Field Strains Of Infectious Bronchitis Virus Research Articles

Molecular epidemiology of infectious bronchitis virus in eastern and southern China during 2021–2023

As a highly infectious and contagious pathogen in chickens, infectious bronchitis virus (IBV) is currently grouped into nine genotypes (GI to GIX). However, the classification of serotypes of IBV is still not clear. In this study, 270 field strains of IBV were isolated from dead or diseased chicken flocks in eastern and southern China during Jan. 2021-Apr. 2023. These isolated IBV strains could be classified into two genotypes, GI (including five lineages GI-1, GI-13, GI-19, GI-22 and GI-28) and GVI based on the complete S1 sequence. Further analysis showed that the GI-19, GI-13, GI-22, GI-28 and GVI were the dominant genotypes with the proportions of 61.48%, 8.89%, 8.89%, 7.78% and 8.89% respectively, and the homology of S1 protein of these isolates ranged from 86.85% to 100% in GI-19, 92.22% to 100% in GI-13, 83.1% to 100% in GI-22, 94.81% to 100% in GI-28 and 90.0% to 99.8% in GVI, respectively. Moreover, cross-neutralization test with sera revealed that these isolates in GI-19 lineage could be classified into at least three serotypes according to the antigenic relationship. In addition, structure assay using PyMOL indicated that one mutation such as S120 in receptor binding site (RBD) of GI-19 might alter the antigenicity and conformation of S protein of IBV. Overall, our data demonstrate that not only multiple genotypes, but also multiple serotypes in a single genotype or lineage have been co-circulated in eastern and southern China, providing novel insights into the molecular evolution of the antigenicity of IBV and highlighting the significance of the selection of the dominant isolate for vaccine development in IBV endemic region.

Histopathological study and molecular characterization of Infectious Bronchitis virus circulating in broilers in Basrah governorate, Iraq

The poultry industry plays an important role in the food economics and the food industry in both their layer and broiler chicken. Poultry diseases on the other way impact in the poultry industry especially diseases from virus sources since it does not have a specific treatment except for vaccines with routine prevention and control. This study was designed for the assessment of histopathological changes, molecular diagnosis and phylogenetic analyses of the Infectious Bronchitis virus circulating in broilers in Basrah province in the south of Iraq. The tissue samples were (trachea, lung and kidney). One hundred samples were studied with clinical signs and lesions related to IB. The histopathological changes caused by the virus were studied with hematoxylin and eosin stain, and the causative agent Infectious Bronchitis Virus (IBV) was identified using a polymerase chain reaction combined with explaining the phylogenetic analysis. The results of molecular diagnosis and identification showed the twelve Iraqi field strains of IBV were closely related and shared a high identity of 98.44 %- 99.69 % of each other. Variant 2(IS/1494 like) was a predominant genotype of IBV in the present study, and has high similarity with the variant 2(IS/1494) like a strain of IBV circulating in Iran and in the Kurdistan region north of Iraq. The histopathological findings were desquamation of the cilia of the mucosa layer of the trachea, infiltration of inflammatory cells, hyperplasia of goblet cells with thickening of mucosal layer in some areas infiltration of inflammatory exudate within capillaries and Para bronchus, where the microscopic results in kidney were showed interstitial hemorrhage, necrosis of the renal tubular epithelium, infiltration of inflammatory cell. The present results provided useful information on target organs damaged by IBV and the genetic epidemiology of such virus strains circulating in Basrah governorate Iraq during this year that would be helpful to develop a local vaccine.

Identification of Host Proteins Interacting with IBV S1 Based on Tracheal Organ Culture.

Infectious bronchitis virus (IBV) belongs to the gamma-coronavirus genus of Coronaviridae and causes serious infectious diseases in the poultry industry. However, only a few IBV strains can infect avian passage cell lines, seriously hindering the progress of basic research on IBV pathogenesis. Whereas IBV field strains can replicate in tracheal ring organ culture (TOC) without any previous adaptation in chicken embryos or primary cells. In this study, to investigate the potential use of TOC as an in vitro infection model for the study of IBV-host interaction, we first established a chicken embryo TOC culture system and carried out an investigation on the IBV replication kinetics in the system. We found that the selected strains of the IBV GI-1, GI-7, GI-13, GI-19, and GI-22 genotypes could successfully replicate in TOC and bring about damage to the infected trachea. Next, we identified host proteins of the chicken embryo trachea that interact with the IBV S1 protein by immunoprecipitation and protein mass spectrometry. A total of 127 candidate proteins were initially identified with major involvement in cell adhesion pathways and apoptosis- and autophagy-related pathways. The heat shock protein 70 (HSP70) was selected for further investigation in the interaction with IBV viral proteins. Our results showed that HSP70 interacted with IBV S1 in both TOC and CEK cells, whereas HSP70 overexpression inhibited viral replication. This study indicates that TOC is a good system for the elucidation of IBV-host interactions and HSP70 is a potential host antiviral factor.

Longitudinal Survey on aMPV Circulation in French Broiler Flocks following Different Vaccination Strategies

In recent years, the impact of respiratory disease resulting from Avian Metapneumovirus (aMPV) infection has been generally rising in the broiler industry in Europe. In this context, in order to investigate aMPV contribution to the clinical picture and the potential benefits of diversified vaccination strategies compared to nonvaccination policies, a longitudinal monitoring was performed, also evaluating Infectious Bronchitis Virus (IBV) presence. Broiler flocks located in Western France, where aMPV has already proven to be a health and productivity issue, were screened by RT-PCR on rhino-pharyngeal swabs, and the viruses were genetically characterized by sequence analysis. For a more comprehensive picture of aMPV molecular epidemiology and evolution in France, aMPV subtype B strains detected from 1985 to 1998 were sequenced and included in the analysis. The survey confirmed the detection of aMPV subtype B in commercial broiler flocks in France, together with a certain heterogeneity demonstrated by the circulation of more recent and historical French field strains. No IBV field strains were detected. The implementation and evaluation of different management choices and vaccine strategies suggests once again that immunization does not prevent infection but contributes greatly to the containment of the clinical manifestations.

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.

Molecular characterization of infectious bronchitis virus in Southwestern China for the protective efficacy evaluation of four live vaccine strains

The high mutation rate of infectious bronchitis virus (IBV) poses a significant threat to the protective efficacy of vaccines. This study aimed at analyzing the S1 genes of IBV field strains isolated in Southwestern China from 2018 to 2020, assessing the pathogenicity of four dominating strains, and evaluating the protective efficacy of four commercial vaccine strains against the endemic representative strains. Thirty-two field strains of IBV were isolated in Southwestern China from 2018 to 2020. Phylogenetic analysis of their S1 genes revealed the nucleotide homology ranged from 64.6% to 100%, and belonged to five genotypes [GI-19 (QX, 53.13%), GI-28 (LDT3-A,15.63%), GI-7 (TW, 12.50%), GI-1 (Mass, 6.23%), GVI-1 (TC07-2, 6.25%)], and two variant groups [variant-3 (3.13%) and variant-5 (3.13%)]. Recombination events between field and vaccine strains or between field strains were identified in the S1 genes of eight IBV field strains. The CK/CH/YNKM/191128 and CK/CH/CQBS/191203 strains of GI-19 showed morbidity rates of 66.7% and 73.7%, respectively, and mortality rates of 13.3% and 33.3%, respectively. Besides, the CK/CH/SCYC/191030 and CK/CH/GZGY/191021 strains of GI-28 caused morbidity rates of 60% and 86.7%, respectively, and mortality rates of 33.3%. The protective efficacy of the four commercial live vaccine strains (4/91, FNO-E55, LDT3-A, and QXL87) ranged from 70% − 100% and reduced tissue lesions against CK/CH/GZGY/191021 and CK/CH/CQBS/191203 strains. LDT3-A strain was the most effective one but still could not completely prohibit IBV shedding. These findings provide a reference for IBV molecular evolution analysis and control of IB.

Real Time Detection of different Variant Strains of Infectious Bronchitis Virus in Trachea, Lung and Kidney of Infected Broiler Chickens

BACKGROUND: Avian infectious bronchitis virus (IBV) has a great potential for genetic variability which leads to generating new virus strains. The changes in IBV genome often cause alterations in the virulence, tissue tropism and viral replication in the host tissues. OBJECTIVES: This study was conducted to identify the virus variant strains in trachea, lung and kidney of infected birds. The possible relationship of IBV variants with the relative quantity of virus in each organ was also investigated.METHODS: IBV variant strains were detected by polymerase chain reaction (PCR) and direct sequencing. Amongst samples collected during one season (spring), from infected commercial broiler flocks located at Golastan and Mazandaran province of Iran, 9 flocks (3 flocks per each variant) were selected based on the identified variants. Trachea, lung and kidney samples of 5 birds per flock were examined for the presence of virus and variants. Virus also quantified in target organs using relative quantitative real time PCR.RESULTS: Based on the PCR and sequencing, three IBV variants were selected (A, B and C). Virus type A and B were detected in all target organs but virus type C detected in trachea and kidney. Virus type C had the highest quantity (17.02 ± 5.22) and virus type A showed the lowest quantity (5.68 ± 2.4) in infected tissues. Relative quantity of virus detected in tissues significantly correlated with IBV variant. CONCLUSIONS: Genetic polymorphism at IBV field strains has revealed significant correlations with viral quantity in lung, trachea and kidney. Our finding is an update of current knowledge on the associations between viral genotype, virulence and pathogenicity.

Genetic and pathogenic characterization of a novel recombinant avian infectious bronchitis virus derived from GI-1, GI-13, GI-28, and GI-19 strains in Southwestern China

Avian infectious bronchitis (IB), caused by avian infectious bronchitis virus (IBV), is an acute and highly contagious disease that is extremely harmful to the poultry industry throughout the world. The cross-using of different attenuated live vaccine strains has led to the occurrence of diverse IBV serotypes. In this study, we isolated an IBV strain from a chicken farm in southwest China and designated it CK/CH/SCMY/160315. Construction of a phylogenetic tree based on full S1 gene sequence analysis suggested that CK/CH/SCMY/160315 bears similarity to GI-28, and further comparison of S1 amino acid residues revealed that CK/CH/SCMY/160315 showed mutations and deletions in many key positions between LDT3-A and other GI-28 reference strains. Importantly, CK/CH/SCMY/160315 was identified as a novel recombinant virus derived from live attenuated vaccine strains H120 (GI-1), 4/91 (GI-13), LDT3-A (GI-28), and the field strain LJL/08-1 (GI-19), identifying at least 5 recombination sites in both structural and accessory genes. Pathogenicity analysis indicated that CK/CH/SCMY/160315 caused listlessness, sneezing, huddling, head shaking, and increased antibody levels in the inoculated chickens. To further describe pathogenicity of this novel strain, we assessed viral load in different tissues and conducted hematoxylin and eosin (HE) staining of the trachea, lungs and kidneys. Our results provide evidence for the continuing evolution of IBV field strains via genetic recombination and mutation, leading to outbreaks in the vaccinated chicken populations in China.

Genetic and antigenic heterogeneity of GI-1/Massachusetts lineage infectious bronchitis virus variants recently isolated in China

Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns, and characteristics of the Mass-type IBV strains.

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation.

Background and Aim:Belonging to the Coronaviridae family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis.Materials and Methods:In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/HRM, we did the sequencing of the partial S1 gene.Results:It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain.Conclusion:Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool.

Molecular epidemiology of infectious bronchitis virus and avian metapneumovirus in Greece

ABSTRACTRespiratory diseases like infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) have been held accountable for major losses for poultry production. Nevertheless, scarce information was present dealing with the prevalence and molecular epidemiology of these infections in Greece and the efficacy of currently applied control strategies. To fill this gap, a specific epidemiological study was designed. A total of 106 broiler and layer farms, including 10 backyard and 96 commercial flocks, were sampled between March 2016 and May 2017, and the obtained tracheal swabs were tested for IBV and aMPV using RT-PCR based techniques followed by sequencing. For each farm, data regarding production type, flock features, clinical signs, and vaccination program were also recorded. Different associations between vaccination protocol, production type, animal category, birds density, age, presence of clinical signs, and IBV and/or aMPV infection were tested. Both IBV and aMPV field strain prevalence were proven high, approximately 20 and 30%, respectively, being the GI-19 lineage (14 out of 19; 73.6%) and B subtype (30 out of 30; 100%), the most commonly detected IBV and aMPV genetic types. Infection with IBV field strains was significantly associated with clinical sign presence (odds ratio = 8.55 [95CI = 2.17–42.90]). Remarkably, only the vaccination protocol involving a double vaccination at 1 D of age was proven protective against IBV-induced symptomatology, with the odds of developing disease being 4.14 [95CI = 1.34–14.51] times lower.No association was demonstrated between aMPV infection and clinical outbreaks or between aMPV and IBV detection, suggesting the marginal role of the former pathogen in poultry farming.Globally, the present study provides the first detailed investigation of the epidemiological scenario of 2 viruses traditionally considered of pivotal relevance in poultry farming and demonstrates that remarkable benefits could be obtained with just minor adjustments in vaccination protocols.

Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure

In this study, a pathogenic avian infectious bronchitis virus (IBV) QX-type strain YN was successfully rescued by vaccinia virus based reverse genetic technology. Ten fragments contiguously spanning the complete IBV genome were amplified and cloned into the vaccinia virus genome by homologous recombination. The full-length genomic cDNA was transcribed in vitro, and its transcript was transfected into BHK-21/N cells that could stably express IBV N protein. At 48 h post transfection, the culture medium was harvested and inoculated into 10-day-old specific-pathogen-free embryonated chicken eggs to replicate the rescued virus. This strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant rYN virus will not require any cell culture adaptations. After only one in ovo passage, the recombinant YN virus (rYN) was successfully recovered and confirmed to possess the introduced silent marker mutation in its genome. Biological characteristics of rYN such as the EID50, TCID50, replication in ovo, and replication kinetcs in vitro were tested and all were similar to its parental strain YN. Our findings demonstrate the successful construction of highly-pathogenic QX-type IBV using a modified rescue procedure, allowing for future studies of the molecular biology and pathogenicity of IBV field strains.

The first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in Algeria.

Background and Aim:Avian infectious bronchitis virus (IBV) frequently infects broilers and is responsible for severe economic losses to the poultry industry worldwide. It has also been associated with kidney damage in the broiler flocks. The aim of the present study is to determine the presence of IBV and its possible involvement in kidney damage of broiler chicks.Materials and Methods:14 clinically diseased broiler flocks from Western and Central Algeria were sampled and analyzed by hemagglutination inhibition (HI) test and reverse transcriptase-polymerase chain reaction (RT-PCR) followed by phylogenic analysis.Results:The QX (100%) and 4/91 (60%) IBV serotypes were the most prevalent in the kidney damaged broilers regardless of vaccination status. The molecular detection of avian IBV by RT-PCR identified six samples as positive, of which only two isolates were typable by sequencing. We identified a novel IBDZ13a genotype which showed 93% sequence homology to the partial-S1 gene sequence of the IB 4/91 commercial vaccine strain. Sequencing analysis characterized this virus as a novel and divergent IB 4/91 field virus with eight amino acid substitutions that might have resulted in altered immunogenicity.Conclusion:The isolation of a new IBV strain (IBDZ13a) from vaccinated broiler flocks may explain the failure of the vaccination programs against IBV field strains. Combination of the HI test and RT-PCR indicated that the nephropathogenic IB outbreaks in broilers are related to this novel strain.

Attenuation, safety, and efficacy of a QX-like infectious bronchitis virus serotype vaccine

Avian infectious bronchitis (IB) is a highly contagious disease caused by avian infectious bronchitis virus (IBV), which is a considerable economic threat to the poultry industry. QX-like IBV strains have increasingly emerged in China in recent years. Hence, development of a specific vaccine to guard against their potential threat is important. In this study, we sought to develop an attenuated vaccine strain. First, attenuated QX-like IBV strain SZ130 was created by continuous passage in chicken embryos for 130 generations, and then its safety was tested. We also evaluated the protective efficacy of different doses of SZ130 against challenge with QX-like IBV field strain SD in chickens. SZ130-infected birds did not experience IB-like signs and organ lesions. Additionally, an excellent protective effect of SZ130 vaccination was observed when vaccinated birds were challenged with SD, with no clinical signs or gross lesions, decreased target tissue replication rates, and lower ciliostasis scores in all immunized groups. These findings indicate that attenuated IBV strain SZ130 is highly safe in chicks and may serve as an effective vaccine against the threat posed by QX-like IBV strains.

Evaluation of 793/B-like and Mass-like vaccine strain kinetics in experimental and field conditions by real-time RT-PCR quantification.

Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response.

Complete genome sequences of two avian infectious bronchitis viruses isolated in Egypt: Evidence for genetic drift and genetic recombination in the circulating viruses

Avian infectious bronchitis virus (IBV) is highly prevalent in chicken populations and is responsible for severe economic losses to poultry industry worldwide. In this study, we report the complete genome sequences of two IBV field strains, CU/1/2014 and CU/4/2014, isolated from vaccinated chickens in Egypt in 2014. The genome lengths of the strains CU/1/2014 and CU/4/2014 were 27,615 and 27,637 nucleotides, respectively. Both strains have a common genome organization in the order of 5′-UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-UTR-poly(A) tail-3′. Interestingly, strain CU/1/2014 showed a novel 15-nt deletion in the 4b-4c gene junction region. Phylogenetic analysis of the full S1 genes showed that the strains CU/1/2014 and CU/4/2014 belonged to IBV genotypes GI-1 lineage and GI-23 lineage, respectively. The genome of strain CU/1/2014 is closely related to vaccine strain H120 but showed genome-wide point mutations that lead to 27, 14, 11, 1, 1, 2, 2, and 2 amino acid differences between the two strains in 1a, 1b, S, 3a, M, 4b, 4c, and N proteins, respectively, suggesting that strain CU/1/2014 is probably a revertant of the vaccine strain H120 and evolved by accumulation of point mutations. Recombination analysis of strain CU/4/2014 showed evidence for recombination from at least three different IBV strains, namely, the Italian strain 90254/2005 (QX-like strain), 4/91, and H120. These results indicate the continuing evolution of IBV field strains by genetic drift and by genetic recombination leading to outbreaks in the vaccinated chicken populations in Egypt.

Investigation of Infectious Bronchitis Virus Strains by Real Time RT-PCR and S1 Gene Sequencing in Broilers

In this study, detection of infectious bronchitis virus (IBV) from chicken tracheal swab samples by real time RT-PCR, which was reported as a rapid and sensitive virus detection method, was aimed. Phylogenetic relations in different field and variant strains of IBV by sequencing of hyper variable regions (HVRs) of S1 gene of IBV was determined. Tracheal swabs were taken from 120 broiler chickens with respiratory signs, randomly. Samples were inoculated into the allantoic fluids (AF) of SPF embryonated chicken eggs. RNAs were extracted by AF. RNAs were translated to ss cDNAs. Real time RT-PCR was performed with double labelled Taqman probe, IBV 5 GU391 and IBV5 GL533 primer pairs. Positive results were obtained in 33 samples of 120 (27.5 %) by real time RT-PCR. cDNAs from positive samples were used for amplification HVRs (Hyper Variable Regions) in S1 gene region with C2U and Ag0723 primers. Samples were amplified by S1 5’mod and CK2 primers, too. RT-PCR products were sequenced directly. Sequencing results have been compared with NCBI’s nucleotide database. Sequencing results were matched to some variant IBV strains in gen banks database at the rate of 72-86 %. These results support that we are faced with variant IBV strains. Also, we observed a lot of SNP mutations in alignment. This study shows that birds can be infected by IBV variant strains at rate of 27.5 %, although they were vaccinated by classic type vaccine. These results demonstrate that vaccination by classical vaccine strains of IBV cannot provide enough cross protection against variant strains. Therefore, outbreaks of the disease still occur in vaccinated flocks. Also, we found these primers are not enough for all IBV field strains to be sequenced and classified. Thus, different primers should be developed and used primer pairs more than two. In this experiment, we found virus load is more in AF than swab samples because of IBV replication in SPF egg AF. We observed dwarfism and hemorrhages at 14 days old chick embryos

Age-dependent immune responses and immune protection after avian coronavirus vaccination

Infectious bronchitis virus (IBV) is an endemic disease of chickens and a major contributor to economic losses for the poultry industry despite vaccination. Recent observations indicated that chicks may have an immature immune system immediately after hatching when vaccinated for IBV. Therefore we hypothesized that early IBV vaccination will generate an immature, poorly protective IBV-specific immune response contributing to immune escape and persistence of IBV. To test this hypothesis the IBV-specific immune response and immune protection were measured in chicks vaccinated at different ages. This demonstrated a delayed production of IgG and IgA plasma antibodies in the 1, 7 and 14-day-old vaccination groups and also lower IgA antibody levels were observed in plasma of the 1-day-old group. Similar observations were made for antibodies in tears. In addition, IgG antibodies from the 1-day-old group had lower avidity indices than day 28 vaccinated birds. The delayed and/or lower antibody response combined with lower IgG avidity indices coincided with increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon IBV field strain challenge. The lack of vaccine-mediated protection was most pronounced in the 1-day-old vaccination group and to a lesser extent the 7-day-old group, while the 14-day-old and older chickens were protected. These data strongly support IBV vaccination after day 7 post hatch.

Dynamic changes of virus load in supernatant of primary CEK cell culture infected with different generations of avian infectious bronchitis virus strains Sczy3 as revealed by real-time reverse transcription-polymerase chain reaction

Infectious bronchitis virus (IBV) can multiply effectively in chick embryo kidney (CEK) cells after adapting to the chick embryo. To investigate the dynamic changes in IBV load in the supernatant of primary CEK cells, we developed an SYBR Green I-based real-time polymerase chain reaction assay to quantify nucleic copy numbers of the IBV-Sczy3 strain. The 20, 54, and 87th generations of CEK-adapted IBV-Sczy3 strains were used to infect CEK cells, and then nucleic copy numbers in the samples of supernatant collected at 12, 24, 36, 48, 60, and 72 h were detected. The results showed that the rapid growth period of the virus load of all the 3 generations was approximately 12-36 h post-infection; the peak of the virus load appeared at 36 h post-infection and then decreased gradually in the order of 20th > 54th > 87th for the 3 generations of CEK-adapted strains; the dynamic change curve of the IBV load in the supernatant of primary CEK cells showed a single peak. The results of this study provide a useful reference for CEK-adapted IBV field strains and the production of CEK-attenuated IBV vaccine.

Molecular characterisation of membrane glycoprotein and 5b protein of nephropathogenic infectious bronchitis virus

Background: Infectious bronchitis virus (IBV) affects the upper respiratory tract and the reproductive tract, and some strains can cause nephritis. Large number of serotypes and genotypes of the virus have been identified and for the most part do not cross-protect. Identifying the genotype or serotype of IBV field strains is empirical for selecting an appropriate candidate to serve as vaccine strain for prevention of infectious bronchitis (IB) disease in chickens. The variant strains of IBV could be circulating among chickens in India and recently nephropathogenic IB was reported. Hence, the present work was aimed to carry out molecular characterization of nephropathogenic IBV isolates obtained from two different geographical locations of south India, involving relatively conserved regions. Materials and Methods: MIBVPCR and NIBVPCR primers were used to amplify partial M and N gene after synthesizing cDNA. Isolates Ind/KA/07/1 and Ind/TN/07/2 were subjected to direct sequencing as these two isolates are two different geographical regions and scored better in induction of lesions during pathotyping. Results: Amino acids of membrane glycoprotein varied on five occasions for the isolate Ind/KA/07/1 when aligned with that of M 41 strain and isolate Ind/TN/07/2. Eight amino acids of both isolates in 5b protein were different from that of M41 strain. Few point mutations, short deletions and insertions were noticed in the amplified genome, based on the membrane protein nucleotide sequence comparison. Conclusion: Prevalence of IBV strains with few modifications in conserved regions indicated that there was presence of variant IBV in south India.