AbstractClinically, Landrace pigs are more susceptible to porcine circovirus-associated diseases (PCVADs) than Piétrain pigs. We previously found that porcine circovirus type 2 (PCV2) can infect T-lymphoblasts. The present study examined the replication kinetics of six PCV2 strains in the lymphoblasts of Landrace and Piétrain pigs. The results showed that T-lymphoblasts from Landrace pigs are much more susceptible to PCV2 infection than those from Piétrain pigs. In addition, PCV2 replication was strain-dependent. PCV2 binding to T-lymphoblasts was partially mediated by chondroitin sulfate (CS) and dermatan sulfate (DS). Phosphacan, an effective internalization mediator in monocytes that contains several CS chains, was also demonstrated to be involved in PCV2 internalization. Viral binding and internalization were not different between the two breeds, however, the subsequent step, the disassembly was. Although inhibition of serine proteases blocked PCV2 replication in both Landrace and Piétrain pigs, this only occurred at a neutral pH in Piétrain pigs, whereas this occurred also at a low pH in Landrace. This suggested that more proteases can cleave PCV2 in Landrace lymphoblasts than in Piétrain lymphoblasts, explaining the better replication. Through co-localization studies of viral particles with endo-lysosomal markers, and quantitative analysis of organelle sizes during viral internalization, it was observed that PCV2 may exhibit a higher propensity for viral escape from late endosomes in Landrace pigs (smaller) compared to Piétrain pigs. These results provide new understandings of the different PCV2 susceptibility in Landrace and Piétrain pigs.