Kyllinga nemoralis is an ignored plant distributed in tropical, subtropical, and warm temperature regions around the world. K. nemoralis plant is rich with bioactive molecules exhibiting antiherpetic properties. This study determined the antiviral activities of K. nemoralis stem with leaves aqueous extract against herpes simplex virus type 1 (HSV-1). The stem with leaves of K. nemoralis plant was extracted using aqueous extraction method. The cytotoxicity of K. nemoralis extract was evaluated using a cell viability assay. Plaque reduction assays were carried out to estimate the antiviral activity of K. nemoralis extract against HSV-1. Antiviral studies consisted of post-treatment, pre-treatment, and virucidal assay. Cell viability assay revealed K. nemoralis stem with leaves aqueous extract is non-toxic toward Vero cells. K. nemoralis stem with leaves aqueous extract inhibited HSV-1 replication in Vero cells with 50% effective concentration (EC 50 ) = 0.004 mg/mL and selective index (SI) = 14 when given after adsorption to the cells. The EC 50 against HSV-1 was 0.013 mg/mL and SI = 4.31 when cells were treated 24 h before virus infection. K. nemoralis stem with leaves aqueous extract exhibited direct virucidal activity against HSV-1 with EC 50 = 0.0065 mg/mL and SI = 8.62. The results presented the potential of K. nemoralis as an anti-HSV-1 agent by the following modes: direct damage to viral particle, interruption of virus attachment, and suppression of viral replication cycle.

Human Herpesvirus 1 known as herpes simplex virus type 1 (HSV-1) is belonged to the Herpesviridae family, Alphaherpesvirinae subfamily, and Simplexvirus genus. Kyllinga nemoralis, known as whitehead spike sedge, white kyllinga, white globose spike, or poverty grass, is a monocotyledonous flowering graminoid similar to grasses and rushes. Acyclovir (ACV) did not eradicate HSV-1 infection as the drug only focuses on inhibiting the production of new viral genomes by interfering with viral DNA synthesis. This study aimed to identify the cytotoxic concentration (CC50) of K. nemoralis roots aqueous extract and evaluate its antiviral activities against HSV-1.    Cytotoxicity assay involved the incubation of Vero cells with 10 concentrations of extract ranging between 0.002 mg/mL and 1.000 mg/mL. MTT solution was added and absorbance was measured at 540 nm [1]. The CC50 of extract was determined from the graph of cell viability (%) against extract concentrations. Antiviral assays of post-treatment, pre-treatment and virucidal tests determined the mode of antiviral activity. The 50 % effective concentration (EC50) of extract was derived from the graph of plaque inhibition (%) against concentrations. Selectivity index (SI) was evaluated as the ratio of CC50 to EC50 [2].    Post-treatment assay: Cells were inoculated with HSV-1 for 2 hours. Extract with (2% DMEM and 1% MCS) was added into cells and incubated for 48 hours.  Pre-treatment assay: Cells were treated with extract in 5% DMEM for 24 hours and inoculated with HSV-1 for 1 hour 30 minutes. Then, cells were overlaid with (2% DMEM and 1% MCS) and incubated for 48 hours.  Virucidal assay: HSV-1 was incubated with extract for 30 minutes. Then, virus-extract suspension was inoculated into cells and overlaid with 2% DMEM and 1% MCS for 48 hours.    After 48 hours incubation, cells were stained with crystal violet solution and incubated at room temperature for 30 minutes. The plaques were counted and percentage of plaque inhibition (%) was calculated.  The CC50 revealed aqueous extract concentration was able to kill at 50% of cell population. Post-treatment assay showed that 0.062 mg/mL of the extract inhibited intracellular viral replication. Pre-treatment study indicated that 0.02 mg/mL of extract produced protective effects towards cells. Finally, virucidal assay demonstrated that 0.222 mg/mL of extract initiated extracellular interaction with virus in inhibiting infection [3] as shown in Table 1.  The cytotoxicity study showed that CC50, 0.72 mg/mL was toxic on cells. Therefore, lower concentrations between 0.016 mg/mL to 0.5 mg/mL, that were least or non-toxic to the cells were utilized in the subsequent antiviral screenings [4]. The extract showed good inhibition of intracellular virus replication with an EC50 of 0.062 mg/mL and SI more than 10 which is 11.61. Similarly, in pre-treatment investigation, the extract produced excellent antiviral activity with an EC50 of 0.02 mg/mL and corresponding SI of 36.00. This finding highlight that the extract is most effective when administered as pre-treatment. Antiviral activity exhibited by roots extract probably results from the inhibition of host cell glycoprotein receptors or due to prevention of binding between HSV-1 virions and host cells [5]. Virucidal assay revealed a weak extracellular interaction between HSV-1 and extract. It was supported by the low SI, 3.24 as shown in Figure 1.1. The finding suggests that 30 minutes of virus-extract exposure could be insufficient for roots extract to show an effective inhibition of virus [2]. In conclusion, the present study revealed that K. nemoralis roots aqueous extract inhibits in-vitro productive HSV-1 infection through three different modes that are effective prevention of intracellular viral replication, excellent interaction with host cell surface to inhibit HSV-1 infection and moderate direct destruction the virus particles.