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. 2012;7(11):e48335.
doi: 10.1371/journal.pone.0048335. Epub 2012 Nov 6.

Methamphetamine reduces human influenza A virus replication

Yun-Hsiang Chen  1 , Kuang-Lun Wu, Chia-Hsiang Chen
Affiliations

Methamphetamine reduces human influenza A virus replication

Yun-Hsiang Chen et al. PLoS One. 2012.

Abstract

Methamphetamine (meth) is a highly addictive psychostimulant that is among the most widely abused illicit drugs, with an estimated over 35 million users in the world. Several lines of evidence suggest that chronic meth abuse is a major factor for increased risk of infections with human immunodeficiency virus and possibly other pathogens, due to its immunosuppressive property. Influenza A virus infections frequently cause epidemics and pandemics of respiratory diseases among human populations. However, little is known about whether meth has the ability to enhance influenza A virus replication, thus increasing severity of influenza illness in meth abusers. Herein, we investigated the effects of meth on influenza A virus replication in human lung epithelial A549 cells. The cells were exposed to meth and infected with human influenza A/WSN/33 (H1N1) virus. The viral progenies were titrated by plaque assays, and the expression of viral proteins and cellular proteins involved in interferon responses was examined by Western blotting and immunofluorescence staining. We report the first evidence that meth significantly reduces, rather than increases, virus propagation and the susceptibility to influenza infection in the human lung epithelial cell line, consistent with a decrease in viral protein synthesis. These effects were apparently not caused by meth's effects on enhancing virus-induced interferon responses in the host cells, reducing viral biological activities, or reducing cell viability. Our results suggest that meth might not be a great risk factor for influenza A virus infection among meth abusers. Although the underlying mechanism responsible for the action of meth on attenuating virus replication requires further investigation, these findings prompt the study to examine whether other structurally similar compounds could be used as anti-influenza agents.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The cytotoxic effect of meth on human lung epithelial A549 cells.
A549 cells were treated with meth at indicated concentrations at 37°C for 72 h. (A) The trypan blue dye exclusion assay was performed to count the total, viable, and dead cells. (B) The cell survival rate was calculated, and data were expressed as percentages of viable counts in meth-treated groups relative to that in the meth-untreated group (control). The results are means ± SD of five replicates from a representative result of three independent experiments. Significant differences were indicated (***: p < 0.0001 versus control).
Figure 2
Figure 2. Meth reduces influenza A virus propagation in human lung epithelial A549 cells.
A549 cells were left untreated or treated with meth at indicated concentrations for 24 h, followed by infection with human influenza virus strain A/WSN/33 (H1N1) at an MOI of 0.001 PFU/cell (trypsin present) in the presence of meth at respective concentrations. Virus progenies were collected at indicated time points, and subjected to plaque assays in MDCK cells to determine virus titers. Values are means ± SD of three replicates from a representative result of three independent experiments. Significant differences from the meth-untreated control were indicated (**: p < 0.01, ***: p < 0.0001).
Figure 3
Figure 3. Meth reduces synthesis of influenza viral proteins in human lung epithelial A549 cells.
A549 cells were left untreated (control) or treated with meth at indicated concentrations for 24 h, and then infected with human influenza virus strain A/WSN/33 (H1N1) at an MOI of 0.001 PFU/cell in the presence of trypsin (multi-cycle growth) and meth at respective concentrations. Whole cell lysates were prepared at 48 h post-infection and subjected to Western blot analysis using antibodies against viral matrix protein-1 [M1] (A), viral nonstructural protein-1 [NS1] (C), and cellular actin. Mock: meth-untreated cells without influenza infection. A representative result from three independent experiments is shown. The expression levels of detected proteins were measured by densitometric analysis. M1 (B), and NS1 (D) levels were normalized by actin levels, and the relative optical density values were expressed as percentages of control. The results represent mean values ± SD of three replicates from a representative result of three independent experiments. Significant differences from the control were indicated (**: p < 0.01, ***: p < 0.0001).
Figure 4
Figure 4. Meth reduces influenza A virus propagation at specific steps of the virus replication cycle.
The assay to determine the effectiveness of meth in reducing influenza A virus replication with regard to the time-course of meth-exposure was performed in A549 cells as described in Materials and Methods. Cells were exposed to meth at 250 µM or left unexposed as the control (meth was not present in all incubation periods), followed by infection with influenza A/WSN/33 (H1N1) virus at an MOI of 1 PFU/cell in the absence of trypsin (single-cycle growth). Meth was added to (time-of-addition) or removed from (time-of-exclusion) the culture medium at different incubation periods as illustrated in the diagram (A). Virus progenies were collected at 22 h post-infection and subjected to plaque assays in MDCK cells to determine virus titers (B, C). ALL: meth was present in all incubation periods (−24∼22 h). The virus titer is expressed as plaque formation units per milliliter (PFU/ml). The results represent mean values ± SD of three replicates from a representative result of three independent experiments. Significant differences from the control were indicated (**: p < 0.01, ***: p < 0.0001).
Figure 5
Figure 5. Meth does not directly reduce the biological activities of human influenza virus strain A/WSN/33 (H1N1).
Influenza A viruses were incubated with meth at indicated concentrations in serum-free culture medium at 37°C for 24 h, and subjected to plaque assay in MDCK cells. (A) Plaque phenotype. A representative result of three independent experiments with similar results is shown. No obvious differences of plaque size were observed between groups. (B) Relative plaque numbers. The results are means ± SD of three replicates and expressed as percentages of plaque numbers of meth-pretreated viruses relative to that of meth-untreated viruses. No significant differences in plaque numbers were shown between groups.
Figure 6
Figure 6. Meth reduces susceptibility to influenza A virus infections in human lung epithelial A549 cells.
(A) A549 cells grown on glass coverslips were left un-treated, or treated with chloroquine (Clq.; 10 µM; as positive control) or meth at indicated concentrations, followed by the infection with influenza A/WSN/33 (H1N1) virus at an MOI of 1 PFU/cell in the absence of trypsin (single-cycle growth) and presence of the corresponding drugs at indicated concentrations. At 24 h post-infection, cells were fixed with formaldehyde and subjected to immunofluorenscence staining for detecting the infected cells by using an antibody against viral nucleoprotein (NP; green); cellular nuclei were located by DAPI staining (blue). A representative result from three independent experiments is shown. Mock: cells were neither exposed to the drugs nor infected with the virus. Scale bar: 100 µm. (B) NP-positive cells were counted from ten microscopic fields with >90% cell confluence. Data are expressed as mean values ± SD from a representative result of three independent experiments. Significant differences from the drug-untreated infected group (control) were indicated (***: p < 0.0001).
Figure 7
Figure 7. Meth reduces plaque formation in human lung epithelial A549 cells infected with influenza A viruses.
The plaque-reduction assay was performed in A549 cells as described in Materials and Methods. Cells were exposed to meth at indicated concentrations or left unexposed as the control, followed by infection with influenza A/WSN/33 (H1N1) virus in the presence of meth at indicated concentrations. (A) Plaque phenotype. A representative result of three independent experiments with similar results is shown. The size of plaques under conditions of meth treatments at 125 and 250 µM appeared to be smaller than that in the condition without meth treatment. (B) Relative plaque numbers. The results are means ± SD of three replicates, and expressed as relative ratios of plaque numbers in meth-treated groups to that in the meth-untreated group (control). Significant differences from the control were indicated (*: p < 0.05).
Figure 8
Figure 8. Effects of meth on influenza infection-induced IFN responses in human lung epithelial A549 cells.
A549 cells were left untreated (control) or treated with meth at indicated concentrations for 24 h, followed by infection with human influenza virus strain A/WSN/33 (H1N1) at an MOI of 0.001 PFU/cell in the presence of trypsin (multi-cycle growth) and meth at respective concentrations. At 48 h post-infection, whole cell lysates were prepared and subjected to Western blot analysis using antibodies against cellular STAT1 (A), phospho-STAT1 [Tyr701] (C), MxA (E), and actin. Mock: meth-untreated cells without influenza infection. A representative result from three independent experiments is shown. The levels of detected proteins were measured by densitometric analysis. The expression levels of STAT1 (B), phospho-STAT1 [Tyr701] (D), and MxA (F) were normalized by actin levels, and the relative optical density values are expressed as percentage of control. The results represent mean values ± SD of three replicates from a representative result of three independent experiments. Significant differences from the control were indicated (*: p < 0.05, **: p < 0.01, ***: p < 0.0001).
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References

    1. Krasnova IN, Cadet JL (2009) Methamphetamine toxicity and messengers of death. Brain Res Rev 60: 379–407. - PMC - PubMed
    1. Colfax G, Shoptaw S (2005) The methamphetamine epidemic: implications for HIV prevention and treatment. Curr HIV/AIDS Rep 2: 194–199. - PubMed
    1. Barr AM, Panenka WJ, MacEwan GW, Thornton AE, Lang DJ, et al. (2006) The need for speed: an update on methamphetamine addiction. J Psychiatry Neurosci 31: 301–313. - PMC - PubMed
    1. Albertson TE, Derlet RW, Van Hoozen BE (1999) Methamphetamine and the expanding complications of amphetamines. West J Med 170: 214–219. - PMC - PubMed
    1. Ricaurte GA, Schuster CR, Seiden LS (1980) Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: a regional study. Brain Res 193: 153–163. - PubMed

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Source of funding: National Health Research Institutes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.