Cow cells are susceptible to infection with diverse IAV strains.

A). Schematic representation of the anatomical source of immortalised (green) and primary (black) cells used in this study. B) The indicated cells were infected with a variety of viral strains at MOI 0.1 (bMEC) or MOI 0.01 (for the remaining cells) and virus titres measured by plaque assay at 48 hpi. Viruses were subcategorised into lab-adapted, human, swine and avian strains as indicated. Circles represent individual data points from 3 independently performed experiments, except PBMC (n = 1). Lines represent median. Statistical annotations are the result of a 2-two-way ANOVA analysis. Tukey’s multiple comparisons tests were performed against MAC-T (amber asterisks) or bMEC (burgundy asterisks) data sets. C) RNP-reconstitution assays (minireplicons) were performed in MAC-T cells by transfecting pDUAL plasmids encoding segments 1, 2, 3 and 5 from the indicated viruses alongside a firefly luciferase-expressing vRNA-like reporter plasmid. Luminescence levels were measured at 48h post transfection. Data represent mean ± SEM from three independent experiments performed in triplicate.

Cattle Texas is fitter in cow cells than an avian EURL genotype EA-2020-C precursor.

The indicated cow cell systems were infected with 2:6 reassortant viruses with PR8 glycoproteins and internal gene constellations of either AIV07 (blue) or Cattle Texas (yellow) (MOI 0.1 in B, 5000 PFU/explant in C and MOI 0.01 for all other systems). Supernatants were collected at the indicated times post infection and virus titres determined by plaque assay. Data represent mean ± SEM from two-four independent experiments each performed in duplicate. Statistical annotations represent the results of multiple unpaired t-tests performed for each individual time post infection. Cell types were immortalised mammary epithelial (MAC-T; A), primary mammary epithelial (bMEC; B), mammary gland explant (C), immortalised pneumocytes (BAT-II; D), skin fibroblast (E), cardiac fibroblast (F), blood derived macrophage (BDΦ) (G), and 2D enteroids (H).

Increased fitness of Cattle Texas in cow cells correlates with enhanced viral polymerase activity and better NS1-driven inhibition of type I IFN induction.

(A) Schematic representation of AIV07 and Cattle Texas segment origins. Blue and yellow represent European and North American ancestry, respectively. (B) RNP-reconstitution assays (minireplicons) were performed in MAC-T cells by transfecting pDUAL plasmids encoding segments 1, 2, 3 and 5 from AIV07 and Cattle Texas in the indicated combinations alongside a firefly luciferase-expressing vRNA-like reporter plasmid. Luminescence levels were measured at 48h post transfection. Data represent mean ± SEM from three independent experiments each performed in triplicate. Statistical annotations are the result of one-way ANOVA analysis. Multiple comparisons were made against full AIV07 3PNP (blue asterisks) or Cattle Texas 3PNP (yellow asterisks) using a Dunnett’s test (C) MAC-T cells were infected with polymerase-segment reassortment viruses at MOI 0.01. Supernatants were collected at the indicated time points and virus titres measured by plaque assay. Data represent mean ± SEM from three independent experiments each performed in technical duplicate. (D) MAC-T cells were transfected with an IFN-β firefly luciferase-coding reporter alongside AIV07 or Cattle Texas segment 8 plasmids, and poly I:C treated 24h later. Luminescence readings were acquired after a further 24h. Data (left hand panel) represent mean ± SEM from three independent experiments each performed in technical triplicate. Statistical annotations are the result of one-way ANOVA analysis followed by a multiple comparison Dunnett’s test. A western blot (right hand panel) was also performed to detect NS1, using α-Tubulin as a loading control. (E) MAC-T cells were infected with segment 8 reassortant viruses at MOI 0.01. Supernatants were collected at the indicated time points and virus titres measured by plaque assay. For all shown statistical annotations: NS – non significant, *p-value <0.05, **p-value <0.01, ***p-value <0.001 and ****p-value <0.0001.

Cow mammary cells are infectable with viruses of human and avian origin.

(A) MAC-T (MOI 0.01), bMEC (MOI 0.1) cells and mammary explants (5000 PFU/explant) were infected with the indicated PR8 2:6 viruses. Supernatants were collected at the indicated times post infection and virus titres determined by plaque assay. Statistical analysis was performed by a 2-way ANOVA and multiple comparisons were performed against Cattle Texas in specific time points using a Dunnett’s test. (B) Annotated MALDI-TOF spectra of permethylated N-glycans from MAC-T and bMEC cells. Annotations show [M + Na]+ molecular ions. Peak annotation is based on composition, biosynthetic knowledge and MS/MS analysis. (C) Proportions of a-2,3 and a-2,6 linked sialic acid in MAC-T and bMEC N-glycans. (D) MAC-T cells were infected with combinations of Norway 2018 (pH1N1) and/or Belgium H3N1 at MOI 5 for 16h. Cells were fixed, stained with anti-H1 and H3 before quantification was performed by FACS analysis. Data represent mean ± SEM from three independent experiments each performed in technical duplicate. For all shown statistical annotations: ****p-value <0.0001.

Virus strains, abbreviations and uses in this study.

1Laboratory adaptation was defined as viruses with a known history of passage in non-original host animals or cells and/or deliberate adaptation to a new host.

IAV replication kinetics in cow mammary and respiratory cells.

MAC-T (A), bMEC (B), BAT-II (C) and nasal turbinates (D) were infected with the indicated viruses at MOI 0.1 (for bMEC) or 0.01 (for the remaining cells) and supernatants were collected at 0, 24 and 48 hpi. Viral titres were determined by plaque assay. Data represent mean ± SEM from three independent experiments each performed in duplicate.

IAV replication kinetics in cow fibroblasts.

Skin (A), cardiac (B) and brain (C) primary fibroblasts were infected with the indicated viruses at MOI 0.01 and supernatants were collected at 0, 24 and 48 hpi. Viral titres were determined by plaque assay. Data represent mean ± SEM from three independent experiments each performed in duplicate.

IAV replication kinetics in immune cells.

Blood-derived macrophages (BDL) (A) or peripheral blood mononuclear cells (PBMC) (B) were infected with the indicated viruses at MOI 0.01 and supernatants were collected at 0, 24 and 48 hpi. Viral titres were determined by plaque assay. Data represent mean ± SEM from three independent (BDLs) or a single (PBMCs) experiments each performed in duplicate.

Cattle Texas host shutoff activity is similar to that of AIV07.

(A) MAC-T cells were infected at MOI 5 and pulsed with puromycin at the indicated times post infection for 1h. Cells were lysed and new protein synthesis was measured by western blotting for puromycin. NS1 and PB1 were detected to validate infection and α-Tubulin as a loading control. (B) MAC-T cells were transfected with a reporter plasmid constitutively expressing Renilla luciferase under the control of a CMV promoter alongside a dose range of pDUAL plasmids expressing AIV07 or Cattle Texas NS1s. A dilution series of pDUAL plasmid expressing segment 3 of A/Chicken/Rostock/1934 H7N1 (FPV) was used as a positive control. Data represent the mean ± SEM of three independent experiments each performed in triplicate. Statistical annotations are the result of one-way ANOVA analysis performed for each plasmid dosage. Shown multiple comparisons were made between AIV07 and Cattle Texas using a Dunnett’s test at given segment 8 dosages. Immunoblotting was performed to detect increasing levels of PB1 and NS1 expression, using α-Tubulin as loading control. (C) Schematic representation of PA-X and NS1 mutations between AIV07 and Cattle Texas. Open reading frames from frames 1 and 2 are represented by white and mint boxes, respectively. Amino acid differences between AIV07 (blue) and Cattle Texas in PA-X and NS1 are highlighted. (D) MAC-T cells were transfected with a reporter plasmid constitutively expressing Renilla luciferase under the control of a CMV promoter alongside segment 3 pDUAL plasmids (expressing PA-X). Data represent mean ± SEM from three independent experiments each performed in triplicate. Statistical annotations are the result of one-way ANOVA analysis and multiple comparisons were performed using a Dunnett’s test. For (B) and (D) Luminescence readings were measured 48h post transfection.

Cattle Texas is not fitter in chicken cells.

(A) CLEC213 chicken lung epithelial cells were infected with the indicated viruses at MOI 0.01. Supernatants were collected at the indicated times post infection and virus titres were determined by plaque assay. Data represent mean ± SEM from three independent experiments each performed in duplicate. (B) CLEC213 cells were transfected with pDUAL plasmids encoding segments 1-3 and 5 of the indicated combinations of AIV07 and Cattle Texas RNP complexes alongside a firefly luciferase-expressing vRNA-like reporter plasmid. Luminescence levels were measured at 48h post transfection. Data represent mean ± SEM from three independent experiments each performed in duplicate. Statistical annotations are the result of an unpaired t-test. (C) Expression of polymerase components from (B) was examined by western blot. α-Tubulin was used as loading control. (D) CLEC213 cells were transfected with a reporter plasmid constitutively expressing Renilla luciferase under the control of a CMV promoter alongside AIV07 or Cattle Texas segment 8 or empty vector plasmids and luminescence readings measured 48h later. Data represent mean ± SEM from four independent experiments each performed in triplicate. Statistical annotations are the result of one-way ANOVA analysis. (E) CLEC213 cells were transfected with a chicken IFN2 promoter-driven firefly luciferase reporter alongside AIV07 or Cattle Texas segment 8 plasmids, and poly I:C treated 24h later. Luminescence readings were acquired after a further 24h. Data represent mean ± SEM from three independent experiments each performed in duplicate. Statistical annotations are the result of one-way ANOVA analysis. (F) NS1 expression from (A) and (B) was tested by western blot. α-Tubulin was used as loading control. For D and F, multiple comparisons were performed using a Dunnett’s test. For all shown statistical annotations: NS – non significant and ****p-value <0.0001.

Infections in bovine mammary explants.

(A). Schematic representation of the experimental design. (B). Multiple explants were extracted from the mammary glands from 3 donors and infected with 5000 PFU/explant. Supernatants were collected at the indicated times post infection and virus titres determined by plaque assay. Data are from individual explants coloured by donor: donor 1 (n=12), donor 2 (n=12), donor 3 (n=10). (C). Weights of explants at 72 hpi. (D). Explants were homogenised, viral titres measured by plaque assay (left panel) and normalised to individual explant weights (right panel). For C-D, black lines indicate means, circles indicate data for individual explants colour-coded by donor. Statistical annotations are the result of a one-way ANOVA test. Statistical comparisons are given against Cattle Texas using a Dunnet’s test.

Structures of N-glycans from MAC-T cells.

Proposed structures of the N-glycans from MAC-T cells. Structures were based on composition, biosynthetic knowledge and MS/MS analysis. Structures in bold have been confirmed directly by MS/MS. Structures were drawn according to the Symbol Nomenclature for Glycans (SNFG) guidelines.

Structures of N-glycans from bMEC cells.

Proposed structures of the N-glycans from bMEC cells. Structures were based on composition, biosynthetic knowledge and MS/MS analysis. Structures in bold have been confirmed directly by MS/MS. Structures were drawn according to the Symbol Nomenclature for Glycans (SNFG) guidelines.

MALDI-TOF spectra of permethylated N-glycans from MAC-T cells following sialidase digestion.

MALDI-TOF spectra of permethylated N-glycans from MAC-T cells treated with sialidase-S or sialidase-A. Annotations show [M + Na]+ molecular ions and include major structures which were unsialylated (black) and sialylated (red).

MALDI-TOF spectra of permethylated N-glycans from bMEC cells following sialidase digestion.

MALDI-TOF spectra of permethylated N-glycans from MAC-T cells treated with sialidase-S or sialidase-A. Annotations show [M + Na]+ molecular ions and include major structures which were unsialylated (black) and sialylated (red).

Amino acid differences between the internal gene products of avian precursor AIV07 isolate and bovine isolate B3.13.

Complete genome sequences were downloaded from GISAID (EPI_ISL_9012457 and EPI_ISL_19014384 respectively), aligned in SSEv1.4 sequence editing software and annotated manually.