Susceptibilities of Herpes Simplex Viruses to Penciclovir and Acyclovir in Eight Cell Lines

The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. Organotypic epithelial "raft" cultures are tissue culture systems that permit full differentiation of keratinocyte monolayers via culturing of the cells on collagen gels at the air-liquid interface. Previously, raft cultures have been successfully applied to the study of human papillomaviruses (6,14), and more recently they have been used for the study of herpes simplex virus (HSV) infection (10,16,24,25) and adeno-associated virus type 2 (15). We have also shown that the organotypic raft culture of human keratinocytes can be used as a model to evaluate both poxvirus replication and efficacy of antiviral agents (22).Skin lesions are frequently induced by viruses and can be the first sign of a systemic disease, particularly in immunocompromised patients and children. Among the dermotropic viruses, skin lesions induced by the alphaherpesviruses herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) show characteristic histologic features.HSV is typically responsible for mucosal lesions of the mouth and genital organs in humans. Following axonal transport, the virus reaches the dorsal root ganglia, where it establishes and maintains lifelong latent infections. Periodically, the virus reactivates, multiplies, and is transported through the axon back to a portal of entry, where it gives characteristic skin lesions (26,27).Primary infection with VZV results in chickenpox (varicella), a common childhood illness. As with other members of the herpesvirus family, infection with VZV is lifelong and the virus enters a latent state following the acute infection (4). Latency is associated with persistence of the viral DNA in the posterior root ganglia, and reactivation of the virus results in skin lesions characteristic of herpes zoster (shingles). Herpes zoster manifests as a localized rash in a unilateral, dermatomal distribution that is often associated with severe neuropathic pain (11,23).As keratinocytes are the main target cells for productive infection in vivo for both HSV and VZV, characterization of virus replication in organotypic raft cultures of these cells represents a very relevant model for studying virus-host cell interactions and antiviral agents. Here we report the infection of epithelial raft cultures with HSV and VZV, the lesions observed in these cultures...

Section: Discussionmentioning

confidence: 99%

Organotypic Epithelial Raft Cultures as a Model for Evaluating Compounds against Alphaherpesviruses

Andrei

Oord

Fiten

et al. 2005

“…EC 50 was an unknown fitting parameter in all simulations. While EC 50 values are roughly similar for ACV and FCV, estimates based on the results of plaque reduction assays are reliable only for resistant isolates with EC 50 s of Ͼ2 g/ml (8.9 M ACV and 6.2 M FCV): estimates vary by 1 to 2 log units between cell lines for sensitive isolates (20). Moreover, the conditions used for these cell culture-based assays may or may not represent in vivo infection.…”

Section: Fig 1 Mathematical Model (A)mentioning

confidence: 99%

“…Second, we performed global sensitivity analyses of PK/PD parameters to explore which PK/PD values are most likely to impact clinical outcomes. All model parameters (C max , T max , and EC 50 ) were randomly drawn from probability distribution functions (pdf's), which were derived according to a literature review (15,19,20). The Hill coefficient was randomly selected for values between 1 and 2.…”

Section: Fig 1 Mathematical Model (A)mentioning

confidence: 99%

Rapid Viral Expansion and Short Drug Half-Life Explain the Incomplete Effectiveness of Current Herpes Simplex Virus 2-Directed Antiviral Agents

Schiffer

Swan

Corey

et al. 2013

eThe nucleoside analogues acyclovir (ACV) and famciclovir (FCV) reduce the frequency and severity of herpes simplex virus 2 (HSV-2) genital shedding, yet despite their high potency in vitro and a lack of induced drug resistance, frequent episodes of breakthrough mucosal shedding occur. We tested a published stochastic, spatial mathematical model of HSV-2 replication and spread, in concert with pharmacokinetic and pharmacodynamic equations, against virologic data from clinical trials of twice-daily acyclovir and famciclovir suppression. The model reproduced the key features of clinical trial data, including genital shedding episode rate, expansion and decay dynamics, and heterogeneous peak viral production and duration. In simulations, these agents shortened episode duration by limiting the extent of viral production by 1 to 2 log units and limiting the formation of secondary ulcers by ϳ50%. However, drug concentrations were noninhibitory during 42% of the dosing cycle. Even if drug concentrations were high at episode initiation, prolonged episodes often ensued due to drug decay over ensuing hours and subsequent rebound of rapidly replicating HSV-2. The local CD8؉ T-cell density was more predictive of episode viral production (R 2 ‫؍‬ 0.42) and duration (R 2 ‫؍‬ 0.21) than the drug concentration at episode onset (R 2 ‫؍‬ 0.14 and 0.05, respectively), though the model projected that an agent with an equivalent potency but a two times longer half-life would decrease shedding by 80% compared to that of standard twice-daily regimens. Therefore, long half-life is a key characteristic of any agent that might fully suppress HSV-2 reactivations.T he agents currently licensed for treatment of herpes simplex virus 2 (HSV-2) infection, acyclovir (ACV), famciclovir (FCV), and valacyclovir (VCV), decrease genital tract viral shedding and lesion rates (1-3), yet they do not eliminate shedding, even when given three times daily at maximal doses (4). The reason that shedding episodes lasting several days still occur in most treated patients is unknown. Drug resistance does not explain breakthrough shedding and is usually clinically relevant only in immunocompromised hosts (5). Other possibilities include inadequate penetration of drug into tissue or breakthrough replication due to subtherapeutic concentrations. Recent evidence highlights that certain antiretroviral agents bind cooperatively with viral target enzymes (6, 7). A lack of cooperative binding is another possible mechanism for diminished antiviral potency.We recently published a stochastic spatial model of HSV-2 pathogenesis (8) that provides hypotheses for the shedding patterns observed in persons off therapy. In simulations of this model off treatment, viral spread occurs in several stages: neurons release HSV-2 in a slow drip throughout the genital tract. Mucosal replication is initiated when epithelial cells are infected (9). Rapid cellto-cell spread of HSV ensues within a single ulcer, and the extent of infection inversely correlates with the local density of resi...

“…Cytotoxicity assay. The cytotoxicity of the drug for mammalian cells was measured by the neutral red uptake assay (26) with mouse peritoneal macrophages and a transformed cell line (Vero cells) (29). The cells were cultivated in 96-well microtiter plates (150 l containing 10 5 cells/ml in Eagle minimal essential medium [Eagle MEM]/well) at 37°C in a humidified 5% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

Antileishmanial Activity of a Linalool-Rich Essential Oil fromCroton cajucara

Rosa

Mendonça-Filho

Bizzo

et al. 2003

216

119

The in vitro leishmanicidal effects of a linalool-rich essential oil from the leaves of Croton cajucara against Leishmania amazonensis were investigated. Morphological changes in L. amazonensis promastigotes treated with 15 ng of essential oil per ml were observed by transmission electron microscopy; leishmanial nuclear and kinetoplast chromatin destruction, followed by cell lysis, was observed within 1 h. Pretreatment of mouse peritoneal macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these macrophages and L. amazonensis, with a concomitant increase by 220% in the level of nitric oxide production by the infected macrophages. Treatment of preinfected macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these cells and the parasites, which led to a 60% increase in the amount of nitric oxide produced by the preinfected macrophages. These results provide new perspectives on the development of drugs with activities against Leishmania, as linalool-rich essential oil is a strikingly potent leishmanicidal plant extract (50% lethal doses, 8.3 ng/ml for promastigotes and 8.7 ng/ml for amastigotes) which inhibited the growth of L. amazonensis promastigotes at very low concentrations (MIC, 85.0 pg/ml) and which presented no cytotoxic effects against mammalian cells.