Infectious bronchitis virus Mass-type (GI-1) and QX-like (GI-19) genotyping and vaccine differentiation using SYBR green RT-qPCR paired with melting curve analysis

Laconi, Andrea; Berends, Alinda J.; Laat, Esther C.H. de; Urselmann, Tara A.P.M.P.; Verheije, Hélène M.

doi:10.1016/j.jviromet.2019.113771

Cited by 6 publications

(6 citation statements)

References 22 publications

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“…qRT-PCRs were performed using the iTaq universal SYBR Green one-step kit (Bio-Rad Laboratories, Hercules, California, USA). A genotype QX specific SYBR Green qRT-PCR developed and validated in our laboratory [15] based on the S1 gene sequence was used to detect and quantify viral RNA. The limit of detection of the assay was equal to 10 1.31 EID 50 /100 ml.…”

Section: Viral Rna Analysismentioning

confidence: 99%

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation

Laconi

Weerts

Bloodgood

et al. 2020

Vaccine

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a b s t r a c tInfectious bronchitis (IB) is a highly contagious respiratory disease of poultry, caused by the avian coronavirus infectious bronchitis virus (IBV). Currently, one of the most relevant genotypes circulating worldwide is IBV-QX (GI-19), for which vaccines have been developed by passaging virulent QX strains in embryonated chicken eggs. Here we explored the attenuated phenotype of a commercially available QX live vaccine, IB Primo QX, in specific pathogens free broilers. At hatch, birds were inoculated with QX vaccine or its virulent progenitor IBV-D388, and postmortem swabs and tissues were collected each day up to eight days post infection to assess viral replication and morphological changes. In the trachea, viral RNA replication and protein expression were comparable in both groups. Both viruses induced morphologically comparable lesions in the trachea, albeit with a short delay in the vaccinated birds. In contrast, in the kidney, QX vaccine viral RNA was nearly absent, which coincided with the lack of any morphological changes in this organ. This was in contrast to high viral RNA titers and abundant lesions in the kidney after IBV D388 infection. Furthermore, QX vaccine showed reduced ability to reach and replicate in conjunctivae and intestines including cloaca, resulting in significantly lower titers and delayed protein expression, respectively. Nephropathogenic IBVs might reach the kidney also via an ascending route from the cloaca, based on our observation that viral RNA was detected in the cloaca one day before detection in the kidney. In the kidney distal tubular segments, collecting ducts and ureter were positive for viral antigen. Taken together, the attenuated phenotype of QX vaccine seems to rely on slower dissemination and lower replication in target tissues other than the site of inoculation.

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Section: Viral Rna Analysismentioning

confidence: 99%

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation

Laconi

Weerts

Bloodgood

et al. 2020

Vaccine

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“…In molecular diagnostic assays, the S1 gene is widely used in the design of strain-specific primers for IBV detection [11,15,16] because it is associated with strain heterogeneity and biological characteristics [19]. This study selected the specific primers for QX-like and Thai IBV from S1 gene sequences.…”

Section: Discussionmentioning

confidence: 99%

“…In recent years, progress in the field of molecular biology has facilitated the development of rapid and reliable diagnostic assays. Real-time RT-PCR for IBV detection has been developed, which provides highly specific and sensitive results in a timely manner [11][12][13]. However, real-time RT-PCR requires more specialized devices.…”

Section: Introductionmentioning

confidence: 99%

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

Junnu

Pohuang

2023

Vet World

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

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“…AvCoV-IBV is a serious respiratory pathogen, which causes enormous economic losses in the commercial poultry sector worldwide [ 15 , 16 , 17 ]. As a result, rapid and sensitive detection methods were critical for performing epidemiological investigation and immunization effectiveness assessment.…”

Section: Discussionmentioning

confidence: 99%

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum

Song

et al. 2022

IJMS

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Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55–1.65% and 2.58–6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

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Infectious bronchitis virus Mass-type (GI-1) and QX-like (GI-19) genotyping and vaccine differentiation using SYBR green RT-qPCR paired with melting curve analysis

Cited by 6 publications

References 22 publications

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation

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

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum

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