*3.3. Genistein Reduces E*ff*ective Dose of Acyclovir and Ganciclovir against B Virus*

Acyclovir (AVC) is typically the first antiviral drug administered following human B virus infection; however, if the patient shows symptoms of CNS involvement, ganciclovir (GCV) is typically administered. Like ACV, GCV has significant side effects, particular at high doses and with long-term usage. We examined whether genistein, when administered at 50 μM in combination with ACV or GCV, could enhance inhibition of B virus. First, we tested the IC50 dose of genistein with various concentrations of ACV or GCV. Infected cells receiving a combined genistein–ACV/GCV treatment showed marked reduction in plaque formation compared to cells treated with genistein, ACV or GCV alone (Figure 4A,B; Table 1). For example, whereas a 6 μM dose of ACV had little effect on B virus, a co-treatment with genistein at its IC50 value of 50 μM resulted in a 75% decrease in plaque formation (Figure 4A; Table 1). GCV is more effective against B virus than ACV, with an IC50 value of 16 μM versus 55 μM in Vero (Table 1), and, thus, was tested at a smaller range of concentration. Genistein exhibited the same additive effect when given with GCV (Figure 4B; Table 1). The similar level of

plaque reduction was expected since these two nucleoside analogs share similar antiviral actions. The data also showed that increasing the concentration of ACV or GCV produced little changes to the reduction by genistein, suggesting that genistein and these two nucleoside analogs are not necessarily interacting with each other chemically, but may work via independent mechanisms to prevent viral infection. Conversely, we tested the IC50 dose of ACV with varying concentrations of genistein and inhibition in plaque formation was virtually identical as that achieved by a cotreatment of ACV and 50 μM genistein (Figure 4C; Table 1). Thus, neither drug can be concluded as an enhancer, but rather that genistein and ACV interact in a synergistic manner resulting in an enhanced antiviral response.


**Table 1.** Calculated IC50 Values against B Virus.

#### **4. Discussion**

This study tested the hypothesis that the plant flavonoid genistein has antiviral activity against B virus. Using plaque reduction assays and plaque titration assays, we not only showed that genistein can reduce virus replication and spread in a dose-dependent manner, but also that it targets a point in the virus life cycle after DNA replication. Further, we showed that genistein could act synergistically with current antiviral therapies to reduce their effective dose. Finally, we showed that efficacious doses had no cytotoxic effects on primary human and macaque cell lines.

Genistein had IC50 values against B virus of 33, 46, and 55 μM in HFF, RMF, and Vero cell lines, respectively. While the antiviral activity of genistein in HFF and RMF cells has not been previously reported on, our data for Vero is congruent with values reported from other groups. The effective concentration of genistein ranges between 12–50 μM [27–30,32], while toxicity is observed between 25–250 μM, depending on the technique and cell type. The highest value in the toxicity range was reported from Lyu et al. via MTT assay [21]. In our study, cellular toxicity was measured using four complimentary methods and no cytotoxicity was noted in primary fibroblasts at any of the tested doses (12.5–100 μM), however, toxicity was noted above 50 μM in Veros (data not shown). Animal studies of genistein in mice and pigs have shown no adverse effects on growth, reproduction or development [19,31]. Therefore, genistein is an attractive candidate for antiviral therapy.

Our data suggest that genistein is targeting a late-phase event in the life cycle of B virus to block new virion production and prevent viral spread. In our current study, we found that pre-exposure of cells to genistein had no enhanced effect on virus reduction or reduction in viral DNA production, suggesting that genistein targets an intracellular event during the viral life cycle. Productive virus replication proceeds with the organized and temporal expression of three gene family groups—IE, E, and L—with viral DNA replication occurring between E and L phases. Numerous phosphorylation events are involved during each of these phases, which could provide potential targets for a protein kinase inhibitor such as genistein. Some studies have already demonstrated that genistein interferes with late infection by inhibiting phosphorylation of glycoproteins [20,27,30]. In this study, immunohistochemical (IHC) staining for viral antigen revealed a very restricted intracellular distribution with increasing concentrations of genistein. This observation supports the possibility that genistein is interfering with late phase virion assembly and/or trafficking through the endomembrane system. Currently, we are investigating the specific mechanism by which genistein inhibits virion production and viral spread.

The macaque is widely used in the biomedical research community owing, in part, to its high degree of genetic homology to the human. The threat of zoonosis has led to the establishment of specific pathogen-free (SPF) breeding colonies. In the macaque, B virus presents with minimal pathology and macaques in these colonies often show sero-conversion to B virus as they reach sexual maturity [38]. Sporadic sero-conversion, coupled with asymptomatic shedding from infected animals, produces a potentially dangerous situation whereby animal handlers, veterinarians, and researchers can be at risk of exposure. In this study, genistein was equally effective at limiting B virus infection in macaque fibroblasts as human fibroblasts. These observations raise the interesting possibility of using genistein to control outbreaks within these SPF colonies.

Genistein has antiviral activity against several DNA and RNA viruses, though this is the first report of its antiviral activity against B virus in primary human and macaque cell lines [19–32]. Our data suggest that genistein may have therapeutic potential as an augmenter to current antiviral drugs for B virus. Combination therapy has been found to be useful against HSV-1 [39–43]. In most cases, combination therapy against virus infection requires a lower dose of either drug to achieve the same or greater antiviral response. If genistein can synergize with ACV or GCV to reduce the effective dose, then such a combination could greatly reduce negative side effects seen with long-term administration to B virus-infected patients. In a clinical setting, genistein can be provided as a topical ointment to be applied at the site of exposure. This method can enhance the absorption of genistein to the infected site since it bypasses the rapid hepatic metabolism and low bioavailability associated with oral consumption of flavonoids. Furthermore, it can limit toxicity only to the infected area. Topical delivery has been suggested to work well for other flavonoids with demonstrated antiviral activity [44,45]

In conclusion, genistein is a promising new antiviral treatment of human B virus infection and may serve as an augmenter to current, more toxic antiviral treatments.

**Author Contributions:** J.C.L., N.D., P.W.K. and J.K.H. all contributed to the conceptualization of this project. J.C.L. and N.D. performed data curation. J.C.L., N.D. and J.K.H. performed formal analysis of the data. J.C.L., N.D. and P.W.K. performed experimental investigations. J.C.L., N.D., P.W.K. and J.K.H. developed methodologies. J.C.L. and N.D. performed validations of the findings. J.C.L., N.T. and J.K.H. contributed to writing and editing. J.C.L. and J.K.H. contributed to writing the final draft. P.W.K. and J.K.H. provided supervision and project administration. J.K.H. provided resources and funding acquisition.

**Funding:** This research received no external funding.

**Acknowledgments:** This work was supported by the B Virus Resource Center in the Viral Immunology Center at Georgia State University and the Georgia Research Alliance. We thank Mugdha Vasireddi for her contribution to experimental design, advice, and trouble shooting. We thank Chadi Filfili for his support and training in the BSL4. We thank the resource medical technologists and administration specialists from the National B Virus Resource Center for their help, support, and thoughtful conversations. We thank Martin L. Hudson for his support and his assistance in manuscript revision.

**Conflicts of Interest:** The authors declare no conflict of interest.
