MRC-5 cells were obtained from the Korean Cell Line Bank (KCLB no. 10171). The recombinant laboratory pOka strain of VZV (VZV-pOka) expressing green fluorescent protein (VZV-pOka-GFP) and clinical YC01 strain of VZV (VZV-YC01) were kindly provided by Dr Chan-Hee Lee (Chungbuk National University, Cheongju, Korea) (11). Plant material (Lysimachia mauritiana Lam.) and the 70% ethanol extract used in the present study were collected from Jeju Island in Korea and provided by the Jeju Biodiversity Research Institute (Jeju, Korea; specimen no. JBR-324).

Fluorescence was examined and images analyzed using an inverted Nikon TS100-F fluorescence microscope (Tokyo, Japan) equipped with a digital camera and Nikon NIS-Elements microscope imaging software.

Total DNA was isolated using an Accuprep Genomic DNA Extraction kit (Bioneer, Daejeon, Korea), and VZV DNA was amplified and quantified in a StepOnePlus Real-Time PCR system (Applied Biosystem, Foster City, CA, USA) using HOT FIREPol^® EvaGreen quantitative PCR (qPCR) mix Plus (Solis BioDyne, Tartu, Estonia). The following primers were used for qPCR: VZV ORF62 forward, 5′-TCTTGTCGAGGAGGCTTCTG-3′ and reverse, 5′-TGTGTGTCCACCGGATGAT-3′; GAPDH forward, 5′-CATGAGAAGTATGACAACAGCCT-3′ and reverse, 5′-AGTCCTTCCACGATACCAAAGT-3′.

MRC-5 cells were inoculated with serially diluted VZV-YC01-infected MRC-5 cells and treated with either DMSO or LME at concentrations of 5, 10, 25, 50 µg/ml. At 6 days after infection, cells were fixed with 10% formaldehyde at room temperature for 10 min and stained with 0.3% crystal violet. After 5 h, the number of plaques was counted, and the virus titer expressed as plaque-forming units (pfu/ml).

VZV transcript levels were quantified using quantitative reverse transcription PCR (qRT-PCR). Total RNA was isolated using a HiGene™ Total RNA Prep kit (BIOFACT, Daejeon, Korea) and reverse-transcribed into complementary DNA (cDNA) with a QuantiTECT^® reverse transcription (RT) kit, according to the manufacturer's instructions (Qiagen, Hilden, Germany). cDNAs were amplified and quantified in a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using HOT FIREPol^® EvaGreen qPCR mix Plus (Solis BioDyne, Tartu, Estonia). The following primers were used for qRT-PCR: VZV ORF62 (immediate early, IE) forward, 5′-TCTTGTCGAGGAGGCTTCTG-3′ and reverse, 5′-TGTGTGTCCACCGGATGAT-3′; ORF28 (E) forward, 5′-CGAACACGTTCCCCATCAA-3′ and reverse, 5′-CCCGGCTTTGTTAGTTTTGG-3′; gB (L) forward, 5′-GATGGTGCATACAGAGAACATTCC-3′ and reverse, 5′-CCGTTAAATGAGGCGTGACTAA-3′; GAPDH forward, 5′-CATGAGAAGTATGACAACAGCCT-3′ and reverse, 5′-AGTCCTTCCACGATACCAAAGT-3′.

Cells were collected, fractionated and transferred onto nitrocellulose membranes, as described previously (12). Antibodies specific for VZV IE62 and tubulin were purchased from Abcam (Cambridge, UK) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Enhanced chemiluminescence detection reagent (Pierce, Rockford, IL, USA) and secondary peroxidase-labeled anti-mouse immunoglobulin G antibodies (Amersham Biosciences, Piscataway, NJ, USA) were used according to the manufacturer's specifications.

CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) was performed in keeping with the manufacturer's protocol.

To examine the antiviral effects of the 70% ethanol extract of Lysimachia mauritiana (LME) against VZV, uninfected MRC-5 cells were inoculated with cells infected with VZV-pOka-GFP at an MOI of 0.1, and treated with either 50 µg/ml DMSO or LME. At 0, 24, 48 or 72 h after infection, GFP fluorescence was measured under a fluorescence microscope. At 72 h after infection, significantly reduced VZV-pOka-GFP fluorescence was observed in the LME-treated cells (Fig. 1A). For quantitative analysis of VZV-pOka-GFP replication, the relative amounts of viral DNA were measured using qPCR with primers specific for ORF62. Compared to 0 h after infection, VZV DNA was amplified 170,000- and 280,000-fold at 48 and 72 h, respectively (Fig. 1B; compare 0 h with 48 and 72 h). Relative to DMSO-treated cells, VZV replication was downregulated 3.8- and 4.7-fold in LME-treated cells at 48 and 72 h after infection, respectively (Fig. 1B), clearly supporting an inhibitory effect of LME on VZV replication.

The plaque reduction assay was performed to further investigate the inhibitory activity of LME against the clinical isolate of VZV. Uninfected MRC-5 cells were inoculated with serially diluted VZV-YC01-infected MRC-5 cells and treated with either DMSO or LME at concentrations of 5, 10, 25 or 50 µg/ml. Cells were re-treated with DMSO or LME at 3 days after infection. At 6 days post-infection, cells were fixed and stained, and the plaque forming units (pfu) per ml were determined via counting. Interestingly, LME reduced the number of plaques by 2.3- and 17.3-fold at concentrations of 25 and 50 µg/ml, respectively, compared to cells treated with DMSO (0 µg/ml) (Fig. 2). The 50% inhibitory concentration (IC₅₀) of LME for VZV at which the number of plaques was reduced by 50% was calculated as 26.09 µg/ml.

To ascertain whether the inhibitory activity of LME on VZV replication was due to effects on cell viability, MRC-5 cells were treated with different concentrations of LME, and the levels of cellular ATP representing the presence of metabolically active cells were measured at 24, 48 or 72 h after treatment using the CellTiter-Glo assay. With prolongation of incubation time, cellular ATP levels were increased, possibly owing to cell proliferation. Interestingly, at 72 h post-treatment, the viability of MRC-5 cells treated with 25 µg/ml LME was similar to that of DMSO-treated cells (Fig. 3). Treatment with 50 µg/ml LME slightly reduced MRC-5 cell viability by 33 and 39% at 48 and 72 h, respectively (Fig. 3). Since LME exerted no significant adverse effects on MRC5 cell viability at the IC₅₀ value, it is unlikely that its anti-VZV activity of LME is related to cytotoxicity.

The effects of LME on VZV lytic gene expression were determined via qRT-PCR analysis of IE, E or L transcript levels. MRC-5 cells were inoculated with VZV-YC01-infected MRC-5 cells at an MOI of 0.1 and treated with either DMSO or LME at a concentration of 25 µg/ml. At 0, 24, 48 and 72 h after infection, the relative amounts of transcripts for ORF62 encoding IE62 (IE), ORF28 encoding a subunit of viral DNA polymerase (E) and glycoprotein B (gB) (L) were determined (Fig. 4). In DMSO-treated cells, ORF62, ORF28 and gB genes were induced at 24 h and higher levels expressed at 48 and 72 h after infection (Fig. 4). LME had no effect on VZV lytic gene expression at 24 h after infection (Fig. 4). However, at 48 and 72 h, ORF62, ORF28 and gB transcript levels were significantly reduced in the LME-treated cells (Fig. 4).

The protein level of IE62 encoded by ORF62, an essential transactivator of VZV lytic gene expression, was further determined. MRC-5 cells were inoculated with VZV-pOka-infected MRC-5 cells at an MOI of 0.1 and treated with either DMSO or LME at a concentration of 25 µg/ml. At 72 h after infection, IE62 protein expression was assessed by western blot analysis with a specific anti-IE62 antibody. IE62 protein was strongly induced in DMSO-treated cells (Fig. 5; compare lanes 2 and 1), and expression was significantly reduced by LME (Fig. 5; compare lane 4 with 2). Based on the collective results, we propose that LME downregulates IE62 protein expression and, in turn, negatively affecting VZV replication in vitro.