Diagnosis Of Infectious Bronchitis Virus Research Articles

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.

Aptamer-Assisted Proximity Ligation Assay for Sensitive Detection of Infectious Bronchitis Coronavirus.

Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses. IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.

New PA/1220/98-like variant of infectious bronchitis virus in Poland

ABSTRACT The aim of the present study was to report the first detection of a new infectious bronchitis virus (IBV) variant in Polish commercial flocks which is completely different to any previously known in this region. In 2018, samples from Ross 308 breeding hens aged 35 weeks were delivered for IBV diagnosis. IBV presence was detected, but all attempts to amplify the S gene fragment were negative. The field material was analysed using the Illumina MiSeq platform and a 1073-nt fragment of the S1 coding region was obtained. The gCoV/ck/Poland/516/2018 strain shared only 52.7–58.1% nucleotide identity to any known genotype of IBV and shared the highest identity of 81.4% to the unique North American PA/1220/98 variant. Based on the obtained sequence, a specific molecular test was constructed and used for screening of chicken samples from 35 field cases delivered to our laboratory between 2018 and 2019 for IBV diagnosis. Application of this test enabled detection of another three chicken flocks as positive for this new strain. All positives were identified in commercial layers with egg production problems. To date, the virus has not been detected in broiler chickens. Taking into account the proposed criteria for the definition of a new IBV genotype or lineage, it seems that the detected viruses in Poland, together with the unique North American PA/1220/98 variant, may be classified as separate lineages/genotype in the new IBV classification. RESEARCH HIGHLIGHTS The new IBV variant is distantly related to other known GI–GVII IBV genotypes/lineages. It affects long-lived birds causing egg production problems. The detected IBV and the unique North American PA/1220/98 variant are candidates for separate lineages in the new GVIII genotype.

Molecular characterization and genotyping of recent isolate of Infectious Bronchitis Virus (IBV) in Egypt

In this study a total number of 22 organ samples (including trachea, lung and kidney) from 22 broiler farms from northern Upper Egypt were collected from Mars 2017 to June 2018 from chickens showing clear clinical and pathological signs of Infectious Bronchitis. The samples were prepared and examined by real time RT-PCR for diagnosis of IBV. A total number of 11 samples were positive (50%) which were used for further isolation on SPF eggs by three blind serial passages. Positive samples that showed the pathogenic lesions of IB (curling and dwarfing of embryos) were collected and tested with real time RT-PCR (rRT-PCR) for more confirmation then a part from S1 gene sequence was amplified by RT-PCR and the product was sequenced and the data have been compared with other related IBV strains. The results indicate that the Egyptian virus in this study has an identity percent reached up to 89% with other recent Egyptian isolates. However, it reached 67% with classical vaccine strains like H120 and variant I like CR88 strain. The lowest identity was observed with M41 strain (59%) in this study. The phylogenetic tree compared to other isolates from Middle East and worldwide showed that this isolate is related to the IBV variant 2 group closely related to IBVEg/1265B/2012 strain and the Israeli strain IS/1494/06.

The role of infectious bronchitis virus in respiratory and renal problems in broiler chickens

Broiler chickens in Egypt represent the main source of animal protein. Unfortunately, they usually are hit by many disease problems. Respiratory and renal affections are investigated in forty four broiler chicken flocks during 2014-2015 in three large Governorates; Sharkia, Dakahlia and Gharbia. The birds age ranged from 19-54days and suffured from general signs of illness, respiratory troubles with Whitish and greenish diarrhea. Mortality rates ranged from (0.5-30%). On necropsy, tracheitis, bronchitis and congestion of lungs were recorded in all flocks. Caseated plugs at tracheal bifurcation (29/44), fibrinous pericarditis, perihepatitis and air sacculitis(19/44) were also observed. Nepherosis-nephritis (22/44) and General congestion with hemorrhage on proventriculus (n=9) and cecal tonsil (n=4) were seen among affected birds. Chicken embryo inoculation with respiratory and renal tissue suspension revealed reduction in embryo size (n=9), death within 72hrs (n=24) while 5 flocks required successive blind passage to show reduction in embryo size up to 2cm compared to 5cm in negative control. The reverse transcriptase polymerase chain reaction (RT-PCR) was carried out on both original tissues and allantoic fluids using specific primers for S1 gene. Nine positive infectious bronchitis virus (IBV) were detected in original samples compared with only 2 in allantoic fluids of the same samples. It could be concluded that IBV is responsible on 20% of respiratory and renal troubles in broilers under investigation. The direct RT-PCR could be reliable tool for appropriate IBV diagnosis in chickens.

Pathogenesis of infectious bronchitis virus (IBV) and laboratory test methods available to detect IBV in chickens

Infectious bronchitis is a high contagious disease of chicken caused by gamma coronavirus. The disease was characterized by respiratory, renal, urogenital, and alimentary infections. Different strains of IBV may show variable tissue tropisms. For example, the classical strains of Massachussetts serotype (M41) targeted in respiratory and reproductive system while variant serotypes including QX, 793/B, D388 have been detected in respiratory, reproductive, alimentary system and kidney. Because the evolution and mutation of IBV have contributed to the emergence of new genotypes of IBV, the outbreak of IB still occurred in the vaccinated flocks. Therefore, diagnosis of IB plays an important role for limiting the effects caused by IBV infection. Available laboratory tests for diagnosis of IBV include virus isolation, electron microscope, immunohistochemistry, serological tests (virus neutralization, haemagglutination inhibition, enzyme-linked immunosorbent assay –ELISA-, agar gel immunodiffusion), genotype identification (RT-PCR, genome sequencing [using NGS Miseq]).

Molecular Characterization of Infectious Bronchitis Virus in Chicken

INFECTIOUS BRONCHITIS (IB) is a highly contagious ………disease of serious economic importance in the poultry industry worldwide. Diagnosis of Infectious Bronchitis Virus (IBV) infections in Egypt during 2013 - 2016 among poultry flocks. A total of 41 chicken flocks were examined (40 broiler flock and one breeder flock). Results revealed that 21 chicken flocks were found positive IBV by N gene conventional RT-PCR and real time RT-PCR. The samples were isolated and propagated in commercial embryonated chicken eggs (ECE) with result revealed that only one sample that showed curling and dwarfing of the embryo after the second passage while 10 samples were HA positive that suggest mixed infection of IBV with other viruses such as Newcastle Virus or Avian Influenza Virus. Furthermore, the allantoic fluids of the positive samples were subjected to specific S1 PCR to differentiate strains of IBV. The absence of positive reaction of S1gene conventional RT-PCR may confirm the involvements of other respiratory pathogens in chickens as Newcastle disease, avian influenza and mycoplasmosis that masked IBV infections. In conclusion, our results confirmed the detection of IBV in chicken flocks in Egypt which responsible for the respiratory problems in poultry farms and diagnosis with Real-time PCR because it is more rapid, sensitive, and reproducible, and the risk of carryover contamination is minimized compared to conventional PCR.

Development of a Microplate Lectin-Capture RT-PCR (MLC-RT-PCR) for the Detection of Avian Infectious Bronchitis Virus

Rapid, sensitive and specific methods are necessary to confirm the diagnosis of outbreaks of avian infectious bronchitis virus (IBV) infection. The amplification of IBV genome by reverse transcription followed by polymerase chain reaction (RT-PCR) has been one of the most used methods for the detection of this virus in clinical samples. To reduce the time and the number of steps in the molecular diagnosis of IBV, we developed a sensitive and rapid detection method based on viral capture by a lectin (Concanavalin A—Con A) in the microplate wells, followed by RT-PCR to amplify the S1 gene. The detection limit of IBV was 103 EID50/ml for the amplification of 5’part of the S1 gene, and 104 EID50/ml for the amplification of full S1 gene. This technique was specific for IBV detection, and no amplified products were detected for other avian viral pathogens (bursal infectious disease virus, avian metapneumovirus and Newcastle disease virus). The MLC-RT-PCR was as sensitive as conventional RT-PCR, and virus isolation method for the detection of IBV in tissue samples collected from experimentally infected birds. The MLC-RT-PCR technique demonstrated a great potential for the rapid and specific diagnosis of IBV.

Evaluation of sialic acid and acute-phase proteins (haptoglobin and serum amyloids A) in healthy and avian infection bronchitis virus-infected chicks.

Forty-five 24-day-old Cobb chicks infected with infectious bronchitis virus (IBV) and ten healthy 24-day-old Cobb chicks without any clinical signs of IBV as control group were selected for the study. All of the diseased chicks showed some or all of the clinical signs of infectious bronchitis including gasping, coughing and nasal discharge, wet eyes, swollen sinuses, reduction of food consumption and weight gain. Diagnosis of IBV was based on clinical signs and ELISA test. Blood samples were taken from the wing vein into two tubes: one containing ethylenediaminetetraacetic acid (EDTA) and one without EDTA. Haptoglobin (Hp), serum amyloid A (SAA), total sialic acid (TSA), lipid-bound sialic acid (LBSA) and protein-bound sialic acid (PBSA) concentrations were measured. All of the study variables were significantly higher in diseased birds compared with control group. Results showed that there were significant positive correlations between TSA, LBSA and PBSA in both groups. No correlation was observed between Hp and SAA with any other parameters; however, there was significant negative correlation between Hp and SAA in the control group. Results for receiver operating characteristic analysis showed that area under the curve (AUC) for TSA, LBSA, PBSA, Hp and SAA were 0.93, 0.98, 0.90, 0.90 and 0.80, respectively. According to AUC, LBSA was the most sensitive factor to change in the diseased birds. It can be concluded that in naturally occurring IBV infection, significant increases in TSA, LBSA, PBSA, Hp and SAA concentrations are expected and among study variables, LBSA had the most obvious change so it may be considered as the most sensitive parameter.

Development of a real-time TaqMan ® RT-PCR assay for the detection of infectious bronchitis virus in chickens, and comparison of RT-PCR and virus isolation

A sensitive and specific method for the diagnosis of infectious bronchitis virus (IBV) is of great importance. In this study the development of a real-time TaqMan ® RT-PCR targeting the highly conserved nucleocapsid (N) gene of IBV and including an internal PCR control is described. The assay was specific for IBV and did not detect other avian pathogens, including turkey coronaviruses. A comparative limit of detection was determined for M41, an embryo-adapted strain, and IS/885/00, a poorly embryo-adapted variant. For M41 real-time RT-PCR and virus isolation were one or two times more sensitive than RT-PCR targeting the N or spike glycoprotein (S1) genes, respectively. For IS/885/00, real-time RT-PCR was more sensitive by tenfold than virus isolation and 30- or 40-fold than by N gene or S1 gene RT-PCR, respectively. Real-time RT-PCR and virus isolation were 17–75% more sensitive than RT-PCR targeting the S1 gene for testing tracheal swabs directly from experimentally infected chicks. When tracheal and cloacal swabs from clinical specimens were tested directly, 50% more samples were positive by real-time RT-PCR than by the S1 gene RT-PCR. Real-time RT-PCR targeting the N gene is more sensitive than common diagnostic assays, allowing rapid and accurate IBV detection directly from clinical specimens, facilitating differential diagnosis.

Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.

Typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the S1 gene.

A universal primer set was developed that amplifies a region covering hypervariable region (HVR) 1 and HVR 2 in the S1 gene of the infectious bronchitis virus (IBV). The universality of this primer set was confirmed by testing the reference strains of different serotypes or variants of the IBV present in the United States. An approximately 450-bp region containing HVR 1 and HVR 2 of 7 untyped field isolates obtained in 1999 and 2000 was amplified. Direct sequencing followed by phylogenetic analysis on that region allowed us to type those field isolates that were not typable by reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). Furthermore, it was found that typing by phylogenetic analysis of that region correlates with virus neutralization results. Together with RT-PCR and RFLP, this method will serve as a fast typing method for IBV diagnosis.

Isolation and Characterization of a Novel Antigenic Subtype of Infectious Bronchitis Virus Serotype DE072

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.

Detection of infectious bronchitis virus in experimentally infected chickens by an antigen-competitive ELISA

An antigen-competitive enzyme-linked immunosorbent assay (Ag-C-ELISA) was developed for the detection of infectious bronchitis virus (IBV) antigens, M41 strain, in tissues from experimentally infected chickens, or in allantoic fluid harvested from inoculated embryonated eggs. The detection limit of IBV in the Ag-C-ELISA was 104.1 median embryo infective doses (EID50)/well. Tracheal and lung samples from chickens vaccinated with 102.5 EID50 of live attenuated infectious bronchitis (H120) vaccine were negative in the direct detection Ag-C-ELISA. The results indicate that the Ag-C-ELISA has the potential to detect IBV, either directly in tissue samples or when combined with the passage of material in embryonated eggs, thereby constituting an alternative method for the diagnosis of IBV.

Use of reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism to examine the interaction between infectious bronchitis virus strains Massachusetts 41 and JMK in ovo

The reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism (RTPCR-RFLP) technique has been developed and used in the serotype diagnosis of infectious bronchitis virus (IBV) infection. In this report, we first demonstrate that the RT-PCR-RFLP was sensitive in detecting both Massachusetts 41 (Mass 41) and JMK strains of IBV singly; the detection limit for both was approximately 0.15ng viral RNA using gel electrophoresis. Subsequently, the ability of the technique to semi-quantify the relative amounts of Mass 41 and JMK RNA in their mixture was shown by performing RT-PCR-RFLP on various combinations of serially diluted known amounts of Mass 41 and JMK RNA. Results indicated that both Mass 41 and JMK can be detected and semi-quantified when there is less than 103-fold difference between the two viral RNA template inputs, which are above the lower detection limit. Based on this result, the interaction between Mass 41 and JMK in ovo was examined by inoculating 10-day-old embryos with various combinations of different EID50 titers of Mass 41 and JMK. RT-PCR-RFLP results showed that Mass 41 dominates over JMK in an EID50 titerdependent fashion, but not the reverse. To further investigate the underlying mechanism, replication efficiency of Mass 41 and JMK was compared at 6, 12, 18, 24, 36 and 48h after inoculation of embryos with 105.3 EID50 of individual Mass 41 and JMK. No significant difference (P > 0.05) on replication efficiency between these two serotypes was found based on the results of total RNA concentration and RT-PCR results of 10-fold dilution of RNA at the indicated time points. By inoculating heat-inactivated Mass 41 with live JMK into embryos, only JMK was detected by RT-PCR-RFLP and no dominating interference was observed. Data suggest that the dominance is not due to the replication efficiency of Mass 41 and JMK; a receptor-mediated mechanism might be responsible for it.

Oligonucleotide probes in infectious bronchitis virus diagnosis and strain identification

Genomic RNA fingerprints of infectious bronchitis virus (IBV) strains M41 and Conn46 were prepared to identify T1 RNase-resistant oligonucleotides ‘unique’ to each of the two IBV strains. Such oligonucleotides were subsequently eluted from the gels and their nucleotide sequences determined. When oligonucleotide probes of those sequences were synthesized and used in a dot-blot hybridization assay, the probes lacked IBV strain-specificity and reacted with the RNAs of homologous as well as heterologous IBV strains. Based on these results, the methods used in this study need to be applied to a large number of oligonucleotide probes, to find one or a few that might be suitable as IBV strain- or serotype-specific oligonucleotide probes.

A monoclonal antibody blocking ELISA to detect serotype-specific infectious bronchitis virus antibodies

1. A monoclonal antibody (Mab) blocking enzyme-linked immunosorbent assay (B-ELISA) was developed and compared to a conventional indirect ELISA (I-ELISA) and a virus-neutralization (VN) test for detection of specific antibodies to avian infectious bronchitis virus (IBV) serotypes. Sera used in this study were derived from chickens experimentally inoculated with the three most prevalent IBV serotypes, Arkansas (Ark), Connecticut (Conn), and Massachusetts (Mass). 2. Overall, there was good correlation between the results of B-ELISA and the VN test. Both detected serotype-specific antibodies in chicken sera during primary and secondary phases of the immune response. Results of both tests indicated that the antibodies produced during the primary response to IBV serotypes are strongly serotype-specific. Those produced during the secondary response react more strongly with the homologous virus, but do exhibit some level of cross-reactivity with heterologous antigens. I-ELISA detected IBV group-specific and not serotype-specific antibodies. 3. The B-ELISA which both offers the convenience of the conventional I-ELISA and the serotype specificity of the VN test, holds excellent promise for field application in IBV diagnosis and evaluation of response to IBV vaccines.

Screening for Antibodies Against Infectious Bronchitis Virus: Specificity of the Haemagglutination Inhibition Test

A screening of antibodies against strain M-41 and the Dutch variant strains D-274 and D-1466 of infectious bronchitis virus (IBV) using the haemagglutination inhibition test (HI) was carried out in Chile. The presence of these variant strains has not been reported yet and therefore the probability of finding positive flocks is low. On this basis some statistical analysis were made with those data obtained in order to contribute to the problem of the specificity of the HI test for IBV diagnosis. All sera were distributed in different categories of possible results against each strain according with the HI titre obtained and employing different discrimination levels for a positive or negative status against IBV. It was concluded that discrimination levels varying from 4.0 to 5.0 log2 are useful for establishing the presence or absence of antibodies against IBV but if a serotype specific diagnosis is needed, then the discrimination level should be elevated, specially when secondary immune responses are measured.