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

Abstract

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.