|Gene-Eden-VIR: Laboratory and Clinical Studies|
Gene-Eden-VIR includes five natural ingredients. Laboratory and clinical studies showed that
these ingredients have an antiviral effect, specifically, during the latent phase. Some of these studies are
The following section presents detailis of some of the scientific studies that tested the five Gene-Eden-VIR ingredients. The results of these in vitro and in vivo studies show that these ingredients decrease the latent viral load in the infected host.
Licorice, which has been used for thousand of years as a flavoring agent, is derived from the root of Glycyrrhiza glabra. The licorice root contains glycyrrhizic acid (GA), also called glycyrrhizin, or glycyrrhizinic acid.
The following in vitro experiments show that GA treatment induced apoptosis of primary effusion lymphoma (PEL) cells that harbor a latent Kaposi sarcoma-associated herpesvirus (KSHV) [i]. The apoptosis decreases the number of cells harboring latent foreign polynucleotides in an infected host, which decreases the latent viral load in the host.
The experiments used twelve different human cell types. Five cell types, BC-1, BC-2, BC-3, BCBL-1, and BCP-1 are B-cells derived from different body cavity-based lymphomas. BC-3, BCBL-1, and BCP-1 cells harbor a latent infection with KSHV but not. BC-1 and BC-2 cells harbor both KSHV- and EBV. The examples also used primary human keratinocytes from Clonetics at the second passage. CB33 cells lymphoblastoid cells infected with EBV. Ramos cells (ATCC) are Burkitt lymphoma cells that are negative for KSHV and EBV. SLK are a KS-derived cell line negative for KSHV. KS2616 are cells prepared from a KS lesion of a HIV-negative patient. The KS2616 cells are positive for KSHV.
KSHV latent genes determine the virus persistence. Therefore, Northern blot analysis was used to measure the effect of GA treatment on the expression of three KSHV latent genes in four different KSHV-positive B cells. The there genes were the KSHV latency-associated nuclear antigen 1 (LANA-1, ORF73), the KSHV cyclin protein (v-cyclin, ORF72), and the viral FLICE-inhibitory protein (v-FLIP, K13). All KSHV-infected cells, including PEL cells, express LANA-1. This protein enables the KSHV genome to be present as an episome in latently infected cells. LANA-1 binds to p53 and inhibits the p53-induced apoptosis. LANA-1 also binds the retinoblastoma tumor-suppressor protein (Rb), which possibly inhibits the Rb-induced cell cycle arrest. V-cyclin binds to and activates the cyclin-dependent kinase 6 (cdk6), which leads to phosphorylation and inactivation of p53 and Rb.
The cells were treated with two active and nontoxic GA concentrations (3 and 4 mM). The expressions of the viral gene in treated cells were than compared to those in untreated cells. The example used BC-3 and BCBL-1 cells, which are infected with KSHV, and BC-1 and BC-2 cells, which are co-positive with KSHV and EBV.
On the basis of the growth curves, the BCBL-1 cells were treated with GA for 2 days, the BC-3 cells for 3 days, and BC-2 and BC-1 cells for 6 days. Different times were needed since the cells grow in different rates. The filters were hybridized with a probe specific for LANA-1. This probe detects the LT1 transcripts only. The results showed a dose-dependent decrease of the LT1 transcripts in n all GA-treated cells. Two additional probes that are specific for v-FLIP and v-cyclin were then hybridized. These probes detect the LT2 transcripts. The results showed a decrease in LT1 with an increase in LT2 transcripts. As control the transcript level of β-actin in the GA treated cells were assayed. The results showed similar levels of b-actin in these cells.
The decrease in LANA-1 transcripts in the GA-treated cells might suggest a change in the activity of the LT1/LT2 promoter in these cells. To determine the effect of GA treatment on the LT1/LT2 promoter, BJAB cells were transiently transfected with a reporter gene that expresses luciferase under the control of the LT1/LT2 promoter. The results showed similar level of luciferase expression in untreated and GA-treated cells. These results suggest that GA treatment does not affect the LT1/LT2 promoter activity.
Expression patterns of LANA-1, v-cyclin, and v-FLIP proteins. A Western blot and FACS analysis were used to measure the expression of LANA-1, v-cyclin, and v-FLIP proteins in of BCBL-1, BC-3, and BC-1 cells treated with GA for 2, 3, and 6 days, respectively. Untreated cells positive for KSHV show expression of LANA-1. The GA treatment of the KSHV positive cells decreased LANA-1 expression. The effect was reversible.
FACS analyses of 4 KSHV-positive B cells untreated and following treatment with GA were then used. All KSHV-positive B cells constitutively express v-cyclin. However, following treatment with GA, 35-50% of the cells over-expressed v-cyclin.
The analysis also showed similar expression of v-FLIP in GA treated and untreated cells. It has been showed that the v-FLIP protein blocks Fas-mediated apoptosis in cells latently infected with KSHV. However, in the current experiments GA treatment did not affect apoptosis, which indicates that v-FLIP although expressed, is not interfering with apoptosis.
Previous studies showed that over-expression of v-cyclin promotes apoptosis in cells with elevated levels of cdk6, and that cellular Bcl-2 or v-FLIP does not inhibit this apoptosis. Therefore, the concentration of cdk6 in the BC-1, BC-3, and BCBL-1 cells was assayed. The experiment used normal human lymphocytes, liver cells, 293 human epithelial kidney cells, and BJAB cells as controls. A Western blot analysis revealed that untreated BC-1 and BCBL-1 express high concentrations of cdk6, while untreated BC-3 cells express low concentrations of cdk5. Treatment with GA of these cells increased cdk6 expression 8- to 11-fold, which induced apoptosis in the BC-3 cells. The 293 and BJAB control cells also showed high concentrations of cdk6. However, treatment of the BJAB cells with GA did not result in apoptosis, possibly because these KSHV-negative cells show no expression of v-cyclin. The results suggest that over-expression of the v-cyclin/cdk6 complex might contribute to the apoptosis induced by the GA treatment in the KSHV-positive B cells.
The next step was to examine the biological implications of the modified latent gene expression in the KSHV-positive B cells treated with GA. One of the first intracellular changes during the onset of apoptosis is the disruption of the mitochondrial membrane potential. Proteins that are normally localized in the mitochondrial inter membrane space, such as cytochrome c and AIF, translocate to the nucleus and trigger a cascade of catabolic reactions that result in apoptosis. Following the release from the membrane, cytochrome c with Apaf-1 and procaspase-9 form the “apoptosome.” This complex activates the caspase cascade and apoptosis.
In these experiments, KSHV-positive B cells treated with GA showed the typical disruption of mitochondrial membrane, and many cells with condensed or fragmented chromatin typical of apoptosis (using a TUNEL assay). FACS analysis was used to determine the percentage of TUNEL-positive cells. The analysis showed that the GA treatment induced apoptosis in 80-95% of the KSHV-positive cells. This percentage is higher than the 35-50% of cells over-expressing v-cyclin following GA treatment, indicating that other proteins in addition to v-cyclin induce the observed apoptosis. In comparison to the KSHV-positive cells, uninfected B cells treated with GA showed no disruptions of their mitochondrial membranes or DNA condensation. Treatment with a 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which is know to promote the switch from latent to lytic viral cycle, also did not disrupt the mitochondrial membranes or caused DNA condensation. The lack of the apoptotic effect of the TPA treatment indicates that lytic gene expression was not involvement in the observed apoptosis.
Usually, disruption of the mitochondrial membrane induces caspase-cascade activation and DNA fragmentation. ELISA was used to examine the activation of the caspase cascade in KSHV-positive B cells untreated and following treatment with GA. There was no activation in any sample, indicating a caspase-independent apoptosis. The experiment then targeted AIF, a mitochondrial oxidoreductase that translocates from the mitochondria to the nucleus under stress condition causing DNA loss and chromatin condensation, typical changes in apoptosis when caspases are inhibited. To identify translocation of AIF, an immunofluorescence analysis was used in KSHV-positive B cells following treatment with GA for 4 days. A cytoplasmic pattern, characteristic of mitochondrial AIF, was evident in the untreated KSHV-positive B cells and in the KSHV-negative cells (BJAB) when untreated and following treatment with. In contrast, the analysis detected a diffuse nuclear staining, indicating translocation to the nucleus, in KSHV-positive B cells following treatment with GA. These observation suggest that GA induced changes in the mitochondrial membrane potential with AIF translocation to the nucleus and DNA fragmentation only in KSHV-positive B cells. To summarize, these observations suggest that the change in the expression of the KSHV latent genes, that is, the decrease in LANA-1 expression and increase in v-cyclin expression, induces the apoptotic effects.
p53 activation and oxidative stress. Several studies showed that over-expression of the v-cyclin protein promotes cell cycle progression and apoptosis. Other studies showed that LANA-1 prevents apoptosis by inactivating p53. A decrease in p53 expression and loss of function characterizes many human malignancies. Following DNA damage, p53 undergoes phosphorylation at Ser15 or Ser20, which induces cell cycle arrest in G1 and the initiation of DNA repair. If the cell fails to repair the damaged DNA, p53 initiates apoptosis. Therefore, the decrease of LANA-1 expression in KSHV-positive B cells might lead to p53 phosphorylation and apoptosis. To test this idea, the concentration of non-phosphorylated and phosphorylated p53 was assayed in uninfected and KSHV-positive B cells, both untreated and following treatment GA. Following treatment with GA, the experiment detected a high concentration of p53 phosphorylated at Ser15 in KSHV-positive B cells. Then BC-3 cells were transfected with the pLPCX/LANA-1 vector, a mammalian expression vector encoding LANA-1 under the control of the CMV promoter. After 24 hours, the transfected cells were treated with 4 mM GA for 4 days. A Northern blot analysis was used to confirm the presence of the 3.5-kb LANA-1 transcript. A Western blot analysis showed that GA treatment in the presence of high concentrations of LANA-1 resulted in a very low concentration of phosphorylated form of p53 and a high concentration of the non-phosphorylated form of p53. These results confirm that LANA-1 was responsible for the decrease in p53 phosphorylation. These results also support the conclusion that down regulation of LANA-1 by GA was responsible for the increase in p53 phosphorylation.
p53-induces apoptosis is associated with the formation of ROS, including H2O2, O2, and OH. An increase in H2O2 increases the concentration of the H2O2 scavenger catalase. Catalase activity was assayed in KSHV-positive B cells in untreated cells and following treatment with GA. Following treatment with GA, catalase activity increased 4-fold (BC-3), 2-fold (BCBL-1), and 3-fold (BC-1) in KSHV-positive cells relative to the untreated KSHV-positive cells and relative to the KSHV-negative cells (BJAB) either untreated or following treatment with GA.
FACS was used to determine the distribution of cell cycle in KSHV-positive and uninfected B cells both untreated and following treatment with GA. After 6 days, the treatment with GA caused 99% of KSHV-positive B cells to be blocked in G1. In contrast, only 25-50% of untreated KSHV-positive cells and uninfected cells were blocked in G1 when untreated on following treatment with GA. These results indicate that a decrease in LANA-1 expression restores p53 function and induces cell cycle arrest of the latent KSHV-infected cells.
Summary: These results show that GA, while not being toxic at the tested levels, specifically induces apoptosis in latent KSHV-infected cells.
Note that although Curreli, et al. (2005, ibid) showed that GA induces apoptosis in cells carrying a latent infection with KSHV, they did not mention any possible influence of the apoptosis on disease, on microcompetition with foreign DNA, or on the risk of developing a microcompetition-related disease, and the severity of such disease. Specifically, they did not argue against the current misconception that latent infection does not constitute a pathogenic threat (see an expression of such misconception in Babcock 1999[ii]).
The following in vitro experiment shows that GA treatment inhibits Epstein-Barr virus early antigen (EBV-EA) activation in latently infected cells[iii]. Since EBV-EA activation is necessary for persistent replication during the latent phase (Prang 1997[iv]), a decrease in EBV-EA transcripts decreases the latent viral load in the infected host.
The example used the Raji cells, a Burkitt-lymphoma-derived cell line that harbors 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes (Adams 1987[v]) The cells were superinfected with P2HR1 (LS) virus, which results in reactivation of the latent EBV virus and replication in the superinfected cells. Following the superinfection, approximately 95% of the superinfected cells became positive for the EBV early antigen (EA). In the presence of GA, a dose-dependent inhibition of the expression of the EBV-EA was observed. The example also observed a dose-dependent inhibition of viral genome copy number determined by real-time quantitative PCR. The GA concentration required for inhibiting the EBV genome copy number and antigen expression by 50% (EC50) was approximately 5 uM.
Quercetin is a flavonoid and, or more specifically, a flavonol. It is the aglycone form of a number of other flavonoid glycosides, such as rutin and quercitrin, found in citrus fruit, buckwheat and onions. Quercetin forms the glycosides quercitrin and rutin together with rhamnose and rutinose, respectively. Quercetin is classified as IARC group 3 (no evidence of carcinogenicity in humans).
The following in vitro experiment shows that quercetin treatment inhibits EBV-EA activation in latently infected cells[vi] Since EBV-EA activation is necessary for persistent replication during the latent phase (Prang 1997, ibid), a decrease in EBV-EA transcripts decreases the latent viral load in the infected host.
The experiment used Burkitt-lymphoma-derived Raji cells, which harbor 50 to 60 latent, predominantly extrachromosomal Epstein-Barr virus genomes (Adams 1987, ibid). The experiment exposed the Raji cells to EBV-EA positive serum isolated from a patient with nasopharyngeal carcinoma. The serum activated the EBV-EA. Treatment with quercetin derivatives inhibited the EBV-EA activation in the Raji cells without showing cytotoxicity. Quercetin pentaallyl ether (QPA) showed the most significant inhibitory effect on EBV-EA activation (100% inhibition at 1000 mol ratio/TPA and more than 80% inhibition at 500 mol ratio/TPA) and high viability (more than 70% viability at1000 mol ratio/TPA).
The following in vitro experiment shows that quercetin treatment decreases the concentration of the Hepatitis B e antigen (HBeAg) in cells latently infected with the Hepatitis B virus (HBV) [vii]. Since a decrease in HBeAg concentration is associated with a decrease in the concentration of the covalently closed circular DNA (cccDNA) form, which is responsible for viral persistence during latency, a decrease in HBeAg indicates a decrease in the latent viral load in the infected host.
The experiment used the human hepatoma Hep G2.2.15 cell culture system as in vitro model to evaluate the anti-HBV effects of hyperoside, a quercetin derivative (quercetin-3-O-β-D-galactoside). The HBV-producing 2.2.15 cells were obtained from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (Beijing, China). These cultures were derived from HepG2 cells that were transfected with a plasmid vector containing G418-resistance sequences and 2 head-to-tail dimmers of the HBV genome. The cells were found to produce elevated levels of HBeAg, which is expressed in HBV infected cells during the latent phase (Favre 2003[viii]).
The experiment incubated the 2.2.15 cells for 24 hours and then treated them with different concentrations of hyperoside (0.05, 0.025, 0.0125, 0.00625, and 0.003125g/L) in serum-free medium. The results showed that hyperoside decreased the concentration of HBeAg in the cells. The median effective concentration (IC50) of hyperoside on day 4 was about 0.012 g/L, and on day 8 about 0.009 g/L.
HBV cccDNA is responsible for viral persistence during the natural course of chronic HBV infection and serves as the template for the production of HBV pregenomic RNA (pgRNA), the primary step in HBV replication. A study (Laras 2006[ix]) used sensitive and specific quantitative real-time polymerase chain reaction (PCR) assays to measure the intrahepatic concentration, pgRNA production, and replicative activity of cccDNA in liver biopsy samples from 34 non-treated patients with chronic hepatitis B (CHB): 12 HBeAg(+) and 22 HBeAg(-). The results showed that in HBeAg(+) patients, the median values of cccDNA and pgRNA levels were 10-fold and 200-fold higher than in HBeAg(-), respectively. These results indicate that a decrease in HBeAg concentration is associated with a decrease in the concentration of cccDNA, and therefore, a decrease in the HBV DNA copy number during latency. Based on these results, we can conclude that hyperoside treatment decreases the copy number of latent HBV in infected cells.
Epigallocatechin gallate (EGCG), also known as Epigallocatechin 3-gallate, is a type of catechin and is the most abundant catechin in green tea. It is the ester of epigallocatechol and gallic acid.
1. EBV, Rta gene, P3HR1 cells, inhibition of persistence replication
The following in vitro experiment shows that EGCG treatment inhibits the activation of the EBV immediate-early protein Rta in latently infected cells[x]Since Rta is essential to for reactivation from latency and maintenance of the latent pool (Pavlova 2003[xi]), a decrease in Rta expression decreases the viral genome copy number in the latently infected cells, and decreases the latent viral load in the infected host.
P3HR1 is a Burkitt’s lymphoma line cell line that carries a latent infection with EBV. A flow cytometry analysis that used immunostaining of Rta with primary antibody and subsequent detection of the primary antibody with fluorescein isothiocyanate (FITC) or rhodamine-conjugated secondary antibody revealed that a low percentage of untreated P3HR1 cells express the Rta protein. The analysis also revealed that following treatment with TSA, a treatment known to activate the EBV lytic cycle, the population of P3HR1 cells expressing Rta increased to 23.4%, and that treatment with 70 mM EGCG decreased the percentage of cells expressing Rta to 9.8%. Treatment with 100 mM EGCG further decreased the percentage of P3HR1 cells expressing Rta to 0.5%. To summarize: EGCG treatment significantly reduced the expression of EBV immediate-early protein Rta, which, in turn, decreases the EBV latent copy number in infected cells.
The following in vitro experiment shows that EGCG treatment decreases the copy number of the nuclear covalent closed circular DNA (cccDNA) form, which is characteristic of latent HBV[xii]In HBV-positive cells, the viral DNA is transported into the nucleus where it transforms into the cccDNA form. Since the cccDNA form is essential for HBV maintenance during latency, a decrease in cccDNA copy number decreases the viral genome copy number in the latently infected cells, and decreases the latent viral load in the infected host.
The experiment used the human hepatoblastoma cell line HepG2-N10, which was generated by transfecting HepG2 cells with a transfer plasmid which contains a 1.3 unit length of genotype A HBV genome (subtype adw2). Cells were treated with fresh medium containing various concentrations of EGCG. Treatment with a concentration of 22.9ug/ml EGCG reduced the concentration of HBV cccDNA by 60%.
Cinnamic aldehyde or cinnamaldehyde (more precisely trans-cinnamaldehyde) is the chemical compound that gives cinnamon its flavor and odor. Cinnamaldehyde occurs naturally in the bark of cinnamon trees and other species of the genus Cinnamomum like camphor and cassia. These trees are the natural source of cinnamon, and the essential oil of cinnamon bark is about 90% cinnamaldehyde. Most cinnamaldehyde is excreted in urine as cinnamic acid, an oxidized form of cinnamaldehyde.
The following in vitro experiment shows that treatment with cinnamaldehyde or cinnamic acid inhibits EBV-EA activation in latently infected cells[xiii]Since EBV-EA activation is necessary for persistent replication during the latent phase (Prang 1997, ibid), a decrease in EBV-EA transcripts decreases the latent viral load in the infected host.
The experiment used the Raji cells, a Burkitt-lymphoma-derived cell line that harbors 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes (Adams 1987, ibid). The cell were treated with 2-O-tetradecanoylphorbol-13-acetate (TPA), known to promote activation of EBV-EA. The cells were treated with cinnamaldehyde or cinnamic acid and an indirect immunofluorescence technique was used to stain the EBV-EA expressing cells. The inhibition activity of the test compound was estimated by the percentage of positive cells compared to controls. The results showed that cinnamaldehyde and cinnamic acid inhibited EBV-EA activation in a dose dependent manner. The IC50 of cinnamaldehyde or cinnamic acid was 158 and 40, respectively. IC50 represents the mol ratio to TPA that inhibits 50% of positive controls (100%) activated with 32 pmol TPA.
Selenium is a chemical element with the atomic number 34, represented by the chemical symbol Se, and an atomic mass of 78.96. Selenium is a semi metal that rarely occurs in its elemental state in nature. It is toxic in large amounts, but trace amounts of it are necessary for normal cellular function in most, if not all, animals, forming the active center of the enzymes glutathione peroxidase and thioredoxin reductase and three known deiodinase enzymes.
The following in vitro experiment shows that treatment with Se inhibits EBV-EA activation in latently infected cells[xiv]Since EBV-EA activation is necessary for persistent replication during the latent phase (Prang 1997, ibid), a decrease in EBV-EA transcripts decreases the latent viral load in the infected host.
The experiment used the Raji cells, a Burkitt-lymphoma-derived cell line that harbors 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes (Adams 1987, ibid). The experiment stimulated the Raji cells with butyrate and croton oil. The stimulated cells were incubated with Se-rich rice extract. The experiment than used the indirect immunological flurescence method to count the EBV-EA positive expression rate and the inhibition rate. The results showed that Se-rich rice extract significantly inhibited the EBV-EA expression in Raji cells. At extract concentrations of 0.016, 0.078, and 0.388 mg/ml, the inhibition rate of EA expression was 2.85%, 12.88%, and 20.75%, respectively.
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[x] Chang LK, Wei TT, Chiu YF, Tung CP, Chuang JY, Hung SK, Li C, Liu ST. Inhibition of Epstein-Barr virus lytic cycle by (-)-epigallocatechin gallate. Biochem Biophys Res Commun. 2003 Feb 21;301(4):1062-8.
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[xiii] Motohashi N, Yamagami C, Tokuda H, Okuda Y, Ichiishi E, Mukainaka T, Nishino H, Saito Y. Structure-activity relationship in potentially anti-tumor promoting benzalacetone derivatives, as assayed by the epstein-barr virus early antigen activation. Mutat Res. 2000 Jan 24;464(2):247-54.
[xiv] Jian SW, Mei CE, Liang YN, Li D, Chen QL, Luo HL, Li YQ, Cai TY. [Influence of selenium-rich rice on transformation of umbilical blood B lymphocytes by Epstein-Barr virus and Epstein-Barr virus early antigen expression]. Ai Zheng. 2003 Jan;22(1):26-9. [Article in Chinese]