Limits...
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.

Show MeSH

Related in: MedlinePlus

Kinetics of viral RNA and particle production by muscle cells.A, B. The infectious titers in the supernatants of CHQ myotubes (A) or myoblasts (B) were measured by plaque assay at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 or 3 independent experiments with the standard deviation. The detection threshold (grey line) was 25 pfu/ml for myotubes and 250 pfu/ml for myoblasts. For this experiment, the concentration of serum in myoblasts’ medium, which could limit the efficiency of viral multiplication and the sensitivity of the plaque assay, was reduced to 5%. C. The amount of total M RNAs was quantified by RT-qPCR in supernatants (dashed lines) and in cell extracts (solid lines) of CHQ myoblasts at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 independent experiments with the standard deviation.D, E. Supernatant from myotubes were harvested 48h after infection with the A/California/7/2009 or the A/Paris/1149/2008 isolates, incubated with or without TPCK-trypsin, and used to infect myotubes (D) and myoblasts (E) from the same donor (CHQ). Indirect immunofluorescence detection of desmin (red) and NP (green) and staining of the nuclei with DAPI (blue) were performed. Bar: 100µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3818236&req=5

pone-0079628-g004: Kinetics of viral RNA and particle production by muscle cells.A, B. The infectious titers in the supernatants of CHQ myotubes (A) or myoblasts (B) were measured by plaque assay at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 or 3 independent experiments with the standard deviation. The detection threshold (grey line) was 25 pfu/ml for myotubes and 250 pfu/ml for myoblasts. For this experiment, the concentration of serum in myoblasts’ medium, which could limit the efficiency of viral multiplication and the sensitivity of the plaque assay, was reduced to 5%. C. The amount of total M RNAs was quantified by RT-qPCR in supernatants (dashed lines) and in cell extracts (solid lines) of CHQ myoblasts at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 independent experiments with the standard deviation.D, E. Supernatant from myotubes were harvested 48h after infection with the A/California/7/2009 or the A/Paris/1149/2008 isolates, incubated with or without TPCK-trypsin, and used to infect myotubes (D) and myoblasts (E) from the same donor (CHQ). Indirect immunofluorescence detection of desmin (red) and NP (green) and staining of the nuclei with DAPI (blue) were performed. Bar: 100µm.

Mentions: We examined whether influenza A viruses could undergo productive replication following infection of muscle cells at a MOI of 0.3 pfu/plated cell, by measuring the infectious titer in the supernatants of infected cells at various time points (Figure 4A, 4B). To allow the maturation of the viral hemagglutinin HA, samples were treated with 1µg/ml TPCK-trypsin before titration by plaque assay. This treatment was sufficient to allow complete maturation of both viruses, as assessed by testing increasing concentrations of trypsin (data not shown). In the supernatants of myotubes (Figure 4A), the infectious titers increased as early as 8 hPI, and reached a maximum 48h PI before decreasing, probably due to cell death and degradation of virions. The pandemic virus was produced at higher titers than the seasonal virus.


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)

Kinetics of viral RNA and particle production by muscle cells.A, B. The infectious titers in the supernatants of CHQ myotubes (A) or myoblasts (B) were measured by plaque assay at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 or 3 independent experiments with the standard deviation. The detection threshold (grey line) was 25 pfu/ml for myotubes and 250 pfu/ml for myoblasts. For this experiment, the concentration of serum in myoblasts’ medium, which could limit the efficiency of viral multiplication and the sensitivity of the plaque assay, was reduced to 5%. C. The amount of total M RNAs was quantified by RT-qPCR in supernatants (dashed lines) and in cell extracts (solid lines) of CHQ myoblasts at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 independent experiments with the standard deviation.D, E. Supernatant from myotubes were harvested 48h after infection with the A/California/7/2009 or the A/Paris/1149/2008 isolates, incubated with or without TPCK-trypsin, and used to infect myotubes (D) and myoblasts (E) from the same donor (CHQ). Indirect immunofluorescence detection of desmin (red) and NP (green) and staining of the nuclei with DAPI (blue) were performed. Bar: 100µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079628-g004: Kinetics of viral RNA and particle production by muscle cells.A, B. The infectious titers in the supernatants of CHQ myotubes (A) or myoblasts (B) were measured by plaque assay at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 or 3 independent experiments with the standard deviation. The detection threshold (grey line) was 25 pfu/ml for myotubes and 250 pfu/ml for myoblasts. For this experiment, the concentration of serum in myoblasts’ medium, which could limit the efficiency of viral multiplication and the sensitivity of the plaque assay, was reduced to 5%. C. The amount of total M RNAs was quantified by RT-qPCR in supernatants (dashed lines) and in cell extracts (solid lines) of CHQ myoblasts at various time points after infection with the A/California/7/2009 (blue lines) or the A/Paris/1149/2008 (green lines) viruses. Data are the mean of 2 independent experiments with the standard deviation.D, E. Supernatant from myotubes were harvested 48h after infection with the A/California/7/2009 or the A/Paris/1149/2008 isolates, incubated with or without TPCK-trypsin, and used to infect myotubes (D) and myoblasts (E) from the same donor (CHQ). Indirect immunofluorescence detection of desmin (red) and NP (green) and staining of the nuclei with DAPI (blue) were performed. Bar: 100µm.
Mentions: We examined whether influenza A viruses could undergo productive replication following infection of muscle cells at a MOI of 0.3 pfu/plated cell, by measuring the infectious titer in the supernatants of infected cells at various time points (Figure 4A, 4B). To allow the maturation of the viral hemagglutinin HA, samples were treated with 1µg/ml TPCK-trypsin before titration by plaque assay. This treatment was sufficient to allow complete maturation of both viruses, as assessed by testing increasing concentrations of trypsin (data not shown). In the supernatants of myotubes (Figure 4A), the infectious titers increased as early as 8 hPI, and reached a maximum 48h PI before decreasing, probably due to cell death and degradation of virions. The pandemic virus was produced at higher titers than the seasonal virus.

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