Single-step multiplex reverse transcription-polymerase chain reaction for the detection and differentiation of QX-like infectious bronchitis virus from the Thai variant and vaccine strains H120, Ma5, and 4/91

Background and Aim: QX-like infectious bronchitis virus (IBV) is a highly infectious avian coronavirus that causes respiratory and kidney disease. It is linked to increased mortality and loss of performance in infected chickens worldwide, including Thailand. Thus, a simple and rapid diagnostic method for the diagnosis of QX-like IBV is needed. This study aimed to develop a single-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay to detect and differentiate QX-like IBV from Thai IBV and vaccine strains used in the poultry industry (H120, Ma5, and 4/91). Materials and Methods: Primer sets specific for QX-like and Thai IBV were designed to target the S1 gene. The specificity of the technique was verified using nine isolates of QX-like IBV, four isolates of Thai IBV, and other avian viral respiratory pathogens. The detection limit was evaluated using a serial ten-fold dilution of QX-like and Thai IBV. Results: The results showed that single-step mRT-PCR could detect QX-like IBV and differentiate it from Thai IBV and the vaccine strains H120, Ma5, and 4/91. The limit of detection of the developed assay was 102.2 embryo infectious dose (EID)50/mL for QX-like IBV and 101.8 EID50/mL for Thai IBV. Interestingly, the developed assay could identify mixed infection by both IBVs in a single sample. Conclusion: The single-step mRT-PCR assay developed in this study can potentially discriminate QX-like IBV from Thai IBV and the vaccine strains H120, Ma5, and 4/91 in a single reaction. It is also suitable for use in all laboratories with access to conventional PCR equipment. [ FROM AUTHOR] Copyright of Veterinary World is the property of Veterinary World and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Key Aspects of Coronavirus Avian Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) is an enveloped and positive-sense single-stranded RNA virus. IBV was the first coronavirus to be discovered and predominantly causes respiratory disease in commercial poultry worldwide. This review summarizes several important aspects of IBV, including epidemiology, genetic diversity, antigenic diversity, and multiple system disease caused by IBV as well as vaccination and antiviral strategies. Understanding these areas will provide insight into the mechanism of pathogenicity and immunoprotection of IBV and may improve prevention and control strategies for the disease.

Emergence of a genotype I lineage 1 variant avian infectious bronchitis virus in Assam, India

Background: Infectious bronchitis (IB) is an acute and highly contagious viral disease of poultry affecting chicken of all ages. The causative agent IB virus (IBV) is a Gammacoronavirus within the family Coronaviridae. Viral genetic mutations and recombination events particularly in the spike protein (S1) of IBV constantly give rise to emerging IBV variants. Vaccination is considered as the most reliable approach for IBV control, but current vaccines have been found to be ineffective due to constant emergence of new variant viruses. Objective(s): The objective of our study was to detect IBV genotypes prevalent in Assam, India. Material(s) and Method(s): Oro-pharyngeal swabs and tissue samples from unvaccinated broiler chickens showing respiratory symptoms were tested using RT-PCR targeting the N gene of IBV. The virus was isolated from infected swab/tissue samples in 9 days old specific pathogen free embryonated chicken eggs through allantoic cavity route. Phylogenetic studies were done based on the S1 gene of IBV. Results and Conclusion(s): Clinically, the birds showed gasping and tracheal rales. Necropsy revealed distended ureters. Virus was isolated and identified by curling and dwarfing of the dead embryos and further confirmed by RT-PCR. Positive PCR amplicons were sequenced and phylogenetic analysis clustered the IBV isolate from Assam with genotype I lineage 1 IBV prototype sequence belonging to Beaudette and Mass 41 strains but the isolate exhibited a relatively high degree of sequence divergence with reference strains. Our findings suggest that the IBV isolate might have emerged from recombination with the local circulating virus or vaccine strains. This will have important implications for IB prevention strategies.

Nationwide Surveillance for Infectious Bronchitis Virus in South Korea from 2020 to 2021.

Infectious bronchitis virus (IBV) is a major pathogen in poultry. The genotypes of IBV vary considerably, and their antigenicity may differ. Nationwide surveillance in South Korea was performed to determine the prevalence and distribution of IBV and its genotypes. By both active and passive surveillance, a total of 939 samples were collected and tested for IBV detection by pathogen-specific reverse transcriptase-PCR. IBV RNA-positive samples were inoculated in embryonated eggs for virus isolation. IBV was genotyped and analyzed phylogenetically based on a partial nucleotide sequence of the S1 gene. A total of 114 IBV strains were isolated; 34 (30.9%) of the 110 samples obtained by passive surveillance, and 80 (9.7%) of the 829 samples obtained by active surveillance, were positive. Most IBVs in both groups were isolated from broilers. Five genotypes (QX-like, B4-like, KM91-like, K40/09-like, and 20AD17-like) were observed in South Korea, with the QX-like genotype being the most common, and the 20AD17-like genotype being a novel genotype. These findings will help to maximize protection against IBV infection by providing a reference for the selection of an avian vaccine for IBV in South Korea.

Vigilancia nacional del virus de la bronquitis infecciosa en Corea del Sur del año 2020 al 2021. El virus de la bronquitis infecciosa (IBV) es un patógeno importante en la avicultura. Los genotipos del virus de la bronquitis varían considerablemente y su antigenicidad puede ser diversa. Se realizó un estudio de vigilancia a nivel nacional en Corea del Sur para determinar la prevalencia y distribución del virus de bronquitis y sus genotipos. Mediante vigilancia activa como pasiva, se recolectaron un total de 939 muestras y se analizaron para la detección del virus de la bronquitis infecciosa mediante transcripción reversa y PCR específica para este patógeno. Se inocularon muestras positivas para ARN del virus de bronquitis en huevos embrionados para el aislamiento del virus. Los virus de bronquitis se genotipificaron y analizaron filogenéticamente basándose en una secuencia parcial de nucleótidos del gene S1. Se aislaron un total de 114 cepas del virus de bronquitis; 34 (30.9%) de las 110 muestras obtenidas por vigilancia pasiva y 80 (9.7%) de las 829 muestras obtenidas por vigilancia activa resultaron positivas. La mayoría de los virus de bronquitis en ambos grupos se aislaron de pollos de engorde. Se observaron cinco genotipos (similares a QX, similares a B4, similares a KM91, similares a K40/09 y similares a 20AD17) en Corea del Sur, siendo el genotipo similar a QX el más común y el genotipo similar a 20AD17 que ha sido un genotipo de nueva aparición. Estos hallazgos ayudarán a maximizar la protección contra la infección por el virus de la bronquitis infecciosa al proporcionar una referencia para la selección de vacunas aviares para bronquitis infecciosa en Corea del Sur.

A Multiplex Polymerase Chain Reaction for Differential Detection of Turkey Coronavirus from Chicken Infectious Bronchitis Virus and Bovine Coronavirus

A multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis virus (IBV), and bovine coronavirus (BCoV) is presented in this chapter. Primers are designed from the conserved or variable regions of nucleocapsid (N) or spike (S) protein genes of TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by PCR reaction is used to amplify a portion of N or S gene of the corresponding coronaviruses. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, are obtained for TCoV. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene is obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene is obtained for BCoV. Copyright © Springer Science+Business Media New York 2016.

An RT-PCR Assay for Detection of Infectious Bronchitis Coronavirus Serotypes

Avian infectious bronchitis virus (IBV), a chicken Gammacoronavirus, is a major poultry pathogen, and is probably endemic in all regions with intensive poultry production. Since IBV was first described in 1936, many serotypes and variants of IBV have been isolated worldwide. IBV isolates are capable of infecting a large range of epithelial surfaces of the chicken, involving the respiratory, renal, and reproductive systems;however, the clinical signs are usually not specific for differential diagnoses. Virus isolation is commonly used for diagnosis of IBV infection, which was achieved through passage of clinical materials via the allantoic route of embryos. Currently, more sensitive molecular approaches for the detection of avian pathogens have been developed, including reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR, which are more suitable for use in diagnostic laboratories. In this chapter, we describe a one-step RT-PCR which can be used for detecting most of IBV serotypes in the IBV-infected allantoic fluid and has been used routinely in our laboratories for detection of IBVs. Copyright © Springer Science+Business Media New York 2016.

Reverse Genetics of Avian Coronavirus Infectious Bronchitis Virus

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA;that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA dependant RNA polymerase. Copyright © Springer Science+Business Media New York 2016.

Antibody-Capture Enzyme-Linked Immunosorbent Assay for Detection of Antibody to Turkey Coronavirus Using Infectious Bronchitis Virus or Recombinant Nucleocapsid Protein as Coating Antigen

Turkey coronavirus (TCoV) infection continues to threaten turkey industry. Because specific treatment and effective vaccination program are not available, rapid and cost-effective detection of antibodies to TCoV infection is an important control measure to monitor the disease status in the fields. Two antibody-capture enzyme-linked immunosorbent assay (ELISA) procedures for detection of antibodies to TCoV are outlined in this chapter. One ELISA method uses chicken infectious bronchitis coronavirus (IBV) as the coating antigen based on antigenic cross-reactivity between TCoV and IBV. The other method relies on a recombinant TCoV nucleocapsid protein. Both methods are useful for serological diagnosis of TCoV infection in the turkey flocks. Copyright © Springer Science+Business Media New York 2016.

Updates on Coronavirus-Related Ocular Manifestations: From the Past to COVID-19 Pandemic

Context: Coronaviruses are the largest RNA viruses associated with some ocular manifestations. There are various contradictory reports about the ocular manifestations of coronaviruses in humans. Different ocular tissues can affect coronavirus-associated infectious diseases. Evidence Acquisition: All manuscripts were collected from PubMed, Google Scholar, and other relevant databases. All searches were done by specific keywords, including "coronavirus," "ocular disease," and "eye." Results: There are various contradictory reports about the ocular effects of coronaviruses in humans. Different ocular tissues are involved in coronavirus-associated infectious diseases. The ductal connection between the upper respiratory tract and eye mucosa through the nasolacrimal duct can be an entrance to respiratory viruses, such as coronaviruses. The coronavirus can infect the retina, conjunctiva, cornea, and uvea of the eye. The primary SARS-CoV-2 receptor, ACE2, is mainly expressed in the posterior tissues of the eye, such as the retina and RPE. Feline CoV, SARS-CoV, MERS-CoV, and infectious bronchitis virus (IBV) are responsible for conjunctiva involvement in coronavirus-related ocular pathogenesis. Also, various studies are held on COVID-19 and the impact of the conjunctiva on diagnosis and medical complications. Given that the cornea has an acceptable expression of ACE2 and TMPRSS2 genes compared to lung tissue, some studies were done on the impact of the cornea in COVID-19. Feline infectious peritonitis virus (FIPV) is also related to uvea complications. The experiments of human and animal models on the effects of coronaviruses on the retina and cornea in the event of various epidemics of coronaviruses and new and unknown ocular complications can be of great help to future studies. Conclusions: Given the importance of investigating the pathogenesis and other routes of SARS-CoV-2 infection, especially in areas other than the respiratory tract, this report attempts to highlight the importance of eye infections caused by the virus, its role in maintaining the virus transmission chain, and its impact on public health.

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

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

Comparative trachea transcriptome analysis in SPF broiler chickens infected with avian infectious bronchitis and avian influenza viruses.

Infectious bronchitis virus (IBV) and avian influenza virus (AIV) are two major respiratory infections in chickens. The coinfection of these viruses can cause significant financial losses and severe complications in the poultry industry across the world. To examine transcriptome profile changes during the early stages of infection, differential transcriptional profiles in tracheal tissue of three infected groups (i.e., IBV, AIV, and coinfected) were compared with the control group. Specific-pathogen-free chickens were challenged with Iranian variant-2-like IBV (IS/1494), UT-Barin isolates of H9N2 (A/chicken/Mashhad/UT-Barin/2017), and IBV-AIV coinfection; then, RNA was extracted from tracheal tissue. The Illumina RNA-sequencing (RNA-seq) technique was employed to investigate changes in the Transcriptome. Up- and downregulated differentially expressed genes (DEGs) were detected in the trachea transcriptome of all groups. The Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology databases were examined to identify possible relationships between DEGs. In the experimental groups, upregulated genes were higher compared to downregulated genes. A more severe immune response was observed in the coinfected group; further, cytokine-cytokine receptor interaction, RIG-I-like receptor signaling, Toll-like receptor signaling, NOD-like receptor signaling, Janus kinase/signal transducer, and activator of transcription, and apoptotic pathways were important upregulated genes in this group. The findings of this paper may give a better understanding of transcriptome changes in the trachea during the early stages of infection with these viruses.

Animal Coronaviruses

Coronaviruses (CoVs) infect diverse animal species and cause respiratory, enteric, hepatic, renal, neurologic, and even systemic diseases. The majority of CoVs have a narrow host specificity, but a few CoVs have a broad range of host specificity. This chapter provides a brief review of animal CoVs, including SARS-CoV-2 of animals for their receptors, host tropism, and pathogenesis in target animals.

Expression and immunoreactivity of a recombinant multi-epitope antigen designed based on four major structural proteins of avian infectious bronchitis virus.

The development of rapid, simple, and sensitive diagnostic methods for identification of avian infectious bronchitis virus (IBV) is crucial for the effective control of avian infectious bronchitis. In the present study, a tandemly arranged multiepitope peptide (named SEMN) was designed with four antigenic regions derived from four major structural proteins of IBV. Then, we performed codon optimization of SEMN gene by changing the codon-adaptation index from 0.45 to 0.94 and expressed the optimized gene in codon bias-adjusted Escherichia coli Rosetta (DE3), followed by determination of the immunoreactivity of the purified protein. Bioinformatics analysis of SEMN showed a high antigenicity, surface probability and hydrophilicity. The recombinant protein rSEMN was expressed both in soluble forms and as inclusion bodies, and the molecular weight of rSEMN was about 39 kDa. The preliminary diagnostic performance of rSEMN was confirmed by Western blotting analysis using chicken anti-IBV polyclonal antibodies. Further studies are needed to evaluate the immunogenicity in animal models and to give a final assessment of the diagnostic utility of this recombinant multi-epitope antigen.

The Characterization of chIFITMs in Avian Coronavirus Infection In Vivo, Ex Vivo and In Vitro.

The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.