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Type I interferon response is delayed in human astrovirus infections.

Guix S, Pérez-Bosque A, Miró L, Moretó M, Bosch A, Pintó RM - PLoS ONE (2015)

Bottom Line: Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections.Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells.On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses.

View Article: PubMed Central - PubMed

Affiliation: Enteric Virus Group, Department of Microbiology, University of Barcelona, Barcelona, Spain; Nutrition and Food Safety Research Institute (INSA-UB), University of Barcelona, Santa Coloma de Gramanet, Spain.

ABSTRACT
Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections. Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells. Although IFN-β mRNA is detected within infected cells and supernatant from infected cells show antiviral activity against the replication of other well-known IFN-sensitive viruses, these responses occur at late stages of infection once genome replication has taken place. On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses. Finally, different levels of IFN response were observed in cells infected with different HAstV mutants with changes in the hypervariable region of nsP1a/4, suggesting that nsP1a/4 genotype may potentially have clinical implications due to its correlation with the viral replication phenotype and the antiviral responses induced within infected cells.

No MeSH data available.


Related in: MedlinePlus

Analysis of the level of IFN response in HAstV-infected cells.(A) Quantification of IFN-β mRNA levels by qRT-PCR during infection of CaCo-2 cells at a MOI of 1. qRT-PCR values with primers specific for human IFN-β mRNA were normalized to endogenous GAPDH mRNA levels at each time point, and results were expressed as fold induction of IFN-β expression versus 0 hpi. PolyI:C-transfected cells at 24 hpt were used as a positive control. Results shown are the mean values of 2 independent experiments and error bars represent the SEM. (B) Kinetic analysis of IRF3 subcellular localization during HAstV infection at a MOI of 1. PolyI:C transfected cells fixed at 24 h post-transfection and mock-infected cells fixed at 48 and 96 hpi were used as positive and negative controls, respectively. Cells were labeled for HAstV capsid protein (red) and IRF3 (green). White arrows indicate cells with nuclear translocation of IRF3. (C) Percentage of cells with translocation of IRF3 into the nucleus. Data (mean values ± SEM) were calculated after counting the number of cells with nuclear IRF3 from 5 fields from coverslips from 2–3 independent experiments using the Image J software.
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pone.0123087.g002: Analysis of the level of IFN response in HAstV-infected cells.(A) Quantification of IFN-β mRNA levels by qRT-PCR during infection of CaCo-2 cells at a MOI of 1. qRT-PCR values with primers specific for human IFN-β mRNA were normalized to endogenous GAPDH mRNA levels at each time point, and results were expressed as fold induction of IFN-β expression versus 0 hpi. PolyI:C-transfected cells at 24 hpt were used as a positive control. Results shown are the mean values of 2 independent experiments and error bars represent the SEM. (B) Kinetic analysis of IRF3 subcellular localization during HAstV infection at a MOI of 1. PolyI:C transfected cells fixed at 24 h post-transfection and mock-infected cells fixed at 48 and 96 hpi were used as positive and negative controls, respectively. Cells were labeled for HAstV capsid protein (red) and IRF3 (green). White arrows indicate cells with nuclear translocation of IRF3. (C) Percentage of cells with translocation of IRF3 into the nucleus. Data (mean values ± SEM) were calculated after counting the number of cells with nuclear IRF3 from 5 fields from coverslips from 2–3 independent experiments using the Image J software.

Mentions: In order to analyze the magnitude of the IFN response, levels of IFN-β mRNA were quantified by qRT-PCR, and subcellular localization of IRF3 was examined by immunofluorescence during the course of infection at a MOI of 1, which results in more than 90% of infected cells as measured by IF. IFN-β mRNA levels induced by HAstV peaked at 48 hpi but were 3.2-fold lower than those induced by polyI:C transfection (Fig 2A). Since transfection efficiency of CaCo-2 cells is low and polyI:C transfection only resulted in 6 ± 0.7% of cells with nuclear IRF3 producing IFN-β mRNA (Fig 2C), when comparing the total levels of IFN-β mRNA produced by polyI:C-transfected cultures to the levels produced by infected cultures, it would seem that in these latter cultures, either each infected cell produced very low levels of IFN-β mRNA or that IFN-β mRNA would only be produced by a few number of infected cells. Co-staining of viral structural proteins and IRF3 showed that only 2 ± 0.2% of HAstV-infected cells had nuclear IRF3 at 24 hpi (Fig 2B and 2C). At 48 hpi, the percentage of cells with nuclear IRF3 increased up to 10.34 ± 1.8 0%, but in any case was not close to 100%. Percentages were higher when infecting cells with a MOI of 10, with a maximum of 20.4 ± 5.8% at 48 hpi (Fig 2C). At 96 hpi, cell damage due to infection was too strong to allow proper interpretation of IF data (Fig 2B). At 12 hpi, IRF3 was localized to the cytoplasm of all HAstV-infected cells (data not shown). Together these results indicate that, despite the high number of HAstV-infected cells, the IFN response in infected cultures was attenuated.


Type I interferon response is delayed in human astrovirus infections.

Guix S, Pérez-Bosque A, Miró L, Moretó M, Bosch A, Pintó RM - PLoS ONE (2015)

Analysis of the level of IFN response in HAstV-infected cells.(A) Quantification of IFN-β mRNA levels by qRT-PCR during infection of CaCo-2 cells at a MOI of 1. qRT-PCR values with primers specific for human IFN-β mRNA were normalized to endogenous GAPDH mRNA levels at each time point, and results were expressed as fold induction of IFN-β expression versus 0 hpi. PolyI:C-transfected cells at 24 hpt were used as a positive control. Results shown are the mean values of 2 independent experiments and error bars represent the SEM. (B) Kinetic analysis of IRF3 subcellular localization during HAstV infection at a MOI of 1. PolyI:C transfected cells fixed at 24 h post-transfection and mock-infected cells fixed at 48 and 96 hpi were used as positive and negative controls, respectively. Cells were labeled for HAstV capsid protein (red) and IRF3 (green). White arrows indicate cells with nuclear translocation of IRF3. (C) Percentage of cells with translocation of IRF3 into the nucleus. Data (mean values ± SEM) were calculated after counting the number of cells with nuclear IRF3 from 5 fields from coverslips from 2–3 independent experiments using the Image J software.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383485&req=5

pone.0123087.g002: Analysis of the level of IFN response in HAstV-infected cells.(A) Quantification of IFN-β mRNA levels by qRT-PCR during infection of CaCo-2 cells at a MOI of 1. qRT-PCR values with primers specific for human IFN-β mRNA were normalized to endogenous GAPDH mRNA levels at each time point, and results were expressed as fold induction of IFN-β expression versus 0 hpi. PolyI:C-transfected cells at 24 hpt were used as a positive control. Results shown are the mean values of 2 independent experiments and error bars represent the SEM. (B) Kinetic analysis of IRF3 subcellular localization during HAstV infection at a MOI of 1. PolyI:C transfected cells fixed at 24 h post-transfection and mock-infected cells fixed at 48 and 96 hpi were used as positive and negative controls, respectively. Cells were labeled for HAstV capsid protein (red) and IRF3 (green). White arrows indicate cells with nuclear translocation of IRF3. (C) Percentage of cells with translocation of IRF3 into the nucleus. Data (mean values ± SEM) were calculated after counting the number of cells with nuclear IRF3 from 5 fields from coverslips from 2–3 independent experiments using the Image J software.
Mentions: In order to analyze the magnitude of the IFN response, levels of IFN-β mRNA were quantified by qRT-PCR, and subcellular localization of IRF3 was examined by immunofluorescence during the course of infection at a MOI of 1, which results in more than 90% of infected cells as measured by IF. IFN-β mRNA levels induced by HAstV peaked at 48 hpi but were 3.2-fold lower than those induced by polyI:C transfection (Fig 2A). Since transfection efficiency of CaCo-2 cells is low and polyI:C transfection only resulted in 6 ± 0.7% of cells with nuclear IRF3 producing IFN-β mRNA (Fig 2C), when comparing the total levels of IFN-β mRNA produced by polyI:C-transfected cultures to the levels produced by infected cultures, it would seem that in these latter cultures, either each infected cell produced very low levels of IFN-β mRNA or that IFN-β mRNA would only be produced by a few number of infected cells. Co-staining of viral structural proteins and IRF3 showed that only 2 ± 0.2% of HAstV-infected cells had nuclear IRF3 at 24 hpi (Fig 2B and 2C). At 48 hpi, the percentage of cells with nuclear IRF3 increased up to 10.34 ± 1.8 0%, but in any case was not close to 100%. Percentages were higher when infecting cells with a MOI of 10, with a maximum of 20.4 ± 5.8% at 48 hpi (Fig 2C). At 96 hpi, cell damage due to infection was too strong to allow proper interpretation of IF data (Fig 2B). At 12 hpi, IRF3 was localized to the cytoplasm of all HAstV-infected cells (data not shown). Together these results indicate that, despite the high number of HAstV-infected cells, the IFN response in infected cultures was attenuated.

Bottom Line: Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections.Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells.On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses.

View Article: PubMed Central - PubMed

Affiliation: Enteric Virus Group, Department of Microbiology, University of Barcelona, Barcelona, Spain; Nutrition and Food Safety Research Institute (INSA-UB), University of Barcelona, Santa Coloma de Gramanet, Spain.

ABSTRACT
Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections. Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells. Although IFN-β mRNA is detected within infected cells and supernatant from infected cells show antiviral activity against the replication of other well-known IFN-sensitive viruses, these responses occur at late stages of infection once genome replication has taken place. On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses. Finally, different levels of IFN response were observed in cells infected with different HAstV mutants with changes in the hypervariable region of nsP1a/4, suggesting that nsP1a/4 genotype may potentially have clinical implications due to its correlation with the viral replication phenotype and the antiviral responses induced within infected cells.

No MeSH data available.


Related in: MedlinePlus