Table 2. Residues 55 and 269 in VP1 protein play very important role in hemagglutination of RBCs by Viral Like Particles (VLPs).

Viral variant	Minimum HA VLP concentration, ng/ml
WT1	0.08
55F	>200,000
WT2 (Mad-1)	6.25
269F	50,000

Hemagglutination was conducted as described in Materials and Methods using serial dilutions of VLPs starting from 200 µg/ml. VLPs were added to type O RBC and incubated at 4°C for 3 hours. Agglutination is visualized by the lack of a round pellet formed by the settling of RBCs out of suspension. F55 is a VP1 variant with phenylalanine at the position 55 (AAT09831), F269 is a VP1 variant with phenylalanine at the position 269 (BAE0011). WT (AAQ88264) and Mad-1 (P03089) are VP1 variants with leucine and serine at positions 55 and 269 respectively.