Figure 1
A: Schematic diagram showing the construction of the self excisable pRB-1B*6X clone. Top – Genome structure of MDV showing the expanded Us2 locus: Us10, Sorf3, gpt (guanine phosphoribosyl transferase gene), CM (chloramphenicol resistance gene), mini-F plasmid, flanking loxP sites, Us3, Us6 and Us7, as well as the unique restriction sites are shown. Middle – Genome structure of pRB-1B*6X clone showing the restored Us2, the inserted Kan flanked by FRT sites and the SV-cre cassette inside the lox P region. Bottom – Genome structure of vRB-1B*6X virus after the excision of the SV-cre and the Kan cassette in the CEF. B: Southern blotting hybridization of Eco RI-digested DNA prepared from either the BAC clone or CEF infected with wild type or the recombinant viruses, hybridized, stripped and probed sequentially with DIG-labelled MEQ, Us2 or gpt probes. M – Molecular weight markers showing the size of MEQ (2.4 kb), Us2 (5.3 kb) and gpt (1.7 kb) fragments. Lane 1 – pRB-1B5 DNA; Lane 2 – pRB-1B*6X DNA; Lane 3 – vRB-1B5-infected CEF DNA; Lane 4 – vRB-1B*6X-infected CEF DNA; Lane 5 – wtRB-1B-infected CEF DNA. C: Comparison of in vitro and in vivo replication and percentage survival of birds infected with RB-1B viruses (1) in vitro replication kinetics of vRB-1B5 (black square), vRB-1B*6X (black triangle) and wtRB-1B (grey diamond) viruses in CEF calculated as PFU per mL at different time points after infection (2) kinetics of in vivo replication of vRB-1B5 (black square), vRB-1B*6X (black triangle) and wtRB-1B (grey diamond) viruses determined from the viral genome copy numbers in PBL (continuous line) and feather DNA (dotted line) using TaqMan real-time qPCR of meq quantitative PCR. (3) Comparison of the cumulative mortality rates at different time points after infection with vRB-1B5 (black square), vRB-1B*6X (black triangle) and wtRB-1B (grey diamond) viruses. The survival rates showed statistically significant differences between the wtRB-1B and vRB-1B5 (p =< 0.0001) and wtRB-1B virus and vRB-1B*X6 groups (p = 0.0064).