Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSVp53-CC encode VSV matrix protein (M) with ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor p53 fused to a far-red fluorescent protein eqFP650. Each virus was serially passaged 32 times (accounts for more than 60 viral replication cycles) on either SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in non-malignant cells. Surprisingly, two identical VSV glycoprotein (G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein and no deletions or mutations were found in the p53 or eqFP650 portions of virus-encoded transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants encoding large transgenes. [ABSTRACT FROM AUTHOR]