Isolation and phylogenetic analysis of avian infectious bronchitis virus from an imported chicken meat product in Malaysia.

Avian infectious bronchitis (IB), a Gammacoronavirus, is a highly contagious upper respiratory disease, affecting chickens of all ages with a significant economic threat to the poultry industry. In February 2020, a specimen of imported chicken meat product was received and requested for coronavirus testing. The result was positive for the avian coronavirus, the IB virus (IBV) by molecular detection in the pre-screening test. Thus, this study aimed to isolate and characterize the IBV from the specimen. Virus isolation via egg inoculation was attempted and IBV was successfully isolated. The S1 subunit of the spike (S) gene of the IBV was amplified, sequenced, and the Basic Local Alignment Search Tool (BLAST) analysis showed that the IBV has 99% and 98% nucleotide similarity with the Malaysian and China IBVs, respectively. The phylogenetic analysis indicated that the virus belongs to the GI-19 lineage (also known as the QX strain) and is grouped with other IBVs from Malaysia and China. The GI-19 lineage is one of the primary IB strains that circulate in Malaysia. The recovery of the virus may be due to the persistence characteristic of the virus on meat; and the cold chain practices in the imported food product prolong the survival of this coronavirus. Though IBV is not identified as a hazard in chicken meat or meat products, raw food should be cooked thoroughly before being consumed. With the increase in international trade in poultry and poultry products, disease screening at the entry point and import risk analysis is crucial to ensure food safety and prevent the introduction of new viruses into Malaysia.

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.

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.

Infection with IBV DMV/1639 at a Young Age Leads to Increased Incidence of Cystic Oviduct Formation Associated with False Layer Syndrome.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes respiratory disease but can affect the reproductive tract of laying-type chickens. If infection occurs in pullets, false layer syndrome, which is characterized by the development of large, fluid-filled cystic oviducts, can occur. Recently, IBV strain DMV/1639 has been detected in parts of Canada and the U.S., where false layer syndrome has occurred, though it is not clear if IBV is the sole cause or if age at infection is an influencing variable. Our study investigates the role and timing of IBV infection on the development of false layer syndrome, using the IBV types DMV/1639 and Massachusetts (Mass). Six groups of 120 SPF chickens were challenged at either three, seven, or fourteen days of age, using either DMV/1639 or Mass IBV. Cystic oviducts were seen in all the challenged groups, and the pullets challenged at 14 days of age had fewer cystic oviducts than pullets challenged at 3 or 7 days of age. The highest percentage of severe histology lesion scores were seen in the 3-day challenge groups. The data collected in this experiment confirm that IBV DMV/1639 causes cystic oviducts and indicate that age at infection plays a role in the pathogenesis of false layer syndrome.

Improved Subtyping of Avian Influenza Viruses Using an RT-qPCR-Based Low Density Array: 'Riems Influenza a Typing Array', Version 2 (RITA-2).

Avian influenza virus (AIV) variants emerge frequently, which challenges rapid diagnosis. Appropriate diagnosis reaching the sub- and pathotype level is the basis of combatting notifiable AIV infections. Real-time RT-PCR (RT-qPCR) has become a standard diagnostic tool. Here, a total of 24 arrayed RT-qPCRs is introduced for full subtyping of 16 hemagglutinin and nine neuraminidase subtypes of AIV. This array, designated Riems Influenza A Typing Array version 2 (RITA-2), represents an updated and economized version of the RITA-1 array previously published by Hoffmann et al. RITA-2 provides improved integration of assays (24 instead of 32 parallel reactions) and reduced assay volume (12.5 µL). The technique also adds RT-qPCRs to detect Newcastle Disease (NDV) and Infectious Bronchitis viruses (IBV). In addition, it maximizes inclusivity (all sequences within one subtype) and exclusivity (no intersubtypic cross-reactions) as shown in validation runs using a panel of 428 AIV reference isolates, 15 reference samples each of NDV and IBV, and 122 clinical samples. The open format of RITA-2 is particularly tailored to subtyping influenza A virus of avian hosts and Eurasian geographic origin. Decoupling and re-arranging selected RT-qPCRs to detect specific AIV variants causing epizootic outbreaks with a temporal and/or geographic restriction is possible.

Pathogenicity of the Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) on Female Reproductive Tract of Chickens.

Infectious bronchitis virus (IBV) infection causes significant economic losses to various sectors of the poultry industry worldwide. Over the past few years, the incidence of false layer syndrome in Eastern Canadian layer flocks has been associated with the increased prevalence of the IBV Delmarva (DMV)/1639 strain. In this study, 1-day-old specific-pathogen-free (SPF) hens were infected with the Canadian DMV/1639 strain and observed until 16 weeks of age in order to determine if the IBV DMV/1639 strain is causing false layer syndrome. Early after infection, the virus showed a wide tissue distribution with characteristic gross and histopathological lesions in the respiratory tract and kidney. Around 60-70% of the infected hens demonstrated continuous cloacal viral shedding until the end of the experiment (at 16 weeks) which was associated with high IBV genome loads detected in the cecal tonsils. The experiment confirmed the field observations that the Canadian DMV/1639 strain is highly pathogenic to the female reproductive tract causing marked cystic lesions in the oviduct. Moreover, significant histopathological damage was observed in the ovary. Our study provides a detailed description of the pathological consequences of the IBV DMV/1639 strain circulating in an important poultry production sector.

Infectious Bronchitis Virus Prevalence, Characterization, and Strain Identification in California Backyard Chickens.

Infectious bronchitis virus (IBV) causes significant losses in the poultry industry throughout the world. Here we characterize the lesions of infectious bronchitis (IB) and IBV prevalence and identify the circulating strains in small flocks in California. Backyard chickens (BYCs) submitted to the Davis (Northern California; NorCal) and San Bernardino (Southern California; SoCal) branches of the California Animal Health and Food Safety Laboratory System from January through March 2019 were included in the study. Trachea, kidney, and cecal tonsils were collected for real-time reverse transcriptase (qRT)-PCR, histology, immunohistochemistry (IHC), and sequence analysis. A total of 50 chickens out of 169 submissions tested positive for IBV by qRT-PCR. Of these, 16% (20/123) were from NorCal and 65% (30/46) from SoCal laboratory. The cecal tonsil was the most frequently positive tissue by qRT-PCR and IHC. Lymphoplasmacytic tracheitis was the most frequent histopathologic finding in 24 of 39 birds, while the kidney showed interstitial nephritis, tubular necrosis, tubular dilation, and/or gout in 14 of 43 chickens. Infectious bronchitis virus played a primary role or a synergistic effect in the mortality of chickens that succumbed to other infectious diseases. The sequences of IBV detected in 22 birds were analyzed, and 14 strains were most similar to CA1737. One strain each matched Conn46, Cal99, and ArkDPI, and the remaining five did not have a substantial match to any available reference strains. The findings in this study indicate that small flocks can be reservoirs of IBV and might facilitate evolution of new variants as well as reversion of attenuated strains to virulence.

Artículo regular­Prevalencia, caracterización e identificación de cepas del virus de la bronquitis infecciosa en pollos de traspatio de California. El virus de la bronquitis infecciosa (con las siglas en inglés IBV) causa pérdidas significativas en la industria avícola en todo el mundo. En este estudio se caracterizaron las lesiones de la bronquitis infecciosa (IB), la prevalencia del virus y se identificó a las cepas circulantes en pequeñas parvadas en California. Se incluyeron en el estudio pollos de traspatio (BYC) remitidos a las sedes en Davis (norte de California; NorCal) y San Bernardino (sur de California; SoCal) del Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria de California de enero a marzo del 2019. Se recolectaron tráquea, riñón y tonsilas cecales para análisis cuantitativo en tiempo real (qRT)-PCR, histología, inmunohistoquímica (IHC) y análisis de secuencias. Un total de 50 pollos de 169 casos dieron positivo para la presencia del virus de bronquitis infecciosa por qRT-PCR. De estos, el 16% (20/123) provenían del norte de California y el 65% (30/46) del laboratorio del sur de California. Las tonsilas cecales fueron las muestras de tejidos positivos con mayor frecuencia por qRT-PCR e IHC. La traqueítis linfoplasmocítica fue el hallazgo histopatológico más frecuente en 24 de 39 aves, mientras que el riñón mostró nefritis intersticial, necrosis tubular, dilatación tubular y/o gota en 14 de 43 pollos. El virus de la bronquitis infecciosa jugó un papel principal o un efecto sinérgico en la mortalidad de los pollos que murieron por otras enfermedades infecciosas. Se analizaron las secuencias del virus de bronquitis detectadas en 22 aves y 14 cepas fueron muy similares al virus de bronquitis infecciosa CA1737. Tres virus coincidieron con Conn46, Cal99 y ArkDPI, y las cinco restantes no tenían una coincidencia sustancial con ninguna cepa de referencia disponible. Los hallazgos de este estudio indican que las pequeñas parvadas pueden ser reservorios del virus de la bronquitis infecciosa y podrían facilitar la evolución de nuevas variantes, así como la reversión de cepas atenuadas a formas virulentas.

Molecular Biology and Pathological Process of an Infectious Bronchitis Virus with Enteric Tropism in Commercial Broilers.

Infectious bronchitis virus (IBV) induces respiratory and urogenital disease in chickens. Although IBV replicates in the gastrointestinal tract, enteric lesions are uncommon. We have reported a case of runting-stunting syndrome in commercial broilers from which an IBV variant was isolated from the intestines. The isolate, CalEnt, demonstrated an enteric tissue tropism in chicken embryos and SPF chickens experimentally. Here, we determined the full genome of CalEnt and compared it to other IBV strains, in addition to comparing the pathobiology of CalEnt and M41 in commercial broilers. Despite the high whole-genome identity to other IBV strains, CalEnt is rather unique in its nucleotide composition. The S gene phylogenetic analyses showed great similarity between CalEnt and Cal 99. Clinically, vent staining was slightly more frequent in CalEnt-infected birds than those challenged with M41. Furthermore, IBV IHC detection was more evident and the viral shedding in feces was overall higher with the CalEnt challenge compared with M41. Despite underlying intestinal lesions caused by coccidiosis and salmonellosis vaccination, microscopic lesions in CalEnt-infected chickens were more severe than in M41-infected chickens or controls, supporting the enteric tropism of CalEnt. Further studies in SPF chickens are needed to determine the pathogenesis of the virus, its molecular mechanisms for the enteric tropism, and its influence in intestinal health.

Genomic Analysis of Avian Infectious Bronchitis Viruses Recently Isolated in South Korea Reveals Multiple Introductions of GI-19 Lineage (QX Genotype).

Infectious bronchitis virus (IBV) was first identified in the 1930s and it imposes a major economic burden on the poultry industry. In particular, GI-19 lineage has spread globally and has evolved constantly since it was first detected in China. In this study, we analyzed S1 gene sequences from 60 IBVs isolated in South Korea. Two IBV lineages, GI-15 and GI-19, were identified in South Korea. Phylogenetic analysis suggested that there were six distinct subgroups (KM91-like, K40/09-like, and QX-like I to IV) of the South Korean GI-19 IBVs. Among them, QX-type III and IV subgroups, which are phylogenetically different from those reported in South Korea in the past, accounted for more than half of the total. Moreover, the phylogeographic analysis of the QX-like subgroups indicated at least four distinct introductions of GI-19 IBVs into South Korea during 2001-2020. The efficacy of commercialized vaccines against the recently introduced QX-like subgroups should be verified, and continuous international surveillance efforts and quarantine procedures should be enhanced to prevent the incursion of viruses.

Transcriptome analysis of primary chicken cells infected with infectious bronchitis virus strain K047-12 isolated in Korea.

Infectious bronchitis virus (IBV), an avian coronavirus, is highly contagious. Chickens with IBV infection develop acute pathogenesis in multiple organs, including the respiratory and urogenital tracts. Frequent recombination in the spike (S) glycoprotein gene has made vaccine strategies ineffective. To understand IBV pathogenesis, we analyzed the genetic distance between Korean IBV isolates and other coronaviruses, including SARS-CoV-2. To obtain comprehensive information about early immune responses such as innate cytokine production and associated immune regulation during IBV infection, we infected primary chicken embryonic kidney cells and performed transcriptome analysis. We observed that the functional pathways of innate immunity are regulated and confirmed expression of genes that coordinate early immune responses. Understanding the immune profile of the host cell may assist in vaccine development.

Recombination Events Shape the Genomic Evolution of Infectious Bronchitis Virus in Europe.

Infectious bronchitis of chicken is a high morbidity and mortality viral disease affecting the poultry industry worldwide; therefore, a better understanding of this pathogen is of utmost importance. The primary aim of this study was to obtain a deeper insight into the genomic diversity of field infectious bronchitis virus (IBV) strains using phylogenetic and recombination analysis. We sequenced the genome of 20 randomly selected strains from seven European countries. After sequencing, we created a genome sequence data set that contained 36 European origin field isolates and 33 vaccine strains. When analyzing these 69 IBV genome sequences, we identified 215 recombination events highlighting that some strains had multiple recombination breaking points. Recombination hot spots were identified mostly in the regions coding for non-structural proteins, and multiple recombination hot spots were identified in the nsp2, nsp3, nsp8, and nsp12 coding regions. Recombination occurred among different IBV genotypes and involved both field and vaccine IBV strains. Ninety percent of field strains and nearly half of vaccine strains showed evidence of recombination. Despite the low number and the scattered geographical and temporal origin of whole-genome sequence data collected from European Gammacoronaviruses, this study underlines the importance of recombination as a major evolutionary mechanism of IBVs.

Comparison of tracheal and choanal cleft swabs and poultry dust samples for detection of Newcastle disease virus and infectious bronchitis virus genome in vaccinated meat chicken flocks.

This study assessed different methods (tracheal and choanal cleft swabs from individual birds, and poultry dust as a population level measure) to evaluate the shedding kinetics of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) genome in meat chicken flocks after spray vaccination at hatchery. Dust samples and tracheal and choanal cleft swabs were collected from four meat chicken flocks at 10, 14, 21 and 31 days post vaccination (dpv) and tested for IBV and NDV genome copies (GC) by reverse transcriptase (RT)-PCR. IBV and NDV GC were detected in all sample types throughout the study period. Detection rates for choanal cleft and tracheal swabs were comparable, with moderate and fair agreement between sample types for IBV (McNemar's = 0.27, kappa = 0.44) and NDV (McNemar's = 0.09; kappa = 0.31) GC respectively. There was no significant association for IBV GC in swabs and dust samples (R2 = 0.15, P = 0.13) but NDV detection rates and viral load in swabs were strongly associated with NDV GC in dust samples (R2 = 0.86 and R2 = 0.90, P<0.001). There was no difference in IBV and NDV GC in dust samples collected from different locations within a poultry house. In conclusion, dust samples collected from any location within poultry house show promise for monitoring IBV and NDV GC in meat chickens at a population level and choanal cleft swabs can be used for detection of IBV and NDV GC instead of tracheal swabs in individual birds.

Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus.

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

Coronavirus: proteomics analysis of chicken kidney tissue infected with variant 2 (IS-1494)-like avian infectious bronchitis virus.

Avian infectious bronchitis virus is one of the most important gammacoronaviruses, which causes a highly contagious disease. In this study, we investigated changes in the proteome of kidney tissue of specific-pathogen-free (SPF) chickens that were infected with an isolate of the nephrotropic variant 2 genotype (IS/1494/06) of avian coronavirus. Twenty 1-day-old SPF White Leghorn chickens were randomly divided into two groups, each comprising 10 chickens, which were kept in separate positive-pressure isolators. Chickens in group A served as a virus-free control group up to the end of the experiment, whereas chickens in group B were inoculated with 0.1 ml of 104.5 EID50 of the IBV/chicken/Iran/UTIVO-C/2014 isolate of IBV, and kidney tissue samples were collected at 2 and 7 days post-inoculation (dpi) from both groups. Sequencing of five protein spots at 2 dpi and 22 spots at 7 dpi that showed differential expression by two-dimensional electrophoresis (2DE) along with fold change greater than 2 was done by MS-MALDI/TOF/TOF. Furthermore, the corresponding protein-protein interaction (PPI) networks at 2 and 7 dpi were identified to develop a detailed understanding of the mechanism of molecular pathogenesis. Topological graph analysis of this undirected PPI network revealed the effect of 10 genes in the 2 dpi PPI network and nine genes in the 7 dpi PPI network during virus pathogenesis. Proteins that were found by 2DE analysis and MS/TOF-TOF mass spectrometry to be down- or upregulated were subjected to PPI network analysis to identify interactions with other cellular components. The results show that cellular metabolism was altered due to viral infection. Additionally, multifunctional heat shock proteins with a significant role in host cell survival may be employed circuitously by the virus to reach its target. The data from this study suggest that the process of pathogenesis that occurs during avian coronavirus infection involves the regulation of vital cellular processes and the gradual disruption of critical cellular functions.

Infectious Bronchitis Virus Associated with Nephropathy Lesions in Diagnostic Cases from Commercial Broiler Chickens in California.

In March 2019, the California Animal Health and Food Safety Laboratory (CAHFS), Turlock branch, received two submissions of broiler chickens from commercial flocks reporting increased mortality. Submissions consisted of either white or brown broilers. Submitted chickens appeared depressed with ruffled feathers. At necropsy, moderate to severely enlarged and pale kidneys were observed, with gross lesions indicative of dehydration. Microscopically, renal tubules were degenerated and distended with necrotic debris and tubular casts. The kidney parenchyma contained mononuclear inflammatory cell infiltrates and interstitial edema. Infectious bronchitis virus (IBV) was isolated and identified by reverse transcription quantitative PCR from kidney tissue pools and tracheal swab pools from both cases. Partial sequencing of the S1 hypervariable region was most similar to a local California variant, CA1737. The outbreak lasted roughly 1 wk in both flocks, with 2% total mortality in the brown broilers and 20% total mortality in the white broilers. Final proof of the IBV strains causing nephropathy will require fulfillment of Koch postulates. IBV associated with nephropathy has been sporadically reported in California chicken flocks and represents a significant pathogen due to its potential for inducing high flock mortality. The incidence of IBV associated with a nephropathy diagnosis in chicken necropsy submissions to the CAHFS system-wide from 1998 to 2019 is also reviewed.

A label-free electrochemical assay for coronavirus IBV H120 strain quantification based on equivalent substitution effect and AuNPs-assisted signal amplification.

A label-free electrochemical strategy is proposed combining equivalent substitution effect with AuNPs-assisted signal amplification. According to the differences of S1 protein in various infectious bronchitis virus (IBV) strains, a target DNA sequence that can specifically recognize H120 RNA forming a DNA-RNA hybridized double-strand structure has been designed. Then, the residual single-stranded target DNA is hydrolyzed by S1 nuclease. Therefore, the content of target DNA becomes equal to the content of virus RNA. After equivalent coronavirus, the target DNA is separated from DNA-RNA hybridized double strand by heating, which can partly hybridize with probe 2 modified on the electrode surface and probe 1 on AuNPs' surface. Thus, AuNPs are pulled to the surface of the electrode and the abundant DNA on AuNPs' surface could adsorb a large amount of hexaammineruthenium (III) chloride (RuHex) molecules, which produce a remarkably amplified electrochemical response. The voltammetric signal of RuHex with a peak near - 0.28 V vs. Ag/AgCl is used as the signal output. The proposed method shows a detection range of 1.56e-9 to 1.56e-6 µM with the detection limit of 2.96e-10 µM for IBV H120 strain selective quantification detection, exhibiting good accuracy, stability, and simplicity, which shows a great potential for IBV detection in vaccine research and avian infectious bronchitis diagnosis. Graphical abstract.

Immunopathogenesis of infectious bronchitis virus Q1 in specific pathogen free chicks.

The immunopathogenesis of avian coronavirus, infectious bronchitis virus (IBV) Q1, was investigated in specific pathogen free chicks. Following infection, chicks exhibited respiratory clinical signs and reduced body weight. Oropharyngeal (OP) and cloacal (CL) swabs were collected at intervals and found to be RT-PCR positive, with a greater number of partial-S1 amino acid changes noted in CL swabs compared to OP swabs. In tissue samples, IBV viral load peaked 9 days post infection (dpi) in the trachea and kidneys, and 14 dpi in the proventriculus. At 28 dpi, ELISA data showed that 63% of infected chicks seroconverted. There was significantly higher mRNA up-regulation of IFN-α, TLR3, MDA5, LITAF, IL-1ß and IL-6 in the trachea compared to the kidneys. Findings presented here demonstrate that this Q1 isolate induces greater lesions and host innate immune responses in chickens' tracheas compared to the kidneys.

Molecular identification of infectious bronchitis virus isolated from respiratory diseases in some Iranian broiler flocks.

Infectious Bronchitis (IB) is an acute, highly contagious disease associated with respiratory signs in young chickens and reduced egg production and quality in layers. The purpose of this study was to isolate and identify the infectious bronchitis virus in broiler flocks with respiratory diseases in four provinces of Iran. The specimens from forty IB suspected flocks from different regions of Isfahan, East Azerbaijan, Golestan, and Khuzestan provinces were collected, and the trachea, lung, and cecal tonsils were sampled. The samples were inoculated into 9- to 11-day-old embryonated chicken eggs. After collecting the allantoic fluid, RT-PCR was carried out to detect IB viruses. The results showed that IBVs were isolated from 30% of the flocks in these four provinces. The positive samples, according to a partial S1 gene sequence, were more investigated. Comparing nucleotide and amino acid sequences showed that the four isolates had the most similarity to the Pakistani 793/B strain (GI-13 lineage). The three isolates had the most considerable similarity in amino acid and nucleotide sequences to Iraqi and Iranian QX-like viruses (GI-19 lineage). Two isolates had 96 to 98% resemblance to Iranian variant-2 (GI-23 lineage) isolates. One isolate was found to belong to the Massachusetts serotype (GI-1 lineage) having 100% similarity in its amino acid sequence to the Massachusetts serotypes in GenBank. The phylogenetic relationship of the isolates shows complexity and diversity concerning different sequences and geographical regions.

Whole-Genome Sequencing Protocols for IBV and Other Coronaviruses Using High-Throughput Sequencing.

This chapter reports the high-throughput sequencing protocol for sequencing Coronaviruses and other positive strand viruses to produce a dataset of significant depth of coverage. The protocol describes sequencing of infectious bronchitis virus propagated in embryonated eggs and harvested in the allantoic fluid. The protocol is composed of three main steps-enrichment of the allantoic fluid using ultracentrifugation, extraction of total RNA from allantoic fluid, and library preparation from total RNA to DNA sequencing libraries. The workflow will be suitable for all coronaviruses using high-throughput sequencing platforms.