Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses

Abstract

Although H5N1 influenza viruses have been responsible for hundreds of human infections, these avian influenza viruses have not fully adapted to the human host. The lack of sustained transmission in humans may be due, in part, to their avian-like receptor preference. Here, we have introduced receptor binding domain mutations within the hemagglutinin (HA) gene of two H5N1 viruses and evaluated changes in receptor binding specificity by glycan microarray analysis. The impact of these mutations on replication efficiency was assessed in vitro and in vivo. Although certain mutations switched the receptor binding preference of the H5 HA, the rescued mutant viruses displayed reduced replication in vitro and delayed peak virus shedding in ferrets. An improvement in transmission efficiency was not observed with any of the mutants compared to the parental viruses, indicating that alternative molecular changes are required for H5N1 viruses to fully adapt to humans and to acquire pandemic capability.

Fig. 1

Comparison of parental and mutant H5N1 receptor binding preference using a glycan microarray. Glycan specificity was assessed for H5N1 viruses; rHK486 (A), rHK486-LS (B), rHK486-DSLS (C), rHK486-GDSLS (D), rVN1203 (E), rVN1203-RLS (F) and rVN1203-DSLS (G) using glycan microarrays. Viruses, rHK486, rHK486-LS, rHK486-DSLS, rVN1203 and rVN1203-RLS were assessed using 256 HA units while rHK486-GDSLS was assessed using 128 HA units and VN1203-DSLS was assessed using 309 HA units. Due to different imprinting between the chips used for these glycan microarray studies, glycans # 10, 11, 26, 27, 42–45, 48, 49, 60–62, 79–81, 84, and 99 are absent for VN1203 and VN1203-DSLS. Colored bars highlight glycans that contain α2–3 SA (blue) and α2–6 SA (red), α2–6/α2–3 mixed SA (purple), N-glycolyl SA (green), α2–8 SA (brown), β2–6 and 9-O-acetyl SA, and non-SA (gray). Error bars reflect the standard error in the signal for six independent replicates on the array. Structures of each of the numbered glycans are listed in Table 2.

Fig. 2

Replication of parental and mutant H5N1 viruses in Calu-3 cells. Calu-3 cells were grown to confluency on transwell inserts. Cells were infected with wild-type or mutant H5N1 viruses at an MOI of 0.01 for 1 h at 37°C. Unbound virus was removed by washing the cells 3 times and infected cells were cultured in DMEM medium supplemented with 0.3% bovine serum albumin. Calu-3 cells and virus were cultured in the presence (not shown) or absence of trypsin (1 μg/ml; Sigma, St. Louis, MO). Apical supernatants were collected at the indicated times and virus content was determined in a standard plaque assay using MDCK cells. The values shown represent the mean virus titer of fluids from three replicate infected cultures. A. HK486 parental and mutant viruses are shown. B. VN1203 parental and mutant viruses are shown. Error bars represent the standard deviation of three independent wells.

Fig. 3

Replication of parental and mutant H5N1 viruses in ferrets. At least 3 ferrets each were inoculated i.n. with a 10⁶ EID₅₀ or PFU dose of virus. Virus titers in nasal washes collected every other day for 9 days were measured in eggs. A. Mean titers for HK486 parental (solid bar) and mutant (dotted bar) viruses are shown. B. Mean titers for VN1203 parental (solid bar) and mutant (dotted bar) viruses are shown. The limit of detection was 10^1.5 EID₅₀/ml and is indicated by a dotted line.