At each co-culture passage point, surplus human 293T cells were used to prepare genomic DNA. PERV transmission was monitored by subjecting these samples to quantitative (Q)-PCR. PERV-specific pol gene PCR products could be detected in the human genomic DNA samples after approximately two weeks of continuous co-culture ( Figure 1B) . From the point of first detection onward, the total number of PERV transmissions continued to increase, averaging 190 new events per day per 50,000 cells (10 5 beta-actin copies; SEM = 62; n = 5 experiments). Importantly, pig cells did not breach the 293T cell compartment because Q-PCR analyses of the same human genomic DNA samples failed to detect a concomitant transfer of pig genomic DNA ( Figure 1B ; also see Online Figure S1 for PERV pol gene Q-PCR standard curves, representative PERV-specific datasets and human beta-actin controls). Moreover, PERV copy number did not increase over a two week interval when infected 293T cells were grown in isolation, indicating that PERV was not replicating in the human cells and that the majority of the observed transmission events were derived from the PK-15 cells (i.e., new events). These results combine to indicate that the trans-well assay provided a robust system for monitoring bona fide zoonotic PERV transmissions.