**1. Introduction**

Herpes B virus (Macacine herpesvirus-1) is an alphaherpesvirus endemic in macaque monkeys, an animal widely used in biomedical research [1]. B virus is of significant interest because it is capable of potentially deadly human zoonosis and thus poses a major occupational hazard to researchers, veterinarians, and animal handlers. B virus can be spread to humans via direct contact with infectious macaque body fluids and tissues during bites, scratches or splashes. Pathogenesis typically occurs within two weeks post-infection, with symptoms ranging from influenza-like illness, headache, vesicular lesions, ataxia and/or ascending paralysis. In humans, B virus is highly neuroinvasive and can lead to severe neurologic complications, such as transverse myelitis, encephalitis and potentially death. Without timely diagnosis and proper treatment, the mortality rate for infected individuals is as high as 70% [1–5]. Currently, there is no cure for human B virus infection, only life-long antiviral treatments, and patients live under the constant threat of potential virus reactivation.

The survival rate following human B virus infection is 80% when antiviral treatments are started early in infection [4]. Acyclovir (ACV) or ganciclovir (GCV) are typically the first drugs given in most clinical cases of B virus infection [1]. Following infection, antivirals are administered intravenously to put the virus into a latent state; then patients will remain on an oral antiviral regimen throughout their lifetime to prevent virus reactivation. ACV and GCV are attractive antivirals against B virus because they enter the host cell in an inactive form and must be phosphorylated intracellularly by virus-encoded thymidine kinase to become active [6–8]. However, these antivirals have several limitations. ACV, for example, has poor solubility in water, a short plasma half-life, and low blood-brain barrier passage. Following oral administration of ACV, there is a 20% bioavailability of the drug in the plasma and even lower levels in the cerebrospinal fluid [9,10]. In addition, in vitro studies have shown that B virus is approximately 10-fold less sensitive to ACV than the closely related human herpessimplex virus-1 (HSV-1) [8]. Thus, ACV must be administered in significantly greater concentration to control the disease, especially when intravenous administration is required. Increased doses and long-term administration comes with increased risk of negative side-effects. ACV can form crystals in the renal tubules during clearance from the kidney and, consequently, patients on ACV treatment often suffer from neurotoxicity associated with impaired renal function [11–13]. Further, there are reports of resistance of HSV-1 to ACV when patients are infected with virus isolates that carry mutations in either the DNA polymerase and/or the thymidine kinase gene [14]. It is anticipated that similar problems will ultimately arise when using ACV as a prophylactic treatment for B virus infection. Clearly, it is critical that new drugs be identified to provide a safer and more effective intervention against primary and reactivated B virus infections.

Genistein is a flavonoid found in plants of the Fabaceae (bean) family and in soy-based foods. In plants, flavonoids serve many functions critical to plant physiology. In humans, they possess many pharmacologic properties and therapeutic potential, such as antioxidant, anti-microbial, anti-carcinogenic, and anti-inflammatory activity, and have been summarized in several reviews [15,16]. In 1999, the Food and Drug Administration approved the food labeling of health claims of soy-based foods in reducing the risk of coronary heart diseases [17]. Since 1998, there have been 65 Phase I-III clinical trials registered with the National Institute of Health assessing the biological benefits of genistein and its derivatives [18].

Genistein is a general protein kinase inhibitor with selectivity towards tyrosine residues. Tyrosine phosphorylation regulates many cellular processes, including cell proliferation and angiogenesis. Therefore, genistein has been widely recognized as an anticancer agent, though the idea of flavonoids acting as antivirals is far from novel. Genistein has been shown to have antiviral properties against several herpesviruses including HSV-1, cytomegalovirus, and bovine herpesvirus-1, as well as non-herpesviruses, including SV40, human papilloma virus, porcine reproductive and respiratory syndrome virus, African swine fever virus, and human immunodeficiency virus [19–35]. Given the similarities of B virus to other herpesvirus against which genistein exhibits antiviral activities, we hypothesized that genistein would exhibit antiviral activity against B virus and, further, that genistein could be used in conjunction with current antiviral treatments to reduce their effective dose and enhance their antiviral properties.

#### **2. Materials and Methods**

#### *2.1. Cells, Media, and Virus*

Human foreskin fibroblasts (HFFs; ATCC® CCD-1112SK™, Manassas, VA, USA), Rhesus macaque foreskin fibroblasts (RMF; isolated in house), and African Green Monkey kidney cells (Vero; ATCC® CCL-81™, Manassas, VA, USA) were used in this study. The media compositions employed were (1) HFF: minimum essential medium (MEM), 10% inactivated fetal bovine serum (ΔFBS; Atlas Biologicals F-0050-A, Fort Collins, CO, USA), 100 U/mL penicillin-streptomycin (pen-strep; Mediatech/Corning #30-002-CL Tweaksbury, MA, USA), 1% sodium pyruvate (Mediatech/Corning #25-000-CL Tweaksbury, MA, USA), 1% glutaMAX™ (Gibco # 35050-061), and 1% non-essential amino acids (Mediatech/Corning #25-025-CL Tweaksbury, MA, USA); (2) RMF: Dulbecco's modified essential medium (DMEM; Sigma-Aldrich #D-6046, St. LouisMOUSA), 18% ΔFBS, and 100 U/mL pen-strep; and (3) Vero: DMEM, 10% ΔFBS, 100 U/mL pen-strep. For all experiments, cells were grown at 37 ◦C in a 95% O2, 5% CO2 incubator. B virus (laboratory strain E2490) was propagated in Vero cells. Virus stock

titer was determined by standard plaque assay in Vero cells. Propagation and harvesting of B virus isolates were performed in the Georgia State University (GSU) BSL4 Laboratory in accordance with the guidelines of the 5th edition of Biosafety in Microbiological and Biomedical Laboratories.
