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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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Related in: MedlinePlus

Detection of sialic acids at the surface of human muscle cells.A. α2-3 and α2-6 linked sialic acids were detected on non-permeabilized muscle cells (myoblasts and myotubes) from donor CHQ by using the biotinylated lectins MAAII and SNA, respectively. Lectin binding was further revealed by Streptavidin-A488 staining (green), and cells were permeabilized for detection of intracellular desmin (red) by immunofluorescence. B. The MAAII and SNA lectins were used for the detection of the two types of sialic acids by cytofluorometry, on non-permeabilized myoblasts from 3 different donors (CHQ, KM46C38, KM49C) and MDCK-SIAT cells as a control. Cells were then permeabilized for the staining of NCAM. The geometric mean of fluorescence intensity (GeoMFI) for each condition (no lectin, grey; MAAII, light green; SNA, dark green) is given for the total population of MDCK cells (which were NCAM-negative) and for the NCAM-positive population in myoblast cultures. Data are the mean of two duplicates with standard deviation as error bar, and represent two independent experiments. C. Distribution of the lectin staining intensities in the NCAM-positive population of CHQ myoblasts stained with MAAII (light green) and SNA (dark green).
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pone-0079628-g003: Detection of sialic acids at the surface of human muscle cells.A. α2-3 and α2-6 linked sialic acids were detected on non-permeabilized muscle cells (myoblasts and myotubes) from donor CHQ by using the biotinylated lectins MAAII and SNA, respectively. Lectin binding was further revealed by Streptavidin-A488 staining (green), and cells were permeabilized for detection of intracellular desmin (red) by immunofluorescence. B. The MAAII and SNA lectins were used for the detection of the two types of sialic acids by cytofluorometry, on non-permeabilized myoblasts from 3 different donors (CHQ, KM46C38, KM49C) and MDCK-SIAT cells as a control. Cells were then permeabilized for the staining of NCAM. The geometric mean of fluorescence intensity (GeoMFI) for each condition (no lectin, grey; MAAII, light green; SNA, dark green) is given for the total population of MDCK cells (which were NCAM-negative) and for the NCAM-positive population in myoblast cultures. Data are the mean of two duplicates with standard deviation as error bar, and represent two independent experiments. C. Distribution of the lectin staining intensities in the NCAM-positive population of CHQ myoblasts stained with MAAII (light green) and SNA (dark green).

Mentions: We asked whether the difference in susceptibility to influenza A virus infection was related to differences in the expression of viral receptors at the surface of myoblasts and myotubes. Indeed, host range and tissue tropism of influenza viruses are in part determined by the specificity of their surface glycoproteins for their receptors, sialic acids attached to galactose by an α2-6 or an α2-3 linkage [47]. Thus, we investigated the presence of α2-6 and α2-3 linked sialic acids on the surface of muscle cells using specific lectins. Both myoblasts and myotubes stained positive with the lectins MAAII (which binds preferentially to α2-3 linked sialic acids) and SNA (which binds preferentially to α2-6 linked sialic acids), as did MDCK-SIAT cells used as a control (not shown) (Figure 3A). These data suggest that both cells express influenza A virus receptors, and that there are no major differences in the distribution of α2-6 and α2-3 sialic acid receptors at the surface of myoblasts and myotubes. The use of flow cytofluorometry allowed us to confirm that majority of NCAM+ myoblasts from the CHQ donor and two additional donors, KM46C38 and KM49C, were stained with both lectins, as were control MDCK-SIAT cells (Figure 3B and 3C). Unfortunately, flow cytofluorometry analysis of myotubes is precluded by their large size. However, interestingly, we found that the ratio of α2-6 linked over α2-3 linked sialic acids is lower in myoblasts than in MDCK-SIAT cells, which could contribute to their low susceptibility to human influenza A viruses.


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)

Detection of sialic acids at the surface of human muscle cells.A. α2-3 and α2-6 linked sialic acids were detected on non-permeabilized muscle cells (myoblasts and myotubes) from donor CHQ by using the biotinylated lectins MAAII and SNA, respectively. Lectin binding was further revealed by Streptavidin-A488 staining (green), and cells were permeabilized for detection of intracellular desmin (red) by immunofluorescence. B. The MAAII and SNA lectins were used for the detection of the two types of sialic acids by cytofluorometry, on non-permeabilized myoblasts from 3 different donors (CHQ, KM46C38, KM49C) and MDCK-SIAT cells as a control. Cells were then permeabilized for the staining of NCAM. The geometric mean of fluorescence intensity (GeoMFI) for each condition (no lectin, grey; MAAII, light green; SNA, dark green) is given for the total population of MDCK cells (which were NCAM-negative) and for the NCAM-positive population in myoblast cultures. Data are the mean of two duplicates with standard deviation as error bar, and represent two independent experiments. C. Distribution of the lectin staining intensities in the NCAM-positive population of CHQ myoblasts stained with MAAII (light green) and SNA (dark green).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818236&req=5

pone-0079628-g003: Detection of sialic acids at the surface of human muscle cells.A. α2-3 and α2-6 linked sialic acids were detected on non-permeabilized muscle cells (myoblasts and myotubes) from donor CHQ by using the biotinylated lectins MAAII and SNA, respectively. Lectin binding was further revealed by Streptavidin-A488 staining (green), and cells were permeabilized for detection of intracellular desmin (red) by immunofluorescence. B. The MAAII and SNA lectins were used for the detection of the two types of sialic acids by cytofluorometry, on non-permeabilized myoblasts from 3 different donors (CHQ, KM46C38, KM49C) and MDCK-SIAT cells as a control. Cells were then permeabilized for the staining of NCAM. The geometric mean of fluorescence intensity (GeoMFI) for each condition (no lectin, grey; MAAII, light green; SNA, dark green) is given for the total population of MDCK cells (which were NCAM-negative) and for the NCAM-positive population in myoblast cultures. Data are the mean of two duplicates with standard deviation as error bar, and represent two independent experiments. C. Distribution of the lectin staining intensities in the NCAM-positive population of CHQ myoblasts stained with MAAII (light green) and SNA (dark green).
Mentions: We asked whether the difference in susceptibility to influenza A virus infection was related to differences in the expression of viral receptors at the surface of myoblasts and myotubes. Indeed, host range and tissue tropism of influenza viruses are in part determined by the specificity of their surface glycoproteins for their receptors, sialic acids attached to galactose by an α2-6 or an α2-3 linkage [47]. Thus, we investigated the presence of α2-6 and α2-3 linked sialic acids on the surface of muscle cells using specific lectins. Both myoblasts and myotubes stained positive with the lectins MAAII (which binds preferentially to α2-3 linked sialic acids) and SNA (which binds preferentially to α2-6 linked sialic acids), as did MDCK-SIAT cells used as a control (not shown) (Figure 3A). These data suggest that both cells express influenza A virus receptors, and that there are no major differences in the distribution of α2-6 and α2-3 sialic acid receptors at the surface of myoblasts and myotubes. The use of flow cytofluorometry allowed us to confirm that majority of NCAM+ myoblasts from the CHQ donor and two additional donors, KM46C38 and KM49C, were stained with both lectins, as were control MDCK-SIAT cells (Figure 3B and 3C). Unfortunately, flow cytofluorometry analysis of myotubes is precluded by their large size. However, interestingly, we found that the ratio of α2-6 linked over α2-3 linked sialic acids is lower in myoblasts than in MDCK-SIAT cells, which could contribute to their low susceptibility to human influenza A viruses.

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