A198V HA substitution increases receptor binding avidity of F/98
virus, and does not prevent antibody binding physically
HI or MN titers can be decreased by the reduction of antibody binding to
virus or/and increasing receptor binding avidity (Hensley et al., 2009;
Li et al., 2013b). To clear the molecular mechanisms of A198V mutation
for escape from neutralizing-antibodies, the interactions between virus
and receptors on red blood cell surface and antibody binding ELISA were
performed, respectively. The results showed that the virus
rF/HAA198V bound to chicken erythrocytes treated with
32-fold higher α2-3,6,8 neuraminidase concentrations than the F/98
strain. Compared to the virus F/98, rF/HAK131R,
rF/HAS145N, rF/HAG181E, or rF/HA348
possessing HA A198, the viruses rF/HA349, rF/HA389, rF/HA489, or rF/HA47
carrying HA V198 bound to chicken erythrocytes treated with at least
4-fold higher α2-3,6,8 neuraminidase concentrations (Figure 3A),
indicating A198V mutation increased receptor binding avidity of F/98
virus significantly. Antibody binding ELISA confirmed that serum
generated against the F/98 virus bound similarly to either F/98 virus or
rF/HAA198V virus (Figure 3B); serum generated against
the rF/HAA198V virus also bound similarly to either F/98
virus or rF/HAA198V virus (Figure 3C), which suggested
that HA A198V substitution did not cause a significant antigenic change.
Taken together, these data indicated that the A198V mutation promoted
escape from pAb response by increasing viral receptor binding avidity,
but not by preventing antibody binding physically.
HA A198V substitutionelute
from cells at a slower rate
The sialic acid receptor is the target receptor shared with HA and
neuraminidase (NA). We postulated that the residue substitution with
high receptor binding avidity at the position 198 located in the sialic
acid binding domain of HA might affect the viral NA activity. The NA
activity assay was performed, and the result showed that the NA activity
of the rF/HAA198V virus was more strong than that of the
F/98 virus (P <0.05) (Figure 4A).
To further evaluate the effect of the A198V HA mutation on the
functional balances of HA and NA, we measured the elution of virus from
chicken red blood cells (RBCs). rF/HAA198V virus bound
tightly to RBCs as indicated by slow blood elution within 3 h. However,
3 h later, the elution F/98 virus from RBCs was at a similar rate to
that of rF/HAA198V virus with a strong positive
correlation (r =0.9944, R2=0.9887,P <0.01) (Figure 4B).
The HA A198V resulted in a slower release of virus from the RBCs, and we
speculated that the strong receptor binding avidity could affect the
viral release from cells. To investigate whether the
rF/HAA198V virus impacted on the viral infection and
release from cell surface, we detected the amounts of viruses by
detecting M genes with real-time reverse transcription (RT)-PCR assay in
MDCK cells. The results showed that the A198V mutation had no influence
on the viral infection (Figure 4C), and the F/98 virus released from
MDCK cells surface faster than the rF/HAA198V virus
(P <0.01) (Figure 4D). These results indicated that although
A198V mutation enhanced NA activity, the strong receptor binding avidity
was not conducive to the virus release from the cell surface in the
early stages of H9N2 virus infection.