Herpes genitalis, caused by HSV-2, is an incurable genital ulcerative disease transmitted by sexual intercourse. The virus establishes life-long latency in sacral root ganglia and reported to have synergistic relationship with HIV-1 transmission. Till date no effective vaccine is available, while the existing therapy frequently yielded drug resistance, toxicity and treatment failure. Thus, there is a pressing need for non-nucleotide antiviral agent from traditional source. Based on ethnomedicinal use we have isolated a compound 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole (HM) from the traditional herb Ophiorrhiza nicobarica Balkr, and evaluated its efficacy on isolates of HSV-2 in vitro and in vivo. The cytotoxicity (CC 50 ), effective concentrations (EC 50 ) and the mode of action of HM was determined by MTT, plaque reduction, time-of-addition, immunofluorescence (IFA), Western blot, qRT-PCR, EMSA, supershift and co-immunoprecipitation assays; while the in vivo toxicity and efficacy was evaluated in BALB/c mice. The results revealed that HM possesses significant anti-HSV-2 activity with EC 50 of 1.1-2.8 µg/ml, and selectivity index of >20. The time kinetics and IFA demonstrated that HM dose dependently inhibited 50-99% of HSV-2 infection at 1.5-5.0 µg/ml at 2-4 h post-infection. Further, HM was unable to inhibit viral attachment or penetration and had no synergistic interaction with acyclovir. Moreover, Western blot and qRT-PCR assays demonstrated that HM suppressed viral IE gene expression, while the EMSA and co-immunoprecipitation studies showed that HM interfered with the recruitment of LSD-1 by HCF-1. The in vivo studies revealed that HM at its virucidal concentration was nontoxic and reduced virus yield in the brain of HSV-2 infected mice in a concentration dependent manner, compared to vaginal tissues. Thus, our results suggest that HM can serve as a prototype to develop non-nucleotide antiviral lead targeting the viral IE transcription for the management of HSV-2 infections.

Funding: The authors acknowledge financial support from the Department of Biotechnology (BT/PR13759/PBD/ 17/686/2010) and the Indian Council of Medical Research (45/2 /2010/PHA -BMS), New Delhi. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Since 1974, HSV infections are managed with the nucleoside analogue acyclovir (ACV) which phosphorylated in the infected cells by viral thymidine kinase [ 12 ]. However, ACV fails to eradicate the virus from the infected cell or prevent recurrences, as it cannot counteract in the early stage of HSV infection [ 13 ]. Additionally, long term use of ACV or related drugs leads to the frequent development of drug resistant viruses, especially in immunocompromised individuals [ 16 , 17 ], implicating an increasing risk of HSV recurrence and treatment failure [ 18 - 20 ]. Moreover, till date no effective therapy [ 14 ] or vaccine [ 15 ] is available. Thus, new drugs with novel mode of action are required for the management and prevention of HSV infections. Here, we report the isolation of an antiviral compound from Ophiorrhiza nicobarica (International plant index Id: 758538-1), a traditional herb used by the Shompen and Nicobarese tribes of the Nicobar Islands, India, against skin ailments [ 21 ], having antimicrobial and antiinflammatory activities [ 22 , 23 ] with significant anti-HSV-2 activity. Further, we have demonstrated the in vitro mode of action and therapeutic efficacy of the isolated compound in Balb/C mice vaginally infected with HSV-2.

Genital herpes is an incurable genital ulcerative disease, usually caused by herpes simplex virus type 2 (HSV-2), and represent one of the most serious public health concerns globally. The virus is transmitted during sexual intercourse and replicates in the genital epithelium, with life-long latency in dorsal ganglia [ 1 ]. More than 90% of genital herpes are caused by HSV-2 infections; which spread through unprotected sex, periodically reactivated and may cause fatal infections like acute encephalitis and meningitis in neonates and immune deficient patients [ 2 ]. It is reported that the immediate-early (IE) genes of HSV activate HIV-1 [ 3 ], Varicella-zoster virus [ 4 ] and human papillomavirus type 18 [ 5 ] genes, and is a significant risk factor for HIV/AIDS transmission [ 6 ]. Moreover, HSV‐2 can cross the placental barrier and affect foetus in early pregnancy, leading to spontaneous abortion or mental retardation of the foetus [ 7 ]. Furthermore, HSV-2 is known to transform infected cells into tumor cells [ 8 ] and have synergistic relationship with HIV disease progression [ 9 ]. Young women are biologically more vulnerable to genital infections than men to get these infections during vaginal intercourse [ 10 ]. A recent study showed that HSV-suppressive therapy can greatly reduce genital and plasma HIV-1 RNA load in co-infected patients [ 11 ], indicating that the risk of acquisition or transmission of HIV infection can be greatly decreased by reducing the spread of genital herpes.

Materials and Methods

Plant materials Ophiorrhiza nicobarica Balkr. (Rubiaceae), a wild perennial herb, was collected from the Galathia River village, Great Nicobar Islands, India, in which no specific permissions were required as it was within the human habitat and not under any protected area. The herb, not under protected species, was identified by Dr. Sreekumar, Scientist, Botanical Survey of India (BSI), Andaman Nicobar Circle, Port Blair, and deposited in the Herbarium collection (Herbarium No. 9227) of the BSI, Port Blair, India.

Extraction, fractionation and identification of compound Alcoholic extracts were prepared from the dried and coarsely powdered herb (100 g) with 1 L of MeOH (95%) for 48-72 h in a soxhlet extractor, collected, filtered and evaporated in vacuo to a residue at 40-45°C, with a yield of 7.8±0.2% (w/w). A part of the residue was stored in a dessicator for further study and the other part (32 g) was suspended in water and extracted with n-butanol, and then separately monitored by TLC. The n-butanol fraction (24 g) was subjected to Silica-gel CC, eluting with petroleum ether (PE), PE:CHCl 3 , CHCl 3 , and CHCl 3 :MeOH (at different ratios) and MeOH. The eluted fractions A (7 g), B (6 g) and C (8 g) were repeatedly subjected to Silica gel CC and monitored by TLC. Fraction A was positive for terpenoid, fraction B to phytosterol and fraction C for alkaloid. All fractions were separately isolated and subjected to CC by aluminium oxide eluted with PE, PE:CHCl 3 , CHCl 3 , and CHCl 3 :MeOH (at different ratios) on TLC. Fractions A and B were combined, condensed, recrystallized and identified by IR, NMR and mass spectroscopy [24]. The 1H and 13C NMR spectra recorded at 600 and 150 MHz in a Bruker AVANCE600 spectrometer using C 5 D 5 N with TMS as internal standard, and ESI-TOF mass on a Q-TOF-Micromass spectrometer, indicated that the compounds are ursolic acid (fraction A) and β-sitosterol (fraction B). The fraction C was then added with NH 3 (25%) to make it alkaline (pH 9) and then dissolved in chloroform with shaking. Finally, the chloroform phase was evaporated to obtain a total alkaloid extract (8 g), which was chromatographed on a silica gel column (3.5 ∞ 90 cm), using a linear gradient of CHCl 3 -MeOH system, and collected as 5 subfractions: 9.5-0.5, 9-1, 8.5-1.5, 8-2, and 7.5-2.5. These subfractions were filtered, concentrated and subjected to purification by TLC using precoated silica gel plates with CHCl 3 :MeOH:NH 3 (50:50:3) solvent system. TLC separation of fractions demonstrated two bands with R f of 0.33 and 0.63 of which the R f 0.33 band showed antiviral activity and thus, identified by 13CNMR 1,HNMR and mass spectroscopy.

Cells and viruses Vero cells (African green monkey kidney cells; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 5-10% fetal bovine serum (FBS; Invitrogen, USA), 100 U/mL penicillin and 100 µg/mL streptomycin, at 37°C in 5% CO 2 . The viral strains used were HSV-2G (ATCC 734), purchased from the ATCC, along with the clinical isolates 1-4 and TK-deficient HSV-2, which were kindly provided by Professor P.K. Dutta and Professor M. Sengupta, Calcutta Medical College & Hospital, Kolkata, India. Virus stocks were prepared from infected culture at a multiplicity of infection (moi) of 0.5 for 1 h at 37°C. The residual viruses were then washed out with phosphate-buffered saline (PBS) and the cells were cultured for another 48-72 h. The cultured cells were lysed finally by three cycles of freezing and thawing, centrifuged at 1500 g at 4°C for 20 min and the collected supernatant was tittered by plaque assay, and stored at -80°C for further studies.

Cytotoxicity assay To determine the effect of fraction C and its isolated compound on uninfected cells, cultured Vero cells (104 cells/well) in 96 well plates were exposed to various concentrations of the test drugs, in triplicate and incubated at 37°C in 5% CO 2 , using ACV and dimethylsulfoxide (DMSO, 0.1%) as controls. After 48 h, 10% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) was added to each well with DMEM, incubated for 3-4 h, and then mixed with 100 µl of MTT solubilizing solution (Sigma), and the optical density (OD) was read by a plate reader at 570 nm with a reference wavelength of 690 nm. Data were calculated as the percentage of cell viability using the formula: [(sample absorbance-cell free sample blank)/mean media control absorbance)]X100%. The 50% cytotoxic concentration (CC 50 ) causing visible morphological changes in 50% of Vero cells with respect to cell control was determined from the concentration-response curves after viable cell count [24].

Antiviral and dose-response assay In the antiviral assay Vero cell (104 cells/well) culture on 96-well plates were infected with HSV-2G and the clinical isolates (0.5 moi) separately, and then exposed to the various concentrations of the test compound and ACV, in triplicate, and incubated for 72 h at 37°C in 5% CO 2 . The MTT assay was carried out, as described above and viral inhibition rate was calculated as: [(A tv -A cv )/(A cd -A cv )]X100%, where A tv indicates the absorbance of test compound with virus-infected cells. A cv indicates the absorbance of virus control, and A cd the absorbance of the cell control. The antiviral concentration of 50% effectiveness (EC 50 ) was defined as the concentration that achieved 50% inhibition of virus-induced cytopathic effects [24]. For dose response assay the Vero cells were infected with HSV-2 isolates at 100-300 plaque-forming units (pfu) for 1 h at room temperature. Then the test compound at different concentrations (0-50 μg/ml) was added and the mixture was incubated at 37°C for 1-2 h, and virus yields were determined by plaque assay after 48 h of incubation [25]. The 50% inhibitory or effective concentration (EC 50 ) was calculated from dose-response curves. Antiviral activities were also estimated by selectivity index (SI) calculated from CC 50 and EC 50 values.

Plaque reduction assay The Plaque reduction assay was used to evaluate the antiviral efficacy, using ACV and DMSO (0.1%) as positive and negative control respectively. Vero cell monolayers in twelve well plates, infected with wild type and clinical isolates of HSV-2 (100 pfu) were exposed to serial dilutions of test compound and then overlaid with 1% methylcellulose (Fluka, USA) to form plaques. The plaques developed after 72 h of incubation were fixed with 4% paraformaldehyde and stained with methylene blue (0.03%) in 70% methanol. The virus titers were calculated by scoring the plaque-forming units (pfu). The effective concentration of test compound that inhibited the number of viral plaques by 50-100% (EC 50 and EC 100 ) was interpolated from the dose-response curves [25].

Time-of-addition assay Following three different approaches, Vero cells in twelve well plate (4x105) were exposed to the test compound (5.0 µg/ml) before infection, during infection or after infection with HSV-2G (100 pfu/well) at 0-24 h time intervals in triplicate, using DMSO (0.1%) and ACV (5.0 µg/ml) as controls. For pre-infection Vero cells were treated with the test compound either for 1 h or for 3 h, washed with PBS and then infected with HSV-2G in DMEM containing 2% FBS at 37°C. After 1 h adsorption the cells were covered with overlay media for plaque assay. For co-infection Vero cells were subsequently infected and treated with the test compound and after 1 h of incubation the virus-drug mixture was removed, washed with PBS three times, added with fresh media and subjected to the plaque assay. While for post-infection (p.i) the cells were infected with the virus first, allow to adsorb (1 h) and then treated with the test compound at intervals of 2, 3, 4, 5, 6, 8, 12, 24 h post infection. Finally the cells were harvested after 24 h for plaque assay [25].

Immunofluorescence assay The HSV-2G (0.5 moi) infected Vero cell monolayer was treated with two different concentrations of the test compound (1.5 and 5.0 µg/ml) at 2 h and 4 h p.i, and washed twice with PBS to remove the cell debris. The cells were then fixed with paraformaldehyde (4%) and blocked with 1% bovine serum albumin (BSA) in 0.1% PBS-Triton X100 solution. The cells were further washed with PBS, then permeabilized with 0.1% Triton X100 in PBS, and incubated either overnight at 4°C or 1 h at room temperature with FITC-labelled polyclonal rabbit anti-HSV-2 antibody (Dako Cytomation, Denmark) to tag the drug treated virus. The cells were then washed with PBS and mixed with DAPI (Dako Cytomation, Denmark) to visualize both the virus and cell nucleus under Axio Imager M1 (Carl Zeiss, USA) inverted epifluorescence microscope [26].

Attachment and penetration assay To investigate whether the compound have any effect on viral adsorption or attachment, Vero cell monolayer in six well plate (106) were prechilled at 4°C for 1 h and subsequently challenged with HSV-2G (200 pfu/well) in the presence of test compound (5.0 µg/ml), DMSO (0.1%) or ACV (5.0 µg/ml) for 3 h at 4°C. After infection, the wells were washed twice with ice-cold PBS to remove unbound virus, and overlaid with 1% methylcellulose to allow plaque formation. The plaques developed after 72 h of incubation were stained and counted [25,27]. For viral penetration assay prechilled (at 4°C for 1 h) Vero cell monolayer in six well plate were subsequently incubated with HSV-2G (300 pfu/well) for 3 h at 4°C to allow viral adsorption. The infected cells were then incubated with test compound (5.0 µg/ml), DMSO (0.1%) or ACV (5.0 µg/ml) for another 20 min at 37°C to facilitate viral penetration. At the end of the incubation period, extracellular non-penetrated virus was inactivated by citrate buffer (pH 3.0) for 1 min, and then the cells were washed with PBS and overlaid with overlay medium for plaque formation. The viral plaques developed after 48 h of incubation at 37°C were stained and counted [27].

Virus inactivation assay To determine the effect of test compound on the inactivation of virus particles, HSV-2G (104 PFU/ml) was treated with the compound (5.0 µg/ml) at 37°C for 1 h and then, the mixture was 50 times diluted with fresh DMEM containing 2% FCS to yield sub-therapeutic concentration of the test compound. The virus inocula were then added to Vero cell monolayer. As a comparison, HSV-2G was mixed with the test compound, diluted immediately to 50-fold (no incubation period), and added to Vero cells for infection. The 50-fold dilution served to titrate the drugs below their effective doses and prevent meaningful interactions with the host cell surface. After adsorption for 1 h at 37°C, the diluted inocula were discarded, the cells were washed with PBS twice and then overlaid with overlay media and were subjected to the plaque assay, as described above [27,28].

Combined effect of test compound with Acyclovir In order to analyze the combined effect of test compound (HM) and ACV on plaque formation the EC 50 of both the agents as well as at various concentrations of the compounds against HSV-2G were tested and the combined effect was examined by plaque assay. Duplicate culture of Vero cells were infected with 100 PFU/0.2 ml of HSV-2G for 1 h and the cells were overlaid with 5 ml of overlay medium with various concentrations of HM and/or ACV and then incubated at 37°C for 72 h. The cells were then washed and fixed (4% paraformaldehyde) and stained with methylene blue (0.03%) to count the numbers of plaques for the determination of 50% inhibitory concentration of the plaque number from a curve, while the combined treatment was analyzed by isobologram method [29]. The EC 50 was used to calculate the fractional inhibitory concentration (FIC) of the agents in combination. The interaction between test compound and ACV was interpreted according to the combined FIC index (FIC compound + FIC ACV ) as synergy (≤0.5), no interaction (0.5-4) or antagonism (>4).

Western blot analysis The HSV-2G (5 moi) infected Vero cells were treated with HM (5.0 µg/ml) at intervals of 2, 4, 6 and 8 h post-infection. After 24 h, equal amounts of protein (40 µg/sample) extract from whole cell were harvested in buﬀer (200 µl/well) containing 20 mM Tris (pH 7±0.5), 50 mM NaCl, 5% NP-40 and 0.05% DOC. The soluble fraction was then separated by centrifugation at 16000 g for 10 min at 4°C, subjected to SDS-PAGE and blotted to pre-equilibrated PVDF membrane (Thermo Scientific, USA). The membrane was then blocked in 5% NFDM in 1X TBST (20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween 20), rinsed and incubated with monoclonal anti-ICP4 or polyclonal anti-β actin (Shanta Cruz Biotech Inc., USA) antibody in 5% BSA at 4°C overnight. Immunoblotting was performed with peroxidase-labelled anti-rabbit polyclonal antibodies and visualized by ECL Western blot detection kit (Millipore, USA) [30].

Quantitative real-time PCR The HSV-2G (5 moi) infected Vero cells were treated with the test compound (1.5 and 5.0 µg/ml) for 2 h and 4 h intervals, and RNA was isolated immediately using RNeasy Mini kit (QIAGEN) following the manufacturer’s protocol. Then the total RNA (0.1 mg/ml) in RNase-free water in 20 µl of RT mix (containing 5X VILO Reaction Mix, 10X SuperScript Enzyme Mix and DEPC treated water) was subjected to cDNA synthesis using the GeneAmp PCR System 9600 (Perkin Elmer Corp, USA). The real-time PCR was performed with these products by using SYBR Green PCR Master Mix (Qiagen) following manufacturer protocol in a ABI Prism 7000 sequence detection system (Applied Biosystems, CA, USA). The PCRs were amplified at cycling conditions of: 95°C for 10 min and 40 cycles (15 s at 95°C, then 60 s at 60°C) in triplicate [31]. The sequences of primers used were as follows: ICP4 (5'-GACGTTGTGGACTGGGAAG-3' and 5'-ACTTAATCAGGTCGTTGCCG-3'); ICP27 (5'- CCTTTCTCCAGTGCTACCTG-3' and 5'-GCCAGAATGACAAACACGAAG-3') and GAPDH (5'-AAGGTCGGAGTCAACGGATT-3' and 5'-CTGGAAGATGGTGATGATGGGATT-3').

Electrophoretic mobility shift assay Oligonucleotide sequence 5′-GCATGCTAATGATATTCTTTG-3′ of the ICP0 promoter of HSV-2G was biotinylated using Biotin 3' end DNA labelling Kit (Thermo Scientific, USA). The nuclear extracts of HSV-2G infected Vero cells treated with the test compound (5.0 µg/ml) or DMSO (0.1%) for 2 and 4 h intervals were prepared. Reaction mixtures (20 μl) contained, in addition to 3 μg of nuclear extracts, 20 fmol of Biotin 3' end-labelled probe, 50 ng/μl of poly (dI-dC), 2.5% glycerol, 0.05% NP-40 (1%), 5 mM MgCl 2 , and 1X binding buffer. After incubation for 20 min at room temperature, reaction mixtures were applied to 4% polyacrylamide gels in 0.5X Tris-borate-EDTA (TBE) buffer at 4°C. The gel was then transferred to Nylon membranes using Semi Dry Transfer Cell (Bio-Rad, USA), and transferred oligos were immobilized by UV cross-linking for 10 min. For detection of bound oligos, membranes were blocked with blocking buffer followed by addition of Streptavidin-Horseradish Peroxidase conjugate and developed according to the manufacturer’s instructions (Thermo Scientific, USA) [32]. For supershift assays nuclear extracts were pre-incubated with HCF-1 polyclonal antibodies for 30 min on ice.

Co-immunoprecipitation assay The HSV-2G infected, untreated or test compound treated (5.0 µg/ml) cells for 4 h were washed with ice-cold PBS and then lysed in a solution containing 10 mM Tris (pH 8.0), 170 mM NaCl, 0.5% NP40 and protease inhibitors for 30 min on ice with subsequent three freeze/thaw cycles at -80°C to lyse the nuclei. Cell debris was then removed by centrifugation and the supernatants were precleared with protein A-coupled Sepharose beads for 2 h. The lysates were then immunoprecipitated with HCF-1 or LSD1 antibodies as well as isotype-matched control antibodies plus protein A-Sepharose for at least 4 h or overnight. Beads were washed four times with 1 ml of wash buffer (200 mM Tris at pH 8.0, 100 mM NaCl and 0.5% NP-40), once with ice-cold PBS and boiled in 2X loading buffer. Finally the proteins were resolved by SDS-PAGE before probed with indicated antibodies [33].

In vivo Toxicity study Male and female BALB/c mice (18-20 gm), acclimatized for 7-10 days with standard food and water ad libitum, housed in polypropylene cages in Animal House facility were used in accordance with the OECD guidelines accepted by the Committee for the purpose of control and supervision on experiments on animals (CPCSEA), Thiruvanmiyur, Chennai, India and as per the approval of the Institutional Animal Care and Use Committee (IACUC) of the Jadavpur University, Kolkata (Approval No: 367/01/C/CPCSEA). When required, the surgical procedures performed under Ketamine hydrochloride (100 mg/kg i.m.) anesthesia, and all efforts were made to minimize suffering. For acute toxicity studies, different concentrations of the test compound were administered orally to healthy 7-week-old BALB/c male or female mice, three times daily for 7 days, while for subacute toxicity study the animals were feed with the daily doses of the compound for 28 days. The control group (n=10) received normal saline, whereas the experimental groups (six groups, n=6) were administered with different doses of (25-150 mg/kg body weight) of the test compound and observed continuously for 72 h and then daily upto 30 days to record any change in weight, behaviour, sign of clinical toxicity or morbidity, and the LD 50 of test compound was calculated by the method of Reed and Muench [34]. During the acute toxicity study, when required cervical dislocation was used to euthanize animals and the criteria for euthanasia were (i) severe illness or the animals in a moribund state (ii) severe pain and respiratory distress (iii) abnormal vocalisation, aggressiveness, posture and movements (iv) self-induced trauma (v) rapid weight loss, severe dehydration and significant bleeding. The mortality was calculated on the 30th day, using weights and mortality data. Moreover, fresh blood was collected for the estimation of hematological and serum biochemical parameters by cardiac puncture, and then sacrificed to collect liver spleen and kidney for histopathological examination.

Preparation and stability test of ointment formulation The ointment of HM was prepared to evaluate its efficacy, in comparison with ACV ointment (5%, Cipla Ltd., Mumbai, India). The HM (25 and 50 mg) was mixed with simple ointment base, (100 g of petroleum jelly, Vaseline TM), by gentle trituration to get homogenized ointment of 0.25% and 0.5% w/w of active compound. To obtain the most stable ointment, the stability was evaluated at accelerated conditions as per the guidelines of the International Conference on Harmonization on Stability testing of New Drug Substances and Products, 27th October 1993. Further, the ointment samples were kept at different temperatures (40°C, 37°C, room temperature) for 45 days and observed periodically for physical changes like phase separation, development of objectionable color, odour, consistency etc. The accelerated deterioration of ointments was also evaluated by centrifugation at 10,000 rpm for 10 min [35]. The ointment samples resistant towards physical stability and centrifugation were also tested for spreadability by modified wooden block apparatus method [36]. The ointments were freshly prepared on every 5th day.

Skin irritation test To test the dermal hypersensitivity and related allergic manifestation of HM in ointment dosage form, three batches of female Balb/C mice (n=10) were used. About 20 mg of the HM ointment (0.5% w/w) or Vaseline base was applied on the shaved and cleaned dorsal area (100-150 mm2 area) of each animal. After 4 h the residual ointment was removed with warm water and blotted dry to observe the sign of inflammation and related redness, flash, flare and wheel correspond to hypersensitivity. For further confirmation of dermal toxicity of the test compound dorsal hair of Balb/C female mice (n=10) was shaved, removed with hair remover cream (Anne French, Wyeth Ltd., India), cleaned with luke warm water and dried with tissue paper. Then the naked skin (100-150 mm2 area) was abraded with a dermal (Seven-Star) needle and 0.1 g of ointment (0.25, 0.5%) was applied to the abraded area of cohorts of animals (n = 6). After 24 h the ointment was removed, washed with warm water and the animals were examined for erythema and edema within another 1 h. The animals were also observed upto the next 72 h for additional confirmation of toxicity.

HSV-2 genital herpes in mice Seven weeks old BALB/c female mice (n=6) were injected subcutaneously in the neck ruff with 100 µl of depo-medoxyprogesterone acetate (Sigma, USA) at 2 mg/mouse 5-7 days prior to infection to synchronize the estrus-cycle of the animals and facilitate infection. The HSV-2G stock was diluted and 10 µl of diluted suspension was inoculated to the vagina of each mouse by a sterile blunt ended micropipette tips. The mice were observed for 12 days to record the development of vaginitis or lethality. During this study, the intravenous (i.v.) administration of sodium pentobarbital (18% of 200 mg/kg) in a dose of 200 mg/kg was used to euthanize the animals showing the signs of severe illness or a moribund state, considering the following factors: (i) signs of severe pain and respiratory distress (ii) abnormal vocalisation, aggressiveness, posture and movements (iii) self-induced trauma (iv) rapid weight loss, severe dehydration and significant bleeding (v) skin ulceration with pain (vi) severe swelling including redness of vagina and its surrounding tissues along with hair loss in the genital area (vii) hind limb paralysis or other factors related to the animals in severe pain or distress. The median lethal dose (LD 50 ) was determined to be equal to 9 X 104 pfu [37].

Therapeutic Efficacy of Oral and Topical Application of HM in Mice Seven week old acclimatized female BALB/c mice were divided into two separate batches, each with five groups (n=20). First group served as control, while the groups II-V of both the batches were injected subcutaneously in the neck ruff with 100 µl of depo-medoxyprogesterone acetate (Sigma, USA) at 2 mg/mouse, 5-7 days prior to infection to synchronize the estrus-cycle and facilitate infection. The vaginal area of each animal was swabbed with calcium alginate and then inoculated with HSV-2G at 9 X 105 pfu (10 LD 50 ) in 10 µl volume, intravaginally with a blunt ended micropipette tips. After 3-4 h of viral inoculation the animals of group III and IV were treated orally with HM (0.25 and 0.5 mg/kg) and the group V with ACV (5 mg/kg body weight), twice for 7 successive days; while group II was served as infection control. Finally, five animals from each group (I-V) of the first batch were sacrificed on day 2, 4, 6 (during the treatment schedule), and 8th day (one day after complete treatment) for the determination of viral load. The collected samples were homogenized in PBS, centrifuged at 3000 rpm for 15 min and the virus yield in the supernatant was determined by plaque assay. The infected vaginal tissues from untreated and treated animals were collected for histopathological study as described above [37]. On the otherhand, the second batch of five groups (n=20) including control, infected, as well as infected and drug treated animals were observed for another twelve days, after the completion of treatment schedule, for the development of vaginitis or lethality. Genital pathology following HSV‐2 challenge was monitored daily and scored on a five-point scale: no signs of infection (0); slight redness of external vagina (1); swelling and redness of external vagina (2); severe swelling and redness of both vagina and surrounding tissue along with hair loss in the genital area (3); hind-limb paralysis (4); and death (5). To test the efficacy of HM or ACV ointments the animals were infected with HSV-2G (9 X 105 PFU), as described above. Following inoculation, a vaginal sample were collected from each animal by sterile cotton swab and transferred to 0.5 ml of PBS and stored at -20°C. The mice were divided into six groups (n=20) including two test groups (0.25 and 0.5% HM ointment) and one each of ACV control (5% ointment), vehicle control (ointment base), no treatment (virus control) and uninfected control group. Symptoms of viral vaginitis including topical edema of the vaginal tract with turbid secretions were observed on the third day of infection. Treatment starts on day 3 post-infection by applying 2 mg of ointment per mouse, twice daily for 7-days to the vaginal tract with sterile cotton swabs. The infected mice were observed for at least 30 days to determine their mortality and the number of days for mortality. The vaginal swab samples were obtained one day following the completion of the treatment, and from the deceased animals immediately following their death. The samples were then diluted five times in MEM and used to infect Vero cells. Samples that gave positive cytopathic effects were considered positive for HSV-2 [37]. Moreover, to assess the viral shedding, vaginal washes from infected mice were collected, diluted with fresh media, and then infected to Vero cells in six-well plates to determine the virus yield by the plaque reduction assay [37]. During the therapeutic efficacy study of the test compounds, sodium pentobarbital was administered i.v. to euthanize the animals showing the same signs and factors considered during the viral LD 50 determination.