The infection process for many viruses, including HSV-1, it should naturally be an attractive model for an antiviral defensemechanism. And of course, blocking entry has the added advantage of minimizing or eliminating all of the following steps in the infectious cycle. In these experiments SnO2 Epigenetic Reader Domain nanowires have shown an ability to compete for virus at the attachment step by acting like the natural target (HS), similar to what ZnO, Ag-MES, and Au-MES do. Since the amount of virus that enters the cell has a direct relationship with disease severity and reactivation rates, minimizing or blocking the viral load with SnO2 is certainly expected to substantially reduce the distressful and sometimes agonizing results of an untreated infection. In keeping with its emerging biological applications [14] and relatively non-toxic nature under in vitro conditions (Figure 2), the SnO2 nanowires used here were found to be an effective inhibitor of viral entry and cell-to-cell spread. The concentrations of 
SnO2 used in our study were well below any Epigenetic Reader Domain significant cytotoxic levels. The results on average showed a 75 reduction in cell entry, 77 smaller plaques or infected cell clusters and over a 99 drop in cell-to-cell fusion. Reduced entry also translated into reduced replication and spread to other cells. We consider these results very promising for any future development of SnO2 nanowires as antiHSV agents, especially as new and effective prophylactic agents. In this regard it will also be very interesting to test their efficaciesTin Oxide Nanowires as Anti-HSV AgentsFigure 3. SnO2 inhibits HSV-1 entry into HCE cells. HCE cells were mock treated or treated with SnO2 and exposed to HSV-1 at an MOI of 10 for 6 hours. A) After 6 hours of infection cells were washed, permeabilized and incubated with ONPG substrate for quantification of b-galactosidase activity from the viral genome. A dosage dependent decrease in entry was noted in cells as minimal entry occurred. B) X-gal staining of HCE cells. HCE cells grown in a 6-well plated were pretreated with SnO2 before being challenged with HSV-1 for 6 hours. Cells were washed with PBS, fixed, permeabilized and incubated with X-gal, yielding blue cells. Infected cells were imaged at 1081537 106 objective using Zeiss Axiovert microscope. C) The average number of infected cells in SnO2 treated cells is significantly lower than mock treated cells. doi:10.1371/journal.pone.0048147.gagainst other viruses and microbes that use HS for attachment to cells [15]. Although this antiviral mechanism is not unique to SnO2 nanowires it might be more cost effective and tolerable as compared to the other nanoparticles previously mentioned. Future considerations should be made to rank the effectiveness of these antivirals in-vitro, followed by in-vivo studies and formulation trails. Combination therapy could also improve the results as SnO2 and ZnO used together could potentially decrease the cytotoxicity while enhancing the efficacy of the treatment. It might also be possible to present these compounds as virus trappers that stimulate immune response while providing protection from virus infection as microbicides. The combined effect would lead to improved viral clearance and overall antiviral effectiveness. Inconclusion, SnO2 nanowires show the novel promise to prevent and/or 
reduce the complications associated with HSV infection and represent a new class of anti-viral agents that require further testing in animal models and against oth.The infection process for many viruses, including HSV-1, it should naturally be an attractive model for an antiviral defensemechanism. And of course, blocking entry has the added advantage of minimizing or eliminating all of the following steps in the infectious cycle. In these experiments SnO2 nanowires have shown an ability to compete for virus at the attachment step by acting like the natural target (HS), similar to what ZnO, Ag-MES, and Au-MES do. Since the amount of virus that enters the cell has a direct relationship with disease severity and reactivation rates, minimizing or blocking the viral load with SnO2 is certainly expected to substantially reduce the distressful and sometimes agonizing results of an untreated infection. In keeping with its emerging biological applications [14] and relatively non-toxic nature under in vitro conditions (Figure 2), the SnO2 nanowires used here were found to be an effective inhibitor of viral entry and cell-to-cell spread. The concentrations of SnO2 used in our study were well below any significant cytotoxic levels. The results on average showed a 75 reduction in cell entry, 77 smaller plaques or infected cell clusters and over a 99 drop in cell-to-cell fusion. Reduced entry also translated into reduced replication and spread to other cells. We consider these results very promising for any future development of SnO2 nanowires as antiHSV agents, especially as new and effective prophylactic agents. In this regard it will also be very interesting to test their efficaciesTin Oxide Nanowires as Anti-HSV AgentsFigure 3. SnO2 inhibits HSV-1 entry into HCE cells. HCE cells were mock treated or treated with SnO2 and exposed to HSV-1 at an MOI of 10 for 6 hours. A) After 6 hours of infection cells were washed, permeabilized and incubated with ONPG substrate for quantification of b-galactosidase activity from the viral genome. A dosage dependent decrease in entry was noted in cells as minimal entry occurred. B) X-gal staining of HCE cells. HCE cells grown in a 6-well plated were pretreated with SnO2 before being challenged with HSV-1 for 6 hours. Cells were washed with PBS, fixed, permeabilized and incubated with X-gal, yielding blue cells. Infected cells were imaged at 1081537 106 objective using Zeiss Axiovert microscope. C) The average number of infected cells in SnO2 treated cells is significantly lower than mock treated cells. doi:10.1371/journal.pone.0048147.gagainst other viruses and microbes that use HS for attachment to cells [15]. Although this antiviral mechanism is not unique to SnO2 nanowires it might be more cost effective and tolerable as compared to the other nanoparticles previously mentioned. Future considerations should be made to rank the effectiveness of these antivirals in-vitro, followed by in-vivo studies and formulation trails. Combination therapy could also improve the results as SnO2 and ZnO used together could potentially decrease the cytotoxicity while enhancing the efficacy of the treatment. It might also be possible to present these compounds as virus trappers that stimulate immune response while providing protection from virus infection as microbicides. The combined effect would lead to improved viral clearance and overall antiviral effectiveness. Inconclusion, SnO2 nanowires show the novel promise to prevent and/or reduce the complications associated with HSV infection and represent a new class of anti-viral agents that require further testing in animal models and against oth.
