N-methylisatin-beta-thiosemicarbazone derivative (SCH 16) is an inhibitor of Japanese encephalitis virus infection in vitro and in vivo

Background During the early and mid part of 20th century, several reports described the therapeutic effects of N-methylisatin-β-Thiosemicarbazone (MIBT) against pox viruses, Maloney leukemia viruses and recently against HIV. However, their ability to inhibit flavivirus replication has not been investigated. Hence the present study was designed to evaluate the antiviral activity of 14 MIBT derivatives against Flaviviruses that are prevalent in India such as Japanese Encephalitis Virus (JEV), Dengue-2 (Den-2) and West Nile viruses (WNV). Results Amongst the fourteen Mannich bases of MIBT derivatives tested one compound – SCH 16 was able to completely inhibit in vitro Japanese encephalitis virus (JEV) and West Nile virus (WNV) replication. However no antiviral activity of SCH 16 was noted against Den-2 virus replication. This compound was able to inhibit 50% of the plaques (IC50) produced by JEV and WNV at a concentration of 16 μgm/ml (0.000025 μM) and 4 μgm/ml (0.000006 μM) respectively. Furthermore, SCH 16 at a concentration of 500 mg/kg body weight administered by oral route twice daily was able to completely (100%) prevent mortality in mice challenged with 50LD50 JEV by the peripheral route. Our experiments to understand the mechanism of action suggest that SCH 16 inhibited JEV replication at the level of early protein translation. Conclusion Only one of the 14 isatin derivatives -SCH 16 exhibited antiviral action on JEV and WNV virus infection in vitro. SCH 16 was also found to completely inhibit JEV replication in vivo in a mouse model challenged peripherally with 50LD50 of the virus. These results warrant further research and development on SCH 16 as a possible therapeutic agent.


Background
Flaviviruses are considered to be important pathogens responsible for significant human morbidity and mortality. The World Health Organization estimated that more than 50 million Dengue viral infections and 50,000 cases of Japanese encephalitis occur annually worldwide [1]. Severe manifestations of flavivirus disease include hemorrhagic fever, encephalitis and neurological sequelae. Despite the major clinical and public health impact of flaviviruses, there are no drugs available for chemoprophy-laxis or chemotherapy of these infections. The advent of potent combination antiretroviral therapy has been an important breakthrough in the treatment of HIV-1 infection, resulting in marked reductions in HIV-1-related morbidity and mortality [2]. This has rekindled interest in the search for antiviral agents for a variety of viral infections.
Earlier reports have described antiviral activity of some compounds against flaviviruses [3]. However, only a few of them have described both in vitro and in vivo activity of antiviral agents against flaviviruses [3]. Thiosemicarbazones were the first antiviral compounds recognized to have a broad-spectrum antiviral activity against a range of DNA and RNA viruses [4,5]. The use of N-methylisatin-βthiosemicarbazone (methisazone/marboran) as an effective antiviral drug in the chemoprophylaxis of small pox was demonstrated in human volunteers in South India as early as 1965 [6]. In several trials during Indian epidemics methizasone proved its value by reducing the attack rates by 75 to 95% [6]. Similarly, other studies have shown that Methyl isatin-β-diethylthiosemicarbazone inhibits replication of Moloney Leukemia Virus by interfering with the early phase of viral life cycle [7]. However, the antiviral activity of isatin thiosemicarbazone derivatives has not been evaluated against flaviviruses. Therefore, this study was undertaken to investigate if any of the N-methylisatin-β-thiosemicarbazone derivatives could suppress common flavivirus infections encountered in South India such as Japanese Encephalitis, Dengue and West Nile viral infections. The aim was not to develop a clinical protocol for therapy of these infections but rather to investigate the possibility of identifying antiviral agents that could target flavivirus multiplication.

Antiviral screening of compounds in vitro by cytopathic inhibition assay
Initially, the 50% Cytotoxic Concentration (CC 50 ) of the 14 MIBT derivatives and Ribavirin were determined on Porcine Stable kidney (PS) and Baby hamster kidney (BHK 21) cell lines and the results are depicted in Table 1. The antiviral activity of the 14 MIBT derivatives were initially evaluated against JEV, WNV and Den-2 using Cytopathic Effect (CPE) inhibition assay and it was observed that only SCH 16 showed inhibition of CPE. The structure of this MIBT derivative is depicted in Figure 1. Ribavarin, a known inhibitor of flavivirus was used as a control in all the experiments. Although there is no structural similarity between Ribavarin and SCH 16, we opted to use Ribavarin as a positive control in all experiments so that we have a reference value for comparing the results of SCH 16. These two compounds were then subjected to evaluation by the plaque reduction assay at non-cytotoxic concentrations (<CC 50 ). It was noted that SCH 16 and Ribavirin exhib-ited a dose depended reduction of plaques formed by JEV and WNV (Figure 2, Panels A and B) with an IC 50 of 16 µg/ ml (0.000025 µM) and 4 ug/ml (0.000006 µM) for JEV and WNV respectively. On the contrary the IC 50 of Ribavirin was 3.9 µg/ml (0.000016 µM) and 1.7 µg/ml (0.000007 µM) for JEV and WNV respectively. No antiviral activity of SCH 16 was noted against Den-2 although Ribavarin showed a dose dependent inhibition of Den-2 plaque formation ( Figure 2, Panels C and D).
The specificity of the action of an antiviral compound is determined by calculating the Therapeutic Index (TI), which is the ratio of CC50 to IC50. The TI of SCH 16 was 5 and 19 for JEV and WNV respectively while for Ribavirin it was 13 and 29 respectively. This suggests that SCH 16 is moderately active against JEV and highly active against WNV.

The kinetics of action of SCH 16 in relation to the replicative cycle of JEV in vitro
As a first step to understand JEV and SCH 16 interactions, experiments were designed to determine the kinetics of JEV replication in vitro. It was noted that the earliest appearance of JEV antigen in infected PS cells was at 10 hours post-adsorption as detected by IFA (data not presented). However, the first infectious progeny of virus was  Figure 3, Panels D to F).

Effect of SCH 16 on viral translation
To understand the probable action of SCH 16 on the viral replicative cycle and to study the extent of damage caused by the compound on the viral RNA that might result in the inhibition of viral events such as protein synthesis (translation), an in vitro translation experiment was carried out as described in materials and methods. RNA was extracted from drug treated (4 hours and 10 hours post infection) and untreated monolayers of JEV infected cells and subjected to Real Time PCR analysis to confirm the presence of JEV RNA. Subsequently, the viral RNA was subjected to in vitro translation. It was observed that RNA extracted from JEV infected cells treated with SCH 16 for 4 hours failed to translate into JEV proteins in vitro. On the contrary, viral RNA extracted from infected cells treated with SCH 16 at 10 hours as well as RNA from infected cells that were not treated with SCH 16 showed the presence of JEV proteins ( Figure 4).

In vivo evaluation of compounds against JEV using mouse model
After ascertaining the in vivo non-toxic concentrations in preliminary experiments, the therapeutic potential of SCH 16 was evaluated in mice using intracerebral and intraperitoneal challenge routes. In the intracerebral challenge model, mice that were treated with 100 and 200 mg/ kg body weight of SCH 16 showed no protection. However, it was interesting to note that all the mice that were treated with SCH 16 remained healthy up to day 6 postinfection without showing any apparent symptoms of JEV infection (data not presented). The symptoms started appearing in these mice from day 7 post-infection. There was a gradual progression of the symptoms and death occurred on day 9. On the other hand, untreated mice appeared sick by day 3 and succumbed by day 5. This suggests that there was a prolonged survival time of 3 days between the treated and untreated mice.
The prolonged survival time observed in the intracerebral challenge experiments prompted us to make use of a peripheral challenge model (JEV 50LD 50 ) using a multiple dosage regimen wherein 200, 400 and 500 mg/kg body weight of SCH 16 was administered by oral route. It was observed that, there was 25% protection in the group of mice administered with 200 mg/kg body weight of SCH 16, 50% protection observed in the group that received 400 mg/kg body weight and complete protection was observed in the group that were given with 500 mg/kg body weight of SCH 16 ( Figure 5). Mice that survived the challenge post treatment were sacrificed; brains harvested and subjected to virus isolation, detection of viral antigen and viral RNA. Viable virus could not be isolated from the brain tissue of these mice. Further, no viral antigen could be demonstrated in the brain smears by immunofluorescent staining using monoclonal antibodies to JEV. How-Butterfly structure of N-Methylisatin-β-Thiosemicarbazone derivative SCH 16 Figure 1 Butterfly structure of N-Methylisatin-β-Thiosemicarbazone derivative SCH 16.
ever, the RT-PCR products amplified from the brain homogenate suggested that viral RNA was present in the brain of animals that survived JEV infection following treatment with 400 and 500 mg/kg body weight of SCH 16.

Discussion
There is currently no specific antiviral treatment available for Japanese encephalitis, West Nile and Dengue virus infections. Recently there has been renewed interest in the search for antiviral compounds active against a variety of viral infections. For instance, there are several reports describing the in vitro inhibitory effect of compounds such as ribavirin, mycophenolic acid, imino sugars, inhibitors of serine protease, RNA interference and non-steroidal anti-inflammatory drugs against flaviviruses [8][9][10][11][12][13]. N-Methylisatin-β-thiosemicarbazone (MIBT) was one of the first antiviral compounds to be discovered. It exhibits Antiviral activity of Ribavirin and SCH 16 against JEV, WNV and Den-2 evaluated using the plaque reduction assay antiviral activity against a variety of RNA and DNA viruses [14][15][16][17]. Recent studies have demonstrated that thiosemicarbazone and Mannich bases of thiosemicarbazone derivatives exhibit anti-HIV activity in vitro [18][19][20][21][22]. Therefore this study was designed to investigate the antiviral property of isatin β thiosemicarbazone derivatives against JEV, WNV and Den-2 viruses.
In the present study, fourteen Mannich bases of MIBT derivatives were synthesized and evaluated for their ability to inhibit flaviviral replication. However, only one compound (SCH 16) showed antiviral activity against JEV and WNV in vitro with a therapeutic index of 5 and 16 respectively. This compound did not exhibit any virus inactivating property. SCH 16 ( Figure 1) is a mannich base of N-Methylisatin-β-thiosemicarbazone possessing an isatin backbone with modifications made at the side chains. Chemically isatins are diketonic compounds. It has been earlier noted that, heteroaromatic thioamides containing N-substitution at more than one position per heterocyclic ring are worthy of investigation due to its increased antiviral property [4]. It is therefore likely that the antiviral activity of SCH 16 may be due to the N sub-stitution at the 8th position in the heterocyclic benzene ring and a NO2 group attached to the aromatic side chain.
Although SCH16 exhibited antiviral activity against WNV, we did not pursue further experiments with it since WNV is not a public health concern in India. In contrast, JEV is a major public health problem in India and hence we set about to investigate in detail the mechanism of antiviral activity of SCH 16 against JEV. Two crucial questions pertaining to the antiviral activity of SCH16 against JEV were addressed; (i) how long after virus infection can addition of drug be delayed in vitro in order to achieve inhibition of virus replication? and (ii) what is the minimum time required for SCH16 to exert its antiviral activity?. For this purpose we used an experimental approach similar to that described earlier by Baginiski et al and Lammarre et al [23,24]. Our results showed that when the drug was beyond the 8 hour time point thereby suggesting that SCH 16 did not inhibit normal cellular functions (Figure 3, Panels A to C). This suggests that the drug was not toxic to cells and did not inhibit the ability of cells to support virus replication at later time points. To ascertain the minimum time required for SCH16 to exert its antiviral activity, the compound was added at 2 hours post infection and removed at various time points post viral entry. The results revealed that, SCH 16 probably acted as an inhibitor of early protein synthesis. Had SCH 16 been an uncoating inhibitor or a polymerase inhibitor, the drug would have required a contact time of less than 4 hours to bring about its inhibitory effect. Similarly if it were a protease inhibitor the minimum contact period for SCH 16 to bring about inhibition of virus replication would have been greater than 8-10 hours. Since we observed that the minimum contact period of 8 hours was required for SCH16 to completely inhibit virus replication, it probably indicates that the drug is acting at the level of translation. Cooper et al [25] in an earlier study with vaccinia virus had demonstrated that the specific antiviral effect of MIBT was noted 6 hours post-infection thereby indicating inhibition of viral protein synthesis. In order to ascertain whether this was indeed also true for SCH 16 we adopted another approach to investigate the precise role of SCH 16 on translation events in JEV replication. We obtained RNA samples from the experiments that treated JEV infected monolayer's with SCH 16 for 4 hours and SCH 16 added at 10 hours post infection from infected cells treated with SCH 16 as well as cells that were untreated using identical extraction protocols. Subsequently we performed Real Time SYBR Green I PCR using JEV specific primers to confirm the presence of JEV RNA in samples obtained from both drug treated as well as untreated cells. The viral RNA Survival graphs depicting the in vivo effect of SCH 16 against a lethal JEV challenge The therapeutic potential of SCH 16 against JEV was evaluated in vivo in mice using the intracerebral and intraperitoneal challenge studies. The mice that were evaluated in the intracerebral challenge route did not show any protection although there was a delay in appearance of symptoms and death in drug treated mice. The lack of protection by this route may be due to (i) the direct introduction of large amount of infectious virus (50LD 50 ) into the CNS which might have compromised the inhibitory action of SCH 16 and/or (ii) inability to achieve therapeutic concentrations of the drug in the brain either due to delay in the compound reaching the brain from the intraperitoneal compartment or poor penetration of the drug into the brain parenchyma. On the contrary, the drug treated mice challenged by the intraperitoneal route showed a dose dependent reduction in mortality, whilst all the untreated mice succumbed to the challenge with 50LD 50 of JEV by day seven ( Figure 5). Furthermore, neither viable virus nor viral antigen could be demonstrated in the brains of the mice that survived the challenge. However, viral RNA was detected by real-time RT-PCR in all the brain tissues. Since flavivirus RNA dependent RNA polymerases are active within three hours of viral entry this is not a surprise finding [27]. Because, SCH 16 is primarily an early translation inhibitor, it appears that this drug does not interfere with RNA polymerization resulting in accumulation of viral RNA in the brains of drug treated mice that survived the challenge. Alternatively, SCH 16 treatment could have curtailed JEV replication in the periphery resulting in a very small amount of JEV entering the brain. Consequently the virus was unable to establish a productive infection in the brain and the presence of viral RNA could be as a result of residual virus in brains of mice that survived the challenge. In an experimental rat model, with post-encephalitic Parkinsonism induced by JEV infections [28,29] it was observed that, administration of isatin improved the motor neuron activities significantly. Indeed, they attributed that the improvement in the motor weakness was probably due to the MAO inhibitory activity of isatin and suggested that isatin could possibly serve as a new therapeutic agent for Parkinsonism. However, these studies were not designed to address the antiviral action of isatin against JEV but aimed at investigating the neurotransmitter inhibitory effect. It may be argued therefore that the in vivo effect of SCH 16 against JEV noted in this study may also be attributed to the immunomodulating or neuroprotective property of SCH 16.
An intriguing observation in this study was the differential ability of SCH 16 to suppress JEV, WNV and Den-2 multiplication in vitro. It is difficult to hypothesize the differential antiviral property of SCH 16 noted against JEV and WNV in this study as they are structurally similar and we have used the same cell system (PS cells) for evaluating the drug. On the contrary, we used BHK 21 cells for assaying the antiviral activity of SCH 16 against Den-2 virus, which could have contributed to the lack of anti-Dengue activity of SCH 16. Protein synthesis consists of an intricate series of events requiring components that are too numerous to be encoded by viral genomes [30,31]. It has been observed that Den-2 and other flaviviruses, such as WNV, yellow fever, JEV, and Kunjin viruses, are presumed to undergo cap-dependent translation [32,33]. However, evidence exists that under certain conditions that inhibit cap dependent translation, Den-2 viruses can switch to more efficient cap independent translation. Further, mammalian cellular stress response and immune functions, such as the interferon antiviral response [34,35], may compel viral translation by one mechanism over the other. Since we used PS cells for evaluation of JEV and WNV and BHK 21 cells for Den-2 it is possible that the translation pathway adopted by Den-2 against SCH 16 may be due to the presence of certain BHK 21 cell specific factors. However, strong experimental evidence is needed to support this hypothesis and it would be interesting to investigate whether SCH 16 is indeed a cap dependent translation inhibitor.

Conclusion
In conclusion, the findings of this study unequivocally demonstrate that SCH 16 has antiviral activity against JEV and WNV in vitro. Furthermore, SCH 16 was also found to completely inhibit JEV replication in vivo in a mouse model challenged peripherally with 50LD 50 of the virus in a dose dependent manner. This necessitates further investigation into the pharmacokinetcis of the compound. Its moderate therapeutic index (TI = 5) may be a concern. However, further investigation on structure -activity relationships and appropriate modification in the aryl ring of the isatin moiety could provide more effective JEV-inhibitors with improved efficacy in future.

Cells and animals
Aedes albopictus (C6/36) mosquito cell line and Porcine Stable kidney (PS) cells were maintained in Minimum Essential Medium (MEM) with 10% fetal calf serum while Baby Hamster Kidney (BHK-21) cells were maintained in Dulbecco's MEM with 10% fetal calf serum (NCCS, Pune, India). Random bred Swiss albino mice (4-5 week old) were obtained from Central Animal Research Facility, NIMHANS, Bangalore, India, and used for the in vivo evaluation. All animal experiments were conducted after obtaining permission from Institutional Animal Ethics Committee.

N-Methylisatinisatin-β-Thiosemicarbazone (MIBT) derivatives
Fourteen mannich bases of isatin-β-thiosemicarbazone derivatives (Table 1) were obtained from Dr. Sriram, Birla Institute of Technology and Science (BITS), Pilani, India. The compounds were synthesized by Schiff reaction. N, Ndiethyl thiosemicarbazide was condensed with isatin in the presence of glacial acetic acid to form 1H-indole-2, 3dione -3-N, N-diethyl thiosemicarbazone (Schiff base). The N-Mannich bases were further condensed using acidic imino group along with formaldehyde and various secondary amines to obtain isatin thiosemicarbazone derivatives. Ribavirin, which is a known inhibitor of flavivirus replication, was obtained from commercial sources (Sigma, USA) and used as a control drug in this study.

Cytotoxicity of Ribavirin and MIBT derivatives
Cytotoxicity of the antiviral compounds was evaluated using the Trypan blue exclusion assay [36]. Briefly, PS and or BHK-21 cells grown to semi-confluence in 24-well plates were exposed to different concentrations of the compounds for 4 days at 37°C. Following this, the cells were harvested by trypsinization and re-suspended in 0.5 ml of MEM containing 10% FCS. A 100 µl of the cell suspension was mixed with 50 µl of 2.5% Trypan blue and the number of viable cells was enumerated using a hemocytometer. The concentration of compound that reduced cell growth by 50% was estimated as the 50% cytotoxic concentration (CC 50 ). The effect of the compounds on cellular proliferation was also studied. Briefly, the drug treated cells and untreated cells were seeded at a rate of 2 × 10 4 cells per well into 24-well plates and allowed to proliferate for 3 days in MEM, containing 10% FCS. The proliferations of cells were monitored every day microscopically by recording signs of toxicity such as altered morphology presence or absence of vacuoles and/ or dead cells.

Screening for inhibition of virus induced cytopathic effect in vitro
The antiviral activity assay of the Ribavarin and MIBT derivatives against JEV, Den-2 virus or WNV were screened in vitro using the cytopathic effect (CPE) inhibition assay carried out in a 96 well plate. Briefly, monolayers of PS and/or BHK-21 were inoculated with 100 µl of appropriate virus suspension containing 1 MOI of virus and adsorbed for two hours at 37°C. At the end of incubation period, the virus (JEV, Den-2 or WNV) was removed and the monolayers were rinsed with MEM to remove unbound virus. Doubling dilutions of different concentrations of Ribavirin and MIBT derivatives (beginning with CC 50 ) were prepared in MEM, added to the monolayer (100 µl) and incubated at 37°C for 3 days under 5% CO 2 . The experiment was terminated when the virus control showed maximum CPE. The presence or absence of CPE was recorded microscopically every day and the plates were stained using crystal violet at the termination of experiment and compared with the untreated virus controls and drug controls. All the experiments were run in triplicates to ensure reproducibility.

Confirmation of antiviral activity by plaque reduction assay
The compounds that showed inhibition of virus replication in the CPE inhibition assay were further evaluated using plaque-reduction assay. Briefly, PS (4 × 10 4 cells/ well) cells were grown to a confluent monolayer in a 24 well plate and infected with 100 µl of virus suspension containing 1 MOI of JEV and incubation was carried out for 2 hours at 37°C. At the end of adsorption, monolayers were rinsed with sterile PBS and 100 µl MEM containing varying concentrations of the compounds were added. The monolayer was then overlaid with maintenance medium containing 0.2% molten agarose (Sigma-Aldrich, USA). Appropriate controls were included in each run of the assay. Incubation was carried out at 37°C for 3 days. At the end of incubation period monolayers were fixed in 10% formal saline, the agarose was gently removed and the cells were stained using 1% crystal violet. Two independent observers counted the plaques using a hand lens.
All the experiments were run in triplicates. Percentage inhibitions of plaques were determined using the formula given below.
The antiviral activity was expressed as 50% inhibitory concentration (IC 50 ) of the compound, which is the concentration of the compound required to inhibit viral plaques by 50% as compared to virus control. The therapeutic % Inhibition Number of plaques in virus control-Number of p = l laques in drug treated Number of plaques in virus control × 1 100 potential and specificity of action was determined by calculating the Therapeutic Index (TI), which is the ratio of CC 50 to IC 50 (CC 50 /IC 50 ) [37].

Understanding the mechanism of action of SCH 16 in relation to JEV replication
To understand the possible mechanism of action in relation to the replicative cycle of JEV, the compounds that showed 100% inhibition of viral plaques were evaluated by in vitro experiments detailed below.

Determining kinetics of JEV replication in PS cells
A 24 well plate containing sterile cover slips in each well was seeded with 4 × 10 4 cells/well and incubated at 37°C overnight. When the cells were a confluent monolayer, they were infected with JEV (MOI = 1) for 1 hour at 37°C. The monolayer was rinsed thoroughly with sterile PBS and replenished with medium containing 1% FCS. This time point was considered as '0' hour post-infection. Subsequently at 2, 4, 6, 8, 10, 12, 14, 16 and 24 hours postinfection, the medium was harvested to determine the amount of extracellular virus released into the supernatant. At each time point, the cover slip containing cells was also removed, fixed in chilled acetone and stained by Immunofluorescent Assay (IFA) using a monoclonal antibody to envelope protein of JEV to detect the cell bound antigen [38].
Understanding the kinetics of the antiviral activity of SCH 16 A 24 well plate was seeded with 4 × 10 4 cells/well and incubated at 37°C overnight. To this monolayer JEV was added (MOI = 1) and incubated for 1 hour at 37°C. At the end of adsorption, the virus was removed, the monolayer was rinsed 3 -4 times using sterile PBS and replenished with MEM containing 1% FCS. This time point was considered as 0 hour post-infection. Starting from 0 hour time point, 76 ug/ml (IC 50 ) of the compound was added at 2, 4, 6, 8, 10, 12, 14, 16, and 24 hours post-infection and incubated at 37°C. The supernatant fluid was harvested from the respective wells at 48 hours post-infection. The fluid was divided into two parts. One part was used to determine the virus yield in the supernatant fluid (TCID 50 /ml) and the second part of the fluid was used to detect the presence of soluble JEV antigen using an antigen capture ELISA described elsewhere [39]. In order to detect cell bound antigen the cover slip cultures were fixed in chilled acetone for 30 minutes at 4°C and stained using monoclonal antibody to JEV (Clone F2C2) and antimouse IgG FITC conjugate by indirect IFA as described earlier. The cells in each well were treated with 750 µl of TRIzol (Invitrogen, USA) for RNA extraction and reverse transcription was carried out using cDNA archive kit (Applied Biosystems, USA) as described below.

Real Time PCR using Syber Green I chemistry
Detection of viral RNA was carried out by Real Time PCR using Syber Green I chemistry as described by Shu et al [40] with minor modifications. Briefly, a 120 base pair product of the PreM gene of JEV was amplified using the forward primer F1 (gga gcc atg aag ttg tca aat ttc) and reverse primer R1 (ttg ccc gga ccc aac at) based on the prototype standard strain of JEV (P20778) Gen Bank Ac.No.7080251.
A second set of experiments was designed to estimate the minimum time required for the compound to bring about complete inhibition of JEV replication. A 24 well plate was seeded with 4 × 10 4 cells/well in quadruplicates and incubated at 37°C overnight. Confluent PS monolayers were infected with JEV (MOI = 1) and adsorbed for 1 hour at 37°C. Following this, the monolayer was rinsed with sterile PBS and replenished with plain medium containing non-toxic concentration of SCH16. Control wells received plain medium. This time point was considered as '0' hour post-infection. Starting from 0 hour time point, medium containing the compound was removed at 0, 4, 8, 12, and 14 hours post-infection and replenished with MEM containing 1% FCS. At the end of 48 hours incubation, the fluid harvested from one of the quadruplicate set of wells, was evaluated for presence of extracellular virus by titration while soluble antigen was detected using an antigen capture ELISA described earlier. Cells in a second set of wells were trypsinised, re-suspended in maintenance medium and subjected to three freeze thaw cycles to release intracellular virus, which was quantitated by titration. Cells from the third set of wells were stained by an IFA to detect cell bound antigen. The cells in the fourth set of wells were treated with 750 µl of TRIzol (Invitrogen, USA) for RNA extraction and reverse transcription was carried out using cDNA archive kit (Applied Biosystems, USA).

Effect of SCH 16 on the translation of JEV
In order to understand the probable action of SCH 16 on the events of viral replication, an in vitro translation experiment was carried out using commercially available Tran-scend™ non-radioactive translation detection system and rabbit reticulocyte lysate kit (Promega, USA). A 24 well plate was seeded with PS cells (4 × 10 4 /ml), incubated at 37°C for 18 to 24 hrs and the monolayer formed was adsorbed with JEV (MOI = 1) for 1 hour. The infected monolayer was rinsed with sterile PBS to remove the unbound virus. To one set of JEV infected monolayer cultures, SCH 16 at non-toxic concentration was added at '0' hour and incubated for 4 hours. Medium containing SCH 16 was removed at 4 post-infection and replenished. To a second set of monolayer cultures, SCH 16 at the same concentration was added at 10 hours post adsorption. The plates were further incubated for 48 hours at 37°C.
Appropriate virus and cell controls were included in parallel to the test. At the end of incubation, the cells were treated with 750 µl of TRIzol and the viral RNA was extracted as described earlier. The viral RNA thus obtained, was divided in to two equal parts. One part was subjected to RT-PCR using JEV specific primers to confirm the presence of JEV RNA. The second part was subjected to in vitro translation carried out using a commercial kit (Promega, USA) and manufacturer's guidelines. Briefly, a 50 µl reaction containing 35 µl of rabbit reticulocyte lysate, 10 µl of nuclease free water, 1 µl of RNasin (40 U/ µl), 1 µl of complete amino acid mixture (1 mM), 1 µl of Transcend ™ tRNA and 2 µl of RNA template was set up at 30°C and incubated for 60 minutes. After the completion of translation reaction, 1 ul of the product was subjected to SDS -PAGE, electroblotted on to a PVDF membrane, blocked with skimmed milk powder solution, reacted with JEV specific monoclonal antibody to visualize the bands.

In vivo evaluation of SCH 16 against JEV Evaluation of non-toxic concentration of the compounds in mice
In order to determine the in vivo non-toxic concentrations, the compound SCH 16 (100, 200, 400 and 500 mg/kg body weight) were administered either per orally, or intraperitoneally into four different groups of 4 -5 weeks old Swiss albino mice (n = 4). Two groups of mice served as normal controls that received plain medium per orally or intraperitoneally. All mice were observed for a period of 45 days for loss or gain in weight, and other evidences of toxicity as compared to the untreated normal mice.

In vivo evaluation of SCH 16 by intracerebral challenge
To determine the in vivo antiviral potential of SCH 16, three groups of mice (4 mice per group) were injected intracerebrally with 30 µl of virus suspension containing 50 LD 50 of JEV. This was followed by intraperitoneal administration of SCH16 (100 and 200 mg/Kg body weight) into two groups of mice twice daily for 10 days. A third group of mice served as control animals that received virus intracerebrally and plain MEM intraperitoneally. Animals were monitored for the appearance of symptoms for JEV infections such as paralysis and death.

In vivo evaluation of SCH 16 by peripheral challenge
The therapeutic potential of SCH 16 was also evaluated using a peripheral challenge model wherein JEV (50LD 50 ) was injected (200 µl) intraperitoneally into four groups of 4-5 weeks old mice (n = 4). Two hours later, each group received 30 µl of 1% sterile starch intracerebrally to facilitate virus entry into the brain. This was followed by oral administration of SCH 16 in a dose of 200, 400 or 500 mg/kg body weight twice daily into the three respective groups for 12 days. A fourth group of mice (n = 4) served as 'no drug controls' which received virus intraperito-neally, starch intracerebrally and plain MEM orally while a fifth group of mice (n = 4) served as sham controls and received MEM intraperitoneally and starch intracerebrally. Mice were observed every day for 20 days post-infection for the appearance of symptoms and death. At the end of the observation period the mice that survived the infection were sacrificed, brains harvested and subjected to JEV antigen detection by IFA, JEV nucleic acid detection by real-time PCR and virus isolation using shell vial method [41].