Evaluation And Mechanistic Studies Of Natural Product Antiviral Activity Against Herpes Simplex Virus Infection

Type Ⅰ and type Ⅱ herpes simplex virus (HSV-1and HSV-2), also known as human herpesviruses1and2(HHV-1and HHV-2) are two serotypes of HSV that only infect human beings. HSV-1is mainly associated with facial infections with visible cold sores or fever blisters. HSV-2is a major pathogen of sexually transmitted diseases (STD), which infects epithelial cells on or around genitals causing genital lesions and neonatal infections. Studies have demonstrated that HSV infection increases the risk of spontaneous abortion, cervical cancer, Alzheimer’s disease as well as HIV (human immunodeficiency virus) infection. Patients acquire HSV-1at relatively young ages, while initial HSV-2infections occur mainly after puberty, often transmitted after intimate contact. Some studies reported that up to90%of persons are seropositive for HSV-1by the age of60.HSV is an enveloped double-stranded DNA virus that belongs to Herpesviridae family, which can set up primary infection and latent infection. Following the primary infection, virus gains access to sensory nerve termini and be transported via retrograde axona transport to sensory ganglia where the virus establishes latency in neuronal cell bodies. The virus can be reactivated when the body suffering stress such as ultraviolet (UV) light, menstruation, psychological stress and result in frequent recurrence of HSV infection. HSV infection can also cause neurological diseases such as meningitis and encephalitis. There are no vaccines available to prevent HSV infection. The most commonly used drugs are nucleoside analogues, such as acyclovir, fanciclovir. These drugs inhibit HSV replication by targeting HSV thymidine kinase (TK). However, they only decrease virus spreading and cannot cure the HSV infection. Sometimes they can cause serious side effects such as digestive disorders and blood problems. In addition, drug resistance is a challenge in treatment of mutant HSV strain by nucleoside analogues. Therefore, the development of novel alternative therapeutic strategies and antiviral drug against HSV infection has become an important research objective.In this study, we aimed to screen antiviral compounds from naturally occurring compounds and Chinese traditional herbs against HSV infection, and further investigate their antiviral mechanisms. We found that the water extract of Houttuynia cordata Thunb.(HCWE), a medicinal plant generally used in folk medicine in several Asian countries, has significant anti-HSV-2infection activity. The IC50(the half maximal inhibitory concentration) of HCWE against HSV-2infection was estimated at50μg/ml. The plaque forming assay showed that at150μg/ml and450μg/ml, HCWE suppressed infectious HSV-2virions production by more than3and4logs, respectively. Activation of NF-κB (nuclear factor-KB) and Erk/MAPK (extracellular signal-regulated kinases/mitogen-activated protein kinase) pathways are required for HSV replication and growth. Fractionation of nuclear proteins and immunostaining assay showed that HCWE treatment markedly blocked HSV-2induced NF-KB/p65nuclear translocation, while has no effect on HSV-2-induced Erk activation. Furthermore, we found that quercetin, quercitrin, and isoquercitrin, major water extractable flavonoids from H. cordata by HPLC (high performance liquid chromatography) analysis significantly blocked HSV-2infection. Together, our results uncovered that H. cordata blocks HSV-2infection via inhibition of NF-κB activation and quercetin, quercitrin, and isoquercitrin may be the active antiviral components in HCWE.Stilbenoids including resveratrol have the basic structural unit of1,2-diphenylethylene. It is a type of natural phenol widely distributed in plants fulfilling many functions. In an attempt to evaluate bioactive stilbenoids, we have screened a group of dimeric and oligomeric stilbenoids isolated from plants of Dipterocarpaceae family against HSV-1and HSV-2infection. We identified that several trimers and tetramers have significant antiviral activity against both HSV-1and HSV-2. Most of them showed an IC50value lower than5μM against HSV-2infection. Unlike resveratrol, an antioxidant reagent, stilbenoids promoted rapid and transient release of reactive oxygen species (ROS) without affecting NF-κB and Erk/MAPK pathways activation. Addition of N-acetylcysteine (NAC), a scavenger of ROS, reversed the inhibitory effect of those compounds against HSV replication. In addition to the identification of resveratrol derivatives with potent anti-HSV activity, our results uncovered a mechanism of stilbenoid-mediated anti-HSV response through ROS generation.Taken together, our work not only identified antiviral natural compounds against HSV infection, and also provided novel strategies and targets for natural compound in developing anti-HSV infection drugs.