FIG 4.
Mutation of the conserved N1 head glycosylation sites causes slight viral replication defects in eggs. (A) Scatterplots of the HAU titers and NA activities that were measured in the allantoic fluid harvested from eggs infected with recombinant PR8 viruses carrying N1-BR18 with the indicated glycosylation site mutations. The viruses were rescued by reverse genetics and passaged twice in eggs. The data points from individual eggs following the second passage are shown together with the medians (line). P values (95% confidence interval [CI]) were determined with respect to the WT values by a one-way analysis of variance (ANOVA). (B) Representative NA and HA immunoblots of the recombinant PR8N1-BR18 viruses with the indicated mutations in the N1 head glycosylation sites. The allantoic fluid from the second passage was pooled, and the virions were isolated by centrifugation and adjusted to equal total protein concentrations prior to being resolved by NR and reducing (RD) SDS-PAGE. NA activity (below the immunoblots) in the virions was also measured using equal total protein amounts.