Infectious bronchitis virus: an overview of the "chicken coronavirus".

Infectious bronchitis virus (IBV) is a highly contagious avian Gammacoronavirus that affects mainly chickens (Gallus gallus) but can circulate in other avian species. IBV constitutes a significant threat to the poultry industry, causing reduced egg yield, growth and mortality levels that can vary in impact. The virus can be transmitted horizontally by inhalation or direct/indirect contact with infected birds or contaminated fomites, vehicles, farm personnel and litter (Figure 1). The error-prone viral polymerase and recombination mechanisms mean diverse viral population results, with multiple genotypes, serotypes, pathotypes and protectotypes. This significantly complicates control and mitigation strategies based on vigilance in biosecurity and the deployment of vaccination.

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.

Pathological effect of different avian infectious bronchitis virus strains on the bursa of Fabricius of chickens.

Infectious bronchitis is an acute and highly contagious disease caused by avian infectious bronchitis virus (IBV). As well as the typical clinical respiratory signs, such as dyspnoea and tracheal rales, QX genotype strains can also cause damage to the urinary system and reproductive system. Our previous studies found that chickens infected with QX-type IBV also displayed damage to the bursa of Fabricius. To investigate the effects of different genotypes of IBV on the bursa of Fabricius, we challenged one-week-old SPF chickens with Mass, QX and TW genotype IBV strains and compared the clinical signs, gross lesions, histopathological damage, viral loads, and expression levels of inflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-α,ß, γ and TNF-α). The results showed that all three strains caused tissue damage, while significant temporal variations in the viral loads of the different infected groups were detected. IBV infection seriously interfered with the natural immune response mediated by inflammatory cytokines (IFN-α, IFN-ß, IL-6 and IFN-γ) in chickens. Our results suggested that IBV has potential immunological implications for chickens that may lead to poor production efficiency. RESEARCH HIGHLIGHTSAvian coronavirus IBV is an important pathogen of chickens.IBV has potential immunological implications in chickens.The bursal viral load of different IBV strains varies significantly.

Metagenomic sequencing determines complete infectious bronchitis virus (avian Gammacoronavirus) vaccine strain genomes and associated viromes in chicken clinical samples.

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

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.

Genetic and Pathogenic Characterization of QX(GI-19)-Recombinant Infectious Bronchitis Viruses in South Korea.

Infectious bronchitis viruses (IBVs) are evolving continuously via genetic drift and genetic recombination, making disease prevention and control difficult. In this study, we undertook genetic and pathogenic characterization of recombinant IBVs isolated from chickens in South Korea between 2003 and 2019. Phylogenetic analysis showed that 46 IBV isolates belonged to GI-19, which includes nephropathogenic IBVs. Ten isolates formed a new cluster, the genomic sequences of which were different from those of reference sequences. Recombination events in the S1 gene were identified, with putative parental strains identified as QX-like, KM91-like, and GI-15. Recombination detection methods identified three patterns (rGI-19-I, rGI-19-II, and rGI-19-III). To better understand the pathogenicity of recombinant IBVs, we compared the pathogenicity of GI-19 with that of the rGI-19s. The results suggest that rGI-19s may be more likely to cause trachea infections than GI-19, whereas rGI-19s were less pathogenic in the kidney. Additionally, the pathogenicity of rGI-19s varied according to the genotype of the major parent. These results indicate that genetic recombination between heterologous strains belonging to different genotypes has occurred, resulting in the emergence of new recombinant IBVs in South Korea.

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.

Infectious bronchitis virus in Australia: a model of coronavirus evolution - a review.

Infectious bronchitis virus (IBV) was first isolated in Australia in 1962. Ongoing surveillance and characterization of Australian IBVs have shown that they have evolved separately from strains found throughout the rest of the world, resulting in the evolution of a range of unique strains and changes in the dominant wild-type strains, affecting tissue tropism, pathogenicity, antigenicity, and gene arrangement. Between 1961 and 1976 highly nephropathogenic genotype GI-5 and GI-6 strains, causing mortalities of 40% to 100%, predominated, while strains causing mainly respiratory disease, with lower mortality rates, have predominated since then. Since 1988, viruses belonging to two distinct and novel genotypes, GIII and GV, have been detected. The genome organization of the GIII strains has not been seen in any other gammacoronavirus. Mutations that emerged soon after the introduction of vaccination, incursion of strains with a novel lineage from unknown sources, recombination between IBVs from different genetic lineages, and gene translocations and deletions have contributed to an increasingly complex IBV population. These processes and the consequences of this variation for the biology of these viruses provide an insight into the evolution of endemic coronaviruses during their control by vaccination and may provide a better understanding of the potential for evolution of other coronaviruses, including SARS-CoV-2. Furthermore, the continuing capacity of attenuated IBV vaccines developed over 40 years ago to provide protection against viruses in the same genetic lineage provides some assurance that coronavirus vaccines developed to control other coronaviruses may continue to be effective for an extended period.

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.

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.

A highly pathogenic recombinant infectious bronchitis virus with adaptability in cultured cells.

Infectious bronchitis virus (IBV) of GI-19 (QX), GI-7 (TW), GI-13 (4/91) and GI-1 (Mass) lineages have been frequently detected in China in recent years. Here, An IBV strain, referred as GD17/04, was isolated from the dead yellow feather chicken vaccinated with H52 and 4/91 vaccines, whose genome sequence was obtained through high-throughput sequencing. Then it has been confirmed by the RDP and SimPlot analysis that GD17/04 is a recombinant strain deriving from YX10, 4/91, TW 2575/98 and H52 strains. Therein S1 gene of GD17/04 consists of sequences of TW2575/98 and 4/91, the former for the region of 20,371 to 21,072 nt and 21,847 to 21,975 nt, the latter for the sandwiched region of 21,073 to 21,846 nt. Moreover, as a nephropathogenic variant which caused high morbidity of 100 % and mortality of 60 %, unlike most other IBV strains, GD17/04 can cause obvious cell lesion in primary CEK cell, and even in DF-1 cells, without the process of continuous passage. As the few IBV strain can infect avian passage cell line, GD17/04 provides a material basis for further study of the interaction mechanism between IBV and avian host. Collectively, the findings highlight the significance that biological characteristics of novel strain should be studied, in addition to constant epidemiologic and molecular surveillance for IBV.

Molecular characterization of isolated infectious bronchitis viruses from affected vaccinated broiler flocks in Syria.

BACKGROUND: Avian Infectious Bronchitis Virus (IBV) is a highly contagious disease that imposes a huge economic burden on the global poultry industry. IBV contains numerous serotypes and variants with incomplete tenuous cross immunological protection. The failure of currently used vaccines to protect against diverse, circulating IBV strains that are specific to a given region poses a major problem for the poultry industry. Thus, there is an urgent need to conduct studies aimed at genotyping field IB viruses. In this study, we have determined the molecular characteristics of circulating IBV by sequencing the S1 gene of viral isolates from affected previously vaccinated broiler flocks suffering from the disease. RESULTS: Ten isolates propagated in embryonated eggs showed an ability to induce typical IBV lesions after three successive viral passages. We performed a nested RT-PCR assay that targeted the hypervariable region 3 (HVR-3) of the S1 gene, and identified the isolates as IBV through sequence analysis. The IBV isolates showed sequence similarity between the Syrian isolates that vary from 96.20 to 100%, and those being closer to the Variant-2 strain IS/1494/06 (EU780077.2) with 97.5 to 99.4% similarities. However, less nucleotide identity was found with sequences belonging to the used vaccine strains such as H120, Mass5, and 4/91. CONCLUSIONS: This study showed the presence of the Variant-2 strain circulating in Syrian broiler flocks showing signs of IBV disease. Currently, there is no commercial effective vaccine which protects birds against the Variant-2 strain. Continuous monitoring procedures should be taken to control and limit the spread of the IBV Variant-2 strain. This research emphasizes both the importance of epidemiological monitoring in intensive poultry farming for novel pathogens and the use of local isolates as future vaccine targets.

Emerging infectious bronchitis virus (IBV) in Egypt: Evidence for an evolutionary advantage of a new S1 variant with a unique gene 3ab constellation.

Infectious bronchitis virus (IBV), a gamma-coronavirus, causes infectious bronchitis (IB), a major respiratory disease of chicken. Its high mutation rate in conjunction with recombination of the RNA genome constantly creates IBV variants that are difficult to control by currently available vaccines. In this study, we addressed the question whether small-scale holdings might harbor IBV variants that serve as a reservoir for newly emerging variants. Egyptian IBV isolate EGY/NR725/2016 (NR725/16) from a small-scale broiler farm was assigned to genotype I, clade 23 (S1:GI-23), based on partial S1 gene sequences and corroborated by full genome sequencing. Analysis of the S1 gene established three subclades for historical IBV strains (S1:GI-23.1, S1:GI-23.2.1 and S1:GI-23.2.2) and confirmed NR725/16 as being part of a separate fourth subclade (S1:GI-23.3). Samples from the years 2018 and 2019 revealed that the new subclade prevails in Egypt, carrying fixed mutations within the hypervariable regions (HVR) 1-3 of the S1 protein that affect two neutralization sensitive epitopes at sites 294F, 297S and 306Y (48.2) and 329R (62.1). In addition, recombination was recognized in isolate NR 725/16, with intra-subtype mixing for the entire genes 3ab and E and inter-subtype mixing for the entire gene 6b with a close match to QX like viruses of genotype GI-19. Further analysis of gene 3ab detected the homologous gene pool to NR725/16 in samples from 2013 (3ab:C) and closely related 3ab genotypes in IBV Egyptian isolates from 2016, 2018 and 2019. These data prove a flourishing exchange between poultry holdings with a common gene pool. The continued circulation of viruses harboring genes S1:GI-23.3 and 3ab:C indicates an evolutionary advantage of this combination possibly by combining antigenic escape with modulated pathogenicity to facilitate IBV spread in the vaccinated poultry population in Egypt.

Efficacy of Infectious Bronchitis GI-13 (793B) Vaccine Candidate Tested According to the Current European Union Requirements and for Cross-Protection Against Heterologous QX-Like Challenge.

Infectious bronchitis (IB) is a highly contagious viral disease of chickens, known to cause severe economic losses. Vaccination against IB virus (IBV) is an important control measure against the disease. The objective of the present study was to test Avishield IB GI-13, the vaccine candidate against IBV, strain V-173/11 (GI-13 genotype), according to European Pharmacopoeia (Ph. Eur.) efficacy requirements. Laboratory study on specific-pathogen-free (SPF) chickens showed 100% protection against challenge 10 days after vaccination of 1-7 day-old chickens by three recommended routes. Duration of immunity was shown to be at least 8 weeks after vaccination. Chickens with maternally derived antibodies (MDA) were 100% protected against challenge 21 and 35 days after vaccination. Testing of the vaccine candidate in field conditions on commercial broiler and layer farms showed 80-90% protection against homologous challenge after spray (broilers and layers) or oral (broilers) vaccine administration. Serum antibodies were monitored during the studies, and although good seroconversion was observed in MDA-positive chickens 34 days after vaccination or later, the data from SPF chickens indicate that non-humoral immunity is important in protection against challenge. Neutralizing antibodies in tears were detected, however, their level could not be fully linked with individual protection scores. A cross-protection study showed that administration of the combination of Avishield IB H120 vaccine and Avishield IB GI-13 vaccine candidate at day 1, confers good protection against heterologous QX-like challenge. Stability of the vaccine after reconstitution in 0.2% skimmed milk solution or distilled water at room temperature was confirmed over the period of 3 h. The vaccine candidate fully complied with Ph. Eur. requirements, with very good protection levels, indicating that it can be administered already at 1 day of age by spray at the hatchery or at 7 days of age by drinking water on the farm.

Characterization of novel, pathogenic field strains of infectious bronchitis virus (IBV) in poultry in Trinidad and Tobago.

Avian coronaviruses, including infectious bronchitis virus (IBV) and turkey coronavirus (TCoV), are economically important viruses affecting poultry worldwide. IBV is responsible for causing severe losses to the commercial poultry sector globally. The objectives of this study were to identify the viruses that were causing outbreaks of severe respiratory disease in chickens in Trinidad and Tobago (T&T) and to characterize the strains. Swab samples were collected from birds showing severe respiratory signs in five farms on the island of Trinidad. Samples were tested for the presence of IBV, as well as avian influenza virus (AIV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) by real-time reverse transcription polymerase chain reaction (qRT-PCR). All samples from the five farms tested negative for AIV, NDV and aMPV; however, samples from clinically affected birds in all five of the farms tested positive for IBV. Genetic data revealed the presence of TCoV in chickens on two of the farms. Interestingly, these two farms had never reared turkeys. Phylogenetic analysis showed that IBV S1 sequences formed two distinct clusters. Two sequences grouped with vaccine strains within the GI-1 lineage, whereas three sequences grouped together, but separately from other defined lineages, forming a likely new lineage of IBV. Pairwise comparison revealed that the three unique variant strains within the distinct lineage of IBV were significantly different in their S1 nucleotide coding regions from viruses in the closest lineage (16% difference) and locally used vaccine strains (>20% difference). Results also suggested that one of the samples was a recombinant virus, generated from a recombination event between a Trinidad virus of the GI-1 lineage and a Trinidad virus of the newly defined lineage. Many amino acid differences were also observed between the S1 coding regions of the circulating field and vaccine strains, indicating that the IBV vaccines may not be protective. Vaccine-challenge studies are however needed to prove this.

Pathogenicity of a QX-like avian infectious bronchitis virus isolated in China.

Avian infectious bronchitis is a serious and highly contagious disease caused by infectious bronchitis virus (IBV). We isolated a highly virulent IBV strain (CK/CH/JS/TAHY) from kidneys of diseased chickens. Phylogenetic analysis based on the S1 gene revealed that CK/CH/JS/TAHY clustered with the QX-like type. The S1 gene has 1,620 nucleotides and encoded a polypeptide of 540 amino acids with typical coronavirus cleavage recognition sites of HRRR. About 1-day-old specific pathogen-free White Leghorn chickens inoculated with CK/CH/JS/TAHY at 105.5 EID50 exhibited clinical signs including coughing, sneezing, nasal discharge, and tracheal vocalization accompanied by depression with 84% mortality and 100% morbidity. The kidneys of dead birds were swollen and pale and exhibited severe urate deposition. Histopathological examination revealed kidney hemorrhages, multifocal necrosis of the renal tubules and trachea with cilia loss, sloughing of epithelial cells, and edema of the lamina propria. IBV-specific antibodies appeared at 10 D post-infection. Chickens vaccinated with a CK/CH/JS/TAHY oil-emulsion vaccine showed 26.7% morbidity and 3% mortality indicating a protective effect. In conclusion, the IBV strain is a virulent avian IBV and that exhibited severe pathogenicity in chickens and is a vaccine candidate to prevent infection by Chinese QX-like nephropathogenic IBV strains.

A highly pathogenic GI-19 lineage infectious bronchitis virus originated from multiple recombination events with broad tissue tropism.

In the present study, an IBV strain I0305/19 was isolated from a diseased commercial broiler flock in 2019 in China with high morbidity and mortality. The isolate I0305/19 was clustered together with viruses in sublineage D of GI-19 lineage on the basis of the complete S1 sequence analysis. Isolate I0305/19 and other GI-19 viruses isolated in China have the amino acid sequence MIA at positions 110-112 in the S protein. Further analysis based on the complete genomic sequence showed that the isolate emerged through at least four recombination events between GI-19 ck/CH/LJS/120848- and GI-13 4/91-like strains, in which the S gene was found to be similar to that of the GI-19 ck/CH/LJS/120848-like strain. Pathological assessment showed the isolate was a nephropathogenic IBV strain that caused high morbidity of 100 % and mortality of 80 % in 1-day-old specific-pathogen-free (SPF) chicks. The isolate I0305/19 exhibited broader tropisms in different tissues, including tracheas, lungs, bursa of Fabricius, spleen, liver, kidneys, proventriculus, small intestines, large intestines, cecum, and cecal tonsils. Furthermore, subpopulations of the virus were found in tissues of infected chickens; this finding is important in understanding how the virulent IBV strains can potentially replicate and evolve to cause disease. This information is also valuable for understanding the mechanisms of replication and evolution of other coronaviruses such as the newly emerged SARS-CoV-2.

Molecular characterization of infectious bronchitis virus based on RNA-dependent RNA polymerase gene.

Extensive rate of variations in the S1 gene (spike glycoprotein subunit gene) of infectious bronchitis virus (IBV) causes challenges for clinicians in counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance. This study investigated the possibility of using an RNA-dependent RNA polymerase gene (RdRp) as a target for molecular characterization of IBV strains in Iran. Trachea samples were collected from commercial broiler flocks (n = 52) showing respiratory syndrome. Specific PCR primers were designed for a variable region located in the RdRp gene flanked by highly conserved regions. Reverse transcriptase PCR followed by sequence analysis identified eight IBV variants, with an overall prevalence of 44.2%. Deduced nucleotide and amino acid sequences were compared with published sequences for IBV strains. Because of the long-distance similarities, the field samples could be discriminated from vaccine strains. Phylogenetic analysis of RdRp gene sequences resulted in clustering of the IBV strains related to each area. Using RdRp as a genetic marker eliminates the challenges arising from the enormous variations that make it difficult to discriminate between field and vaccine strains as well as affiliate certain variants to various geographical areas.

A novel low virulent respiratory infectious bronchitis virus originating from the recombination of QX, TW and 4/91 genotype strains in China.

In China, variants of infectious bronchitis virus (IBV) evolve continually and diverse recombinant strains have been reported. Here, an IBV strain, designated as ck/CH/LJX/2017/07 (referred as JX17) was isolated from chicken vaccinated with H120 and 4/91 in Jiangxi, China, in 2017. Sequence analysis reveals of the S1 gene of JX17 the highest nucleotide identity of 98.15% with that of GI-7 genotype TW2575/98 strain. Furthermore, whole genome analysis among JX17 and other 18 IBV strains demonstrates that JX17 has the highest nucleotide identity of 95.94% with GI-19 genotype YX10 strain. Among all genes of JX17 except the S1 gene, the N gene and 3' UTR have the highest identity to GI-13 genotype 4/91 strain and the rest genes are the most identical to GI-19 genotype YX10 strain. Analyzed by the RDP and SimPlot, the recombination of JX17 strain was shown to occur in regions which include 5'-terminal S1 gene (20,344 to 22,447 nt), most N gene and 3' UTR (26,163 to 27,648 nt). The pathogenicity study shows that JX17 is a natural low virulent IBV variant which caused respiratory symptoms but no death. Taken together, these results indicate that IBV strains continue to evolve through genetic recombination and three prevalent genotypes in China including QX, TW and 4/91 have started to recombine.