Background: Avian infectious bronchitis (IB) is a highly contagious and important respiratory disease that causes significant economic losses in the poultry industry. So far, several serotypes and genotypes of IB virus (IBV) have been identified, posing challenges to disease prevention and control. Vaccination is the most effective method for controlling the virus. The live attenuated H120 vaccine is one of the common vaccines used in the country against this disease. Today, the determination of the 50% infectious dose in SPF embryonated eggs (EID50%) is used to quantitatively evaluate and determine the titer of the virus for vaccine formulation against IB.
Objectives: This study was conducted to establish a rapid and sensitive real-time polymerase chain reaction (PCR) method for quantitatively evaluating changes in virus titer and the rate of virus replication in embryonated eggs.
Methods: The assay was developed using a universal primer probe set, and the viral load was assessed at 12-hour intervals, beginning immediately after inoculation of embryonated chicken eggs (ECEs) with the H120 vaccine virus and continuing up to 60 hours postinoculation. Harvested allantoic fluids were investigated using the classical EID50 method and a molecular assay.
Results: Viral load dynamics were comparable between the two methods. The lowest titer was observed at 12 hours, and the highest load was detected at 24 and 36 hpi (h post-infection).
Conclusion: The results of this study demonstrate promising potential for using a molecular method instead of the classical chicken embryo infectious dose 50% (EID50) method, which is expensive and time-consuming and has high variability. Nevertheless real-time PCR has not been approved for vaccine production. It is suggested that this method be applied as an in-process control test to reduce the time and expenses associated with manufacturing the H120 IB vaccine while ensuring accurate and precise analysis.