An Attenuated Cytomegalovirus Vaccine with a Deletion of a Viral Chemokine Gene Is Protective against Congenital CMV Transmission in a Guinea Pig Model
Figure 1
Schematic representation of the GPCMV MIP gene and generation of recombinant virus. (a) Map of GPCMV genome. GPCMV MIP (gp1 gene) maps to 2.3 kb EcoR I fragment near left genome terminus. Knockout of gp1 was achieved by introduction of gpt/eGFP cassette into a Stu I collapsed version of plasmid DNA. Following co-transfection of plasmid and viral DNA and gpt selection as described in text, a clonal recombinant virus was obtained by limiting dilution. (b) Southern blot analysis of wild-type (ATCC 22122; left panel) and v545 (recombinant) DNA. Probing with pKTS107 probe revealed presence of restriction polymorphisms demonstrating predicted configuration of recombinant virus. H, Hind III digest; E, EcoR I digest; X, Xba I digest. Molecular weight markers, lamba/Hind III ladder. (c) To further characterize the genome structure of the v545 mutant, viral DNA was analyzed by PCR. The PCR was done using primer pairs cassette F1/R1 (upper panel) and cassette F2/R2 (lower panel) which, respectively, amplify 552 bp and 671 bp products in wild-type GPCMV and vAM403. Insertion in v545 coupled with deletion of MIP gene results in overall shifts in bands to ~2.8-2.9 kb, as predicted. Subsequent sequence analysis of gel-purified products confirmed predicted insertion and genome structure of v545 vaccine virus.
