Molecular basis of mammalian transmissibility of avian H1N1 influenza viruses and their pandemic potential

Abstract

North American wild birds are an important reservoir of influenza A viruses, yet the potential of viruses in this reservoir to transmit and cause disease in mammals is not well understood. Our surveillance of avian influenza viruses (AIVs) at Delaware Bay, USA, revealed a group of similar H1N1 AIVs isolated in 2009, some of which were airborne-transmissible in the ferret model without prior adaptation. Comparison of the genomes of these viruses revealed genetic markers of airborne transmissibility in the Polymerase Basic 2 (PB2), PB1, PB1-F2, Polymerase Acidic-X (PA-X), Nonstructural Protein 1 (NS1), and Nuclear Export Protein (NEP) genes. We studied the role of NS1 in airborne transmission and found that NS1 mutants that were not airborne-transmissible caused limited tissue pathology in the upper respiratory tract (URT). Viral maturation was also delayed, evident as strong intranuclear staining and little virus at the mucosa. Our study of this naturally occurring constellation of genetic markers has provided insights into the poorly understood phenomenon of AIV airborne transmissibility by revealing a role for NS1 and characteristics of viral replication in the URT that were associated with airborne transmission. The transmissibility of these viruses further highlights the pandemic potential of AIVs in the wild bird reservoir and the need to maintain surveillance.

Keywords: H1N1; airborne transmission; avian influenza virus; ferret; nonstructural protein 1.

Fig. 1.

Only those viruses containing all of the putative genetic markers for airborne transmission were transmissible via the airborne route in ferrets. A/ruddy turnstone/Delaware/300/2009 (A) and A/ruddy turnstone/New Jersey/AI09_256/2009 (B) contained all genetic markers for airborne transmission and transmitted via AC. (C) A/shorebird/Delaware/558/2009, which lacked all putative genetic markers, transmitted via DC but not via AC. A/shorebird/Delaware/170/2009 (D) and A/ruddy turnstone/New Jersey/AI09_1299/2009 (E), which contained all putative genetic markers with the exception of those in NS1 or NS1 and PB2, respectively, transmitted by DC but not via AC. (F) A/ruddy turnstone/New Jersey/AI09_841/2009 contained putative genetic markers only in PA-X and NS1, and transmitted by DC but not via AC. Each bar represents the viral titer measured in the nasal wash from a single ferret on the indicated DPI. (G) Summary of transmission data showing putative transmission markers in orange. Viruses that transmitted by AC are shaded in orange, while those that did not are shaded in green (n = 2 inoculated, DC, and AC ferrets per virus). I, inoculated.

Fig. S1.

There were no significant differences in the body temperature or weights of ferrets in experiments detailed in Fig. 1. Overall there were no significant differences in body temperatures (A–C and G–I) or weights (D–F and J–L) between groups of ferrets involved in the experiments whose results are shown in Fig. 1. (A and D) A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300). (B and E) A/ruddy turnstone/New Jersey/AI09_256/2009 (H1N1) (NJ256). (C and F) A/shorebird/Delaware/558/2009 (DE558). (G and J) A/shorebird/Delaware/170/2009 (H1N1) (DE170). (H and K) A/ruddy turnstone/New Jersey/AI09_1299/2009 (H1N1) (NJ1299). (I and L) A/ruddy turnstone/New Jersey/AI09_841/2009 (H1N1) (NJ841). Each data point represents the weight or body temperature of a single ferret at each time point [n = 2 inoculated (blue), DC (green), and AC (red) ferrets per virus].

Fig. 2.

Residue 213 in the NS1 of DE300 was critical for the inhibition of the IFN pathway. The NS1 of DE170, which was not airborne-transmissible in ferrets, inhibited the IFN-β promoter significantly more than the NS1 of DE300, which was airborne-transmissible in ferrets. Residue 213 was critical for this difference, as L7S and G227E did not alter the activity of DE300 NS1 but S213P changed the activity of DE300 NS1 to that of DE170 NS1. The mean ± SEM is shown. Values are normalized to the activity of the IFN-β promoter in the absence of NS1 (i.e., IFN-β promoter activity without inhibition). ***P < 0.001.

Fig. S2.

Activity of the polymerase complex of A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300) with NS1 from DE300 or A/shorebird/Delaware/170/2009 (H1N1) (DE170) was similar. In an in vitro polymerase activity assay, the NS1 of A/ruddy turnstone/DE/300/2009 (DE300), which was airborne-transmissible in ferrets, did not enhance the activity of the DE300 polymerase complex compared with the NS1 of A/shorebird/DE/170/2009 (H1N1) (DE170), which was not airborne-transmissible in ferrets. The DE300 polymerase complex showed significantly less activity compared with the polymerase complex of A/California/04/2009 (H1N1) (CA/04) and A/Puerto Rico/8/1934 (H1N1) (PR8). Data points show the ratio of firefly and Renilla luciferase activity. The mean ± SEM is shown. ***P < 0.001.

Fig. 3.

NS1 was critical for AC, but not DC, transmission in ferrets. (A) The rg virus A/ruddy turnstone/Delaware/300/2009 (H1N1) (rgDE300) transmitted to DC and AC ferrets, as per the wild-type DE300 virus. (B) Introduction of S213P into the NS1 of rgDE300 abrogated AC, but not DC, transmission. (C) NS1 of A/gull/Delaware/AI09_438/2009 (H1N1), which contained S213 in conjunction with S7 and E227, transmitted to DC ferrets, but not to AC ferrets. Each bar represents the viral titer measured in the nasal wash from a single ferret on the indicated DPI. (D) Summary of transmission data showing putative transmission markers in orange. Viruses that transmitted by AC are shaded in orange, while those that did not are shaded in green (n = 2 inoculated, DC, and AC ferrets per virus). I, inoculated.

Fig. S3.

There were no significant differences in the body temperature or weights of ferrets in experiments detailed in Fig. 3. Overall, there were no significant differences in body temperatures (A, C, and E) or weights (B, D, and F) between groups of ferrets involved in the experiments whose results are shown in Fig 3. (A and B) The rg virus A/ruddy turnstone/Delaware/300/2009 (H1N1) (rgDE300). (C and D) The rgDE300 containing NS1 S213P (rgDE300 P213). (E and F) A/gull/Delaware/AI09_438/2009 (H1N1) (DE438). Each data point represents the weight or body temperature of a single ferret at each time point [n = 2 inoculated (blue), DC (green), and AC (red) ferrets per virus].

Fig. S4.

Growth kinetics of A/ruddy turnstone/Delaware/300/2009 (H1N1) (DE300), DE300 generated by rg (rgDE300), rgDE300 containing S213P in NS1 (rgDE300 P213), and A/gull/Delaware/AI09_438/2009 (H1N1) (DE438) were similar. Data points show the mean and SEM of two independent experiments (n = 2 in each experiment). TCID₅₀, tissue culture infectious dose, 50%.

Fig. 4.

Virus at the mucosal surfaces of the respiratory epithelium and olfactory neuroepithelium was critical for airborne transmission. More virus staining was observed at the mucosal surfaces of the nasal respiratory epithelium and olfactory neuroepithelium of ferrets 5 DPI with the rg virus A/ruddy turnstone/Delaware/300/2009 (H1N1) (rgDE300) (A) compared with ferrets inoculated with rgDE300 containing P213 in NS1 (rgDE300 P213) (B) or A/gull/Delaware/AI09_438/2009 (H1N1) (DE438) (C). The rgDE300 P213 and DE438 were found predominantly in the nucleus of infected cells, while rgDE300 was found in the cytoplasm and the nucleus. Apoptotic/necrotic cells were numerous in these tissues in ferrets inoculated with rgDE300 (D and G), while they were rare in ferrets inoculated with rgDE300 P213 (E and H) or DE438 (F and I). Sections were stained with anti-H1N1 influenza virus antibody (brown staining) (A–C), with hemotoxin and eosin (D–F), or with caspase 3 (brown staining) and hematoxylin (blue staining) (G–I) (n = 2 ferrets per virus). (Scale bars: 50 μm.)

Fig. S5.

No significant differences were evident in cytokine gene expression in the URTs of inoculated ferrets. Quantitative real-time PCR on RNA isolated from the nasal turbinates (A–C) or tracheas (D–F) of ferrets inoculated with A/ruddy turnstone/Delaware/300/2009 (H1N1) derived by rg (rgDE300), A/gull/Delaware/AI09_438/2009 (H1N1) (DE438), or rgDE300 containing S213P in NS1 (rgDE300 P213) did not reveal any significant differences in the expression of IFN-α (A and D), IFN-β (B and E), or IFN-γ (C and F) genes at 5 DPI (n = 2 ferrets per virus). RQ, relative quantitation.

Fig. S6.

No significant differences were evident in cytokine gene expression in four different lobes of the lungs of inoculated ferrets. Quantitative real-time PCR on RNA isolated from four different lobes of the lungs (shown as black, gray, orange, and red data points) of ferrets inoculated with A/ruddy turnstone/Delaware/300/2009 (H1N1) derived by rg (rgDE300), A/gull/Delaware/AI09_438/2009 (H1N1) (DE438), or rgDE300 containing S213P in NS1 (rgDE300 P213) did not reveal any significant differences in the expression of the following cytokine genes at 5 DPI: IFN-α, IFN-β, and IFN-γ (A–C); TNF-α (D); or IL-2, IL-6, IL-8, and IL-10 (E–H). (E) Note that data were not obtained for IL-2 gene expression from one lobe (n = 2 ferrets per virus). RQ, relative quantitation.

Fig. S7.

Influenza virus gene segments in airborne-transmissible viruses did not appear to have unique origins. Gene segments in airborne-transmissible viruses did not appear to have origins unique from those in non–airborne-transmissible viruses, and they were not more closely related to prior pandemic H1N1 viruses or putative swine precursor viruses to the 2009 pandemic viruses. (A–H) Neighbor-joining trees were constructed using the full nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, matrix (M) and nonstructural (NS) gene segments, respectively. Branches show the percentage of replicate trees in which associated taxa clustered together in the bootstrap test using 1,000 replicates, and evolutionary distances were calculated using the Poisson correction method. The 36 H1N1 avian influenza A viruses (AIVs) isolated in Delaware Bay, USA, from 1990 to 2015, inclusive, as shown in Table S1, are labeled in red in these trees. Viruses that were airborne-transmissible are labeled with orange dots, and viruses that were not airborne-transmissible are labeled with green dots.

Fig. S7.

Influenza virus gene segments in airborne-transmissible viruses did not appear to have unique origins. Gene segments in airborne-transmissible viruses did not appear to have origins unique from those in non–airborne-transmissible viruses, and they were not more closely related to prior pandemic H1N1 viruses or putative swine precursor viruses to the 2009 pandemic viruses. (A–H) Neighbor-joining trees were constructed using the full nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, matrix (M) and nonstructural (NS) gene segments, respectively. Branches show the percentage of replicate trees in which associated taxa clustered together in the bootstrap test using 1,000 replicates, and evolutionary distances were calculated using the Poisson correction method. The 36 H1N1 avian influenza A viruses (AIVs) isolated in Delaware Bay, USA, from 1990 to 2015, inclusive, as shown in Table S1, are labeled in red in these trees. Viruses that were airborne-transmissible are labeled with orange dots, and viruses that were not airborne-transmissible are labeled with green dots.

Fig. S7.

Influenza virus gene segments in airborne-transmissible viruses did not appear to have unique origins. Gene segments in airborne-transmissible viruses did not appear to have origins unique from those in non–airborne-transmissible viruses, and they were not more closely related to prior pandemic H1N1 viruses or putative swine precursor viruses to the 2009 pandemic viruses. (A–H) Neighbor-joining trees were constructed using the full nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, matrix (M) and nonstructural (NS) gene segments, respectively. Branches show the percentage of replicate trees in which associated taxa clustered together in the bootstrap test using 1,000 replicates, and evolutionary distances were calculated using the Poisson correction method. The 36 H1N1 avian influenza A viruses (AIVs) isolated in Delaware Bay, USA, from 1990 to 2015, inclusive, as shown in Table S1, are labeled in red in these trees. Viruses that were airborne-transmissible are labeled with orange dots, and viruses that were not airborne-transmissible are labeled with green dots.

Fig. S7.

Influenza virus gene segments in airborne-transmissible viruses did not appear to have unique origins. Gene segments in airborne-transmissible viruses did not appear to have origins unique from those in non–airborne-transmissible viruses, and they were not more closely related to prior pandemic H1N1 viruses or putative swine precursor viruses to the 2009 pandemic viruses. (A–H) Neighbor-joining trees were constructed using the full nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, matrix (M) and nonstructural (NS) gene segments, respectively. Branches show the percentage of replicate trees in which associated taxa clustered together in the bootstrap test using 1,000 replicates, and evolutionary distances were calculated using the Poisson correction method. The 36 H1N1 avian influenza A viruses (AIVs) isolated in Delaware Bay, USA, from 1990 to 2015, inclusive, as shown in Table S1, are labeled in red in these trees. Viruses that were airborne-transmissible are labeled with orange dots, and viruses that were not airborne-transmissible are labeled with green dots.

Fig. S7.

Influenza virus gene segments in airborne-transmissible viruses did not appear to have unique origins. Gene segments in airborne-transmissible viruses did not appear to have origins unique from those in non–airborne-transmissible viruses, and they were not more closely related to prior pandemic H1N1 viruses or putative swine precursor viruses to the 2009 pandemic viruses. (A–H) Neighbor-joining trees were constructed using the full nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, matrix (M) and nonstructural (NS) gene segments, respectively. Branches show the percentage of replicate trees in which associated taxa clustered together in the bootstrap test using 1,000 replicates, and evolutionary distances were calculated using the Poisson correction method. The 36 H1N1 avian influenza A viruses (AIVs) isolated in Delaware Bay, USA, from 1990 to 2015, inclusive, as shown in Table S1, are labeled in red in these trees. Viruses that were airborne-transmissible are labeled with orange dots, and viruses that were not airborne-transmissible are labeled with green dots.