Identification, characterization, and natural selection of mutations driving airborne transmission of A/H5N1 virus

Abstract

Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.

Figure 1. Summary of results to determine a minimal set of substitutions required for airborne A/H5N1 virus transmission between ferrets

(A) Starting with virus V1 that represents the airborne transmitted virus with the lowest number of amino acid substitutions (9) as compared to wildtype A/H5N1 (Herfst et al., 2012), the requirement of substitutions in the PB1 and NP segments was investigated. Recombinant viruses V1–V5 are shown with 8 gene segments, in which colored squares represent the presence or absence of indicated substitutions. The proportion of animals positive by virus isolation is indicated with ‘v’. (B) All substitutions of virus V5 were omitted individually and transmission was again tested in ferrets for viruses V6–V12. Virus shedding patterns in donor and recipient ferrets for V1–V12 are provided in Figure S1.

Figure 2. Single nucleotide variations that emerged upon repeated passaging of influenza virus A/H5N1_{HA Q222L, G224S PB2 E627K} in ferrets, as detected by deep sequencing

Passage number is indicated on the x-axis. Left and right y-axes indicated proportion of the mutant among all reads (white bars) and the sequencing depth in number of reads (black lines) respectively. The grey areas indicate that no virus sequences were amplified from these samples. S: virus stock used to inoculate the first ferret (P1); NT: nasal turbinate; NS: nasal swab; NW: nasal wash.

Figure 3. Receptor binding properties of wildtype and mutant A/H5N1 HA proteins

(A) Attachment patterns of viruses expressing wildtype or mutant H5 HA, to tissue sections of ferret and human nasal turbinates. Red color represents binding of influenza viruses. Images were chosen to reflect representative attachment patterns. (B) Direct binding of viruses expressing wildtype or mutant H5 HA to fetuin containing either α2,3-SA (red bars) or α2,6-SA (blue bars). A/dk/Bav/1/77 and A/HK/1/68 represent avian and human prototype strains A/duck/Bavaria/1/1977 (A/H1N1) and A/HongKong/1/1968 (A/H3N2) respectively. Error bars represent the standard deviation of the mean values (n=2). See also Figure S2. (C) Agglutination of TRBCs by viruses expressing wildtype or mutant H5 HA. TRBCs were left untreated, stripped from SA using Vibrio cholerae neuraminidase (VCNA), or modified to contain either α2,3-SA or α2,6-SA. Numbers show the hemagglutination titers determined with the indicated TRBCs using various mutant viruses. A/Netherlands/213/03 served as a typical human virus with α2,6 SA preference.

Figure 4. Analysis of pH threshold for fusion and thermostability of wildtype and mutant A/H5N1 HA proteins

(A) Syncytium formation in MDCK cells upon expression of wildtype or mutant HA proteins after exposure to different pH. The red line marks the range of pH values at which fusion was detected microscopically. HA of A/HongKong/156/97 (H5N1) and A/Netherlands/213/03 (H3N2) were included as typical avian and human control viruses. (B–D) Quantification of fusion as measured by the expression of a CAT reporter gene in a cell content mixing assay for: (B) influenza virus A/Indonesia/5/05 HA _wildtype (solid line) and HA_H103Y (dotted line), (C) HA _{T156A, Q222L, G224S} (solid line) and HA_{H103Y, T156A, Q222L, G224S} (dotted line), and (D) A/HongKong/156/97 (A/H5N1, solid line) and A/Netherlands/213/03 (A/H3N2, dotted line). Arrows indicate the pH threshold value at which syncytia were detected visually in (A). (E) HA protein stability as measured by the ability of viruses to agglutinate TRBCs after incubation at indicated temperatures for 30 minutes. Colors indicate the hemagglutination titers upon treatment at various temperatures for 30 minutes as shown in the legend.

Figure 5. Effect of H99Y in PB1 and E627K in PB2 on polymerase activity and virus replication

(A) Minigenome reporter assay. Plasmids encoding PB2, PB1, PA, and NP were cotransfected with a vRNA reporter encoding firefly luciferase. Luminescence of firefly luciferase was standardized using a plasmid constitutively expressing Renilla luciferase. Results are calculated as relative light units (firefly luciferase/Renilla luciferase), and plotted as fold increase over wildtype. Error bars indicate the standard deviation from the average of two independent experiments. (B) Primer extension assay. MDCK cells were inoculated at an MOI of 1 with wildtype or mutant A/Indonesia/5/05 viruses. At 3 hours post inoculation, cells were lysed and viral RNA levels were determined by primer extension analysis using primers specific for mRNA, cRNA or vRNA of PB1. Determination of the 5S RNA levels served as an internal loading control. (C) Plaque assay. MDCK cells were inoculated with A/Indonesia/5/05 viruses containing the indicated substitutions. After 48 hours, plaque formation was visualized by influenza NP-specific staining. Digital images were analyzed using ImageQuant TL software to determine plaque size. The surface of individual plaques in pixels is plotted using boxplots indicating the median value and quartiles. Differences between individual groups were significant (p<0.01) in Student’s t-test. In all panels, the mutations in PB1 and PB2 are indicated, with dashes representing the wildtype sequences.