key: cord-319253-8bssrn9o
authors: OKINO, Cintia Hiromi; MONTASSIER, Maria de Fátima Silva; de OLIVEIRA, Andressa Peres; MONTASSIER, Helio José
title: Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I
date: 2018-02-27
journal: J Vet Med Sci
DOI: 10.1292/jvms.17-0566
sha: 
doc_id: 319253
cord_uid: 8bssrn9o

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

strains [11] . Alternatively, a RT-qPCR based on the 3′-UTR of IBV (RT-PCR/HRM curve analysis model), could detect and rapidly distinguish novel and vaccine-related IBV strains from Australia [15] . Finally, a duplex SYBR Green I based RT-qPCR [1] and a RT-qPCR using specific probes [13, 23] were able to detect and differentiate Mass and non-Mass strains.

Finally, considering that HVR I and HVR II of S1 gene were thought to be closely associated with major neutralization epitopes and consequently a reliable target for genotyping method, we developed a diagnostic method using Melting Temperature analysis based on these regions aiming to rapid detect and differentiate Mass and non-Mass IBV strains in samples previously isolated and directly in clinical samples. Analytical specificity and sensitivity were also evaluated, and the method was compared to the standard method of virus isolation in embryonated eggs.

Seven reference IBV strains (M41, H120, H52, Ma5, JMK, SE17 and Connecticut) and six Brazilian field isolates (IBVSC01, IBVSC02, IBVPR02, IBVPR03, IBVPR05 and IBVPR07) were provided by Empresa Brasileira de Pesquisa Agropecuária-Embrapa (Concórdia, SC, Brazil) and previously molecularly characterized [26] (Table 1) . Three other non-related viruses (commercial vaccines) were also used to confirm specificity of our method: Newcastle disease virus (NDV, La Sota vaccine strain), infectious bursal disease (IBDV, Lukert vaccine strain) and avian metapneumovirus (AMPV, PL-21 vaccine strain).

Ninety-six one-day-old chicks (Cobb-Vantress Lineage) from a commercial broiler chick hatchery were separated in three positive pressure isolators. At 28 days of age, first group (n=42) was experimentally infected by intra-ocular and intranasal routes with 10 5.0 EID 50 (50% Embryo Infective Dose) of M41 strain. A negative control group (n=42) was mock infected and maintained under the same conditions. Remaining group (n=12) was inoculated with H120 live attenuated vaccine. Three chickens from the first and second groups were euthanized at 1-10, 12, 15, 17 and 20 days post infection (dpi), while birds from third group were euthanized at 3, 5 e 12 dpi. Trachea and lung samples were collected and kept at −70°C until processing by RNA extraction or virus isolation. Throughout the experimental period, birds received water ad libitum and feed once a day, the room temperature was adapted according with bird's age. All procedures have been approved by the Veterinary Sciences Animal Care Committee of Universidade Estadual Paulista, according with ethical principles of animal experimentation adopted by Brazilian College of Experimentation.

Virus isolation in Specific Pathogen Free (SPF) embryonated chicken eggs from tissue samples was performed as previously described [28] . Three passages were conducted to confirm negative samples.

Extraction of RNA from virus strains and tissue samples were performed using Trizol Reagent (Invitrogen, Carlsbad, CA, U.S.A.). The cDNA was synthesized according to instructions provided with SuperScript III Reverse Transcriptase (Invitrogen) and Random Primer (Invitrogen). qPCR was carried out in an Applied Biosystems Real-time 7500 Instrument (Applied Biosystems, Foster City, CA, U.S.A.) using 10 µl of Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 10 pmol of each primer (Table 2), 2 µl of cDNA, in a final volume of 20 µl. The amplification reaction included a preincubation step at 95°C for 8 min to activate the HotStart Taq DNA polymerase, followed by 40 cycles of amplification including denaturation at 95 for 30 sec, annealing at 49.2°C for 30 sec and extension at 72°C for 30 sec [27] . After amplification by RT-qPCR, a melting analysis curve was performed by raising the incubation temperature from 65 to 95°C in 0.1°C increments with a hold of 1 sec at each increment.

The analytical specificity of RT-qPCR was assessed using three non-related RNA viruses including Newcastle disease virus (NDV, La Sota vaccine strain), infectious bursal disease (IBDV, Lukert vaccine strain) and avian metapneumovirus (AMPV, PL-21 vaccine strain).

The analytical sensitivity of RT-qPCR was assessed using seven serial ten-fold dilutions of plasmidial DNA containing the orf of S1 gene fragment (1.6 kb) of M41 strain of IBV cloned into the PYES 2.1/v5-His TOPO vector (Invitrogen), using two combinations of oligonucleotides to evaluate the analytical sensitivity. Cq (Cycle quantification) results were used to calculate the Log of IBV copies (Log10) using linear equation from a standard curve. The reproducibility was determined by testing in duplicate and in four independent runs, followed by calculation of mean, standard deviation (S.D.) and coefficient of variation (C.V.). Tm data of the real-time RT-PCR assay was analyzed statistically by the Scott-Knott test [30] , and the level of significance was set at P≤0.05.

The detection limit of RT-qPCR was 1.32 × 10 2 copies, R 2 =0.997, efficiency=93.06% (Fig. 1) . The reproducibility ranged from 0.1575 to 0.7585 cycles, while the C.V. inter-assay ranged from 0.56 to 2.64%. All IBV strains were successfully detected and no positive signal was recorded for the three non-related avian RNA-viruses tested.

The RT-qPCR developed in this study detected the virus in tracheal samples from birds experimentally infected with M41, or with H120 strains, in the period from 1 to 20 dpi (entire post-infection period evaluated) or from 3 to 12 dpi, respectively, while in pulmonary samples IBV detection varied from 8 to 15 dpi, or only at 3 dpi in M41-infected or H120-infected birds, respectively. Conversely, the standard method of virus isolation in SPF embryonated eggs detected the virus only from 2 to 9 dpi or at 3 dpi for both types of tissue samples from birds infected with M41 or H120, respectively. No positive signal of amplification and no positive results by virus isolation were found in tissues samples from negative control birds ( Table 3) .

The absence of amplification signal in other non-related RNA viruses and in negative control samples, characterized the specificity of RT-qPCR. F=Forward, R=Reverse. a) Modified, b) Annealing site on S1in M41 reference strain of IBV (sequence accession number: AY851295). All tested IBV strains were amplified by RT-qPCR using both primer sets (HVR I and HVR II). Amplicons of expected size for S1 gene of IBV using HVR I or HVR II primer sets were also visualized by gel electrophoresis analysis. In the first-derivative melting curve analysis, the S1 gene amplicons displayed melting temperature (Tm) values ranging from 80.30 to 83.70°C, for HVR I amplicons, and from 76.16 to 78.25°C for HVR II amplicons.

All Mass related IBV strains (reference strains and field isolates) tested in this study presented the same Tm profile (81.38 ± 0.28°C) for melting temperature analysis of HVR I amplicons, as no significant differences were observed, while all the remaining tested strains presented significant differences in their Tm profiles (Fig. 2) . Interestingly, the IBVPR05 strain, showed two melting peaks, with the highest peak at 83.17°C, and another one at the same Tm profile as Mass group (Fig. 2) . We hypothesized that this sample of Brazilian field isolate was composed by two populations or quasispecies, as described previously [17] for a sample of Beaudette strain of IBV amplified by RT-qPCR using another pair of primers.

However, when the melting temperature analysis was performed using HVR II region, the Mass genotype differentiation was not possible, though the Tm profiles for HVR II amplicons of JMK and SE-17 strains and one Brazilian variant isolate (IBVPR05) were distinct one each other and from the remaining tested IBV strains. A similar distinction was observed in the Tm profile of HVR II amplicon for M41 strain with regard to H120 strain, in spite of these viruses are classified in the Massachusetts genotype. The detection limit of our method is similar to that recorded for a real-time PCR assay based on 5′-UTR gene of IBV genome [3] . However our assay is less sensitive than another real-time technique targeting an ORF1b of replicase gene of IBV [11] . This difference might be due to the fact that targeting the most conserved and abundant genes of coronavirus, like replicase gene or nucleoprotein gene, can enhance the sensitivity of conventional and RT-qPCR methods [2, 11, 24] . Nonetheless, more conserved target genes do not allow differentiation between different genotypes of IBV [24] , as S1 gene RT-PCR does, which remains an important tool for genotyping IBV isolates.

There are several reports demonstrating the use of melting temperature analysis to differentiate genotypes of virus pathogens [4, 20, 29, 31, 32] . Similarly, the method used here provided differentiation of Mass genotype using HVR I region, as all tested strains previously genotyped belonging to Mass genotype presented no significant different Tm values, while the other strains previously genotyped differently from Mass presented significant different Tm values (Table 1 ). In addition, previous study [16] using primers flanking similar HVRs of S1 gene in RT-qPCR with EVAGREEN succeeded in discriminating IBV strains from Taiwan and H120 from M41 strain of IBV.

Currently, IBV genotype differentiation has been performed using separated molecular assays, which have increased both time and cost. Assays for genotyping based on conventional molecular methods such as PCR, RFLP, hybridization assay and direct sequencing are time-consuming, more complex and expensive. Alternatively, the method described here has allowed simultaneous detection of IBV genome and Mass genotype identification in a single reaction. The specificity of Mass genotyping by our method was confirmed, comparing with the results obtained previously [21, 26] .

In conclusion, the method described here provides a practical and effective screening test for simultaneous detection and Mass genotyping of IBV. Moreover, this rapid, simple, specific and sensitive test is applicable for both IBV previously isolated and directly in clinical samples.

CONFLICT OF INTEREST. The authors declare that they have no conflicts of interest.

A duplex SYBR Green I-based real-time RT-PCR assay for the simultaneous detection and differentiation of Massachusetts and non-Massachusetts serotypes of infectious bronchitis virus

Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction

Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens

Dengue typing assay based on real-time PCR using SYBR Green I

LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens

The long view: 40 years of infectious bronchitis research

Assessment of cross-immunity dm chickens to strains of avian infectious bronchitis virus using tracheal organ cultures

Infectious bronchitis virus variants: a review of the history, current situation and control measures

Increased level of protection of respiratory tract and kidney by combining different infectious bronchitis virus vaccines against challenge with nephropathogenic Brazilian genotype subcluster 4 strains

Characterization of infectious bronchitis viruses isolated from outbreaks of disease in commercial flocks in Brazil

SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses

Nephritis associated with S1 variant brazilian isolate of infectious bronchitis virus and vaccine protection test in experimentally infected chickens

A Real-Time Reverse-Transcription Polymerase Chain Reaction for Differentiation of Massachusetts Vaccine and Brazilian Field Genotypes of Avian Infectious Bronchitis Virus

Vaccine efficacy against Ontario isolates of infectious bronchitis virus

Rapid detection and non-subjective characterisation

of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model

The genotyping of infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction

Detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the Beaudette strain

Review of infectious bronchitis virus around the world

Serotype identification of avian infectious bronchitis virus by RT-PCR of the peplomer (S-1) gene

Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus

Differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis

A new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism

Development of RT-qPCR assays for the specific identification of two major genotypes of avian infectious bronchitis virus

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

Molecular epidemiology and evolution of avian infectious bronchitis virus

Genetic grouping of avian infectious bronchitis virus isolated in Brazil, based on RT-PCR/RFLP analysis of the S1 gene

Inflammatory and cell-mediated immune responses in the respiratory tract of chickens to infection with avian infectious bronchitis virus

Detection of viral antigen following exposure of one-day-old chicken to the Holland-52 strain of IBV

Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis

Cluster analysis method for grouping means in the analysis of variance

Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: effect of amplicon size, melt rate, and dye translocation

Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan

Massachusetts live vaccination protects against a novel infectious bronchitis virus S1 genotype DMV/5642/06

Antigenic and immunogenic characterization of infectious bronchitis virus strains isolated in China between

ACKNOWLEDGMENTS. The authors thank FAPESP (process number 04/12267-2 and 01/14950-3) and CNPq (process number 01/14950-3) for the financial support; Embrapa (Concórdia, SC, Brazil) for supplying IBV strains; Merial (Campinas, SP, Brazil) for provided the embryonated chickens eggs SPF and Frango Sertanejo (Ipiguá, SP, Brazil) for provided the commercial broilers. We also thank Professor Dr Joao Ademir de Oliveira for statistical advice.