The avian infectious bronchitis virus is classified into serotypes or genotypes (or both) in different poultry-producing countries of the world. In Brazil, Massachusetts type (Mass), used as a live vaccine, and local field Brazilian variants (genotypes; BR) predominate in the commercial poultry flocks. This study describes the development and validation of two real-time reverse-transcription polymerase chain reactions (RT-qPCR) for the specific detection of Mass and BR genotypes in allantoic fluids and clinical samples. Genotype-specific primers, combined with a generic probe targeted to the S1 gene, originated Mass RT-qPCR and BR RT-qPCR–specific assays. Analytical sensitivity and linearity of these assays were determined in comparison with an IBV generic real-time RT-PCR based on the 5′ untranslated region (5′UTR RT-qPCR). Mass RT-qPCR detected five Mass field isolates, three vaccine samples, and one coinfected sample (BR and Mass) while BR RT-qPCR detected 16 BR field isolates. Both assays were linear (R2 > 0.98), reproducible, and as sensitive as the classical 5′UTR RT-qPCR used to detect IBV. In the analysis of 141 IBV clinical samples, 8 were positive for Mass RT-qPCR, 76 for BR RT-qPCR, and 2 for both assays. In the remaining 55 samples, 25 were positive only for 5′UTR RT-qPCR and 30 were negative for the three assays. In conclusion, both assays were able to detect Mass and BR genotypes, allowing rapid and easy IBV molecular typing from allantoic fluids and clinical samples.
Avian infectious bronchitis virus (IBV) belongs to the genus Gammacoronavirus, family Coronaviridae, and is responsible for a highly contagious disease of great economic impact in the poultry industry (19,20). Infectious bronchitis (IB) has a broad clinical presentation, primarily affecting the respiratory tract but also generating lesions in the urinary, digestive, and reproductive systems (6). IBV has an RNA genome with a high genetic diversity, mainly in the S glycoprotein gene (31). The S gene has specific RNA sequences in the different serotypes-genotypes, including the vaccine strains and field variant viruses from different poultry-producing regions (20).
Historically, Massachusetts (Mass) and Connecticut (Conn) serotypes were first described in the 20th Century (21). Afterwards, several new field variants were detected and associated with different IB clinical signs in poultry-producing regions around the world (9,11,22). In South America, from 2001 to 2008 two studies showed the occurrence of Mass and Conn in addition to some local variants in Argentina (28) and Colombia (2). The predominance of local variants was also demonstrated in Brazil (7,14,15,33). Brazilian variants (named BR genotypes) are present in all important poultry producing regions (Center-West, Northeast, South, and Southeast), but the Mass genotype is also found due to the intensive vaccination to prevent IB in industrial poultry-producing flocks (4,7,15,33). Other previously identified European and North American genotypes (D274, Arkansas, Conn, and 4/91) were only rarely found in field samples (14,33,34).
Different methods are used for direct IBV detection (10). Isolation of the virus in specific-pathogen-free (SPF) embryonated chicken eggs is considered the gold standard method, but it is laborious and time-consuming, sometimes requiring several egg passages. Immunologic assays include ELISA, immunofluorescence, immunoperoxidase, and agar gel precipitating test (AGPT) (21). Molecular biology techniques based on reverse-transcription polymerase chain reaction (RT-PCR, nested-RT-PCR, and real-time RT-PCR) are also used for rapid and sensitive detection of IBV in clinical samples (5,13,25,26,36).
Furthermore, molecular assays have been developed to detect specific IBV strains. Some of these assays include a generic detection by RT-PCR and subsequent characterization by other assays such as restriction fragment length polymorphism (RFLP), sequencing, and microsphere-based assay (15,23,25,24,29,35). Others are genotype-specific RT-PCR assays (classical or real-time) for rapid molecular typing of vaccine and field variants strains (1,8,18,30). The present study aimed to develop and validate an RT-qPCR to differentiate the Mass vaccine and Brazilian field genotypes of IBV.
MATERIALS AND METHODS
Vaccine and field isolates strains.
Three vaccine strains were provided by the manufacturers: Cevac® NB L (CevaSaúde Animal Ltda, Paulínia, SP, Brazil); BioBronkVet® H-120 (LaboratórioBiovet, Vargem Grande Paulista, SP, Brazil); and Bioral® H-120 (MerialSaúde Animal Ltda, Campinas, SP, Brazil). Twenty IBV field isolates (in allantoic fluid) were available after isolation from broiler, breeder, and layer flocks located in the main poultry-producing regions of Brazil from 2010 to 2013 (15). In addition, other viral and bacterial species were obtained from vaccine producers and reference laboratories (Table 1).
IBV strains and other pathogen species used in this study
Samples of 141 flocks from the main poultry-producing regions of Brazil were used to evaluate the performance of the assays. These samples included chicken tissue and organs (including tracheal swabs, lungs, kidneys, cecal tonsils, and oviducts), 34 of them impregnated in FTA cards, and all were collected from 2012 to 2014. They were originated from broiler, breeder, and layer flocks with IB clinical signs and were stored at −20 C. Further tracheal swab samples were obtained from 50 poultry flocks without IB signs.
Primers-probe design and synthesis.
The S1 gene sequences of IBV vaccine strains and strains from the BR genotype were obtained and aligned using Bioedit software ( http://www.mbio.ncsu.edu/bioedit). The alignment contained the sequences of 10 Mass genotype strains including five field isolates (JX559785, JX559790, JX559815, JX559826, and JX559828) and five vaccine samples marketed in Brazil and previously sequenced in our laboratory (New LS Mass I®, Cevac NB L, Bio-Bronk-Vet H-120, Bioral H-120, and Nobilis® IB Ma5); eleven BR-I genotype field isolates (DQ448273, DQ492308, DQ448275, GU383095, GU383105, GU383109, JX559786, JX559792, JX559800, JX559808, and JX559820); five BR-II genotype field isolates (HM561897, JX559817, JX559819, JX559821, and JX559822); and reference strains 4/91 (AF093794), D274 (X15832), H-120 (M21970), and M41 (AY851295). The specificity of the oligonucleotide sequences was in silico verified by comparison to the GenBank database using Basic Local Alignment Search Tool (BLAST) analysis ( www.blast.ncbi.nlm.nih.gov). All primers and the probe were synthesized by a commercial company (Integrated DNA Technologies – IDT, Coralville, IA; Table 2).
Sequences of primers and probe selected for this study
RNA was extracted with commercial reagents (NewGene) according to the supplier protocol (SimbiosBiotecnologia, Cachoeirinha, RS, Brazil). Briefly, swabs and macerated organs pools were placed in 1 ml of lysis solution and incubated at 60 C for 10 min. After centrifugation for 1 min (8609 × g), 0.5 ml was removed and added to a new tube containing 20 μl of silica suspension. The tube was centrifuged again for 1 min (8609 × g). The supernatant was discarded and the pellet washed with 150 μl of wash solution A, B, and C (Prep). After the last wash, the silica was dried at 60 C and total RNA was eluted with 50 μl of elution buffer.
IBV detection by RT-qPCR and sequence analysis.
All viruses and bacterial strains used in this study were tested by a real-time TaqMan® RT-PCR (5′UTR RT-qPCR) as previously described (5). Assays were carried out in the Step One Plus™ Real Time PCR System (Applied Biosystems, Norwalk, CT). Selected IBV isolates were submitted to a nested-RT-PCR and sequencing of the S1 gene region (15). Nucleotide sequences from both strands were edited, assembled, and analyzed using the ClustalW method (available in the Bioedit software package). Phylogenetic analysis was performed using the neighbor-joining method with 1000 bootstrap replicates (MEGA software version 5.0; available at http://www.megasoftware.net/) and all samples were classified in genotypes as previously described (15).
IBV molecular typing by specific RT-qPCR.
Two independent real-time TaqMan RT-PCRs were performed for specific detection of Mass (Mass RT-qPCR) and Brazilian field variants (BR RT-qPCR). Reactions systems were performed in a total volume of 30 μl including 2 μl of viral RNA template, 2.5 mM of dithiothreitol, 0.07 mM of each deoxynucleotide triphosphate, 0.25 μM of each primer, 0.5 μM of IBV probe, 25 U of MMLV reverse transcriptase (Promega, Madison, WI), 1.5 U of Taq DNA polymerase, and 6 μl of a 5× concentrate RT buffer (CenbiotEnzimas, Porto Alegre, RS, Brazil RT was carried out at 37 C for 30 min and then the RNA-DNA strands were denatured at 94 C for 3 min. PCR was performed for 40 cycles with the following conditions: 94 C for 20 sec, 55 C for 40 sec, and 72 C for 1 min. All reactions were performed in a StepOnePlus Real-Time PCR System (Applied Biosystems). Amplification plots were recorded, analyzed, and the threshold cycle (Ct) determined with the StepOne software, version 2 (Applied Biosystems). Amplicons were also submitted to electrophoresis in 10% polyacrylamide gels and visualized after silver nitrate staining.
Analytical sensitivity and specificity.
To assess the analytical sensitivity (limit of detection) and linearity of the RT-qPCR assays, one Mass and one BR IBV strain were separately 10-fold diluted and submitted to RNA extraction. Each RNA sample was 4-fold diluted nine times (from 10−1 to 3.8 × 10−7 dilution) and submitted to amplification by 5′UTR RT-qPCR (5), Mass RT-qPCR, and BR RT-qPCR. Eleven replicates (analyzed in four different runs) were carried out to determine the mean Ct values for each template. The detection limit of the developed assays (Mass RT-qPCR and BR RT-qPCR) was compared with 5′UTR RT-qPCR (5). The end point of detection was considered as the last dilution wherein it was possible to detect all tested replicates. The mean Ct values from replicate tests were plotted against the log values of dilution point, and the linear equation correlation coefficient (R2) was calculated with the use of Excel 2007 (Microsoft Corporation, Redmond, WA). Reproducibility was determined by testing in four different days.
Genotype specificity of the RT-qPCR assays were assessed by the analysis of nucleic acid extracted from other pathogens causing poultry respiratory diseases (Table 1). In addition, 50 avian tracheal swab samples, obtained from the poultry flocks without any IB signs, were also tested with all three assays.
Mass and BR RT-qPCR.
All viral and bacterial samples were first tested with the 5′UTR RT-qPCR. IBV isolates and vaccine strains presented a positive result with Ct values between 16.3 and 28.0 and an amplified length of approximately 143 bp. Negative results were observed in the other viral and bacterial samples (Table 1).
The Mass RT-qPCR presented a positive result in the seven IBV samples of the Mass type (the three commercial vaccines and the following four allantoic fluid isolates: SB-A0297, SB-A0395, SB-A2308, and SB-A3513), with Ct values ranging from 16.8 up to 24.9. Eight out of the nine strains from the BR genotype (BR-I strains SB-A1140, SB-A1971, SB-A2240, SB-A2360, SB-A2479 and BR-II strains SB-A2401, SB-A2960, and SB-A2962) were negative in the Mass RT-qPCR. One BR-I genotype strain (SB-A2400) was positive in the assay with a low Ct value (21.4). The expected 119-bp amplification product was observed in all positive samples.
The BR RT-qPCR was positive in all nine BR genotype isolates (with Ct values ranging from 21.4 up to 28.9). All Mass vaccine and isolate strains were negative in the BR RT-qPCR (Table 1). The expected amplified fragments of 91 or 95 bp were observed in all the tested BR strains.
Seven other IBV isolates available in the laboratory (without any previous genotype information) were also tested with a Mass and BR RT-qPCR. All of them were previously positive with the 5′UTR RT-qPCR (Ct values ranging from 13.6 up to 14.9). One strain (SB-21) was positive in the Mass RT-qPCR and negative in the BR RT-qPCR, while six strains (SB-22, SB-24, SB-25, SB-26, SB-27, and SB-28) were positive in the BR RT-qPCR and negative in the Mass RT-qPCR (Table 1). The S1 gene of these seven isolates was sequenced. The first isolate presented similarity with Mass sequences while the other six isolates presented identity to BR-1 genotype sequences.
Specificity of the assays.
All other viral and bacterial avian pathogens samples were tested with 5′UTR RT-qPCR, Mass RT-qPCR, and BR RT-qPCR, all presenting negative results (Table 1). In addition, fifty IBV-negative tracheal swab samples also showed negative results (no amplification curve) in the three assays.
Linearity and sensitivity of the assays.
The Mass RT-qPCR was linear and reproducible. This assay was able to detect viral RNA consistently up to the dilution 2.4 × 10−6 with a high correlation (R2 > 0.98) between the samples with a different viral load and the corresponding Ct. A dynamic range of analysis was observed across the different viral concentrations.
The BR RT-qPCR presented similar performance. This assay was also able to detect viral RNA up to the dilution 2.4 × 10−6 with a very good correlation (R2 > 0.99) and the same dynamic range of the Mass RT-qPCR. This assay was also as sensitive as the 5′UTR RT-qPCR (Table 3).
Sensitivity of the RT-PCR methods for Mass (SB-21) and BR (SB-24) IBV viruses
The 141 clinical samples were evaluated with the three RT-qPCRs. A total of 86 (61%) samples were positive for 5′UTR RT-qPCR and at least one other molecular typing test: 8 for the Mass RT-qPCR, 76 for the BR RT-qPCR, and 2 for both. In the remaining 55 samples, 25 (17.7%) were positive only for 5′UTR RT-qPCR and 30 (21.3%) were negative for the three assays (Table 4).
Results of the analysis of the 141 clinical samples with the three molecular biology assays
RT-PCR molecular typing methods of the S gene have largely replaced the classical methods of serotyping with a good agreement of data (3,25). First, typing techniques were performed by RFLP after RT-PCR of the hypervariable region of the S1 gene. Specific banding patterns for each genotype were observed in the gel and compared with expected patterns of known genotypes (25). Afterwards, nucleotide sequencing of this same genetic region became the most useful technique for IBV molecular typing. RT-PCR product cycle sequencing could be used to identify previously characterized strains and also unrecognized field isolates and variants (15,21,23).
This study developed and validated two TaqMan RT-qPCRs for the specific detection of the two main IBV genotypes previously detected in Brazilian poultry flocks: Mass and BR. The Mass and BR RT-qPCRs were specific to detect Mass and BR strains, respectively, and presented high correlation to S1 sequencing in all IBV isolates and vaccine strains. Furthermore, they presented a high analytical specificity and sensitivity when compared to the 5′UTR RT-qPCR (5). These novel assays were also able to detect and differentiate these genotypes directly from clinical samples.
Molecular assays based on S1 genes were already successfully developed for IBV genotypes detection; for example, the specific RT-PCR for identification of prevalent IBV strains in Europe and the United States (18), a multiplex RT-PCR assay for detection of IBV strains prevalent in Taiwan (8), and a duplex SYBRHGreen (Roche Applied Science, Mannhein, Germany) real-time RT-PCR for the simultaneous detection and differentiation of Mass and non-Mass genotypes in Cuba (1). More recently, real-time PCR assays have been developed for the detection of Mass, Conn, Arkansas, and Delaware genotypes with specific primers and probes targeted to the S1 gene (29,30). Interestingly, this last assay was also able to detect mixed infections in clinical samples, a similar finding of our study (sample SB-A2400 was positive in the two assays, probably indicating a mixed infection of the Mass and BR genotypes).
The developed molecular methods detected all the IBV vaccine strains and positive allantoic fluids (field isolates). In comparison with the 5′UTR RT-qPCR, the Mass RT-qPCR presented almost the same sensitivity (Table 3). However, the Mass and BR RT-qPCR assays did not detect a significant number of positive clinical samples (n = 25, representing 17.7%) in the 5′UTR RT-qPCR assay. These samples could be infected with other S1 genetic IBV variants, classical IBV strains (as for example 4/91, Conn), or even other avian coronavirus (from turkey, pigeon, duck, quail, etc.). These viruses of other bird hosts can be detected with the 5′UTR RT-qPCR (5). Further, previous experimental studies demonstrated that chickens are susceptible to turkey coronavirus infection (16,17), and viral strains of this pathogen are present in Brazilian turkey flocks (27). An IB virus-like Gammacoronavirus was also detected in quail samples in Brazil (32). New studies should be conducted to investigate these possibilities.
The methods presented here have also the potential to evaluate vaccination programs in poultry flocks in Brazil. IBV control has been attempted using live attenuated and inactivated vaccines (6). In Brazil, only Mass strains must be used in commercial live attenuated vaccines as regulated by the Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento) (12). As a consequence, only Mass strains have been observed in the field, together with the vastly disseminated BR variant genotypes (4,7,15,14,33,34). In this sense, the Mass and BR RT-qPCRs could detect these two IBV genotypes in vaccinated flocks, assisting in the prevention and control programs of IB.
TaqMan RT-qPCR assays targeting the S1 gene region were developed to detect the Mass vaccine and Brazilian field genotypes. The assay is rapid, specific, and was able to detect more than one IBV genotype in the same sample. In addition, the RT-qPCR assay shows good potential as a diagnostic tool for IBV molecular typing in clinical samples.
We thank the veterinarians who submitted clinical samples and the technicians from SimbiosBiotecnologia and Laboratório de Diagnóstico Molecular who performed technical support. This work was financially supported by Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), grant 11/1203-1, and Financiadora de Estudos e Projetos (FINEP), grant 01.09.0240.00.
Brazilian variants (genotypes)
avian infectious bronchitis virus
restriction fragment length polymorphism
real-time reverse-transcription PCR