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Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

Baquero-Perez B, Kuchipudi SV, Ho J, Sebastian S, Puranik A, Howard W, Brookes SM, Brown IH, Chang KC - J. Virol. (2014)

Bottom Line: Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines.Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells.Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Science, University of Nottingham, Loughborough, Leicestershire, United Kingdom.

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Influenza virus-infected chicken and duck myotubes show extensive cytopathic damage. Chicken and duck myotubes were infected with LPAI H2N3 virus at a MOI of 1.0 for 24 h. (A and E) Uninfected control chicken and duck myotubes show extensive expression of the muscle-specific intermediate filament desmin (brown). (B and F) Cytopathic damage of chicken and duck myotubes is seen as widespread rounding and detachment of cells (arrows). (C, C′, G, and G′) Whole chicken and duck myotubes appear to have degenerated into numerous small membrane-lined blebs (apoptotic bodies) (myotube boundaries are delineated). (D, D′, H, and H′) Commonly, sections of chicken and duck myotubes contained aggregates of apoptotic blebs with concentrated viral NP, as evident by immunocytochemical NP detection. Morphologically intact myonuclei were found in the sarcoplasm of the duck myotube (H′, arrows). (I and K) Phase-contrast microscopy of mock-infected chicken (I) and duck (K) myotube cultures show typical myotubes. (J and L) In contrast, similar myotube detachment and rounding (arrows) were evident in HPAI H5N1 50-92 virus-infected (MOI of 1.0) chicken (J) and duck (L) myotubes at 24 h p.i. (M to P) Translocation of PS to the outer membrane was detected (green fluorescence) by annexin V-EGFP binding to chicken and duck muscle cells (myotubes and myoblasts) infected with LPAI H2N3 virus (MOI of 1.0) for 24 h. Infected chicken (M) and duck (N) myotubes were extensively positive for PS translocation; some cells had lost membrane integrity, as evident by the nuclear uptake of PI (red fluorescence). Myonuclei labeled with PI displayed chromatin condensation and/or fragmentation in chicken and duck myotubes, whose boundaries are outlined (M and N, respectively). A morphologically intact nucleus with condensed and/or fragmented nuclear contents is highlighted (M, arrows). Chromatin condensation and fragmentation were also seen in chicken (O) and duck (P) myoblasts. (Q) Chicken and duck myotube cultures infected with LPAI H2N3 virus at a MOI of 1.0 significantly activated the effector caspases 3 and 7 at 24 h and 48 h p.i. (*, P < 0.05; **, P < 0.01 [determined by an unpaired t test]) (RLU/s, relative light units per second). (Q′) Results are also represented as fold changes. There was, however, no significant difference in the activation of caspases 3 and 7 between the two avian species. Data points are the means of data from three wells from a 96-well plate, with error bars indicating standard deviations.
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Figure 4: Influenza virus-infected chicken and duck myotubes show extensive cytopathic damage. Chicken and duck myotubes were infected with LPAI H2N3 virus at a MOI of 1.0 for 24 h. (A and E) Uninfected control chicken and duck myotubes show extensive expression of the muscle-specific intermediate filament desmin (brown). (B and F) Cytopathic damage of chicken and duck myotubes is seen as widespread rounding and detachment of cells (arrows). (C, C′, G, and G′) Whole chicken and duck myotubes appear to have degenerated into numerous small membrane-lined blebs (apoptotic bodies) (myotube boundaries are delineated). (D, D′, H, and H′) Commonly, sections of chicken and duck myotubes contained aggregates of apoptotic blebs with concentrated viral NP, as evident by immunocytochemical NP detection. Morphologically intact myonuclei were found in the sarcoplasm of the duck myotube (H′, arrows). (I and K) Phase-contrast microscopy of mock-infected chicken (I) and duck (K) myotube cultures show typical myotubes. (J and L) In contrast, similar myotube detachment and rounding (arrows) were evident in HPAI H5N1 50-92 virus-infected (MOI of 1.0) chicken (J) and duck (L) myotubes at 24 h p.i. (M to P) Translocation of PS to the outer membrane was detected (green fluorescence) by annexin V-EGFP binding to chicken and duck muscle cells (myotubes and myoblasts) infected with LPAI H2N3 virus (MOI of 1.0) for 24 h. Infected chicken (M) and duck (N) myotubes were extensively positive for PS translocation; some cells had lost membrane integrity, as evident by the nuclear uptake of PI (red fluorescence). Myonuclei labeled with PI displayed chromatin condensation and/or fragmentation in chicken and duck myotubes, whose boundaries are outlined (M and N, respectively). A morphologically intact nucleus with condensed and/or fragmented nuclear contents is highlighted (M, arrows). Chromatin condensation and fragmentation were also seen in chicken (O) and duck (P) myoblasts. (Q) Chicken and duck myotube cultures infected with LPAI H2N3 virus at a MOI of 1.0 significantly activated the effector caspases 3 and 7 at 24 h and 48 h p.i. (*, P < 0.05; **, P < 0.01 [determined by an unpaired t test]) (RLU/s, relative light units per second). (Q′) Results are also represented as fold changes. There was, however, no significant difference in the activation of caspases 3 and 7 between the two avian species. Data points are the means of data from three wells from a 96-well plate, with error bars indicating standard deviations.

Mentions: Differentiated chicken and duck myotubes were infected with a LPAI H2N3 virus at a MOI of 1.0. Uninfected control chicken and duck cultures typically displayed extensive swirls of myotubes immunopositive for muscle-specific intermediate desmin filaments (Fig. 4A and E, respectively). At 24 h p.i., severe cytopathic damage was evident, with widespread rounding or detachment of chicken and duck myotubes (Fig. 4B and F, respectively). There were numerous sarcoplasmic blebs, which appeared as small membrane-lined vesicles associated with degenerating myotubes (Fig. 4C to D′ and G to H′, respectively). These blebs appeared to be apoptotic bodies, a feature well recognized in other cell types but not previously observed in myotubes undergoing apoptosis in vitro (41, 42). Concentrations of viral NP colocalized with sarcoplasmic blebs in both infected chicken and duck myotubes (Fig. 4D and D′ and H and H′, respectively). Similar cytopathic changes were observed in chicken and duck myotubes infected with HPAI H5N1 50-92 virus (Fig. 4J and L, respectively). To further examine the cytopathic changes in chicken and duck myotubes infected with LPAI H2N3 virus at a MOI of 1.0 for 24 h, PS membrane translocation (apoptotic change) was localized by annexin V-EGFP binding, visualized as green fluorescence (Fig. 4M to P). Some annexin V-EGFP-positive cells had lost their membrane integrity, as demonstrated by the uptake of the red nuclear fluorochrome PI, indicating late apoptosis or even necrosis. PI-labeled myonuclei showed chromatin condensation and/or fragmentation in chicken and duck myotubes (Fig. 4M and N, respectively). Similar apoptotic changes were also detected in infected chicken and duck myoblasts (Fig. 4O and P, respectively). Both chicken and duck myotube cultures displayed similar and significant activation of caspases 3 and 7 at 24 h and 48 h after infection (Fig. 4Q and Q′). In summary, extensive and severe chicken and duck myotube damage from LPAI and HPAI virus infections was accompanied by clear hallmarks of apoptosis and evidence of necrosis.


Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

Baquero-Perez B, Kuchipudi SV, Ho J, Sebastian S, Puranik A, Howard W, Brookes SM, Brown IH, Chang KC - J. Virol. (2014)

Influenza virus-infected chicken and duck myotubes show extensive cytopathic damage. Chicken and duck myotubes were infected with LPAI H2N3 virus at a MOI of 1.0 for 24 h. (A and E) Uninfected control chicken and duck myotubes show extensive expression of the muscle-specific intermediate filament desmin (brown). (B and F) Cytopathic damage of chicken and duck myotubes is seen as widespread rounding and detachment of cells (arrows). (C, C′, G, and G′) Whole chicken and duck myotubes appear to have degenerated into numerous small membrane-lined blebs (apoptotic bodies) (myotube boundaries are delineated). (D, D′, H, and H′) Commonly, sections of chicken and duck myotubes contained aggregates of apoptotic blebs with concentrated viral NP, as evident by immunocytochemical NP detection. Morphologically intact myonuclei were found in the sarcoplasm of the duck myotube (H′, arrows). (I and K) Phase-contrast microscopy of mock-infected chicken (I) and duck (K) myotube cultures show typical myotubes. (J and L) In contrast, similar myotube detachment and rounding (arrows) were evident in HPAI H5N1 50-92 virus-infected (MOI of 1.0) chicken (J) and duck (L) myotubes at 24 h p.i. (M to P) Translocation of PS to the outer membrane was detected (green fluorescence) by annexin V-EGFP binding to chicken and duck muscle cells (myotubes and myoblasts) infected with LPAI H2N3 virus (MOI of 1.0) for 24 h. Infected chicken (M) and duck (N) myotubes were extensively positive for PS translocation; some cells had lost membrane integrity, as evident by the nuclear uptake of PI (red fluorescence). Myonuclei labeled with PI displayed chromatin condensation and/or fragmentation in chicken and duck myotubes, whose boundaries are outlined (M and N, respectively). A morphologically intact nucleus with condensed and/or fragmented nuclear contents is highlighted (M, arrows). Chromatin condensation and fragmentation were also seen in chicken (O) and duck (P) myoblasts. (Q) Chicken and duck myotube cultures infected with LPAI H2N3 virus at a MOI of 1.0 significantly activated the effector caspases 3 and 7 at 24 h and 48 h p.i. (*, P < 0.05; **, P < 0.01 [determined by an unpaired t test]) (RLU/s, relative light units per second). (Q′) Results are also represented as fold changes. There was, however, no significant difference in the activation of caspases 3 and 7 between the two avian species. Data points are the means of data from three wells from a 96-well plate, with error bars indicating standard deviations.
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Figure 4: Influenza virus-infected chicken and duck myotubes show extensive cytopathic damage. Chicken and duck myotubes were infected with LPAI H2N3 virus at a MOI of 1.0 for 24 h. (A and E) Uninfected control chicken and duck myotubes show extensive expression of the muscle-specific intermediate filament desmin (brown). (B and F) Cytopathic damage of chicken and duck myotubes is seen as widespread rounding and detachment of cells (arrows). (C, C′, G, and G′) Whole chicken and duck myotubes appear to have degenerated into numerous small membrane-lined blebs (apoptotic bodies) (myotube boundaries are delineated). (D, D′, H, and H′) Commonly, sections of chicken and duck myotubes contained aggregates of apoptotic blebs with concentrated viral NP, as evident by immunocytochemical NP detection. Morphologically intact myonuclei were found in the sarcoplasm of the duck myotube (H′, arrows). (I and K) Phase-contrast microscopy of mock-infected chicken (I) and duck (K) myotube cultures show typical myotubes. (J and L) In contrast, similar myotube detachment and rounding (arrows) were evident in HPAI H5N1 50-92 virus-infected (MOI of 1.0) chicken (J) and duck (L) myotubes at 24 h p.i. (M to P) Translocation of PS to the outer membrane was detected (green fluorescence) by annexin V-EGFP binding to chicken and duck muscle cells (myotubes and myoblasts) infected with LPAI H2N3 virus (MOI of 1.0) for 24 h. Infected chicken (M) and duck (N) myotubes were extensively positive for PS translocation; some cells had lost membrane integrity, as evident by the nuclear uptake of PI (red fluorescence). Myonuclei labeled with PI displayed chromatin condensation and/or fragmentation in chicken and duck myotubes, whose boundaries are outlined (M and N, respectively). A morphologically intact nucleus with condensed and/or fragmented nuclear contents is highlighted (M, arrows). Chromatin condensation and fragmentation were also seen in chicken (O) and duck (P) myoblasts. (Q) Chicken and duck myotube cultures infected with LPAI H2N3 virus at a MOI of 1.0 significantly activated the effector caspases 3 and 7 at 24 h and 48 h p.i. (*, P < 0.05; **, P < 0.01 [determined by an unpaired t test]) (RLU/s, relative light units per second). (Q′) Results are also represented as fold changes. There was, however, no significant difference in the activation of caspases 3 and 7 between the two avian species. Data points are the means of data from three wells from a 96-well plate, with error bars indicating standard deviations.
Mentions: Differentiated chicken and duck myotubes were infected with a LPAI H2N3 virus at a MOI of 1.0. Uninfected control chicken and duck cultures typically displayed extensive swirls of myotubes immunopositive for muscle-specific intermediate desmin filaments (Fig. 4A and E, respectively). At 24 h p.i., severe cytopathic damage was evident, with widespread rounding or detachment of chicken and duck myotubes (Fig. 4B and F, respectively). There were numerous sarcoplasmic blebs, which appeared as small membrane-lined vesicles associated with degenerating myotubes (Fig. 4C to D′ and G to H′, respectively). These blebs appeared to be apoptotic bodies, a feature well recognized in other cell types but not previously observed in myotubes undergoing apoptosis in vitro (41, 42). Concentrations of viral NP colocalized with sarcoplasmic blebs in both infected chicken and duck myotubes (Fig. 4D and D′ and H and H′, respectively). Similar cytopathic changes were observed in chicken and duck myotubes infected with HPAI H5N1 50-92 virus (Fig. 4J and L, respectively). To further examine the cytopathic changes in chicken and duck myotubes infected with LPAI H2N3 virus at a MOI of 1.0 for 24 h, PS membrane translocation (apoptotic change) was localized by annexin V-EGFP binding, visualized as green fluorescence (Fig. 4M to P). Some annexin V-EGFP-positive cells had lost their membrane integrity, as demonstrated by the uptake of the red nuclear fluorochrome PI, indicating late apoptosis or even necrosis. PI-labeled myonuclei showed chromatin condensation and/or fragmentation in chicken and duck myotubes (Fig. 4M and N, respectively). Similar apoptotic changes were also detected in infected chicken and duck myoblasts (Fig. 4O and P, respectively). Both chicken and duck myotube cultures displayed similar and significant activation of caspases 3 and 7 at 24 h and 48 h after infection (Fig. 4Q and Q′). In summary, extensive and severe chicken and duck myotube damage from LPAI and HPAI virus infections was accompanied by clear hallmarks of apoptosis and evidence of necrosis.

Bottom Line: Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines.Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells.Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Science, University of Nottingham, Loughborough, Leicestershire, United Kingdom.

Show MeSH
Related in: MedlinePlus