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Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1) viruses.

Desdouits M, Munier S, Prevost MC, Jeannin P, Butler-Browne G, Ozden S, Gessain A, Van Der Werf S, Naffakh N, Ceccaldi PE - PLoS ONE (2013)

Bottom Line: The pathogenesis of these influenza-associated myopathies (IAM) remains unkown, although the direct infection of muscle cells is suspected.Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase.Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

View Article: PubMed Central - PubMed

Affiliation: Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France ; UMR 3569, CNRS, Paris, France ; Cellule Pasteur, Université Paris Diderot, Sorbonne Paris Cité, Paris, France.

ABSTRACT
Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1) in 2009. The pathogenesis of these influenza-associated myopathies (IAM) remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1) isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes) were highly susceptible to infection by both influenza A(H1N1) isolates, whereas undifferentiated cells (i. e. myoblasts) were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP) expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

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Virus budding from infected myotubes and myoblasts.A to J. Transmission electron microscopy (TEM) was performed on non-infected myotubes (A, B), on myotubes infected for 20h with the A/California/7/2009 (C, D, E, F) or the Paris/1149/2008 (G, H) isolates, and on MDCK-SIAT cells, 16h after infection with the A/California/7/2009 isolate (I, J). Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: A to E and G to J: 2µm; F: 100nm.K to T. Correlative light and electron microscopy (CLEM) was carried out on non-infected myoblasts (K, L) and on myoblasts infected for 20h with the A/California/7/2009 (M, N, O, P) or the A/Paris/1149/2008 (Q, R, S, T) isolates. K, M, Q. Immunofluorescence imaging of myoblasts expressing desmin (red) and/or NP (green). L. Transmission electron microscopy imaging of a non-infected myoblast observed in K showing an intact nucleus. N, O, P and R, S, T. Transmission electron microscopy imaging of the cells observed in M and Q, respectively, showing budding virions or nuclear aggregates. Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: K, M, Q: 50µm; N, R: 20 µm; L, O, P, S: 2µm; T: 200nm.
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pone-0079628-g005: Virus budding from infected myotubes and myoblasts.A to J. Transmission electron microscopy (TEM) was performed on non-infected myotubes (A, B), on myotubes infected for 20h with the A/California/7/2009 (C, D, E, F) or the Paris/1149/2008 (G, H) isolates, and on MDCK-SIAT cells, 16h after infection with the A/California/7/2009 isolate (I, J). Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: A to E and G to J: 2µm; F: 100nm.K to T. Correlative light and electron microscopy (CLEM) was carried out on non-infected myoblasts (K, L) and on myoblasts infected for 20h with the A/California/7/2009 (M, N, O, P) or the A/Paris/1149/2008 (Q, R, S, T) isolates. K, M, Q. Immunofluorescence imaging of myoblasts expressing desmin (red) and/or NP (green). L. Transmission electron microscopy imaging of a non-infected myoblast observed in K showing an intact nucleus. N, O, P and R, S, T. Transmission electron microscopy imaging of the cells observed in M and Q, respectively, showing budding virions or nuclear aggregates. Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: K, M, Q: 50µm; N, R: 20 µm; L, O, P, S: 2µm; T: 200nm.

Mentions: The low infectivity and M RNA quantity in supernatants from infected myoblasts prompted us to check whether viral budding occurred in muscle cells, by means of transmission electron microscopy (TEM), 20 hours after infection at a MOI of 30 pfu/plated cell. In myotubes, we were able to observe both filamentous and spherical extracellular and budding virions, mostly at the cell surface, that were absent in non-infected cell samples (Figure 5A, 5C, 5G, arrowheads.). Virions were also rarely observed in seemingly intracellular vesicles, which are most likely extracellular spaces between the cells and the surface of the culture plate, due to the local invagination of the plasma membrane (Figure 5C, 5E, arrowheads). These particles exhibited the typical morphology of influenza viruses: a diameter of 80 to 100 nm and spikes (Figure 5F), and for some of them, a dense area at one pole that suggested the presence of ribonucleoparticles (RNPs). Most analyzed infected cells presented nuclear inclusions of dense material that were never observed in uninfected cells (Figure 5B, 5D, 5H, arrows) and were distinct from nucleoli (Figure 5B, 5D, 5H, stars). Virions and nuclear aggregates were also observed in control cultures of MDCK-SIAT infected with the A/California/7/2009 virus (Figure 5I and 5J).


Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1) viruses.

Desdouits M, Munier S, Prevost MC, Jeannin P, Butler-Browne G, Ozden S, Gessain A, Van Der Werf S, Naffakh N, Ceccaldi PE - PLoS ONE (2013)

Virus budding from infected myotubes and myoblasts.A to J. Transmission electron microscopy (TEM) was performed on non-infected myotubes (A, B), on myotubes infected for 20h with the A/California/7/2009 (C, D, E, F) or the Paris/1149/2008 (G, H) isolates, and on MDCK-SIAT cells, 16h after infection with the A/California/7/2009 isolate (I, J). Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: A to E and G to J: 2µm; F: 100nm.K to T. Correlative light and electron microscopy (CLEM) was carried out on non-infected myoblasts (K, L) and on myoblasts infected for 20h with the A/California/7/2009 (M, N, O, P) or the A/Paris/1149/2008 (Q, R, S, T) isolates. K, M, Q. Immunofluorescence imaging of myoblasts expressing desmin (red) and/or NP (green). L. Transmission electron microscopy imaging of a non-infected myoblast observed in K showing an intact nucleus. N, O, P and R, S, T. Transmission electron microscopy imaging of the cells observed in M and Q, respectively, showing budding virions or nuclear aggregates. Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: K, M, Q: 50µm; N, R: 20 µm; L, O, P, S: 2µm; T: 200nm.
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getmorefigures.php?uid=PMC3818236&req=5

pone-0079628-g005: Virus budding from infected myotubes and myoblasts.A to J. Transmission electron microscopy (TEM) was performed on non-infected myotubes (A, B), on myotubes infected for 20h with the A/California/7/2009 (C, D, E, F) or the Paris/1149/2008 (G, H) isolates, and on MDCK-SIAT cells, 16h after infection with the A/California/7/2009 isolate (I, J). Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: A to E and G to J: 2µm; F: 100nm.K to T. Correlative light and electron microscopy (CLEM) was carried out on non-infected myoblasts (K, L) and on myoblasts infected for 20h with the A/California/7/2009 (M, N, O, P) or the A/Paris/1149/2008 (Q, R, S, T) isolates. K, M, Q. Immunofluorescence imaging of myoblasts expressing desmin (red) and/or NP (green). L. Transmission electron microscopy imaging of a non-infected myoblast observed in K showing an intact nucleus. N, O, P and R, S, T. Transmission electron microscopy imaging of the cells observed in M and Q, respectively, showing budding virions or nuclear aggregates. Arrowheads: budding virions or virions in close contact with the cell. Arrows: dense nuclear aggregates. Stars: nucleoli. Bars: K, M, Q: 50µm; N, R: 20 µm; L, O, P, S: 2µm; T: 200nm.
Mentions: The low infectivity and M RNA quantity in supernatants from infected myoblasts prompted us to check whether viral budding occurred in muscle cells, by means of transmission electron microscopy (TEM), 20 hours after infection at a MOI of 30 pfu/plated cell. In myotubes, we were able to observe both filamentous and spherical extracellular and budding virions, mostly at the cell surface, that were absent in non-infected cell samples (Figure 5A, 5C, 5G, arrowheads.). Virions were also rarely observed in seemingly intracellular vesicles, which are most likely extracellular spaces between the cells and the surface of the culture plate, due to the local invagination of the plasma membrane (Figure 5C, 5E, arrowheads). These particles exhibited the typical morphology of influenza viruses: a diameter of 80 to 100 nm and spikes (Figure 5F), and for some of them, a dense area at one pole that suggested the presence of ribonucleoparticles (RNPs). Most analyzed infected cells presented nuclear inclusions of dense material that were never observed in uninfected cells (Figure 5B, 5D, 5H, arrows) and were distinct from nucleoli (Figure 5B, 5D, 5H, stars). Virions and nuclear aggregates were also observed in control cultures of MDCK-SIAT infected with the A/California/7/2009 virus (Figure 5I and 5J).

Bottom Line: The pathogenesis of these influenza-associated myopathies (IAM) remains unkown, although the direct infection of muscle cells is suspected.Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase.Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

View Article: PubMed Central - PubMed

Affiliation: Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France ; UMR 3569, CNRS, Paris, France ; Cellule Pasteur, Université Paris Diderot, Sorbonne Paris Cité, Paris, France.

ABSTRACT
Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1) in 2009. The pathogenesis of these influenza-associated myopathies (IAM) remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1) isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes) were highly susceptible to infection by both influenza A(H1N1) isolates, whereas undifferentiated cells (i. e. myoblasts) were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP) expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

Show MeSH
Related in: MedlinePlus