To confirm the specificity of DNA replication and the identity of the DpnI-resistant DNA bands, we disrupted the ORFs encoding viral proteins NS1, NP1, VP1 and VP2 in pIHBoV1; knockout of expression of the corresponding viral protein was confirmed by Western blot analysis. When the NS1 ORF was disrupted, no DpnI digestion-resistant DNA was detected ( Figure 2D , lane 4), confirming that replication of this DNA requires NS1. Notably, when the NP1 ORF was disrupted, an RF DNA band was detected but it was very weak ( Figure 2D , lane 6), suggesting that NP1 is also involved. When the VP2 ORF was knocked out, the ssDNA band disappeared, but this was not the case when VP1 was disrupted (VP2 was still expressed; Figure 2D , compare lanes 7 to 9), these findings are consistent with a role for