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.

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.

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.

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.

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.

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 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.

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.

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.

An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.

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.

Extensive genetic and biological characterization of infectious bronchitis virus strain D2860 of genotype GVIII.

Infectious bronchitis virus (IBV) strains of genotype GVIII have been emerging in Europe in the last decade, but no biological characterization has been reported so far. This paper reports the extensive genetic and biological characterization of IBV strain D2860 of genotype GVIII which was isolated from a Dutch layer flock that showed a drop in egg production. Whole genome sequencing showed that it has a high similarity (95%) to CK/DE/IB80/2016 (commonly known as IB80). Cross-neutralization tests with antigens and serotype-specific antisera of a panel of different non-GVIII genotypes consistently gave less than 2% antigenic cross-relationship with D2860. Five experiments using specified pathogen-free chickens of 0, 4, 29 and 63 weeks of age showed that D2860 was not able to cause clinical signs, drop in egg production, false layers or renal pathology. There was also a distinct lack of ciliostasis at both 5 and 8 days post-inoculation at any age, despite proof of infection by immunohistochemical (IHC) staining, RT-PCR and serology. IHC showed immunostaining between 5 and 8 days post inoculation in epithelial cells of sinuses and conchae, while only a few birds displayed immunostaining in the trachea. In vitro comparison of replication of D2860 and M41 in chicken embryo kidney cells at 37°C and at 41°C indicated that D2860 might have a degree of temperature sensitivity that might cause it to prefer the colder parts of the respiratory tract.

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.

Mitigación del síndrome de la falsa ponedora mediante anticuerpos maternos contra el virus de la bronquitis infecciosa. En este estudio se investigó la relación entre la inmunidad pasiva y el desarrollo del síndrome de la falsa ponedora (FLS) y sus lesiones asociadas comparando los efectos reproductivos a largo plazo de una cepa de tipo silvestre DMV/1639 del virus de la bronquitis infecciosa (IBV) y la cepa vacunal GA08, en aves con y sin anticuerpos maternos. Hubo un claro efecto protector proporcionado por los anticuerpos maternos tanto contra la vacunación temprana como contra el desafío. También se observó que la vacunación a una edad temprana, en ausencia de anticuerpos maternos, puede inducir problemas reproductivos, como una reducción de la producción de huevo y lesiones asociadas al síndrome de la falsa ponedora (p. ej., oviducto quístico y celomitis de yema de huevo). Esto podría indicar que los anticuerpos maternos y el momento de la infección por el virus de la bronquitis infecciosa son más importantes en la generación del síndrome de la falsa ponedora que el tipo de cepa del virus de la bronquitis infecciosa.

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.

The efficacy of simultaneous successive classic and variant infectious bronchitis virus vaccines versus circulating variant II Egyptian field virus.

Background: Being a ubiquitous, highly contagious virus with a continuous mutation and a large number of evolutions worldwide, the infectious bronchitis virus (IBV) continues to wreak problems among Egyptian chickens and generate economic losses. The commonly applied IBV vaccination protocols in broilers include alternatives to classic and/or variant attenuated live virus vaccines. Aim: The current study targeted to assess the protective efficacy of concurrent and successive Ma5 and 4/91 vaccine strain regimens against the field variant II IBV strain (IBV-EGY-ZU/Ck-127/2021) in chickens. Methods: Commercial broiler chickens were vaccinated with Ma5 and 4/91 strains simultaneously at 1 and 14 days of age. The evaluation parameters included clinical protection and humoral and early innate immunity aspects in the renal tissues of vaccinated and infected birds. Results: The vaccine regimen ameliorated the clinical and histopathological lesions against variant II IBV and enhanced body gain as well as succeeded in preventing tracheal shedding and minimizing cloacal shedding of the field virus. The IL-1ß mRNA gene expression was evident as early as 24 hours, with highly significant upregulation at 48 hours post vaccination and 24 hours post challenge (PC) in vaccinated birds. Remarkable upregulation was observed in oligoadenylate synthetases (OAS) expression 48 hours PC in vaccinated and unvaccinated infected birds. The vaccinated birds developed a significant antibody titer of 704.0 ± 111.98 at 28 days of age, with a consistent antibody titer increase after the challenge. Conclusion: Overall, a combination of heterologous protectotype commercial vaccines achieved good protection against the Egyptian variant II IBV strain. This vaccine program could be an effective protocol against the threat posed by IBV viruses circulating in the Egyptian field.

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.

Differential Impact of Massachusetts, Canadian 4/91, and California (Cal) 1737 Genotypes of Infectious Bronchitis Virus Infection on Lymphoid Organs of Chickens.

Infectious bronchitis virus (IBV) induces severe economic losses in chicken farms due to the emergence of new variants leading to vaccine breaks. The studied IBV strains belong to Massachusetts (Mass), Canadian 4/91, and California (Cal) 1737 genotypes that are prevalent globally. This study was designed to compare the impact of these three IBV genotypes on primary and secondary lymphoid organs. For this purpose, one-week-old specific pathogen-free chickens were inoculated with Mass, Canadian 4/91, or Cal 1737 IBV variants, keeping a mock-infected control. We examined the IBV replication in primary and secondary lymphoid organs. The molecular, histopathological, and immunohistochemical examinations revealed significant differences in lesion scores and viral distribution in these immune organs. In addition, we observed B-cell depletion in the bursa of Fabricius and the spleen with a significant elevation of T cells in these organs. Further studies are required to determine the functional consequences of IBV replication in lymphoid organs.

Investigating the uses of machine learning algorithms to inform risk factor analyses: The example of avian infectious bronchitis virus (IBV) in broiler chickens.

Infectious bronchitis virus (IBV) is a contagious coronavirus causing respiratory and urogenital disease in chickens and is responsible for significant economic losses for both the broiler and table egg layer industries. Despite IBV being regularly monitored using standard epidemiologic surveillance practices, knowledge and evidence of risk factors associated with IBV transmission remain limited. The study objective was to compare risk factor modeling outcomes between a traditional stepwise variable selection approach and a machine learning-based random forest Boruta algorithm using routinely collected IBV antibody titer data from broiler flocks. IBV antibody sampling events (n = 1111) from 166 broiler sites between 2016 and 2021 were accessed. Ninety-two geospatial-related and poultry-density variables were obtained using a geographic information system and data sets from publicly available sources. Seventeen and 27 candidate variables were screened to potentially have an association with elevated IBV antibody titers according to the manual selection and machine learning algorithm, respectively. Selected variables from both methods were further investigated by construction of multivariable generalized mixed logistic regression models. Six variables were shortlisted by both screening methods, which included year, distance to urban areas, main roads, landcover, density of layer sites and year, however, final models for both approaches only shared year as an important predictor. Despite limited significance of clinical outcomes, this work showcases the potential of a novel explorative modeling approach in combination with often unutilized resources such as publicly available geospatial data, surveillance health data and machine learning as potential supplementary tools to investigate risk factors related to infectious diseases.