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(A) rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) 2D PCoA of SI microbiota mock-treated (blue symbols) or IAV-infected mice (red symbols) at indicated time points post infection. (C) Mean UNIFRAC distances for the comparison of indicated experimental groups. Statistical significance was determined by two-tailed student’s T test. (D) 2D PCoA of SI microbiota mock-treated (blue symbols) or IAV-infected mice (red symbols) at indicated time points post infection. Each symbol represents one mouse. Each symbol type refers to an individual cage. (E) Shannon index, relative abundances of indicated bacterial classes determined by 16S rRNA gene NGS, and relative 16S/18S levels of animals 7 dpi are shown. Each symbol represents one animal. Each symbol type refers to an individual cage. (F) Relative abundance of total bacteria based on qPCR (16S/18S) normalized to median of mock samples is shown for individual mice and indicated time points treated with mock (black symbols) or indicated viruses (red symbols) for for H1N1 7 dpi (n = 8 per group) pH1N1 6 dpi (n = 5 per group). (G) Most abundant OTU on genus level. Median relative abundance (%) is depicted. (PDF 1956 kb)

(A) rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) Most abundant OTU found in baseline, mock or IAV-infected LRT samples 7 dpi on genus level. Median relative abundance (%) is depicted. (C) N-fold changes of 16S/18S DNA levels normalized to mean of mock samples of each day examined. Individual mice and median are depicted (n = 18 per group per time point, two independent experiments, representative data shown). (D) Individual lung titers of mock treated (black circles) and IAV (pH1N1, Neth/602) infected mice (red squares), median titers (pfu/organ) are indicated. Limit of detection (LoD) 50 pfu (n = 5 per group). (E) Relative abundance of total bacteria and Firmicutes based on qPCR (16S/18S) normalized to mean of mock samples is shown for individual mice and indicated time points treated with mock (black symbols) or indicated viruses (red symbols for pH1N1 6 dpi) (n = 5 per group). (F) Bacterial titers of cultivatable URT microbiota 7 dpi grown on indicated agar plates (n = 6 per group). (G) Sequence alignment of Sanger sequencing results of 16S rRNA gene V4 region of Enterobacteriaceae (OTU ID 1111294) colonies. (H) Sequence alignment of Sanger sequencing results of 16S rRNA gene V4 region of Lactobacillaceae (OTU ID 1107027) colonies. (PDF 1691 kb)

(A) Rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) Alpha diversity depicted as shannon index for fecal microbiota of baseline (gray), mock-treated (black) or IAV-infected (red) mice. Individual values per mouse and median are depicted. (C) 2D PCoA plots of fecal microbiota mock-treated (blue) or IAV-infected mice (red) at indicated time points post infection. Each symbol represents one mouse. (D) Most abundant OTU on genus level. Median relative abundance (%) is depicted. (E) Relative abundance (%) of significantly changing OTUs of individual mice and median are depicted (mock red, IAV black). (F) Cultivatable bacterial cfu/fecal pellet of mock-treated (black) or IAV-infected (H5N1 red, H1N1 green) 7 dpi grown under aerobic or anaerobic conditions are depicted (n = 6 per group per time point, two independent experiments). (PDF 1343 kb)

(A) rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) 2D PCoA of SI microbiota mock-treated (blue symbols) or IAV-infected mice (red symbols) at indicated time points post infection. (C) Mean UNIFRAC distances for the comparison of indicated experimental groups. Statistical significance was determined by two-tailed student’s T test. (D) 2D PCoA of SI microbiota mock-treated (blue symbols) or IAV-infected mice (red symbols) at indicated time points post infection. Each symbol represents one mouse. Each symbol type refers to an individual cage. (E) Shannon index, relative abundances of indicated bacterial classes determined by 16S rRNA gene NGS, and relative 16S/18S levels of animals 7 dpi are shown. Each symbol represents one animal. Each symbol type refers to an individual cage. (F) Relative abundance of total bacteria based on qPCR (16S/18S) normalized to median of mock samples is shown for individual mice and indicated time points treated with mock (black symbols) or indicated viruses (red symbols) for for H1N1 7 dpi (n = 8 per group) pH1N1 6 dpi (n = 5 per group). (G) Most abundant OTU on genus level. Median relative abundance (%) is depicted. (PDF 1956 kb)

(A) Rarefection plot: number of sequences is plotted against Chao1 estimator. Error bars indicate SE. (B) Alpha diversity depicted as shannon index for fecal microbiota of baseline (gray), mock-treated (black) or IAV-infected (red) mice. Individual values per mouse and median are depicted. (C) 2D PCoA plots of fecal microbiota mock-treated (blue) or IAV-infected mice (red) at indicated time points post infection. Each symbol represents one mouse. (D) Most abundant OTU on genus level. Median relative abundance (%) is depicted. (E) Relative abundance (%) of significantly changing OTUs of individual mice and median are depicted (mock red, IAV black). (F) Cultivatable bacterial cfu/fecal pellet of mock-treated (black) or IAV-infected (H5N1 red, H1N1 green) 7 dpi grown under aerobic or anaerobic conditions are depicted (n = 6 per group per time point, two independent experiments). (PDF 1343 kb)

phylum-specific qPCR for streptomycin vs. mock-treated mice. Individual relative levels of Bacteroidetes or Firmicutes normalized to 18S and median are depicted. Pooled data from two independent mouse experiments are shown (n = 5 per group). (PDF 717 kb)

mean copy number of minus strand (vRNA) and plus strand (mRNA/cRNA) RNA copies ± SD per organ sample are depicted as determined by specific RT-qPCR; n.d. not detected. (PDF 215 kb)

mean copy number of minus strand (vRNA) and plus strand (mRNA/cRNA) RNA copies ± SD per organ sample are depicted as determined by specific RT-qPCR; n.d. not detected. (PDF 215 kb)