*3.2. Genistein Reduces B Virus Spread and Replication in a Dose-Dependent Manner*

We used three methods to assay for antiviral properties of genistein against B virus. Initially, we performed high input infections (MOI 5) in the presence or absence of genistein for 24 h, then collected cells/supernatants and back-titered these suspensions on Vero cells via a standard plaque assay. As shown in Figure 2A, genistein significantly reduced plaque formation in a dose-dependent manner. Since this assay required passaging the virus through a second cell type, we next asked if similar results could be obtained by assaying the virus directly into fibroblasts using either cell-ELISA or plaque reduction assays. Cell-ELISA also revealed a clear trend of reduced virus replication with

genistein in a dose-dependent manner with an IC50 value of 33 μM in HFF (Figure 2B) and 46 μM in RMF (Figure 2C). Next, we performed direct plaque reduction assays. Genistein reduced plaque formation and plaque size in a dose-dependent manner (Figure 2C). Of note, at high doses, virus antigen was rarely detected in the cytoplasm of cells, instead localized to the nucleus. These data suggest that genistein can reduce both productive virus replication and spread, not only in cell-to-cell scenarios but also within the infected cell. Further, our findings support that genistein is effective in restricting B virus infection in both human and macaque cell lines.

**Figure 2.** Genistein has antiviral activity against B virus in human and macaque fibroblasts. HFFs and RMFs were left untreated (negative control) or treated with either 1% DMSO (experimental control) or increasing concentrations of genistein and infected with 150 PFU of B virus for 48 h. (**A**) Indirect virus yield assay in HFFs. After 48 h, cells/supernatants were harvested and back-titered on Veros. Data shows total PFU counted in Veros. Cell-based Elisa in HFFs (**B**) and RMFs (**C**). After 48 h, cells were fixed with 100% methanol and assayed via ELISA for amount of virus antigen. An in-assay standard curve was generated to calculate virus titer and IC50 values were calculated using a logarithmic regression line. Statistical analysis performed via one-way ANOVA with Dunnet's correction for multiple comparisons. N = 9. Error bars show standard error of the mean. \* *p* = <0.05, \*\* *p* < 0.01, \*\*\* *p* =< 0.001. Direct virus yield assay in HFFs (**D**–**I**) and RMFs (**J**–**O**). After 48 h, cells were fixed with 100% methanol and virus antigen was visualized via IHC with DAB detection. Scale bars shown at 200 μM.3.3. Genistein Targets B Virus Post-Viral Entry and Genomic Replication.

Antiviral agents may act directly to inactivate viral particles or, following virus entry into cells, there are several steps that antivirals can target: Immediate-early and early gene synthesis, DNA replication, late gene synthesis, virus assembly and virus budding. Direct plaque reduction assays are an effective means of calculating the reduction in virus titer; however, as shown in Figure 2C, B virus does not produce a clear plaque in primary fibroblasts, making it difficult to obtain reliable quantitative data using this methodology. For these reasons, we performed plaque reduction assays in Veros, as this cell line is sensitive to B virus infection and B virus produces clear plaques in these cells. We verified that genistein could effectively reduce B virus replication in a dose-dependent manner in Veros. Genistein inhibited plaque formation with an IC50 value of 56 μM for B virus (data not shown). To examine if genistein could directly inactivate B virus, 50 μM of genistein was pre-incubated with the virus for 15, 30, 60, 90 or 120 min, and then cells were infected with the virus/drug mix. To verify that the effect of genistein was only on the virus and not the cell, the mix was diluted to obtain a 5 μM final concentration of genistein prior to cellular infection. Our results showed that pre-incubation of B virus with 50 μM genistein for up to 2 h prior to infection had no effect on plaque formation, suggesting that genistein does not directly inactivate the virus and its antiviral activity is due to interference in post-infection processes (Figure 3A).

**Figure 3.** Genistein does not directly inactivate B virus; instead, it inhibits the virus after virus replication. (**A**) Direct inactivation assay. Veros were infected with 150 PFU of B virus following pre-incubation of B virus with 50 μM genistein for 0–120 min. A standard plaque assay was performed and the total PFU counted. (**B**) Plaque reduction assay. Veros were infected with 150 PFU of B virus and 50 μM of genistein was added at various times post-infection. A standard plaque assay was performed and the total PFU counted. RT-PCR results for total genomic virus post-48 h infection of HFFs (**C**) or RMFs (**D**) following B virus infection in the presence of increasing concentrations of genistein or 1% DMSO (control).

B virus' life cycle is ordered into three distinct phases of virus gene transcription/translation: Immediate-early (IE), early (E), and late (L), wherein each phase is dependent on the previous. The full replicative cycle is ~12 h in Vero, with IE genes expressed by 2 hpi, E genes expressed between 2–4 hpi and followed by genomic replication, and L gene synthesis up to 8 hpi. From 8–12 hpi, virions are assembled, glycoproteins undergo glycosylation, and virions are released from the infected cell. To look at the effect of genistein at all phases of the viral life cycle, we added 50 μM genistein at 1, 3, 5, 7, and

9 hpi. There was no difference in virus replication at any time point (Figure 3B). Further, there was no statistically significant difference when genistein was added at the same time as B virus (data not shown; Figure 4C), suggesting that genestein does not impact viral entry. To verify that HFF and RMF would behave in a similar fashion, DNA was isolated from cells infected with increasing concentrations of genistein. No significant difference in the total viral DNA levels in infected cells at any concentration was found (Figure 3C,D). Collectively, these data suggest genistein targets a viral event post-genomic replication and L gene synthesis and, further, that this is not a species-dependent mechanism.

**Figure 4.** Genistein acts synergistically with antiviral agents to reduce effective dose. (**A**) and (**B**) show the results of virus yield assays, wherein Vero cells were infected with 150 PFU of B virus and 50 μM of genistein with increasing concentrations of ACV (**A**) or GCV (**B**). (**C**) Vero cells were infected with 150 PFU of B virus and 50 μM ACV with increasing concentrations of genistein. Total PFU was determined by standard plaque assay.
