In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity

Abstract

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

FIG. 1.

Attachment of viruses with altered receptor specificity to tissues of the ferret upper and lower respiratory tracts. The attachment pattern of viruses NL03 (H3N2) and IND05 (H5N1) and the five mutant viruses IND05-HA_N182K, IND05-HA_Q222L, IND05-HA_G224S, IND05-HA_Q222L,G224S, and IND05-HA_{N182K,Q222L,G224S} to ferret nasal turbinates, trachea, bronchus, bronchiole, and alveoli was determined. Virus attachment is shown in red. The panels were chosen to reflect the attachment pattern in the whole tissue section as much as possible, but small differences between the single panels and overall view may exist.

FIG. 2.

Attachment of viruses with altered receptor specificity to tissues of the human upper and lower respiratory tract. The attachment pattern of viruses NL03 (H3N2) and IND05 (H5N1) and the three mutant viruses IND05-HA_N182K, IND05-HA_Q222L,G224S, and IND05-HA_{N182K,Q222L,G224S} to human nasal turbinates, trachea, bronchus, bronchiole, and alveoli was determined. Virus attachment is shown in red. The panels were chosen to reflect the attachment pattern in the whole tissue section as much as possible, but small differences between the single panels and overall view may exist.

FIG. 3.

Replication kinetics of viruses with IND05-HA with altered receptor specificity and different NA genes in MDCK (A to D) and A549 (E to H) cells. Cells were inoculated at an MOI of 0.01 TCID₅₀ of virus/cell with the IND05-HA (•), IND05-HA_N182K (▪), IND05-HA_Q222L,G224S (▴), or IND05-HA_{N182K,Q222L,G224S} (⧫) in combination with NA of influenza virus PR8 (A and E), NA of influenza virus IND05 (B and F), NA of influenza virus IND05 with a stalk insertion (NA_stalk) (C and G), or NA of influenza virus NL07 (H1N1) (D and H). Remaining gene segments originated from influenza virus PR8. Supernatants were harvested at 6, 12, 24, and 48 h after inoculation and titrated by endpoint dilution in MDCK cells. Geometric mean titers were calculated from two independent experiments; error bars indicate the standard deviation.