Hemagglutination test
Liver samples collected from three infected rabbits were frozen and stored at −70°C. Liver samples were homogenized (20% in phosphate-buffered saline [PBS]), frozen at –70°C, and thawed twice. The hemagglutination test (Hu et al., 2016) was carried out in U-shaped microtiter plates containing 50 μL of PBS (pH 6.5). Fifty-microliter suspensions of homogenized liver samples were two-fold serially diluted and placed in U-shaped plates; they were then further incubated with an equal volume of 1% human O erythrocytes at 4°C, 25°C, or 37°C. The hemagglutination was visually determined 30 min later. RHDV isolate WF/China/2007 (GenBank accession number: FJ794180) was used as a positive control.