Construction of infectious bronchitis virsus endoribonucleasensp15 defective recombinant virus by reverse genetic system

ABSTRACT

Infectious Bronchitis Virus (1BV) belongs to the y coronavirus, however, the function of IBV encoded endoribonuclease (non - structural protein 15, nsp15) has not been determined yet. To explore the function of nsp15 in the process of IBV replication, we mutated the IBV nsp15 endonuclease core residue His238 to Ala, constructed the nsp15-defective recombinant virus rIBV-nsp15-H238A, via in vitro ligation and recombination technology. Plaque assay and TCID50 were performed to measure virus titer, virus plaque size and growth curve. The IBV Beaudette-R genome was cloned as 5 fragments in vectors, BsaI or BsmBI restriction sites were added to the end of each fragment. After plasmid amplification, the cDNA fragments were obtained by enzymatic digestion, followed with in vitro ligation and transcription. Full - length genomic RNA was electroporated into Vero cells, together with N transcript, to rescue the recombinant viruses rIBV and rIBV - nsp15- H238A. Plaque assay was performed to detect and compare the viral titer and plaque size of these two recombinant viruses. Results showed that the virus titer of rIBV -nsp15 -H238A was 2.71x106PFU/mL, 3 times lower than that of rIBV (9.4x106PFU/mL). The plaque size of rIBV-nsp15-H238A was much smaller than that of rIBV, indicating that rIBV-nsp15-11238A replicates and spreads slower than rIBV. The growth curve of rIBV-nsp15-H238A was slower than that of rIBV. Our study demonstrates that nsp15 I-1238 is the key amino acid and plays an important role in the replication and spread of IBV. The construction of nsp15 defective recombinant virus provides a powerful tool for the study of the function of nsp15.