QX-like Infectious Bronchitis Virus Research Articles

Spike Glycoprotein 1 Partial Gene Analysis of GI-19 (QX-like) Infectious Bronchitis Virus Isolated and Propagated from Breeder, Broiler, and Layer Chickens in Java Region

This study aims to identify and characterize receptor binding sites (RBS) and antigenic sites (HVR-I and II) of the S1 gene fragment of the infectious bronchitis virus (IBV) isolated and propagated from commercial chickens in Java Region to monitor recent circulating virus. The samples in this study were the organs which indicated infectious bronchitis infection. The stages of this research consisted of making virus suspensions, isolation, and propagation, as well as molecular detection and characterization of viruses. Virus isolation and propagation were carried out on chicken embryonated eggs aged 11 days via the allantoic route. Culture confirmation was performed by RT-PCR of the S1 gene fragment, followed by sequencing and bioinformatics analysis. The 168-hour propagation was observed in both dwarfed and curled embryos of two isolates from 11 isolates detected as IBV-positive. Phylogenetic tree construction resulted in all isolates being grouped as GI-19 genotype (QX-like). Amino acid identity among QX-like strains was calculated at 87–100%. A total of 210 predicted amino acid residues were observed, including 31 substitutions and 2 deletions. Conclusions of this study were identified and characterized as GI-19 genotype (QX-like) IBV with amino acid changes on S1 fragment from breeder, broiler, and layer chickens in Java Region.

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

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

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

QX-like infectious bronchitis virus (IBV) is a highly infectious avian coronavirus that causes respiratory and kidney disease. It is linked to increased mortality and loss of performance in infected chickens worldwide, including Thailand. Thus, a simple and rapid diagnostic method for the diagnosis of QX-like IBV is needed. This study aimed to develop a single-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay to detect and differentiate QX-like IBV from Thai IBV and vaccine strains used in the poultry industry (H120, Ma5, and 4/91). Primer sets specific for QX-like and Thai IBV were designed to target the S1 gene. The specificity of the technique was verified using nine isolates of QX-like IBV, four isolates of Thai IBV, and other avian viral respiratory pathogens. The detection limit was evaluated using a serial ten-fold dilution of QX-like and Thai IBV. The results showed that single-step mRT-PCR could detect QX-like IBV and differentiate it from Thai IBV and the vaccine strains H120, Ma5, and 4/91. The limit of detection of the developed assay was 102.2 embryo infectious dose (EID)50/mL for QX-like IBV and 101.8 EID50/mL for Thai IBV. Interestingly, the developed assay could identify mixed infection by both IBVs in a single sample. The single-step mRT-PCR assay developed in this study can potentially discriminate QX-like IBV from Thai IBV and the vaccine strains H120, Ma5, and 4/91 in a single reaction. It is also suitable for use in all laboratories with access to conventional PCR equipment.

Efficacy of commercial live vaccines against QX-like infectious bronchitis virus in Japan

Infectious bronchitis, an acute and highly contagious disease that affects chickens, is caused by the infectious bronchitis virus (IBV). The antigenic variant QX-like IBV was first reported in China in 1996 and is now endemic in many countries. Our previous study reported the first detection and isolation of QX-like IBVs in Japan and that they were genetically related to the recently detected strains in China and South Korea. The pathogenicity of 2 Japanese QX-like IBV strains (JP/ZK-B7/2020 and JP/ZK-B22/2020) was evaluated by inoculating specific pathogen-free (SPF) chickens with 102 to 106 median embryo infectious dose. Both strains caused clinical signs of respiratory symptoms, gross tracheal lesions, and moderate-to-severe suppression of tracheal ciliostasis. To evaluate the efficacy of commercial IBV live vaccines against the JP/ZK-B7/2020 strain, vaccinated SPF chickens were challenged with the JP/ZK-B7/2020 strain at 104 EID50 (median embryo infectious dose). Only the JP-Ⅲ vaccine provided high levels of protection (reduced suppression of tracheal ciliostasis and reduced viral loads in organs), whereas the Mass vaccine showed little protective effect. Virus neutralization test results and comparisons between IBV genotypes based on the S1 gene suggested that QX-like and JP-III genotypes were closely related. These results suggest that the JP-III IBV vaccine, which has relatively high S1 gene homology with QX-like IBVs, is effective against Japanese QX-like IBV strain.

Effect of monovalent and bivalent live attenuated vaccines against QX-like IBV infection in young chickens

Since 1999, QX-like (GI-19) avian infectious bronchitis viruses have been the predominant strains in China till now. Vaccination is the most effective way to control the disease, while live attenuated vaccine is widely used. In the current research, we evaluated the effect of several monovalent and bivalent live IBV vaccines in young chickens against the QX-like (GI-19) IBV infection. The results showed that monovalent 4/91 and bivalent Ma5+LDT3 vaccines could provide efficient protection in day-old chickens that reduced morbidity and mortality, ameliorated histopathology lesions, and reduced viral loads were observed. These data suggest that vaccination through nasal route with monovalent 4/91 or bivalent Ma5+LDT3 in day-old chickens could serve a safe and effective vaccination strategy for controlling QX-like (GI-19) infectious bronchitis virus.

Receptor binding motif surrounding sites in the spike 1 protein of infectious bronchitis virus have high susceptibility to mutation related to selective pressure.

To date, various genotypes of infectious bronchitis virus (IBV) have co-circulated and in Korea, GI-15 and GI-19 lineages were prevailing. The spike protein, particularly S1 subunit, is responsible for receptor binding, contains hypervariable regions and is also responsible for the emerging of novel variants. This study aims to investigate the putative major amino acid substitutions for the variants in GI-19. The S1 sequence data of IBV isolated from 1986 to 2021 in Korea (n = 188) were analyzed. Sequence alignments were carried out using Multiple alignment using Fast Fourier Transform of Geneious prime. The phylogenetic tree was generated using MEGA-11 (ver. 11.0.10) and Bayesian analysis was performed by BEAST v1.10.4. Selective pressure was analyzed via online server Datamonkey. Highlights and visualization of putative critical amino acid were conducted by using PyMol software (version 2.3). Most (93.5%) belonged to the GI-19 lineage in Korea, and the GI-19 lineage was further divided into seven subgroups: KM91-like (Clade A and B), K40/09-like, QX-like (I-IV). Positive selection was identified at nine and six residues in S1 for KM91-like and QX-like IBVs, respectively. In addition, several positive selection sites of S1-NTD were indicated to have mutations at common locations even when new clades were generated. They were all located on the lateral surface of the quaternary structure of the S1 subunits in close proximity to the receptor-binding motif (RBM), putative RBM motif and neutralizing antigenic sites in S1. Our results suggest RBM surrounding sites in the S1 subunit of IBV are highly susceptible to mutation by selective pressure during evolution.

Development of SYBR green RT-qPCR assay for titrating bivalent live infectious bronchitis vaccines

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID50). However, this method cannot effectively distinguish or quantify different genotypic strains in a mixture of different viruses, especially in the absence of neutralizing monoclonal antibodies. In this study, quantitative real-time PCR (RT-qPCR) was applied using specific primers for the QX- and Mass-like strains to quantitate IBV infection and for comparison with the conventional virus titration quantitative method. A strong positive correlation was observed between RT-qPCR cycle threshold values and the different EID50 concentrations. This method was further used to titrate bivalent IB vaccines, and the amount of individual genotype virus was determined based on specific primers. Thus, this RT-qPCR assay may be used as a highly specific, sensitive, and rapid alternative to the EID50 assay for titering IBVs.

Establishment and Cross-Protection Efficacy of a Recombinant Avian Gammacoronavirus Infectious Bronchitis Virus Harboring a Chimeric S1 Subunit.

Infectious bronchitis virus (IBV) is a gammacoronavirus that causes a highly contagious disease in chickens and seriously endangers the poultry industry. A diversity of serotypes and genotypes of IBV have been identified worldwide, and the currently available vaccines do not cross-protect. In the present study, an efficient reverse genetics technology based on Beaudette-p65 has been used to construct a recombinant IBV, rIBV-Beaudette-KC(S1), by replacing the nucleotides 21,704–22,411 with the corresponding sequence from an isolate of QX-like genotype KC strain. Continuous passage of this recombinant virus in chicken embryos resulted in the accumulation of two point mutations (G21556C and C22077T) in the S1 region. Further studies showed that the T248S (G21556C) substitution may be essential for the adaptation of the recombinant virus to cell culture. Immunization of chicks with the recombinant IBV elicited strong antibody responses and showed high cross-protection against challenges with virulent M41 and a QX-like genotype IBV. This study reveals the potential of developing rIBV-Beau-KC(S1) as a cell-based vaccine with a broad protective immunity against two different genotypes of IBV.

The N1038S Substitution and 1153EQTRPKKSV1162 Deletion of the S2 Subunit of QX-Type Avian Infectious Bronchitis Virus Can Synergistically Enhance Viral Proliferation.

The S2 subunit of infectious bronchitis virus (IBV) plays a critical role in the process of IBV infection. A comparison between the S2 subunit sequence of chicken embryo kidney cell (CEK) adapted virulent QX-like IBV strain SczyC30 (hereafter referred to as zy30) and its CEK-attenuated strain, SczyC100, revealed an N1038S substitution in S2 subunit and a 1154EQTRPKKSV1162 residue deletion in the C-terminus of the S2 subunit. In order to explore whether these two mutations are related to changes in the biological characteristics of IBV, we firstly constructed an infectious clone of zy30 using a bacterial artificial chromosome (BAC), which combines the transcription of infectious IBV genomic RNA in non-susceptible BHK-21 cells with the amplification of rescued virus rzy30 in CEK cells. Then, three recombinant viruses, including an rzy30S2-N1038S strain that contained the N1038S substitution, an rzy30S2-CT9△ strain that contained the 1154EQTRPKKSV1162 deletion, and an rzy30S2-N1038S-CT9△ strain that contained both mutations, were constructed using rescued virus rzy30 as the backbone. The results showed that each mutation did not significantly affect the replication titer in CEK cells but reduced pathogenicity in chickens, while in combination, the N1038S substitution and 1154EQTRPKKSV1162 deletion improved the proliferation efficiency in CEK cells and reduced pathogenicity, compared to rzy30 strain. The contribution made by the 1154EQTRPKKSV1162 deletion in reducing pathogenicity was higher than that of N1038S substitution. Our results revealed that the N1038S substitution and 1154EQTRPKKSV1162 deletion in S2 subunit were deeply involved in the replication efficiency of IBV and contributed to reduction of viral pathogenicity.

Molecular investigation of S2-3a/3b-E-M-4b/4c-5a/5b-N gene of QX-like and variant genotype infectious bronchitis virus isolated in Thailand reveals a distinct E gene.

The QX-like infectious bronchitis virus (IBV) and variant genotype have been discovered worldwide including Thailand. In order to know the origin of QX-like and variant genotype IBV in Thailand, the genetic analysis on multiple genes was investigated. Seven IBVs including four QX-like and three variant genotype were randomly selected from IBVs isolated in Thailand during 2008 and 2010. Phylogenetic analysis of the S2-3a/3b-E-M-4b/4c-5a/5b-N gene showed that Thai QX-like and variant genotype IBV were grouped together in a separate branch from other IBV strains. The isolated IBVs shared nucleotide identities of 96-99.9% with each other. They exhibited a high level of similarity (93.8%) with KM91 strain in South Korea. Phylogenetic analysis of the S2 and 3a/3b gene showed a relationship to KM91 strain. The E gene was distinct from other IBV strains. The M, 4a/4b and 5a/5b gene were closely related to Massachusetts type. The N gene was classified into two groups which were a group of unique to Thailand (variant genotype) and a relationship with Massachusetts type (QX-like). Recombination analysis identified the occurrence of recombination events in the genome of viruses. These findings demonstrated that the QX-like IBV and variant genotype isolates in Thailand were the recombinant viruses. Thai QX-like IBV had a genetic relationship with KM91 strain, Massachusetts type and unknown IBV whereas variant genotype had a genetic relationship with Thai QX-like IBV and Connecticut strain.

Detection and isolation of QX-like infectious bronchitis virus in Japan.

The antigenic variant QX-like infectious bronchitis virus (IBV) is endemic in several countries. In Japan, the QX-like genotype is classified as the JP-III genotype based on the partial S1 gene and as the GI-19 genotype based on the complete S1 gene. This study showed that QX-like IBVs and JP-III IBVs can be identified based on the amino acid polymorphism of the S1 glycoprotein. Furthermore, genetic analysis of several IBV field strains detected in commercial broiler farms across the Kyushu area in 2020 revealed Japanese QX-like IBVs, which are highly homologous to the QX-like IBVs recently detected in China and South Korea. Herein, QX-like IBV field strains were isolated for evaluating commercial vaccine efficacy in our future studies.

Circulation of three genotypes and identification of unique mutations in neutralizing epitopes of infectious bronchitis virus in chickens in Bangladesh.

Infectious bronchitis (IB) is a highly contagious respiratory disease caused by a gammacoronavirus that has been circulating for many years in chickens in Bangladesh, resulting in significant economic losses. The aim of this study was to detect and characterize infectious bronchitis virus (IBV) from clinical outbreaks and surveillance samples. Real-time RT-PCR was used to detect IBV in pooled lung and tracheal tissue samples (n = 78), oropharyngeal swabs (n = 19), and pooled fecal samples (n = 13) from live-bird markets. Both respiratory and nephropathogenic forms of IB were suspected at necropsy (n = 7) from clinical outbreaks. Sequencing of hypervariable regions (HVR1-2 and HVR3) of the region of the spike gene (S) encoding the S1 subunit of five isolates revealed circulation of the Mass-like, QX-like, and 4/91-like genotypes of IBV in Bangladesh. Each genotype was extremely variable, as shown by separate clustering of the viruses in a phylogenetic tree and high nucleotide (nt) sequence divergence (38.8–41.2% and 25.7–37.4% in the HVR1-2 and HVR3 sequence, respectively). The unique mutation G65E was observed in each Mass-like isolate, and Y328S was observed in each 4/91-like Bangladeshi isolate. Three neutralizing epitope sites were predicted within the HVRs that differed significantly among the three genotypes. In addition, one Bangladeshi isolate carried fixed mutations at 294F and 306Y, like other pathogenic QX-like IBVs, which could affect epitopes involved in neutralization, facilitating virus circulation among vaccinated flocks. Therefore, continuous screening and genotype characterization will be necessary to track the epidemiology of IBV and control IB infection in Bangladesh.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05227-3.

An attenuated TW-like infectious bronchitis virus strain has potential to become a candidate vaccine and S gene is responsible for its attenuation

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.

Pathogenic characteristics of a QX-like infectious bronchitis virus strain SD in chickens exposed at different ages and protective efficacy of combining live homologous and heterologous vaccination

Continued reports of infections with infectious bronchitis virus (IBV) variants have occurred since its first isolation in the 1930s. Currently, QX-like IBVs are the predominant circulating genotype around the world. Here, the pathogenicity of QX-like IBV strain SD was characterized in chickens at different ages of exposure to the virus, and the protection efficacy of available vaccine combinations against IBV was evaluated. The results revealed that QX-like IBV strain SD was severely pathogenic in chickens, causing respiratory, urinary and reproductive infections, irrespective of age, based on clinical observations, viral distribution in tissues and a ciliostasis study. Severe respiratory signs, tracheal cilia injury, nephritis and abnormal development of the oviduct and ovarian follicles were evident throughout the experiment. A challenge experiment demonstrated that the homologous QX vaccine showed superior protection efficacy compared with other available vaccines, confirming the importance of IBV vaccine seed homology against the circulating IBV strains. Our findings aid an understanding of the pathogenicity of QX-like IBVs that may help to further control the infection.

Pathogenicity and genome changes in QX-like infectious bronchitis virus during continuous passaging in embryonated chicken eggs

Infectious bronchitis (IB) remains a major problem in the global poultry industry despite the many available vaccines. Live attenuated vaccines are the most effective means of preventing IB and are traditionally generated by serial passaging of a wild strain in embryonated chicken eggs. In this study, the SZ isolate of the QX-like infectious bronchitis virus (IBV) was continuously passaged in chicken embryos for 250 passages. We compared the pathogenicity of different passages (SZ50, SZ100, SZ150, SZ200 and SZ250) of strain SZ by clinical signs, gross lesions, viral load, tissue tropism, weight gain and tracheal ciliary activity. As the passaging increased in the chicken embryos, the strain lost its ability to infect many organs, and the viral pathogenicity gradually decreased. We also found 23 genomic variations of the QX-like strain SZ throughout the passaging process by further analyzing its complete genome sequence. This work offers valuable insight for IBV vaccine development and further research on the IBV attenuation mechanisms.

Evaluation of the antigen relatedness and efficacy of a single vaccination with different infectious bronchitis virus strains against a challenge with Malaysian variant and QX-like IBV strains.

BackgroundThe predominant infectious bronchitis virus (IBV) strains detected in chickens in Malaysia are the Malaysian variant (MV) and QX-like, which are associated with respiratory distress, nephropathy, and high mortality. On the other hand, the antigenic relatedness and efficacy of IBV vaccines against these 2 field IBV strains are not well characterized.ObjectivesThis study aimed to determine the antigen relatedness and efficacy of different IB vaccine strains against a challenge with MV and QX-like strains.MethodsThe antigen relatedness and the ability of different IB vaccine strains in conferring protection against MV and QX-like were assessed based on the clinical signs, macroscopic lesions, and ciliary activity.ResultsThe MV strain IBS037A/2014 showed minor antigenic subtype differences with the vaccine virus Mass H120 and 4/91 strains but showed major antigenic subtype differences with the K2 strain. The Malaysian QX-like strain IBS130/2015 showed major antigenic subtype differences with the MV strain IBS037A/2014 and the vaccine strains except for K2. Chickens vaccinated once with Mass (H120) or with non-Mass (4/91 and K2) developed antibody responses with the highest antibody titer detected in the groups vaccinated with H120 and 4/91. The mean ciliary activities of the vaccinated chickens were between 56 to 59% and 48 to 52% in chickens challenged with IBS037A/2014 and IBS130/2015, respectively. The vaccinated and challenged birds showed mild to severe lesions in the lungs and kidneys.ConclusionsDespite the minor antigenic subtype differences, a single inoculation with Mass or non-Mass vaccines could not protect against the MV IBS037A/2014 and QX-like IBS130/2015.

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.

Molecular characteristics and pathogenicity analysis of QX-like avian infectious bronchitis virus isolated in China in 2017 and 2018

ABSTRACTProportions of QX-like genotype infectious bronchitis virus (IBV) isolates have increased over time. Here, to better understand the epidemiology and pathogenicity of IBV in China and control the spread of infectious bronchitis (IB), we conducted sequence analyses and examined the pathogenicity of 5 field isolates from diseased flocks in 2017 and 2018. Sequence analyses revealed that all the 5 strains, as well as many recent field isolates from other researchers, belonged to the QX-like IBV genotype, which were distantly related to commercial vaccine strains. Viral pathogenicity experiments showed that the isolates caused high morbidity and severe ciliostasis in chickens, although they caused milder lethality. This provides further evidence that QX-like IBV emergence remains a major problem in the poultry industry, and information on IBV epidemiology and pathogenicity may help to control IB.

Pathogenicity of a QX-like strain of infectious bronchitis virus and effects of accessory proteins 3a and 3b in chickens

QX-like genotype infectious bronchitis virus (IBV) has become prevalent in recent years. Few studies have reported the effects of accessory proteins 3a and 3b on pathogenicity in vivo. We developed a reverse genetics system to manipulate the genome of a QX-like IBV strain IBYZ. Recombinant viruses rIBYZ-ScAUG3a, rIBYZ-ScAUG3b and rIBYZ-ScAUG3ab were generated. These viruses do not express the accessory proteins 3a, 3b, or 3ab due to a mutation in the AUG start codons. In SPF embryonated eggs, the recombinant viruses grew to the same viral load as parental strain rIBYZ. The pathogenicity of rIBYZ and recombinant viruses was examined in 1-day-old SPF chickens. In SPF chickens, rIBYZ-ScAUG3a had a lower mortality than rIBYZ. The clinical signs, gross lesions and histopathological changes of rIBYZ-ScAUG3a group were comparable to those of rIBYZ group. However, viral distribution and viral shedding showed that the viral loads of rIBYZ-ScAUG3a were lower than those of rIBYZ in tissue samples and swab specimens. The rIBYZ-ScAUG3b and rIBYZ-ScAUG3ab strains showed attenuated pathogenicity compared to rIBYZ, as no chickens died and all the parameters tested were considerably low. This study indicates that the absence of accessory proteins 3a and 3b in IBV lead to attenuated pathogenicity in chickens. Protein 3b has a greater effect on pathogenicity than protein 3a. These findings may be used in vaccination trials for the development of a new live-attenuated vaccine.

Prevalence and molecular characterization of infectious bronchitis virus isolated from chicken in Bangladesh.

Aim:The present study was aimed to determine the prevalence of infectious bronchitis virus (IBV) as well as virus isolation, identification, and molecular characterization of various strains circulating in Bangladesh.Materials and Methods:A total of 371 swabs and organ samples were collected from four types of chicken including layer, Sonali (local), broiler, and broiler breeder under eight districts (Rangpur, Bogura, Tangail, Dhaka, Gazipur, Mymensingh, Jamalpur, and Cumilla) during 2014-2016 in Bangladesh.Results:Out of 371 samples, 65 samples were positive in reverse transcriptase polymerase chain reaction (RT-PCR) for molecular identification of IBV. The overall prevalence was 17.52% recorded and among the selected types of chicken, the highest prevalence of IBV was found in layer that was 42.22% followed by 17.24% in Sonali, 14.93% in broiler breeder, and lowest prevalence was 11.94% in broiler chicken, respectively. Moreover, the prevalence of IBV was recorded highest in aged chicken at 41-60 weeks, which was 54.55% in layer, 27.27% in Sonali, and, afterward, 14.68% was found in broiler breeder, respectively. Frequency of IBV more frequently in winter (22.67%) followed by rainy (15.87%) and summer season (11.58%). The highest prevalence of IBV was found Tangail district (41.67%) followed by Mymensingh (24.42%), Gazipur (19.32%), Dhaka (15.38%), Jamalpur (16.67%), Bogura (13.68%), Cumilla (5.88%), and Rangpur (9.26%), respectively. Samples that were found high positive in IBV RT-PCR (Ct value below 30) were subjected to inoculation into chicken egg embryo to observe characteristic changes in chicken embryo. Swabs and organ samples were processed and passaged in 9-day-old embryonated chicken eggs through allantoic cavity route. IBV virus suspected samples inoculated into chicken egg embryos after 3-5 passages showed dwarfing and curling of the embryos which are characteristic lesions of IBV. Allantoic fluid was collected from all inoculated eggs and performed partial sequencing of S1 gene for three isolates. After sequencing, the phylogenetic tree was constructed from the nucleotide sequences of IBV isolates. Two of the isolates are 4/91 IBV and another one matched with QX-like IBV.Conclusion:The results revealed that the three isolates from different places in Bangladesh were identified for the 1st time as which will help for IBV control strategy.