Herpes simplex virus type 1 and type 2 are classified under the genus Simplexviruses, subfamily Alphaherpesvirinae and family Herpesviridae according to the classification by the International Committee on the Taxonomy of Viruses (ICTV) (Taylor et al., 2002). HSV can be transmitted via direct contact of the infected secretions and can establish lesions at any site of human body (Xiong et al., 2011). HSV infection can manifest themselves from an asymptomatic infection to a mucocutaneous infection or life-threatening central nervous system infection (Fatahzadeh and Schwartz, 2007). HSV-1 is the most commonly acquired HSV and the common cause of oral herpes and contributed to 30% of genital infection case, while HSV-2 is the major cause of genital herpes infection and is the major cause of sexually transmitted disease (Chen et al., 2012). More than 60% of the world's population was found to be seropositive for HSV while approximately 1 billion of the global human population is affected by genital herpes caused by HSV-1 and HSV-2 (Chen et al., 2012). Furthermore, HSV-2 infection has been reported to be a risk factor in acquiring HIV infection (Celum et al., 2010).
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Nucleoside analogues such as the acyclovir (ACV) or its subsequent derivatives with better bioavailability such as famciclovir, valacyclovir and penciclovir remain as the mainstay for HSV infection treatment. However, due to the ability of HSV to establish latent infection and persists throughout the lifetime of the infected individuals, current antiviral treatment shortens the cause and decrease the severity of symptomatic episodes but not eradicating the virus (Vilhelmova et al., 2011). Moreover, the efficacy of these nucleoside analogues has been compromised by the emergence of ACV-resistant mutant strain in immunocompromised patients such as the organ transplant patient or patient with AIDS (Morfin and Thouvenot, 2003; Vilhelmova et al., 2011). Therefore, there is an urgent need for the discovery or development of novel alternative compound with an alternate mechanism of action (Chen et al., 2012; Vilhelmova et al., 2011; Xiong et al., 2011).
Medicinal plants have been traditionally used for the treatment of ailments (Mukhtar et al., 2008). With the advancement of isolation techniques and the emergence of drug resistance mutant strain among the microbes, investigations around the world are attempting to develop novel therapeutic compounds from the medicinal plants. Phyllanthus is the genus under the Euphorbiaceae family and is widely distributed in most tropical and subtropical regions (Calixto et al., 1998; Joseph and Raj, 2011). It has been traditionally used to reduce fever and to treat jaundice and liver diseases (Unander et al., 1995). Phyllanthus niruri was first shown to inhibit the hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) both in vitro and in vivo by Venkateswaran et al (Venkateswaran et al., 1987). Antiviral testing was carried out using different members of the Phyllanthus family and various species members such as the P. amarus, P. niruri, P. urinaria and P. orbicularis have reported to demonstrate potential inhibitory effect against against broad spectrum of viruses, such as the hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV) and herpes simplex virus (HSV) (Khan et al., 2005; Xiang et al., 2008). Apart from the reported antiviral activities, other reported activities include anti-cancer (Lee et al., 2011; Tang et al., 2010), hepatoprotective (Rajeshkumar and Kuttan, 2000), lipid lowering (Khanna et al., 2002) and blood glucose lowering activity (Ali et al., 2006), suggesting the broad spectrum activities in this plant species and as a potential drug discovery candidate.
Although P. amarus and P. niruri have been demonstrated to possess inhibitory action against HBV and HIV, their inhibitory effect was not studied against HSV. Acetone, methanol and ethanolic extract of P. urinaria and phytochemicals isolated from these extracts have been shown to inhibit both HSV-1 and HSV-2 in vitro (Yang et al., 2005; Yang et al., 2007). Therefore, our study aimed to determine the antiviral activities of four local Phyllanthus species, P. amarus, P. niruri, P. urinaria and P. watsonii that are locally found in Malaysia against HSV-1 and HSV-2 in vitro and to study the proteome changes in Vero cells in response to virus infection and extract treatment.
Materials and methods
Preparation of aqueous plant extracts
The aqueous extracts of four Phyllanthus species (P. amarus, P. niruri, P. urinaria and P. watsonii) were kindly prepared and provided by the Biotechnology Centre, MARDI, Malaysia. The extracts were prepared as previously described (Lee et al., 2011; Tang et al., 2010). The four plant species were grown in MARDI under semi-controlled environment. Briefly, freshly harvested plants were washed and dried in room temperature prior freeze-drying. The dried plant samples were then soaked with ultra pure water and homogenized with extraction buffer. The supernatants were collected after three (3) rounds of extraction. The samples were stored at -20Â°C until further use. The extracts were weighted and dissolved in sterile PBS solution to reach a final concentration of 10mg/mL. It was aliquoted into sterile 1.5mL tubes and stored at -20Â°C until further use.
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African green monkey kidney Vero cells (ATCC CCL-81) was purchased from the American Type Culture Collection (ATCC). Vero cells were propagated and maintained in Dulbecco's Modified Eagle Medium (DMEM) (Hyclone, USA), supplemented with 5% heat inactivated fetal bovine serum (FBS) in humidified 37Â°C, 5% CO2 incubator. Clinical isolated HSV-1 and HSV-2 were obtained from Dr. Shamala Devi's laboratory, Department of Medical Microbiology, University of Malaya and propagated in Vero cells. The virus titer was titrated by plaque assay as described by (Chattopadhyay et al., 2009) with slight modification and was expressed as plaque forming units per mL (PFU/mL). The virus stocks were stored in -80Â°C until further use.
The cytotoxic effect of the aqueous extracts of Vero cells was determined by using the CellTiter 96Â® AQueous Non-Radioactive Cell Proliferation Assay kit (Promega, USA) according to the manufacturer's instructions. Vero cells were seeded onto 96-well culture plates at cell density of 1Ã-104 cells per well and allowed for overnight incubation for cell attachment. Serial dilution of aqueous extracts were prepared and added into each well to reach a final concentration of 62.5, 125, 250 and 500Âµg/mL respectively. The plates were incubated in 37Â°C, 5% CO2 for 72 hours. After 72 hours incubation, the MTS/PMS solution was added into each well and incubated for one hour in dark at 37Â°C. The absorbance was measured using the GloMax Multi Detection System (Promega, USA) at 490nm with reference wavelength of 750nm. The maximum non toxic dose (MNTD) and the 50% cytotoxic concentration (CC50) of each extract were determined from the dose-response curve and used in subsequent experiments.
Vero cells were seeded onto 24-well culture plates at cell density of 5Ã-104 cells per well and allowed for overnight incubation for cell attachment. Vero cells were infected with HSV-1 and HSV-2 at MOI of 0.05 except for the cell control. Extracts were added into each designated well at 3 hours before infection (pre-treatment), 0 hours after infection (simultaneous treatment) and 3 hours after infection (post-treatment) to reach a final concentration of 100Âµg/mL. The plates were incubated in 37Â°C, 5% CO2 for 24, 48 and 72 hours respectively. The antiviral effect of the extracts was also tested at 12.5, 50 and 100Âµg/mL respectively at 72 hours with simultaneous treatment approach. The plates were subjected to two (2) freeze-thaw cycle at the end of incubation and the supernatants were collected and stored at -80Â°C until further analysis.
Quantitative real-time polymerase chain reaction assay
Quantitative real-time polymerase chain reaction (qPCR) assay was used to determine the HSV viral load. The HSV viral DNA was extracted from the supernatant by using the Accuprep Genomic DNA extraction kit (Bioneer, South Korea) according to the manufacturer's protocol and the extracted DNA was stored at -80Â°C until analysis. An in-house qPCR with primers differentiating HSV-1 and HSV-2 were developed. The forward primer sequence for HSV-1 is 5'-TGGGACACATGCCTTCTTGG and the reverse sequence is 5'-ACCCTTAGTCAGACTCTGTTACTTACCC, with amplicon size of 147bp. The forward primer sequence for HSV-2 is 5'-GTACAGACCTTCGGAGG while the reverse primer sequence is 5'-CGCTTCATCATGGGC, with amplicon size of 227bp. The assay was performed using the Bio-rad CFX 96 under the following conditions: 15 min activation at 95Â°C, followed by 35 cycles of 30s at 95Â°C, 30s at 60Â°C and 1min at 72Â°C and final elongation at 72Â°C for 10min. Standard curves were prepared by qPCR using serial dilution of known copies number of purified amplification product for both HSV-1 and HSV-2. The copy number of the samples was calculated from the standard curves. Percentage of reduction was defined as [copy no of infected-copy number of treated]/[copy number of infected]Ã-100 and the half inhibitory concentration (IC50) was determined from the dose response curve. The selective index (SI) value (SI=CC50/IC50) of each extract was calculated and the extract showing the highest SI value was selected for the 2D-GE analysis.
Plaque reduction assay
The inhibitory effect of the aqueous extract of Phyllanthus species on HSV infection was also determined by plaque reduction assay as described by Cheng et al. (2011). Vero cells were seeded onto 24-well culture plates at cell density of 2Ã-105 cells per well and incubated overnight. The medium was discarded and the cell monolayer was infected with 100pfu of HSV-1 or HSV-2 in the absence or presence of the extracts at 50Âµg/mL and 100Âµg/mL respectively and incubated for one hour for virus adsorption. The cell monolayer was then overlaid with overlay medium containing 1% methylcellulose and further incubated at 37Â°C, 5% CO2 for 3 days. The overlay medium was discarded and the plate was rinsed gently with PBS before stained with 0.1% naphthalene blue black and the virus titre was determined.
Sample preparation for two-dimensional gel analysis
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Vero cells were seeded onto T-25 cell culture flasks at cell density of 7.5Ã-106 cells per flask and allowed for cell attachment overnight. Vero cells were mock-infected (designated CO), treated with extract (designated EO), infected with HSV-1 and HSV-2 (designated V1 and V2 respectively) respectively or infected and treated with 100Âµg/mL of extract (designated EV1 and EV2 respectively). The flasks were incubated in 37Â°C, 5% CO2 for 72 hours. The cells were trypsinized, washed twice with PBS, pelleted and resuspended in 200Î¼L of lysis buffer (7M urea, 2M thiourea, 2% CHAPS, 2% IPG Buffer pH 3-11NL (GE, Sweden), and 40mM DTT) and incubated at room temperature for 30 minutes with occasional vortexing to ensure complete lysis. The lysate was then centrifuged at 14,000 rpm for 15 minutes at 4Â°C and the supernatant was aliquoted and stored at -80Â°C. Protein was precipitated using the conventional acetone precipitated by mixing the supernatant with four times volume of cold acetone and stored at -20Â°C for at least 2 hour. The suspension was then centrifuged at 4Â°C, 14,000 rpm for 15 minutes to remove the acetone and the protein pellet was air-dried. The protein pellet was resuspended in the sample rehydration solution (7M urea, 2M thiourea, 4% CHAPS, 2% IPG buffer range pH 3-11NL (GE, Sweden)). The protein concentration of the sample was then quantified using the 2D-Quant Kit (GE, Sweden) according to the manufacturer's protocol.
Two dimensional gel electrophoresis analysis
The samples were made up to 250ÂµL with rehydration solution (7M urea, 2M thiourea, 2% CHAPS, 0.5% IPG buffer pH3-11NL (GE, Sweden), 0.002% bromophenol blue) containing 500Âµg protein. 13cm IPG strip with pH3-10NL (GE, Sweden) was rehydrated overnight with the samples. Isoelectric focusing was carried out for total 17.0kVh. Upon completion, the strips were stored in a clean glass tube and kept at -80Â°C until use or was equilibrated in equilibration solution (6M urea, 75mM Tris-HCl (pH8.8), 29.3% glycerol, 2% SDS and 0.002% bromophenol blue) containing 100mg DTT per 10mL for 15 minutes and 250mg iodoacetamide per 10mL for 15 minutes. Proteins were separated by 12.5% SDS-PAGE gels using 15mA per gel for 30 minutes and 30mA per gel for 2 hours. Following electrophoresis, the gels were fixed in 45% v/v ethanol and 10% v/v acetic acid and stained with hot Commassie blue stain for one hour. The gels were destained in 45% v/v ethanol and 10% v/v acetic acid and scanned with Image Scanner II (Amersham Bioscience, UK). Scanned images were analysed with basic version PD Quest 2.0 (Biorad, USA). Gels from mock, HSV-1 infected, HSV-2 infected, treated HSV-1 and treated HSV-2 were compared respectively. Differentially expressed spots were excised from the gels and subjected to in-gel trypsin digestion according to the protocol.
In-gel trypsin digestion and mass spectrometry analysis
The excised gel spots were destained with 50% acetonitrile (ACN) in 50mM ammonium bicarbonate until the gel spots became clear. Reduction of the gel spots was carried out with 10mM DTT in 100mM ammonium bicarbonate for 30 min at 60Â°C, followed by alkylation with 55mM iodoacetamide in 100mM ammonium bicarbonate for 20 min in dark. The gel spots were washed three times with 50% ACN in 100mM ammonium bicarbonate before dehydrated with 100% ACN and vacuum dried in speed vacuum. Trypsin digestion was then carried out on the gel spots by incubating with 6ng/uL Trypsin Gold (Promega, USA) in 50mM ammonium bicarbonate overnight at 37Â°C. 50% ACN was added and the liquid was transferred to a new tube. 100% ACN was added into the gel spots and the liquid was transferred to the previous new tube. The liquid was completely dried using the speed vacuum at low speed and stored at -20Â°C. The samples were re-constituted with 0.1% formic acid and desalted by using C18 ZipTip (Millipore, USA) according to manufacturer's protocol and mix with matrix at an equal ratio before spotted on the MALDI plate. The MALDI plate was then analyzed by using 4800 Plus MALD-TOF/TOF Analyzer (AB Sciex, USA). Raw mass spectra were processed and were searched against Swiss-Prot and NCBInr using MASCOT servers (Matrix Science, UK). Searches were restricted to Primates and Viruses taxonomies respectively.
Antibodies and western blot analysis
Anti-HSV1 antibody was obtained from a known positive HSV-1 infected donor (consent obtained) and was used at 1/200 dilution. HRP conjugated anti-human IgG was used at 1/1000. HRP-conjugated anti-HSV-2 antibody (Cat no: ab19989) was purchased from Abcam (Cambridge, UK) and used at 1/500 dilution. Protein samples from the two-dimensional gel electrophoresis were used for the Western blot analysis. Western blot was carried out as described by (Towbin et al., 1979) with slight modifications. 50Âµg of proteins were loaded into each well and were resolved using 12.5% SDS-PAGE gels at 100V for 1.5 hour. The resolved proteins were transferred onto nitrocellulose membrane (Cat no: RPN3032D, GE, Sweden) at 200mA for one hour. Protein transfer was determined by staining the membrane with Ponceau S stain. The membrane was blocked with 5% skim milk for one hour, followed by incubation with the primary antibody for overnight. The membrane was washed three times in PBS before incubated with the secondary antibody for 2 hours. 4-chloro-1-napthol was used as the substrate for detection of band of interest. The membrane was scanned and the band intensity was analysed using ImageJ software.
In order to determine the cytotoxic effect of the four Phyllanthus extracts before subsequent assays, the maximal non toxic dose (MNTD) and the half-maximal cytotoxic concentration (CC50) of the four aqueous Phyllanthus extracts, P. amarus, P. niruri, P. urinaria and P. watsonii were determined on Vero cells by using the conventional MTS assay. Table 1 summarizes the MNTD and the CC50 values for the four Phyllanthus aqueous extracts on Vero cells. Among the four species, P. watsonii demonstrated the strongest cytotoxic effect, followed by P. urinaria, P. amarus and P. niruri. On the contrary, P. amarus and P. niruri demonstrated higher MNTD values compared to P. urinaria and P. watsonii. The percentage of cell viability was found to be 90% at 100Î¼g/mL for all the extracts. The anti-HSV activity was therefore determined at concentration of 100Î¼g/mL or lower for the four Phyllanthus extracts.
The dose-response activity of the four extracts against HSV-1 and HSV-2 were determined by testing the extract at 12.5, 50 and 100Âµg/mL respectively by simultaneous treatment at 72 hours by using quantitative real-time PCR approach. The half-maximal inhibitory concentration (IC50) was determined and the selective index (SI) of each extract against HSV-1 and HSV-2 were tabulated in Table 1. The antiviral activity of the extracts against HSV-1 and HSV-2 were confirmed by conventional plaque reduction assay. Among the four Phyllanthus species, P. urinaria was found to have the highest SI value among the four extracts against both HSV-1 and HSV-2, hence it was chosen for the subsequent 2D-GE analysis and Western blot assay.
In order to study at which stage the extracts exerting their effect against the viruses, the antiviral activity of the four Phyllanthus species against HSV-1 and HSV-2 were also determined at pre-treatment, simultaneous treatment and post-treatment respectively by using quantitative real time PCR approach. Figure 1 and Figure 2 summarized the antiviral activity of 100Âµg/mL of the four Phyllanthus extracts against HSV-1 and HSV-2 respectively by pre-, simultaneous and post-treatment at 24, 48 and 72 hours. At 100Î¼g/mL, all four aqueous extracts were found to be most effective in inhibiting both HSV-1 and HSV-2 viral replication by simultaneous treatment but less effective for post-treatment and pre-treatment. This suggested the extracts might affect the events in the early infection and/or late infection. Furthermore, the four aqueous extracts demonstrated more potent inhibitory activity against HSV-2 than HSV-1 at pre-, simultaneous and post-treatment.
Two dimensional gel electrophoresis and mass spectrometry
In order to understand how the HSV infection and the extract treatment affected the proteome of the host cells, two dimensional gel electrophoresis followed by mass spectrometry protein identification was carried out (Figure 3 and 4). 54 spots and 52 spots were found to be differentially expressed in response to HSV-1 and HSV-2 infection in Vero cells respectively. Simultaneous treatment of P. urinaria extract was found to correct the change back to normal expression level as compared to the uninfected and the extract-treated cells.
Total of 20 proteins were identified by mass spectrometry protein identification based on the Mascot score algorithm. The identified proteins were tabulated in Table 2 and Table 3 respectively. The identified proteins were found to involve primarily in the cellular cytoskeletal maintenance (8), metabolism (7), protein folding (2) and heat shock proteins (2). One viral protein, the major DNA binding protein (ICP8) of HSV-1 was identified.
Western blot analysis
The HSV viral proteins expression was studied by performing an immunoprecipitation Western blot (Figure 5). Viral proteins including viral glycoprotein B (gB) [100kDa], glycoprotein D (gD) [40-43kDa], VP16 (or known as Î±-TIF) [50kDa] and VP22 [38kDa] which played important role in determining efficient viral attachment and entry into the cells were identified based on their respective molecular weight. It was found that simultaneous treatment of P. urinaria at 100Âµg/mL reduced the expression of the four viral proteins by more than 90%. Meanwhile, it was shown that the extract treatment completely inhibited the HSV-2 viral proteins expression.
The Phyllanthus genus of the family Euphorbiaceae consists of about 750-800 species that are widely distributed in the tropical and subtropical region (Joseph and Raj, 2011). However, despite the extensive antiviral studies that was carried out on Phyllanthus species, only P. urinaria, P. embilica and P. orbicularis have been tested for their antiviral activities against HSV, whereas other species such as P. amarus and P. niruri that demonstrate potent antiviral activity against HIV and HBV have not been tested against HSV. Hence this study would be the first preliminary study that determine and compare the anti-HSV activities of the four Phyllanthus species, namely P. amarus, P. niruri, P. urinaria and P. watsonii that can be locally found in Malaysia by using quantitative real-time PCR.
Our findings indicated that the extracts were most effective when added either simultaneously with the initiation of virus infection or post infection but not when given pre-infection, suggesting the extract may act at the early stage of infection such as during viral attachment and entry as well as viral replication. Among the four Phyllanthus species, P. urinaria demonstrated the strongest antiviral activities against HSV-1 and HSV-2, with a SI value of more than 33.6, followed by P. watsonii, P. amarus and P. niruri. This study was also the first antiviral study that determined the antiviral activities of P. watsonii, which can be found endemically in the Peninsular Malaysia. Numerous studies have highlighted the potential anticancer activities of P. watsonii against a variety of human cancer cell lines, such as prostate cancer, skin melanoma, breast cancer and lung cancer cell lines (Lee et al., 2011; Tang et al., 2010), but there is no antiviral study that determine the antiviral activities of P. watsonii till date.
Reviews have suggested that the phytochemicals present in the prepared extracts contributed to the antiviral activities of the plants (Khan et al., 2005; Mukhtar et al., 2008; Naithani et al., 2008; Xiang et al., 2008). Previous studies by Lee et al. (2011) and Tang et al. (2010) have identified the presence of several phytochemicals in the extracts prepared from Phyllanthus including geraniin, rutin, gallic acid, caffeolquinic acid, corilagen, galloylglucopyronoside, digalloylglucopyronoside, trigalloylglucopyronoside, quercetin rhamnoside and quercetin glucoside. The presence of trigalloylglucopyronoside in P. urinaria might have contributed to the stronger antiviral activity observed. Different mode of inhibition was also observed in antiviral study that tested the isolated phytochemicals against HSV-1 and HSV-2, suggesting possible difference in mechanism of inhibition and possible synergistic or antagonistic effect. For instance, geraniin and 1,3,4,6-TODG which were isolated from acetone extract of P. urinaria, demonstrated serotype specific inhibition against HSV-1 and HSV-2 respectively (Yang et al., 2007). Another pure compound, excoecarianin, isolated from the acetone P. urinaria extract selectively inhibited HSV-2 but not HSV-1 with a SI value of 20.0 against HSV-2 infection (Cheng et al., 2011). Furthermore, it was found to synergize the antiviral effect of acyclovir when combination of excoecarianin and acyclovir was added onto infected Vero cells (Cheng et al., 2011). Thus, synergism may exist between the phytochemicals in the extracts in which one phytochemical may potentiate or compensate the loss of action of other phytochemicals.
HSV replication cycle takes place in a complicated yet highly regulated manner. Before any viral replication takes place, the virus must be able to attach itself to the host and enter the cells. There are two important HSV viral glycoproteins, namely glycoprotein B (gB) and glycoprotein D (gD) that are essential for facilitating efficient virus entry via the interaction with the host heparan sulphate receptors and associated co-receptors. After gaining entry into the cells, the VP16 (or Î±-TIF) which is brought in with the virion tegument stimulates the transcription of the immediate early genes (Roizman et al., 2006). In this study, the P. urinaria treatment resulted in the lowered expression of the gB, gD and VP16, thereby leading to a reduction in the virus replication.
After gaining entry, the viral nucleocapsid gets propelled along the microtubule toward the nucleus for DNA replication. In this study, we found that proteins that are involved in maintaining the cellular cytoskeletal integrity such as the cytoplasmic actin was down regulated while the cytoskeletal keratin was found to be up regulated in response to HSV infection as depicted in Figure 6 and Figure 7. Similar observation was reported by Antrobus et al. (2009) in which cytoplasmic actin was down regulated in response to HSV-1 infection. The addition of extract treatment was shown to correct the expression level of both cytoplasmic actin and keratin to normal level.
In this study, another protein, alpha-actinin-1, which is involved in the maintenance of the cytoskeletal integrity was found to be up regulated in response to the HSV infection. Alpha-actinin-1 is one of the four isoforms of the alpha-actinin, a cytoskeletal protein that can be found ubiquitously in "non-muscle" cell (Oikonomou et al., 2011). Alpha-actinin is a multifunctional protein, besides linking the cytoskeleton to various transmembrane proteins, it also regulates the activity of various receptors and the downstream signalling pathways (Oikonomou et al., 2011). Alpha-actinin has been demonstrated to be essential for the hepatitis C virus replication (Lan et al., 2003). Moreover, alpha-actinin interacts with known regulators of detachment steps of cell migration, suggesting similar signalling interaction may occurs during virus infection leading to formation of cytopathic effect of infected cells such as rounding up and detachment prior to cell death (Otey and Carpen, 2004). There is limited information currently on how and why HSV infection induced the up regulation of alpha-actinin in Vero cells. It is possible that the infection induced the up regulation of the alpha-actinin-1 which then activated the downstream signalling pathway leading to the disruption of the stability of the host cytoskeleton dynamic. The mechanism underlying this observation is currently unclear and it is unknown whether this applicable to other cell types or is unique to Vero cells. The expression level of alpha-actinin-1 was shown to be lowered to normal expression level when P. urinaria was added to the HSV-2 infected cells, but did not exhibit any lowering effect on the expression level of alpha-actinin-1 in HSV-1 infected cells. This could indicate that although both HSV-1 and HSV-2 are two closely related viruses, the viruses may manipulate the cells via different mechanisms and thus the extract may act differently, as observed in the study by Cheng et al. (2011) and Yang et al. (2005).
The viral replication and the increased expression of the viral proteins as well as the introduction of the extract may induce cellular stress to the host cell and trigger the increased expression of the heat shock proteins (HSPs) such as the heat shock protein 70kDa and the mitochondrial heat shock protein 60kDa, together with the chaperone proteins including protein disulfide isomerase to respond to the accumulation of unfolded or misfolded viral or host proteins. The expression of host proteins that are involved in the cellular metabolism process such as alpha-enolase was found to be up regulated in response to HSV-1 and HSV-2, whereas peroxiredoxin-1 was down regulated in response to HSV-2 infection. A study by Antrobus et al. (2009) also reported the up regulation of alpha-enolase and it has been demonstrated that HSV-1 infection induced the activation of the glycolytic pathway (Abrantes et al., 2012). It is unknown for now why HSV-2 infection leads to the down regulation of peroxiredoxin-1, but expression of peroxiredoxin-1 has been shown to be protective against RSV infection (Jamaluddin et al., 2010).
Hence, based on the current findings of this study, we could postulate that despite HSV-1 and HSV-2 are two closely related viruses, they manipulate their host via mechanisms with slight differences. The antiviral activities exhibited by P. urinaria extract could be due to the multiple action of the extract on the virus, including (1) down regulates the viral gB, gD and VP16 that are essential for viral attachment and entry; (2) inhibits the manipulation of the host actin-cytoskeleton dynamics by the virus; (3) prevents manipulation of the host proteins by the virus. In conclusion, Phyllanthus species emerges as a potential candidate in the development of effective antiviral drugs against HSV-1 and HSV-2, although further in-depth studies are needed to provide an insight into the mechanism involved and the identification of responsible target.