Fig. 1

Classification of the replication capacity of transposition mutants of MeHV-1 in cell culture. Gene requirements of MeHV-1 were assigned based on the replication capacity of the respective transposition mutants in cell culture when compared to parental virus (MuAΔ65 day 5 post-transfection a: brightfield × 100; and b: fluorescent microscopy × 100). Clones were classified as ‘non-essential, no attenuation’ (MuAΔ72 day 5 post-transfection c: brightfield × 100; and d: fluorescent microscopy × 100), ‘non-essential, severe attenuation’ (MuAΔ68 day 7 post-transfection e: brightfield × 100; and f: fluorescent microscopy × 100), or ‘essential, complete attenuation’ (MuAΔ64 day 7 post-transfection g: brightfield × 100; and h: fluorescent microscopy × 100). BAC DNA encoding the MeHV-1 genome and containing a single transposon insertion within either the BAC vector backbone, therefore reflecting parental virus (a and b), UL48 (c and d), UL53 (e and f) or UL27 (g and h) was transfected into CEFs. Monolayers were passaged every five to eight days and were observed for the development of CPE using light microscopy (a, c, e and g) and, whenever possible, for expression of a marker gene (eGFP) using fluorescent microscopy (b, d, f and h)