| Popis: |
There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells. |
