To determine the cut-off values for the ELISA, we determined the mean values and standard deviation of negative sera for HBoV1, 2, 3, and 4 using HBoV VLP ELISA at dilutions of 1:200. We used the mean absorbance at 450 nm of the negative sera plus threefolds the standard deviation as the cut-off values, as previously described [19, 31] . For HBoV1, 2, 3 and 4, the cut-off values were 0.344, 0.304, 0.321, and 0.31, respectively. A sample was considered positive for HBoV1, 2, 3, or 4 if its absorbance at 450 nm was above the cut-off value of the respective species in ELISA.