Rescue of the recombinant infectious bronchitis virus with the ectodomain region of H120 spike glycoprotein

To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus(IBV)Beaudette strain,the ectodomain region of the Spike gene(1,302bp)of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA.This recombinant was designated as BeauR-H120(S1).BeauR-H120(S1)was directly used as the DNA template for the transcription of viral genomic RNAin vitro.Then,the transcription product was transfected into Vero cells by electroporation.At 48hpost-transfection,the transfected Vero cells were harvested,and passaging continued.A syncytium was not observed until the recombinant virus had passed through four passages.The presence of rBeau-H120(S1)was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR.Western blot analysis of rBeau-H120(S1)-infected Vero cell lysates demonstrated that the nucleocapsid(N)protein was expressed,which implied that rBeau-H120(S1)could propagate in Vero cells.The TCID50 and EID50 data demonstrated that the titer levels of rBeau-H120(S1)reached 105.90±0.22 TCID50/mL and 106.13±0.23EID50/mL in Vero cells and9-day-old SPF chicken embryos,respectively.Protection studies showed that the percentage of antibodypositive chickens,which were vaccinated with rBeau-H120(S1)at 7days after hatching,rose to 90%at 21 days post-inoculation.Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41strain(103EID50/chicken).This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.