Innate immune control of influenza virus interspecies adaptation

Abstract

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigated whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We found that IFITM3-deficient mice and human cells could be infected with low doses of avian influenza viruses that failed to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious viral dose threshold. Remarkably, influenza viruses passaged through Ifitm3^-/- mice exhibited enhanced host adaptation, a result that was distinct from passaging in mice deficient for interferon signaling, which caused virus attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses.

Fig. 1.. IFITM3 deficiency lowers the minimum infectious dose threshold for avian influenza viruses in vivo.

WT and Ifitm3^−/− mice were intranasally infected with (a, b) 1, 10, or 50 TCID50 of H5N1 avian influenza strain (2 independent experiments for doses 1 and 10 (n=10 mice) and 1 experiment for dose of 50 (n=5 mice)) or with (c, d) 1 or 10 TCID50 of H7N3 avian influenza strain (n=5 mice). a, c Viral titers from lung homogenates at day 3 post infection. b, d ELISA quantification of IL-6 levels in lung homogenates at day 3 post infection. a-d Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between WT and Ifitm3^−/− mice for each dose are shown.

Fig. 2.. IFITM3 limits zoonotic influenza virus infection of human cells.

(a) Schematic of in vitro infection with potentially zoonotic influenza viruses and representative example from infected A549 human lung cells. (b) The indicated A549 cells or THP-1 differentiated macrophages were treated +/− IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. Percent infection was determined by flow cytometry and normalized to respective shControl or WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between shControl versus shIFITM3 and WT versus IFITM3^−/− are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). (c, d) Western blots of cell lysates at 18 hours +/− IFNβ treatment. Note that commercial IFITM3 antibodies weakly detect IFITM2 in addition to IFITM3. (e) The indicated A549 cells were infected with indicated viruses at a range of 0.001 to 10 MOI for 24 hours. Percent infection was determined by flow cytometry. Data are representative of 3 independent experiments each performed in triplicate (n=9). Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between control and knockdown cells are shown at each dose.

Fig. 3.. Influenza virus adapts to a new species more readily in the absence of IFITM3.

(a) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. (b) Schematic of WT mouse challenge with parental or passaged viruses. (c-h and j-l) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Ifitm3^−/− mice and compared to the parent virus (passage 0). (c, e) Viral titers from lung homogenates taken at day 7 (c represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test, (d, f) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection (d represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. (g, j) Viral titers from lung homogenates taken at day 7 (g) or day 6 (j) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. (h, k) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 (h) or day 6 (k) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. (i, m) Mutations found in the segments of A/California/04/2009 (H1N1) after serial passage through WT or Ifitm3^−/− mice. (I) Weight loss. Skull and crossbones indicate humane euthanasia of all animals infected with KO passage 10. Error bars represent SD of the mean, comparisons were made using the Mann-Whitney test (* P = 0.0022, ** P < 0.0001).