Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells

Abstract

Influenza A viruses of the H9N2 subtype are endemic in poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans. While some avian H9N2 viruses have glutamine (Q) at amino acid position 226 of the hemagglutinin (HA) receptor-binding site, an increasing number of isolates have leucine (L) at this position, which has been associated with the establishment of stable lineages of the H2 and H3 subtypes of viruses in humans. Little is known about the importance of this molecular trait in the infection of H9N2 viruses in humans. We show here that during the course of a single cycle of infection in human airway epithelial (HAE) cells cultured in vitro, the L-226-containing H9N2 viruses displayed human virus-like cell tropisms (preferentially infecting nonciliated cells) different from the tropisms showed by Q-226-containing H9N2 isolates (which infect both ciliated and nonciliated cells at ratios of 1:1 to 3:2) or other waterfowl viruses (which preferentially infect ciliated cells). During multiple cycles of replication in HAE cultures, L-226-containing H9N2 isolates grew consistently more efficiently and reached approximately 100-fold-higher peak titers than those containing Q-226, although peak titers were significantly lower than those induced by human H3N2 viruses. Our results suggest that the variation in residue 226 in the HA affects both cell tropism and replication of H9N2 viruses in HAE cells and may have implications for the abilities of these viruses to infect humans.

FIG. 1.

Identification of cell types in fully differentiated HAE cultures. HAE cultures were stained for cellular markers of different cell types as described in Materials and Methods. (A) Ciliated-cell marker β-tubulin (gray) and goblet-cell marker Muc5AC (red). (B) Ciliated-cell marker (gray) and Clara cell marker Clara cell secretory protein (red).

FIG. 2.

Expression of SAα2,3Gal and SAα2,6Gal receptors on HAE cells. HAE cultures were stained for the presence of cilia and further probed with specific lectins to show the presence of SAα2,3 receptors (A) and SAα2,6 receptors (B). Cilia, gray; receptors, red. The proportions of ciliated and nonciliated cells expressing SAα2,3Gal or SAα2,6Gal receptors were determined and are shown in panel C. Data show the results from three different lots of HAE cells.

FIG. 3.

Cell tropisms of prototypic H9N2 influenza viruses in HAE cells. HAE cultures were infected with viruses at an MOI of 1.0 and fixed at 7 h p.i. The cilia (gray) and viral antigen (red) were visualized by double immunostaining. H9N2 viruses with L-226 are more efficient at infecting nonciliated cells (A and B), and those with Q-226 display dual cell tropisms for both ciliated and nonciliated cells (C and D). A human H3N2 virus with a preference for nonciliated cells (E) and a mallard H7N3 virus with a preference for ciliated cells (F) were included as controls. (G) Graph representing the proportions of ciliated versus nonciliated cells infected by H9N2 viruses during a single round of replication (7 h p.i.). Each bar represents the average for two independent experiments.

FIG. 4.

Receptor specificities of prototypic H9N2 viruses measured with resialylated CRBCs. Native CRBCs were desialylated with Vibrio cholerae neuraminidase and resialylated with α2,3- or α2,6-sialyltransferase in the presence of CMP-SA. The titers of each virus were measured with HA assays using resialylated CRBCs and native CRBCs.

FIG. 5.

Effect of mutations at the HA receptor-binding site on the cell tropisms of H9N2 viruses in HAE cells. HAE cells were infected and stained as described in Materials and Methods. (A) mWF10 (RGWF10 with an L226Q mutation in the HA) infected both ciliated and nonciliated cells. (B) mQa88 (RGQa88 with a Q226L mutation in the HA) targeted predominantly nonciliated cells. (C) dmWF10 (RGWF10 with H/E-to-N/A mutations at HA positions 183 and 190) displayed a cell tropism similar to that of RGWF10 as shown in Fig. 3. (D) tmQa88, with additional H/E-to-N/A mutations at HA positions 183 and 190 and L-226, retained the preference for nonciliated cells. (E) Graph representing the proportions of ciliated versus nonciliated cells infected by the mutant H9N2 viruses during a single round of replication (7 h p.i.). Each bar represents the average for two independent experiments.

FIG. 6.

Effect of amino acid position 226 in the HA on the spread of H9N2 viruses in HAE cells. The cells were inoculated with viruses at an MOI of 0.02, fixed at 24 h p.i., and stained for cilia (gray) and viral antigen (red). RGWF10 (A) spread to some extent, as indicated by the foci consisting of continuous infected cells, and mWF10 (B) and RGQa88 (C) showed very limited spread, whereas mQa88 spread in a manner similar to that of RGWF10. (E and F) Magnified versions of RGWF10- and mQa88-infected cultures showing the more evident spread among the nonciliated cells.

FIG. 7.

Growth kinetics of H9N2 viruses in HAE cells. (A) HAE cultures were inoculated via the apical side with each virus at an MOI of 0.2. The progeny viruses released into the apical side were collected at the indicated time points and titrated in primary CEK cells (avian viruses) or MDCK cells (human viruses) by performing a TCID₅₀ assay (upper panel). Each bar represents the average for two independent experiments run with duplicate HAE cultures. One set of samples from the duplicates in each experiment was selected and titrated with an immune plaque assay. The titers were averaged and are shown in the lower panel. The 1-h time point represents the initial inoculum that remained after washing. The dashed lines indicate the limit of detection in the immune plaque assay. (B) Growth of H9N2 viruses RGQa88 and mQa88 in CEK cells is significantly better than that in MDCK cells, whereas that of RGWF10 and mWF10 as well as human H3N2 viruses is similar in these two cell types. Data show the averages for two independent titrations with the same stock of each virus. (C) At the end of the sampling (60 h p.i. for avian viruses and 48 h p.i. for human viruses) described for panel A, the cultures were fixed and stained for cilia (gray) and distribution of viral antigen (red) to show the relative cytopathic effects induced by infection with different influenza viruses.