Antiviral properties of blue laser in an in vitro model of HSV‐1 infection

Herpes simplex virus type-1 is known to cause lifelong infections in humans. The initial infection is characterized by gingival stomatitis and pharyngitis, whereas reactivation of the virus causes recurrent herpes labialis with ulceration on the intraoral mucosa, mouth, or external facial skin 1. Laser therapy set at red and infrared wavelengths can reportedly reduce the frequency of HSV-1 recurrence and duration of herpetic sores 2. Despite evidence that the blue wavelength is effective in killing different strains of bacteria, it has never been tested on viruses 3. We explored the antiviral properties of blue light in an in vitro model in which we infected human keratinocytes cell line (HaCaT) with HSV-1. The experimental settings employed are reported in Figure 1a 4. Blue LT is effective against HSV-1: we measured an increment in cell vitality and a decrease in HSV-1 viral load in supernatants of cells infected with virus previously irradiated respect to cells infected with virus not irradiated (P = 0.0002 and P = 0.0007 respectively, Setting 1, Fig. 1a–c). Cytotoxicity tests to detect the most effective LT protocol tolerated, indicated that the following parameters are optimal: 0.15 W/cm2, 30 J/cm2, 5 Hz. Using this protocol, blue LT decreased the viral load without affecting the cells’ vitality (P = 0.02, Setting 2, Fig. 1a,d,e). In contrast, Settings 3 and 4 (irradiation of cells prior to infection and irradiation of cells that had been infected 24 hr previously, respectively; Fig. 1a) had no effect on viral load or cell vitality (data not shown). Finally, in cells infected 1 hr prior to irradiation, LT reduced viral load, but not to a statistically significant degree (Setting 5, Fig. 1a,f,g). The results were confirmed by plaque-forming assay (Fig. 1h). Our findings show the blue laser has antiviral activity against HSV-1 and it is more effective against virus irradiated alone, whereas it had only a slight effect on infected cultures, suggesting that LT inactivates the virus prior to cell entry. In contrast, when the virus is already inside the cells, the effect of LT is less evident. Finally, LT does not increase cells’ resistance to infection. Various studies have reported the ability of LT to both reduce frequency of HSV-1 recurrence and facilitate healing of oral vesicles 2. In only one study, which was on HaCaT cells, was infrared laser light found to reduce viral replication and increase pro-inflammatory molecules in HSV-1 infected and irradiated cells, indicating that LT may activate anti-viral immune responses and inhibit spread of virus 5. Unfortunately, the exact mechanism of LT anti-viral effect is still unknown; researchers have hypothesized that it involves an immuno-modulatory effect rather than a direct anti-viral effect. Our results show that blue LT has a direct inhibitory effect on the virus itself. Thus, further studies are warranted to determine how blue LT exerts its anti-viral effect, the aim being to move from an in vitro to a clinical setting, thus promoting its use on HSV-1 infected patients. Trieste University (U22SCFRA15) and IRCCS Burlo Garofolo (RC 15/2017) supported this study. G. Ottaviani has part-time employment in K-Laser d.o.o. (Sežana, Slovenia). The other authors have no conflicts of interest. Additional supporting information may be found in the online version of this article at the publisher's web-site. Fig. S1. Thermal images acquired during irradiation using the following protocols: left 445 nm, 0.3 W/cm2, 60 J/cm2, CW; right 445 nm, 0.15 W/cm2, 30 J/cm2, 5 Hz. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.