Characterization of predominant genotype (QX) of avian infectious bronchitis virus isolated from layer chickens in Bangladesh.

Avian infectious bronchitis (AIB) is a highly transmissible infection that affects the poultry industry globally. This study aims to isolate and characterize emerging strains of infectious bronchitis virus (IBV) from field samples of layer chickens in Bangladesh. A total of 108 samples (trachea, lung, and kidney) were taken from dead and sick layer chickens from 18 farms in 4 areas detecting outbreaks in Bangladesh. The samples were processed and inoculated in embryonated chicken eggs (ECEs) and finally screened by the trypsin-induced hemagglutination (THA) test. Using various techniques such as hemagglutination inhibition (HI), agar gel immuno-diffusion (AGID), virus neutralization test (VNT), reverse transcription-polymerase chain reaction (RT-PCR), and nucleotide sequencing, we were able to identify and confirm the isolated IBV viruses. The study also determined the hemagglutination (HA) pattern of isolated virus using avian and mammalian red blood cells. The pathogenicity of the isolated IBV was determined using embryonated chicken eggs and day-old chicks. The study found that 8 samples were positive for IBV using ECEs, and 4 were positive by the THA test. These isolates were confirmed using HI, AGID, and VN tests. S1 gene-based RT-PCR confirmed all four isolates as IBV, with the recent isolates belonging to the genotype-QX and being similar to IBV isolates from Thailand, Saudi Arabia, and India. The HA pattern of the recent isolates showed that the isolated IBV was virulent. The pathogenicity test also revealed that the four isolates were highly pathogenic. The study indicated that the prevalent genotype (QX) of the IBV strain is present in the layer chicken population of Bangladesh.

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.

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.

Prevalence, Genotype Diversity, and Distinct Pathogenicity of 205 Gammacoronavirus Infectious Bronchitis Virus Isolates in China during 2019-2023.

Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious disease in chickens and seriously endangers the poultry industry. The emergence and co-circulation of diverse IBV serotypes and genotypes with distinct pathogenicity worldwide pose a serious challenge to the development of effective intervention measures. In this study, we report the epidemic trends of IBV in China from 2019 to 2023 and a comparative analysis on the antigenic characteristics and pathogenicity of isolates among major prevalent lineages. Phylogenetic and recombination analyses based on the nucleotide sequences of the spike (S) 1 gene clustered a total of 205 isolates into twelve distinct lineages, with GI-19 as a predominant lineage (61.77 ± 4.56%) exhibiting an overall increasing trend over the past five years, and demonstrated that a majority of the variants were derived from gene recombination events. Further characterization of the growth and pathogenic properties of six representative isolates from different lineages classified four out of the six isolates as nephropathogenic types with mortality rates in one-day-old SPF chickens varying from 20-60%, one as a respiratory type with weak virulence, and one as a naturally occurring avirulent strain. Taken together, our findings illuminate the epidemic trends, prevalence, recombination, and pathogenicity of current IBV strains in China, providing key information for further strengthening the surveillance and pathogenicity studies of IBV.

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.

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.

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.

Molecular Prevalence Study of QX-like Infectious Bronchitis Virus in Japan from 2016 to 2023.

Infectious bronchitis is an acute and highly contagious disease in chickens caused by the infectious bronchitis virus (IBV), which has caused significant economic losses to the poultry industry worldwide. The antigenic variant, the QX-like genotype (GI-19 lineage), has been currently reported in epidemics in East Asia, Southeast Asia, the Middle East, Europe, and Africa. We first reported an epidemic of Japanese QX-like IBVs genetically related to Chinese and South Korean strains in the Kyushu region of Japan in 2020. However, because their nationwide prevalence was unknown, we performed a nationwide survey. The testing of 419 reverse transcription (RT)-PCR-positive samples (376 layers and 43 broilers) of IBV field strains between April 2016 and March 2022 detected two QX-like IBVs in 2019 and 2021 broiler samples from one region. A survey of fecal samples collected from 122-layer farms nationwide between November 2022 and January 2023 detected QX-like IBV genes from seven farms in various regions. Phylogenetic tree analysis on the basis of the S1 gene showed that all QX-like IBVs detected in Japan were genetically related to recent Chinese and South Korean strains. A new RT-PCR assay was developed to distinguish between QX-like IBV and other IBV variants prevalent in Japan, whose results were consistent with those of previously reported identification methods. These results suggest that QX-like IBV is rapidly spreading in Japan and that countermeasures are necessary.

Estudio de la prevalencia molecular del virus de la bronquitis infecciosa similar a la cepa QX en Japón entre los años 2016 al 2023. La bronquitis infecciosa es una enfermedad aguda y altamente contagiosa del pollo causada por el virus de la bronquitis infecciosa (IBV), que ha causado importantes pérdidas económicas a la industria avícola en todo el mundo. Actualmente se ha reportado la variante antigénica, el genotipo similar a la cepa QX (cepa GI-19), en brotes en el este de Asia, el sudeste de Asia, el Medio Oriente, Europa y África. Primeramente, se reportó un brote con virus japoneses similares a QX que eran genéticamente relacionados con cepas chinas y surcoreanas en la región de Kyushu en Japón en el 2020. Sin embargo, debido a que se desconocía su prevalencia a nivel nacional, se realizó una encuesta a nivel nacional. Mediante el análisis de 419 muestras positivas a la presencia de cepas de campo por transcripción reversa (RT) y PCR (376 de ponedoras y 43 de pollos de engorde) entre abril del 2016 y marzo del 2022 se detectaron dos virus de bronquitis infecciosa similares a la cepa QX en el 2019 y en el 2021 en muestras de pollos de engorde de una región. Una encuesta de muestras fecales recolectadas de granjas de 122 ponedoras en todo el país entre noviembre del 2022 y enero del 2023 detectó genes de bronquitis infecciosa similares a la cepa QX de siete granjas en varias regiones. El análisis del árbol filogenético basado en el gene S1 mostró que todos los virus similares a la cepa QX detectados en Japón estaban relacionados genéticamente con cepas recientes de China y Corea del Sur. Se desarrolló un nuevo ensayo de RT-PCR para distinguir entre los virus de bronquitis infecciosa similares a la cepa QX y otras variantes del IBV prevalentes en Japón, cuyos resultados fueron consistentes con los de los métodos de identificación reportados anteriormente. Estos resultados sugieren que virus de bronquitis infecciosa similares a la cepa QX se están extendiendo rápidamente en Japón y que se necesitan medidas de control.

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.

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.

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.

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.