Development And Characterization Of Recombinant Infectious Bronchitis Viruses And The Role Of CD59 In IBV Proliferation

Infectious bronchitis?IB?,caused by infectious bronchitis virus?IBV?,is a highly contagious chicken disease,and can lead to serious economic losses in poultry enterprises.IBV is worldwide in distribution and exists as many different serotypes.The continual introduction of new IBV serotypes requires alternative strategies for the production of timely and safe vaccines against the emergence of variants.Modification of the IBV genome using reverse genetics is one way to generate recombinant IBVs as the candidates of new IBV vaccines.In this study,infectious clone of a Vero cell-adapted IBV Beaudette strain?p65?was constructed.Briefly,five fragments spanning the entire IBV genome were amplified by RT-PCR of total RNA extracted from Vero cells infected with IBV Beaudette strain,each fragment was cloned into either pGEM-T Easy or pCR-XL-TOPO vectors.Restriction sites for either BsaI or BsmBI were introduced into both the 5'and 3'ends of the fragments by specific primers.A19-nucleotide sequence corresponding to the T7 RNA promoter was added at the 5'end of fragment A to facilitate transcription using the T7 polymerase in vitro.The five fragments were obtained by digestion of the corresponding recombinant plasmids with either BsaI or BsmBI,and full-length cDNA of IBV genome was assembled by ligation of the 5 purified fragments.The full-length transcripts were synthesized in vitro using the mMessage mMachine T7 kit.The transcripts of IBV N gene were generated from a linearized plasmid containing N gene and the 3'-UTR region.The full-length transcripts and the N transcripts were introduced into Vero cells by electroporation,and infectious virus was recovered.To explore the expression potential of heterogeneous gene using backbone of IBV Beaudette strain,the ectodomain region of Spike gene?1,302 bp,S1+partial sequence of S2?of IBV H120 strain was amplified by RT-PCR and used to replace the corresponding region in the full-length cDNA of IBV Beaudette strain,this recombinant full-length cDNA was directly used as template for full-length transcription of viral genome in vitro.The full-length transcription products together with the N transcripts were transfected into Vero cells by electroporation.At 48h post transfection,the transfected Vero cells was harvested,and continued to passage.Syncytium was not observed until the recombinant virus was proceeded 4 passages.The rBeau-H120?S1?was verified by detection of the replaced ectodomain region of H120 Spike gene using RT-PCR.Western-blot analysis of rBeau-H120?S1?-infected Vero cell lysates demonstrated that the N protein was expressed,which implied that rBeau-H120?S1?was able to propagate in Vero cells.The data of TCID₅₀ and EID₅₀demonstrated that the titer levels of rBeau-H120?S1?reached to 10^5.90±0.22TCID₅₀/mL and 10^6.13±0.23EID₅₀/mL in Vero cells and 9-day-old SPF chicken embryos,respectively.Protection studies showed that the percentage of antibody positive chickens,which were vaccinated with rBeau-H120?S1?at 7-day after hatch,had risen to 90%at 21 days post inoculation,and it provided 85%rate of immune protection against challenge with 10³ EID₅₀ of the virulent IBV M41 strain.The recombinant virus constructed by reverse genetics could be further developed as a novel genetic engineered vaccine against IB.Meanwhile,another recombinant IBV is developed by replacing the ectodomain region of the S1gene of the IBV Beaudette strain with the corresponding fragment from 4/91 strain,designated as BeauR-4/91?S1?.In Vero cells,the virus proliferates as its parental virus,IBV Beaudette strain,and can cause syncytium formation.The peak titer would reach 10^5.75 TCID₅₀/mL at 32h post-infection.After inoculation of chickens with the recombinant virus,it demonstrated that BeauR-4/91?S1?remained nonpathogenic and was restricted in its replication in vivo.Protection studies showed that vaccination with BeauR-4/91?S1?at 7-day after hatch provided 80%protection rate after challenge with 10³EID₅₀ of wild type IBV 4/91 strain.These results indicate that BeauR-4/91?S1?has the potential to be an alternative vaccine against IBV 4/91-like serotype.This finding might help in providing further information that replacement of the ectodomain fragment of the IBV Beaudette S1 gene with that from a present field strain is promising for IBV vaccine development.To extend lifespan in vivo of the recombinant IBV after vaccination,the effect of complement regulatory protein CD59 on the life cycle of IBV was investigated.CD59 protein is a unique complement regulatory protein that prevents the formation of membrane attack complexes after complement activation.The results showed that CD59 associates with IBV virions,and abrogation of CD59 function increases the sensitivity of IBV particles to antibody-dependent complement mediated lysis,resulting in a significant reduction of IBV infectivity.Furthermore,IBV infection down-regulates CD59 expression on the cell surface and overexpression of CD59 increases viral titer in the supernatant of infected cells,knockdown or cleavage of CD59 decreases viral titer in the supernatant of infected cells,these observations implied that CD59 protein would be involved in IBV release.These findings give us a clue of modifying IBV vaccine strain by insertion of CD59 gene into downstream of transcriptional regulatory sequences in viral genome,aim to evade complement-mediated destruction.