Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death

Figure 1. Flowchart showing all the experimental sets. C-MRI, cardiac magnetic resonance imaging; dpi, days post-infection; ECG, electrocardiogram; FA, formaldehyde treatment; hpi, hours post-infection; LV, left ventricle; n, number of inoculated animals; N, total number of animals in each experimental set; n, number of animals in each group; PFU, plaque-forming units; RV, right ventricle.

Figure 2. Premature death upon influenza A virus (IAV) infection correlates with high levels of infectious particles, active viral replication, and the presence of spliced viral NEP mRNA in the heart. BALB/c AnNHsd mice (n = 4) were intranasally infected with 106 pfu of the indicated viruses. (A) Body weights represented as percentage of body weight at inoculation (time 0). Red numbers denote body weight of animals with premature death. The experiment was performed twice and the results were similar. (B) BALB/c AnNHsd mice (n = 5) were intranasally infected with 103 pfu with the indicated viruses. Prevalence of IAV in the heart of infected animals shown as percentage of total animals. (C) Correlation of heart viral titres in each IAV-infected mouse with lung viral titres at different days post-infection (dpi). NA, not applicable, all values below detection limit in the heart. (D) Scheme of influenza virus spliced NEP mRNA and NS1 mRNA collinear to full-length NS segment. Red and green arrows indicate localization of primers used in PCR to detect NEP mRNA. (E) Sequence of the spliced NEP mRNA sequence (grey) within the full-length viral NS segment sequence. Red and green rectangles…

Figure 3. Active replication of influenza A virus in cardiac tissue. PAmut virus replicates faster than attenuated PB2mut virus in HL-1 cells. (A) Detection of viral nucleoprotein (NP) by confocal microscopy of immunofluorescences in heart tissue from control animals (MOCK; upper panels, a–d), PAmut virus- (middle panels, e–i) or PB2mut virus- (lower panels, j–n) infected animals. The boxed areas in merged images (h and m) are shown enlarged in the insets (i and n). (B) 3D reconstructions of cytoplasmic (left panel) or nuclear (right panel) NP viral protein in PAmut-infected heart tissue (see Supplementary material online, Videos S1 and S2). Magenta, auto-fluorescence in cardiomyocytes; NP viral protein is showed in white for better visualization in 3D reconstructions. (C) Quantification of NP signal in infected heart tissues at 2-day post-infection (dpi). A minimum of 27 images from two different hearts have been quantified for each group. *P < 0.05 by Student’s t-test. (D) Detection of viral NP in endothelial cells surrounding a heart blood vessel from animals infected with PAmut virus. (E) Viral replication kinetics of control CAL, PAmut, or PB2mut virus in cultured HL-1-infected cells…

Figure 4. Histopathology and tissue changes in infected mice. (A) Representative heart samples of H&E staining in the three groups of infected mice. (B) Representative heart samples of Masson Trichrome staining from the three groups of infected mice. (C) Fibrosis quantification in heart samples obtained 4 days after infection (n = 3). (D–G) mRNA quantification and statistical comparisons of lysil oxidase (LOX) (D), BNP (E), IL-1ß (F), and IL-6 (G) among Mock (n = 4), PA mut (n = 6) and PB2mut (n = 7) groups of infected mice. Significance was determined by two-way ANOVA (*P < 0.05; **P < 0.01). BNP, brain natriuretic peptide.

Figure 5. ECG alterations in mice infected with influenza virus. (A) Randomly selected tracings from control, PB2mut, and PAmut mice at Days 0 and 2 after virus inoculation. The tracings show overt prolongation of RR intervals (slower heart rate) and PR intervals in PAmut-infected mice at Day 2. Blue asterisks indicate R waves. (B–E) Surface ECG-based quantification of cardiac rhythm and conduction alterations in virus-infected animals and control mice. *, #, †, and ‡ show P < 0.05 for PAmut vs. PAmut Day0 (*), PB2mut vs. PB2mut Day0 (#), PAmut vs. controls at that time point (†), and PB2mut vs. controls at that time point (‡). Animals (n = 10) were intranasally infected with 106 pfu of PB2mut, or PAmut recombinant virus or were Mock-infected, and were sequentially euthanized for collection of samples . Number of animals for comparisons: Days 0, 1: n = 10 (PB2 and PA) and n = 9 (Mock. The 10th animal was excluded due to poor signal quality); Day 2: n = 7 per group; Day 3: n = 5 per group; and Day 4: n = 5 (PB2, Mock) and n = 4 (PA. The fifth animal died on Day 3). Significance was determined by two-way repeated-measures ANOVA with Bonferroni post hoc test (*P < 0.05; **P < 0.01). (F) ATP…

Figure 6. Active replication of influenza A virus in Purkinje cells. (A) Detection of the NP viral protein using immunofluorescence confocal microscopy in heart tissue from Cx40eGFP MOCK-infected mice (upper panels, a–c) and animals infected with 106 pfu of either PAmut virus (middle panels, d–f) or PB2mut virus (lower panels, g–i). Right panels (j–k) show 3D reconstructions of the NP viral protein in Purkinje cells of PAmut- (j) or PB2mut-infected hearts (k) from Cx40eGFP animals (see also Supplementary material online, Videos S3 and S4). Blue, nuclear staining (DAPI); green, GFP; red, laminin; white, NP viral protein staining. (B and C) H&E staining (B) and Masson Trichrome staining (C) of representative heart samples from Cx40eGFP-infected mice.

Figure 7. Influenza A virus replication and electrophysiological phenotype of infected hiPSC-CMs. hiPSC-CMs were Mock-infected or infected with the indicated virus (3 moi) Samples were used to detect the indicated proteins by western blot at the specified hours post-infection (hpi). (A) Viral PB1 protein. (C) Mitochondrial protease YMEL1. (E) Kir2.1 and NaV1.5 ion channels and Connexin43. GAPDH or α-actinin were used as loading control and healthy human ventricle tissue (H—ventricle) was loaded as human ion channels and Connexin43 control. MW denotes molecular weight markers. The experiment was performed in triplicates (duplicates for YMEL1); one representative sample is shown. Mock data are shown as 100% in one dot. B, D, F, G, and H indicate protein quantification and statistically significant analysis (*P < 0.05, **P < 0.01, ***P < 0.001 by the Student’s t-test). moi, multiplicity of infection.