Mutational analysis of early region 4 of bovine adenovirus type 3

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccinatio n of humans and animals. A series of BAV3 early region 4 (E4) deletion-muta nt viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovi ne retinal cells with E4-modified genomic DNA. Each of these mutants was fu rther analyzed for growth kinetics, viral DNA accumulation, and early/late protein synthesis. Mutant viruses carrying deletions in Orf1, Orf2, Orf3, o r Orf4 showed growth characteristics similar to those of the E3-deleted BAV 3 (BAV302). DNA accumulation and early/late protein synthesis were also ind istinguishable from those of BAV302. However, mutant viruses carrying a del etion in Orf5, Orfs 1-3 (BAV429), or Orfs 3-5 (BAV430) were modestly compro mised in their ability to grow in bovine cells and express early/late prote ins. E4 mutants containing larger deletions, Orfs 1-3 (BAV429) and Orfs 3-5 (BAV430), were further tested in a cotton rat model. Both mutants replicat ed as efficiently as BAV3 or BAV302 in the lungs of cotton rats. BAV3-speci fic IgA and IgG responses were detected in serum and at the mucosal surface s in cotton rats inoculated with mutant viruses. In vitro and in vivo chara cterization of these E4 mutants suggests that none of the individual E4 Orf s are essential for viral replication. Moreover, successful deletion of a 1 .5-kb fragment in the BAV3 E4 region increased the available insertion capa city of replication-competent BAV3 vector (E3-E4 deleted) to similar to4.5 kb and that of replication-defective BAV3 vector (E1a-E3-E4 deleted) to sim ilar to5.0 kb. This is extremely useful for the construction of BAV3 vector s that express multiple genes and/or regulatory elements for gene therapy a nd vaccination. (C) 2001 Academic Press.