To address the persistence of the vectors in the injected joints, synovial tissues from animals injected with HAdV5-PUMA and BVCARHAdV5-PUMA and sacrificed at day 21 were analysed. Synovial tissues from four individual rats from each group were chosen at random and the total DNA extracted. PCR assays, performed using primers recognizing the hexon gene of HAdV5, amplified the expected hexon band of 465 bp in all the samples (Fig. 8a; arrowhead). However, no specific baculovirus band of 1.1 kbp, corresponding to a sequence within the baculovirus CAR gene, was detected in any of the synovium samples (Fig. 8b). These results implied the persistence of the HAdV5-PUMA genome in the synovial tissues at least until day 21 post treatment, but not that of the BVCAR genome.Fig. 8Analysis of vector persistence and immune response to vectors. a The HAdV5-PUMA and BVCAR DNA genomes in synovial tissues were extracted from synovial tissues samples of rats killed at day 21 after intra-articular administration of HAdV5-PUMA alone (B2, B4, B5 and B11) or BVCARHAdV5-PUMA (B10, B12, B13 and B14), and used for PCR analysis to detect the viral genomes. a The 465-bp amplified fragment corresponding to the HAdV5 hexon gene (a, arrowhead) was found in all the samples and in the positive control. Note the occurrence of a non-specific amplified fragment of 1500 bp (black dots). b, For the BVCAR genome, no specific band of 1.1 kbp, corresponding to the baculovirus CAR gene was detected in any of the samples, but was present in the positive control. b To evaluate the immune response of the treated animals to BVCAR and HAdV5-PUMA vectors, the sera of the animals treated for 4 days and 21 days with HAdV5-PUMA alone or with BVCAR HAdV5-PUMA were analysed by ELISA for the presence of antibodies against adenovirus and baculovirus. A single serum dilution (1:10) was used for all samples. No significant adenovirus or baculovirus antibodies were detected in the sera of the animals treated for 4 days (grey bars, c,d). In the animal group treated for 21-days, adenovirus antibodies were detected in the sera of 5/8 animals (black bars, c) and baculovirus antibodies detected in the sera of 2/8 animals (black bars, d). Similar results were obtained in an independent experiment