As controls, human parvovirus B19 VP2 was expressed, as described previously [38] , using the construct pFastBac1B19VP2 provided by Dr. Xiaohui Zou at the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease control and Prevention. Additionally, the PARV4 VP2 gene (1,659 bp in length, nt 3,464-5,122 based on the NC_007018 reference sequence) was synthesized by Sangon Biotech (Shanghai, China) and cloned into baculovirus vector pFBGP67-His [39] . The recombinant baculoviruses were generated in Sf9 cells using the Bac-to-BacH Baculovirus Expression System (Invitrogen) protocol provided by the manufacturer. High Five cells were infected with recombinant baculovirus expressing PARV4 VP2 gene at a MOI of five. The infected cells were collected three days post-infection and purified using a HisTrap HP 1 ml column (GE Healthcare, Waukesha, WI). The concentrations of all purified protein were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) and stored at 280uC prior to use.