Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-7

E extracted from control, Se- and Se+ Vero and SK-N-SH cells and GPx1 enzyme activity was measured at days 3, 7 and 10 post-induction of Se deficiency by using the cGPx1 assay kit. Data are reported as mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.005 compared to control cells. Analyses of GPx1 protein by Western blot. 50 μg of total protein extracted from Vero and SK-N-SH cells grown in control, Se- and Se+ media were separated on PAGE, followed by immunoblotting with anti-GPx1. Equal loading was confirmed by re-blotting the same membranes with anti-β-actin.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-1

E extracted from control, Se- and Se+ Vero and SK-N-SH cells and GPx1 enzyme activity was measured at days 3, 7 and 10 post-induction of Se deficiency by using the cGPx1 assay kit. Data are reported as mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.005 compared to control cells. Analyses of GPx1 protein by Western blot. 50 μg of total protein extracted from Vero and SK-N-SH cells grown in control, Se- and Se+ media were separated on PAGE, followed by immunoblotting with anti-GPx1. Equal loading was confirmed by re-blotting the same membranes with anti-β-actin.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-4

2 hr at day 7 post-induction of Se deficiency. After adsorbtion, the cells were washed and maintained in control M199 medium, Se- and Se+ media. Caspase 3/7 activity was analyzed using fluorogenic substrate at day 2 after infection in WNV-infected and mock-infected cells. The data are expressed as percentage increase of caspase activity in infected cells grown in control, Se-, and Se+ media as compared to corresponding mock-infected cells. *p < 0.05 and **p < 0.05 as compared to cells grown in control and Se- media, respectively. loss of mitochondrial membrane potential is represented as ratio of fluorescence at 590 and 535 nm measured by JC-1 staining at 48 and 72 hr after infection, and expressed as percentage decline in infected cells grown in control, Se-, and Se+ media as compared to corresponding mock-infected cells. *p < 0.05 and **p < 0.05 as compared to cells grown in control and Se- media, respectively. Cell viability of infected and mock-infected Vero cells at day 2 after infection was assessed by cell proliferation assay and percentage cell viability of WNV-infected control, Se- and Se+ cells was calculated by comparing to their respective mock-infected cells. *p < 0.05 as compared to WNV-infected Vero cells grown in control media. WNV-infected Vero cells grown in control, Se-, and Se+ media were analyzed for LDH levels at day 2 after infection and expressed as fold-change over levels present in mock-infected cells. *p < 0.05 and **p < 0.05 as compared to cells grown in control and Se- media, respectively. All the data are presented as mean ± SD of at least two independent infections performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-2

Se- and Se+ media at days 7 and 10 post-induction of Se deficiency were separated on PAGE, transferred onto nitrocellulose membranes and immunoblotted with antibodies specific to catalase, CuZnSOD and MnSOD. Equal loading of protein was validated by re-blotting the same membranes with anti-β-actin. The data is representative of three independent experiments. Increase in the expression of MnSOD in Se- and Se+ Vero cells was confirmed by qRT-PCR. cDNA template was synthesized from total RNA extracted from control, Se- and Se+ Vero cells at days 3, 7 and 10 post-induction of Se deficiency as described in the materials and methods and subjected to qRT-PCR using primers specific for MnSOD and β-actin. Changes in the levels of MnSOD transcripts in Se- and Se+ Vero cells were first normalized to β-actin and then the fold-change as compared to controls was calculated. Data are reported as mean ± SD of triplicate experiments.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-5

induction of Se deficiency and cell supernatants were harvested every 24 hr for 5 days. Viral RNA extracted from the cell supernatant was used to determine viral copy number by qRT-PCR and expressed as viral copy number per mL. Data represents mean ± SD of three independent infections. WNV-infected control, Se- and Se+ Vero cells grown and fixed on coverslips at day 2 post infection were incubated with monoclonal human anti-WNV env antibody and then with Alexa Fluor 488 conjugated goat anti-mouse secondary antibody. Mock infected Vero cells and infected Vero cells stained with secondary antibody alone , were used as a negative control. The experiments were performed in triplicate and , and represents WNV antigen staining in Vero cells grown in control, Se- deficient and Se- adequate media, respectively. Scale bar represents 10 μm at a magnification of 63× in all pictures.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-6

Ions as described in the materials and methods. Equal number of cells were seeded in 96-well plates and growth curve was measured by cell counting of control, Se-, and Se+, Vero and SK-N-SH cells for 5 days post-seeding. Cell viability of Vero and SK-N-SH cells at day 7 of the induction of Se deficiency was assessed by cell proliferation assay and percentage cell viability of Se- and Se+ cells was calculated by comparing to control cells. Data are expressed as mean ± SD from two separate experiments performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p