuable guidance for the efficient development of live attenuated vaccines.

A New Dual Fluorescence Method for Rapid Detection of Infectious Bronchitis Virus at Constant Temperature.

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I-V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/µL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I-V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype.

Co-infection of H9N2 subtype avian influenza virus and QX genotype live attenuated infectious bronchitis virus increase the pathogenicity in SPF chickens.

Avian influenza virus (AIV) infection and vaccination against live attenuated infectious bronchitis virus (aIBV) are frequent in poultry worldwide. Here, we evaluated the clinical effect of H9N2 subtype AIV and QX genotype aIBV co-infection in specific-pathogen-free (SPF) white leghorn chickens and explored the potential mechanisms underlying the observed effects using by 4D-FastDIA-based proteomics. The results showed that co-infection of H9N2 AIV and QX aIBV increased mortality and suppressed the growth of SPF chickens. In particular, severe lesions in the kidneys and slight respiratory signs similar to the symptoms of virulent QX IBV infection were observed in some co-infected chickens, with no such clinical signs observed in single-infected chickens. The replication of H9N2 AIV was significantly enhanced in both the trachea and kidneys, whereas there was only a slight effect on the replication of the QX aIBV. Proteomics analysis showed that the IL-17 signaling pathway was one of the unique pathways enriched in co-infected chickens compared to single infected-chickens. A series of metabolism and immune response-related pathways linked with co-infection were also significantly enriched. Moreover, co-infection of the two pathogens resulted in the enrichment of the negative regulation of telomerase activity. Collectively, our study supports the synergistic effect of the two pathogens, and pointed out that aIBV vaccines might increased IBV-associated lesions due to pathogenic co-infections. Exacerbation of the pathogenicity and mortality in H9N2 AIV and QX aIBV co-infected chickens possibly occurred because of an increase in H9N2 AIV replication, the regulation of telomerase activity, and the disturbance of cell metabolism and the immune system.

Avian Coronavirus Infectious Bronchitis Virus Activates Mitochondria-Mediated Apoptosis Pathway and Affects Viral Replication by Inducing Reactive Oxygen Species Production in Chicken HD11 Cells.

Infectious bronchitis virus (IBV), a coronavirus that causes severe respiratory and gastrointestinal illness in poultry, leads to substantial economic losses. According to earlier research, IBV infection causes chicken macrophage HD11 cells to undergo cell apoptosis. Reactive oxygen species (ROS) and the IBV-activated intrinsic apoptotic signaling pathway were examined in this work. The findings demonstrate that IBV infection causes ROS to accumulate. Moreover, IBV infection decreased the mitochondrial transmembrane potential in HD11 cells, which could be blocked by ROS antioxidants (PDTC and NAC). The two antioxidants significantly affected the expression of Bcl-2 and Bax and further inhibited the activation of caspase-3 and apoptosis in HD11 cells. Additionally, IBV replication was decreased by blocking ROS accumulation. Pretreating HD11 cells with ammonium chloride (NH4Cl) prevented IBV from entering the cells and reduced the oxidative stress which IBV causes. The ability to accumulate ROS was also lost in UV-inactivated IBV. The IBV N protein induces cell apoptosis through the activation of ROS. These findings provide an explanation for the processes of IBV infection in immune cells by indicating that IBV-induced ROS generation triggers cell apoptosis in HD11 cells.

Heterologous maternal antibodies derived from infectious bronchitis vaccines prevent the development of lesions associated with false layer syndrome.

Infectious bronchitis virus (IBV) strains of the Delmarva (DMV)/1639 genotype have been causing false layer syndrome (FLS) in the Eastern Canadian layer operations since the end of 2015. FLS is characterized by the development of cystic oviducts in layer pullets infected at an early age. Currently, there are no homologous vaccines for the control of this IBV genotype. Our previous research showed that a heterologous vaccination regimen incorporating Massachusetts (Mass) and Connecticut (Conn) IBV types protects layers against DMV/1639 genotype IBV. The aim of this study was to investigate the role of maternal antibodies conferred by breeders received the same vaccination regimen in the protection against the development of DMV/1639-induced FLS in pullets. Maternal antibody-positive (MA+) and maternal antibody-negative (MA-) female progeny chicks were challenged at 1 day of age and kept under observation for 16 weeks. Oviductal cystic formations were observed in 3 of 14 birds (21.4 %) in the MA- pullets, while the lesions were notably absent in the MA+ pullets. Milder histopathological lesions were observed in the examined tissues of the MA+ pullets. However, the maternal derived immunity failed to demonstrate protection against the damage to the tracheal ciliary activity, viral shedding, and viral tissue distribution. Overall, this study underscores the limitations of maternal derived immunity in preventing certain aspects of viral pathogenesis, emphasizing the need for comprehensive strategies to address different aspects of IBV infection.

A novel highly virulent nephropathogenic QX-like infectious bronchitis virus originating from recombination of GI-13 and GI-19 genotype strains in China.

Infectious bronchitis virus (IBV) is one of the most widely spread RNA viruses, causing respiratory, renal, and intestinal damage, as well as decreased reproductive performance in hens, leading to significant economic losses in the poultry industry. In this study, a new IBV strain designated as CK/CH/GX/LA/071423 was successfully isolated from the 60-day-old Three-Yellow chicken vaccinated with H120 and QXL87 vaccines. The complete genome sequence analysis revealed that the CK/CH/GX/LA/071423 strain shared a high similarity of 96.7% with the YX10 strain belonging to the GI-19 genotype. Genetic evolution analysis based on the IBV S1 gene showed that the CK/CH/GX/LA/071423 isolate belonged to the GI-19 genotype. Recombination analysis of the virus genome using RDP and Simplot software indicated that CK/CH/GX/LA/071423 was derived from recombination events between the YX10 and 4/91 vaccine strains, which was supported by phylogenetic analysis using gene sequences from the 3 regions. Furthermore, the S1 protein tertiary structure differences were observed between the CK/CH/GX/LA/071423 and the QXL87 and H120 vaccine strains. Pathogenicity studies revealed that the CK/CH/GX/LA/071423 caused death and led to pale and enlarged kidneys with abundant urate deposits, indicative of a nephropathogenic IBV strain. High virus titers were detected in the trachea, kidneys, and cecal tonsils, demonstrating broad tissue tropism. Throughout the experimental period, the virus positive rate in throat swabs of the infected group reached to 100%. These findings highlight the continued predominance of the QX genotype IBV in Guangxi of China and the ongoing evolution of different genotypes through genetic recombination, raising concerns about the efficacy of current IBV vaccines in providing effective protection to poultry.

Molecular epidemiology of infectious bronchitis virus in eastern and southern China during 2021-2023.

As a highly infectious and contagious pathogen in chickens, infectious bronchitis virus (IBV) is currently grouped into nine genotypes (GI to GIX). However, the classification of serotypes of IBV is still not clear. In this study, 270 field strains of IBV were isolated from dead or diseased chicken flocks in eastern and southern China during January 2021 to April 2023. These isolated IBV strains could be classified into 2 genotypes, GI (including 5 lineages GI-1, GI-13, GI-19, GI-22, and GI-28) and GVI based on the complete S1 sequence. Further analysis showed that the GI-19, GI-13, GI-22, GI-28, and GVI were the dominant genotypes with the proportions of 61.48, 8.89, 8.89, 7.78, and 8.89% respectively, and the homology of S1 protein of these isolates ranged from 86.85 to 100% in GI-19, 92.22 to 100% in GI-13, 83.1 to 100% in GI-22, 94.81 to 100% in GI-28 and 90.0 to 99.8% in GVI, respectively. Moreover, cross-neutralization test with sera revealed that these isolates in GI-19 lineage could be classified into at least 3 serotypes according to the antigenic relationship. In addition, structure assay using PyMOL indicated that one mutation such as S120 in receptor binding site (RBD) of GI-19 might alter the antigenicity and conformation of S protein of IBV. Overall, our data demonstrate that not only multiple genotypes, but also multiple serotypes in a single genotype or lineage have been co-circulated in eastern and southern China, providing novel insights into the molecular evolution of the antigenicity of IBV and highlighting the significance of the selection of the dominant isolate for vaccine development in IBV endemic region.

Dissecting infectious bronchitis virus-induced host shutoff at the translation level.

Viruses have evolved a range of strategies to utilize or manipulate the host's cellular translational machinery for efficient infection, although the mechanisms by which infectious bronchitis virus (IBV) manipulates the host translation machinery remain unclear. In this study, we firstly demonstrate that IBV infection causes host shutoff, although viral protein synthesis is not affected. We then screened 23 viral proteins, and identified that more than one viral protein is responsible for IBV-induced host shutoff, the inhibitory effects of proteins Nsp15 were particularly pronounced. Ribosome profiling was used to draw the landscape of viral mRNA and cellular genes expression model, and the results showed that IBV mRNAs gradually dominated the cellular mRNA pool, the translation efficiency of the viral mRNAs was lower than the median efficiency (about 1) of cellular mRNAs. In the analysis of viral transcription and translation, higher densities of RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) reads were observed for structural proteins and 5' untranslated regions, which conformed to the typical transcriptional characteristics of nested viruses. Translational halt events and the number of host genes increased significantly after viral infection. The translationally paused genes were enriched in translation, unfolded-protein-related response, and activation of immune response pathways. Immune- and inflammation-related mRNAs were inefficiently translated in infected cells, and IBV infection delayed the production of IFN-ß and IFN-λ. Our results describe the translational landscape of IBV-infected cells and demonstrate new strategies by which IBV induces host gene shutoff to promote its replication. IMPORTANCE: Infectious bronchitis virus (IBV) is a γ-coronavirus that causes huge economic losses to the poultry industry. Understanding how the virus manipulates cellular biological processes to facilitate its replication is critical for controlling viral infections. Here, we used Ribo-seq to determine how IBV infection remodels the host's biological processes and identified multiple viral proteins involved in host gene shutoff. Immune- and inflammation-related mRNAs were inefficiently translated, the translation halt of unfolded proteins and immune activation-related genes increased significantly, benefitting IBV replication. These data provide new insights into how IBV modulates its host's antiviral responses.

Adding yeast extract to culture medium enhances replication of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells.

Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures, the first two of which are commonly used for viral isolation. Previous studies have suggested that foetal bovine serum (FBS) can inhibit coronavirus replication in cell cultures. In this study, the replication of IBV in chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was assessed using two different cell culture media containing FBS or yeast extract (YE) and two different IBV strains. The highest concentrations of viral genomes were observed when the cell culture medium (CEK) contained YE. Similar results were observed in LMH cells. Examination of the infectivity by titration demonstrated that the cell lysate from CEK cell cultures in a medium including YE contained a higher median embryo infectious dose than that from CEK cell cultures in a medium containing FBS. These results indicate that improved replication of IBV in cell cultures can be achieved by replacing FBS with YE in the cell culture medium.

Protection Against Infectious Bronchitis Virus Vaccine Recombinants and Chicken-Selected Vaccine Subpopulations.

Outbreaks of infectious bronchitis (IB) continue to occur from novel variants of IB virus (IBV) emerging from selection of vaccine subpopulations and/or naturally occurring recombination events. S1 sequencing of Arkansas (Ark) -type viruses obtained from clinical cases in Alabama broilers and backyard chickens shows both Ark Delmarva Poultry Industry (ArkDPI) vaccine subpopulations as well as Ark vaccine viruses showing recombination with other IB vaccine viruses. IB Ark-type isolates AL5, most similar to an ArkDPI vaccine subpopulation selected in chickens, AL4, showing a cluster of three nonsynonymous changes from ArkDPI subpopulations selected in chickens, and AL9, showing recombination with Massachusetts (Mass) -type IBV, were examined for pathogenicity and ability to break through immunity elicited by vaccination with a commercial ArkDPI vaccine. Analysis of predicted S1 protein structures indicated the changes were in regions previously shown to comprise neutralizing epitopes. Thus, they were expected to contribute to immune escape and possibly virulence. Based on clinical signs, viral load, and histopathology, all three isolates caused disease in naïve chickens, although AL9 and AL5 viral loads in trachea were statistically significantly higher (30- and 40-fold) than AL4. S1 gene sequencing confirmed the stability of the relevant changes in the inoculated viruses in the chickens, although virus in some individual chickens exhibited additional S1 changes. A single amino acid deletion in the S1 NTD was identified in some individual chickens. The location of this deletion in the predicted structure of S1 suggested the possibility that it was a compensatory change for the reduced ability of AL4 to replicate in the trachea of naïve chickens. Chickens vaccinated with a commercial ArkDPI vaccine at day of hatch and challenged at 21 days of age showed that vaccination provided incomplete protection against challenge with these viruses. Moreover, based on viral RNA copy numbers in trachea, differences were detected in the ability of the vaccine to protect against these IBV isolates, with the vaccine protecting the most poorly against AL4. These results provide additional evidence supporting that IBV attenuated vaccines, especially ArkDPI vaccines, contribute to perpetuating the problem of IB in commercial chickens.

Protección contra los virus de la bronquitis infecciosa vacunales recombinantes y las subpoblaciones de vacunas seleccionadas en pollos. Los brotes de la bronquitis infecciosa aviar continúan presentándose a partir de nuevas variantes de dicho virus, que surgen de la selección de subpoblaciones de vacunas y/o eventos de recombinación que ocurren naturalmente. La secuenciación del gene S1 de virus tipo Arkansas (Ark) obtenidos de casos clínicos en pollos de engorde y de traspatio de Alabama muestra que tanto las subpoblaciones de la cepa vacunal Arkansas Delmarva Poultry Industry (ArkDPI) así como los virus de la vacuna Arkansas muestran recombinación con otros virus vacunales de la bronquitis infecciosa. Los aislamientos del virus de la bronquitis infecciosa Arkansas tipo "AL5", más similares a una subpoblación de vacuna ArkDPI seleccionada en pollos, "AL4", que muestra un grupo de tres cambios no sinónimos de subpoblaciones de ArkDPI seleccionadas en pollos y el tipo "AL9", que muestra recombinación con el serotipo Massachusetts, se examinaron para determinar su patogenicidad y capacidad para traspasar la inmunidad generada por la vacunación con una vacuna comercial ArkDPI. El análisis de las estructuras predichas de la proteína S1 indicó que los cambios se produjeron en regiones que previamente se había demostrado comprendían epítopos neutralizantes. Por lo tanto, se esperaba que contribuyeran al escape inmunológico y posiblemente a la virulencia. Con base en los signos clínicos, la carga viral y la histopatología, los tres aislados causaron enfermedad en pollos sin exposición previa, aunque las cargas virales de AL9 y AL5 en la tráquea fueron estadísticamente significativamente mayores (30 y 40 veces) en comparación con AL4. La secuenciación del gene S1 confirmó la estabilidad de los cambios relevantes en los virus inoculados en los pollos, aunque el virus en algunos pollos individuales exhibió cambios adicionales en el gene S1. Se identificó una deleción de un solo aminoácido en el dominio terminal N del gene S1 (NTD S1) en algunos pollos individuales. La ubicación de esta eliminación en la estructura predicha del gene S1 sugirió la posibilidad de que se tratara de un cambio compensatorio por la capacidad reducida de AL4 para replicarse en la tráquea de pollos sin exposición previa. Los pollos vacunados con una vacuna comercial ArkDPI el día de la eclosión y desafiados a los 21 días de edad mostraron que la vacunación proporcionó una protección incompleta contra el desafío con estos virus. Además, basándose en el número de copias del ARN viral en la tráquea, se detectaron diferencias en la capacidad de la vacuna para proteger contra estos aislados del virus de la bronquitis infecciosa, siendo la vacuna con la protección más deficiente contra AL4. Estos resultados proporcionan evidencia adicional que respalda que las vacunas atenuadas contra el virus de la bronquitis infecciosa, especialmente las vacunas ArkDPI, contribuyen a perpetuar esta enfermedad en los pollos comerciales.

Assessing a respiratory toxic infectious bronchitis virus (IBV) strain: isolation, identification, pathogenicity, and immunological failure insights.

Infectious bronchitis virus (IBV) is caused by avian coronavirus and poses a global economic threat to the poultry industry. In 2023, a highly pathogenic IBV strain, IBV/CN/GD20230501, was isolated and identified from chickens vaccinated with IBV-M41 in Guangdong, China. This study comprehensively investigated the biological characteristics of the isolated IBV strain, including its genotype, whole genome sequence analysis of its S1 gene, pathogenicity, host immune response, and serum non-targeted metabolomics. Through the analysis of the S1 gene sequence, serum neutralization tests, and comparative genomics, it was proven that IBV/CN/GD20230501 belongs to the GI-I type of strain and is serotype II. One alanine residue in the S1 subunit of the isolated strain was mutated into serine, and some mutations were observed in the ORF1ab gene and the terminal region of the genome. Animal challenge experiments using the EID50 and TCID50 calculations showed that IBV/CN/GD20230501 possesses strong respiratory pathogenicity, with early and long-term shedding of viruses and rapid viral spread. Antibody detection indicated that chickens infected with IBV/CN/GD20230501 exhibited delayed expression of early innate immune genes, while those infected with M41 showed rapid gene induction and effective viral control. Metabolomics analysis demonstrated that this virus infection led to differential expression of 291 ions in chicken serum, mainly affecting the citric acid cycle (tricarboxylic acid cycle).IMPORTANCEThis study identified an infectious bronchitis virus (IBV) strain isolated from vaccinated chickens in an immunized population that had certain sequence differences compared to IBV-M41, resulting in significantly enhanced pathogenicity and host defense. This strain has the potential to replace M41 as a more suitable challenge model for drug research. The non-targeted metabolomics analysis highlighting the citric acid cycle provides a new avenue for studying this highly virulent strain.

Two mutations on S2 subunit were critical for Vero cell tropism expansion of infectious bronchitis virus HV80.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR690/S motif at the S2' cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR690/S motif at the S2' cleavage site induced low-level cell-cell fusion, while H855Q substitution hindered cell-cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines.

A low-endotoxic Salmonella enterica Gallinarum serovar delivers infectious bronchitis virus immunogens via a dual-promoter vector system that drives protective immune responses through MHC class-I and -II activation in chickens.

An effective vaccine strategy is indispensable against infectious bronchitis virus (IBV) and fowl typhoid (FT), both of which threaten the poultry industry. This study demonstrates a vector system, pJHL270, designed to express antigens in prokaryotic and eukaryotic cells. The vector system stimulates immune responses via synchronized antigen presentation to MHC class-I and -II molecules to produce balanced Th1/Th2 responses. The vaccine antigens were crafted by selecting the consensus sequence of the N-terminal domain of the spike protein (S1-NTD) and a conserved immunogenic region of the nucleocapsid protein (N321-406 aa) from IBV strains circulating in South Korea. The vaccine antigen was cloned and transformed into a live-attenuated Salmonella Gallinarum (SG) strain, JOL2854 (∆lon, ∆cpxR, ∆rfaL, ∆pagL, ∆asd). Western blot analysis confirmed concurrent antigen expression in Salmonella and eukaryotic cells. Oral immunization with the SG-based IBV vaccine construct JOL2918 induced IBV antigen and Salmonella-specific humoral and cell-mediated immune responses in chickens. PBMCs collected from immunized chickens revealed that MHC class-I and -II expression had increased 3.3-fold and 2.5-fold, respectively, confirming MHC activation via bilateral antigen expression and presentation. Immunization induced neutralizing antibodies (NAbs) and reduced the viral load by 2-fold and 2.5-fold in the trachea and lungs, respectively. The immunized chickens exhibited multifaceted humoral, mucosal, and cell-mediated responses via parallel MHC class-I and -II activation as proof of a balanced Th1/Th2 immune response. The level of NAbs, viral load, and gross and histological analyses provide clear evidence that the construct provides protection against IBV and FT.

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus.

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/µL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.