Cutaneous HSV-1 Infection Research Articles

Pluronic F127-tailored lecithin organogel of acyclovir: preclinical evidence of antiviral activity using BALB/c murine model of cutaneous HSV-1 infection.

Herpes is a well-known contagious infection equally affecting both sexes. Among many antiviral drugs employed for its treatment, acyclovir (ACY) is the drug of choice. The currently available therapies of ACY suffer from limitations like poor oral bioavailability (10-15%) and high-dose requirement. The present scientific study aims to explore pluronic lecithin organogel (PLO) as a novel drug delivery platform for ACY to bring an improvement in its delivery through topical route. The properties of organogel like biocompatibility and amphiphilic nature which facilitates dissolution of various drugs of different solubility characteristics along with enhancing the permeation potential of active molecules make it a favorable drug delivery platform for the management of topical diseases. The developed PLO formulations were characterized for micromeritic characteristics, viz., zeta potential, percentage drug content, organogel morphology, skin permeation, retention, and stability studies. The selected topical formulation was further compared with the marketed one for its therapeutic efficacy by inducing cutaneous Herpes simplex virus type 1 infection followed by confirmation of viral load by immunofluorescence and PCR analyses. The developed formulation showed significant improvement over the marketed product as reflected in lesion scoring index and PCR analysis. Further, it proved better to the marketed formulation in t.i.d. treatment regimen in comparison to control. The improvement in overall performance leading to enhanced bioavailability and safety is attributed to the synergism between excipient properties and formulation characteristics. The drug ACY in this micro environment not only finds an improved delivery vehicle but it also offers enhanced drug-target interactions.

S100A9 plays a pivotal role in a mouse model of herpetic neuralgia via TLR4/TNF pathway

Herpetic neuralgia is a painful condition following herpes zoster disease, which results from Varicella-zoster virus reactivation in the dorsal or trigeminal sensory ganglia. Nevertheless, the pathophysiological mechanisms involved in herpetic neuralgia are not well understood. Recently, we identified, that neuroimmune-glia interactions in the sensory ganglion is a critical mechanism for the development of herpetic neuralgia. Here, we investigate the contribution of S100A9, a well-known pro-inflammatory molecule produced by myeloid cells, for the development of herpetic neuralgia using a murine model of HSV-1 infection. We found that cutaneous HSV-1 infection results in an increase of S100A9 expression in the Dorsal Root Ganglia (DRGs). Infiltrating neutrophils into the DRGs were the main source of S100A9 post HSV-1 infection. Functionally, genetic or pharmacological inhibition of S100A9 impairs the development of HSV-1 infection-induced mechanical pain hypersensitivity. Finally, we found that the pronociceptive role of S100A9 in herpetic neuralgia depends on the TLR4/TNF pathway. These results unraveled previously unknown mechanisms involved in the pathophysiology of herpetic neuralgia and indicate that S100A9 might be an important target for novel therapies aiming acute herpetic neuralgia.

Fabrication of acyclovir-loaded flexible membrane vesicles (FMVs): evidence of preclinical efficacy of antiviral activity in murine model of cutaneous HSV-1 infection.

The present investigation focuses on the development and evaluation of acyclovir-loaded flexible membrane vesicles (ACY-FMVs) and evaluates their targeting potential to localize the drug into skin layers. The drug-loaded FMVs were prepared by thin-film hydration method and characterized for various attributes including micromeritics, entrapment efficiency, vesicle shape, size, and degree of deformability. The values of particle size and zeta potential of the developed carrier system were found to be 453.7nm and -11.62mV, respectively. The system was further incorporated into a hydrogel and evaluated for skin permeability and retention characteristics in comparison to marketed formulation. The developed formulation demonstrated enhanced retention of drug deep inside the skin layers which can probably decrease the frequency of application of the drug, thereby reducing its adverse effects. Skin irritancy studies performed on Laca mice skin proved the safety and non-irritant nature of ACY-FMVs. The pharmacodynamic studies on murine model for HSV-1 infection demonstrated immense potential and safety of topically applied ACY-FMVs. However, more intensive studies need to be pursued to explore and exploit the potential of lipid-based systems in anti-viral therapeutics. These preclinical findings provide a hope for corroborating the efficacy in clinical situations.

Enhanced acyclovir delivery using w/o type microemulsion: preclinical assessment of antiviral activity using murine model of zosteriform cutaneous HSV-1 infection

The present study was aimed to develop and evaluate a microemulsion-based dermal drug delivery of an antiviral agent, acyclovir. A water-in-oil microemulsion was prepared using isopropyl myristate, Tween 20, Span 20, water and dimethylsulphoxide. It was characterized for drug content, stability, globule size, pH, viscosity and ex vivo permeation through mice skin. In vivo antiviral efficacy of optimized formulation was assessed in female Balb/c mice against herpes simplex virus-I (HSV-I)-induced infection. It was observed that optimized formulation when applied 24-h post-infection could completely inhibit the development of cutaneous herpetic lesions vis-à-vis marketed cream.

Down-RANKing the Threat of HSV-1: RANKL Upregulates MHC-Class-I-Restricted Anti-Viral Immunity in Herpes Simplex Virus Infection

Herpes simplex virus (HSV-1) is a major cause of viral skin infection in humans. Klenner and colleagues now show that the epidermal receptor activator of NFκB ligand (RANKL) is critical for the induction of anti-viral CD8(+) effector T cells (CTL) during cutaneous HSV-1 infection. Activation via RANKL prevents Langerhans cell apoptosis, thus leading to enhanced antigen transport to regional lymph nodes, increasing the CTL-priming capacity of lymph node dendritic cells.

Herpes murine model as a biological assay to test dialyzable leukocyte extracts activity.

Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v) the protective effect of Transferon in vivo correlates with changes in serum cytokines.

Interleukin-1α released from HSV-1 infected skin keratinocytes initiates protective immunity against systemic infection (VIR1P.964)

Abstract Herpes simplex virus-1 (HSV-1) is a common human pathogen that typically causes self-limiting disease. However, neonates and patients with compromised immune systems are at risk of developing potentially fatal systemic disease. Previously we reported that IL-1α is released by keratinocytes treated with poly(I:C), a double stranded RNA analogue often utilized to simulate virus infections. The objective of this study was to examine whether IL-1 signaling plays a role in initiating protective immunity against HSV-1 infections. Extracellular release of IL-1α protein could be detected 6-24 hours after HSV-1 infection of primary human keratinocytes (4-5-fold increase, p<0.01). This release was not associated with a correlating change in mRNA levels suggesting that the cells released a pre-formed pool of protein. HSV-1 triggered activation of inflammatory caspases-1/4 as determined using fluorescence labeled peptides (6-fold, p<0.001); however, the cellular release of IL-1α was independent of caspases-1/4. IL-1R knockout (KO) mice exhibited an increased mortality following cutaneous HSV-1 infection (60 versus 25%, p<0.01). Caspase-1/4 deficiency did not affect survival rates; hence, the protective function of IL-1R must be exclusively due to IL-1α signaling. Recruitment of leukocytes to the infected epidermis was delayed in IL-1R KO mice. Hence, HSV-1 triggered IL-1α release from infected cells appears important for initiating protective immunity against systemic HSV-1 infections.

Topical cream-based oxyresveratrol in the treatment of cutaneous HSV-1 infection in mice

Anti-herpes simplex virus (HSV) activities of oxyresveratrol in vitro and topical administration in cutaneous HSV-1 infection in mice were examined. The inhibitory concentrations for 50% plaque formation (IC 50) of oxyresveratrol against HSV-1 clinical isolates and HSV-2 clinical isolates were 20.9–29.5 and 22.2–27.5 μg/ml, respectively. In topical administration in cutaneous HSV-1 infection in mice, 2.5%, 5%, 10% and 20% oxyresveratrol in cream vehicle applied three times daily for 7 days after infection were evaluated and 10% and 20% oxyresveratrol cream were significantly effective in delaying the development of skin lesions and protection from death ( P < 0.01). The concentration of 10% oxyresveratrol in cream was significantly more effective than that of 30% oxyresveratrol in vaseline applied three times daily ( P < 0.01). Oxyresveratrol cream at 20% was as effective as 5% ACV cream applied three times daily ( P < 0.01). Both 10% and 20% oxyresveratrol cream were as effective as that of 5% ACV cream applied two times daily ( P > 0.05). Therapeutic efficacy of oxyresveratrol in cream vehicle was dose-dependent and the maximum efficacy observed on day 6 after infection was shown at 10% oxyresveratrol in cream applied three times daily. The frequency of application of 10% oxyresveratrol cream at three, four and five times daily was as effective as that of 5% ACV cream applied five times daily ( P > 0.05). These results demonstrated that topical administration of oxyresveratrol in novel cream vehicle reduced the concentration of oxyresveratrol to 10% and was suitable for cutaneous HSV infection.

Anti-herpes simplex virus (HSV-1) activity of oxyresveratrol derived from Thai medicinal plant: Mechanism of action and therapeutic efficacy on cutaneous HSV-1 infection in mice

Oxyresveratrol, a major compound purified from Artocarpus lakoocha, a Thai traditional medicinal plant, was evaluated for its mechanism of action and therapeutic efficacy on cutaneous herpes simplex virus (HSV) infection in mice. The inhibitory concentrations for 50% HSV-1 plaque formation of oxyresveratrol, three clinical isolates, thymidine kinase (TK)-deficient and phosphonoacetic acid (PAA)-resistant HSV-1 were 19.8, 23.3, 23.5, 24.8, 25.5 and 21.7 μg/ml, respectively. Oxyresveratrol exhibited the inhibitory activity at the early and late phase of viral replication and inhibited the viral replication with pretreatment in one-step growth assay of HSV-1 and HSV-2. Oxyresveratrol inhibited late protein synthesis at 30 μg/ml. The combination of oxyresveratrol and acyclovir (ACV) produced synergistic anti-HSV-1 effect, as characterized by the isobologram of plaque inhibition. Mice orally treated with oxyresveratrol (500 mg/kg/dose) dose at 8 h before and three times daily had significant delay in herpetic skin lesion development ( P < 0.05). Topical application of 30% oxyresveratrol ointment five times daily significantly delayed the development of skin lesions and protected mice from death ( P < 0.0001).

Efficacy of Thai medicinal plant extracts against herpes simplex virus type 1 infection in vitro and in vivo

Twenty Thai medicinal plant extracts were evaluated for anti-herpes simplex virus type 1 (HSV-1) activity. Eleven of them inhibited plaque formation of HSV-1 more than 50% at 100 μg/ml in a plaque reduction assay. Aglaia odorata, Moringa oleifera, and Ventilago denticulata among the 11 were also effective against thymidine kinase-deficient HSV-1 and phosphonoacetate-resistant HSV-1 strains. These therapeutic efficacies were characterized using a cutaneous HSV-1 infection in mice. The extract of M. oleifera at a dose of 750 mg/kg per day significantly delayed the development of skin lesions, prolonged the mean survival times and reduced the mortality of HSV-1 infected mice as compared with 2% DMSO in distilled water ( P<0.05). The extracts of A. odorata and V. denticulata were also significantly effective in limiting the development of skin lesions ( P<0.05). There were no significant difference between acyclovir and these three plant extracts in the delay of the development of skin lesions and no significant difference between acyclovir and M. oleifera in mean survival times. Toxicity of these plant extracts were not observed in treated mice. Thus, these three plant extracts may be possible candidates of anti-HSV-1 agents.

Stress-induced changes in pathophysiology and interferon gene expression during primary HSV-1 infection

Herpes simplex viruses (HSV) are the cause of the most common clinically recognized herpesvirus infections. The severity and duration of the primary HSV infection have been correlated with the frequency and severity of subsequent recurrences. Reactivation of latent HSV-1 can occur as a result of physical or emotional stress; however, the effects of stress on the modulation of the clinical pathophysiology of primary HSV-1 infections are not well understood. Although it is known that stress can be immunosuppresive, the immunological mechanisms by which stress modulates early immune responses, such as type I interferon gene expression during a primary HSV-1 infection are still not understood. It was hypothesized that due to suppressed early immune responses, stress would increase the severity of a cutaneous primary HSV-1 infection. In this investigation, a cutaneous HSV-1 model in the SKH-1 mouse was characterized and utilized to study the effect of restraint stress on the modulation of the clinical pathophysiology of primary HSV-1. Despite prolonged viral replication at the site of primary infection, restraint stress decreased the clinical severity of primary HSV-1 in the skin of SKH-1 mice. A decrease in type I and type II IFN expression was found in the skin of acutely infected restrained mice when compared to controls at day 3 post-infection using competitive RT-PCR. Using the glucocorticoid-receptor antagonist RU486, IFN-β and INF-γ expression were restored in restrained animals to control levels. Treatment with RU486 also increased the clinical severity of the cutaneous infection to control levels in restrained mice. Thus, RST masked the severity of an HSV-1 infection by decreasing its clinical signs while impairing the ability of the host to control viral replication prolonging the infectious period.

Effect of Azone upon the in vivo antiviral efficacy of cidofovir or acyclovir topical formulations in treatment/prevention of cutaneous HSV-1 infections and its correlation with skin target site free drug concentration in hairless mice

The purpose of this study is to examine the influence of Azone upon the skin target site free drug concentration ( C ∗) and its correlation with the in vivo antiviral efficacies of cidofovir (HPMPC) and acyclovir (ACV) against HSV-1 infections. Formulations of HPMPC and ACV with or without Azone were used. The in vitro skin flux experiments were performed and the C ∗ values were calculated. For the in vivo efficacy studies, hairless mice cutaneously infected with HSV-1 were used and three different treatment protocols were carried out. The protocols were chosen based upon when therapy is initiated and terminated in such a way to assess the efficacy of the test drug to cure and/or prevent HSV-1 infections. A finite dose of the formulation was topically applied twice a day for the predetermined time course for each protocol and the lesions were scored on the fifth day. For ACV formulation with Azone, the C ∗ values and hence the in vivo efficacy were much higher than those for that without Azone. In protocol #1, however, early treatment did not increase the in vivo efficacy of ACV when compared with the standard treatment protocol #3. In protocol #2 where the treatment was terminated on the day of virus inoculation, the efficacies for both ACV formulations were completely absent. Although the estimated C ∗ values for HPMPC formulations with and without Azone were comparable, formulation with Azone was much more effective than that without Azone in all treatment protocols. HPMPC formulations with Azone at similar flux values were much more effective in “treating and preventing” HSV-1 infections than those without Azone. For ACV formulations, in contrast, addition of Azone has failed to show any effect on the preventive in vivo antiviral efficacy and the enhancement of ACV in vivo antiviral efficacy was merely the skin permeation enhancement effect of Azone.

Efficacy of Gel Formulations Containing free and Liposomal Foscarnet in a Murine Model of Cutaneous HSV-1 Infection

The efficacy of gel formulations containing free and liposomal foscarnet has been evaluated in a murine model of cutaneous Herpes simplex virus type-1 infection. Both formulations were applied topically 3 times daily for 4 days and initiated 24 h post-infection. The penetration of liposomes incorporated into the gel in infected skin tissues was better than that of liposomes dispersed in buffer. Therein, their localization mostly matched that of viral antigen detected by immunoperoxydase staining. Despite these facts, the efficacy of gel formulations of both free and liposomal foscarnet in preventing the development of a zosteriform rash in mice was similar. Electron microscopic examination revealed that liposomes incorporated into the gel formed aggregates together with the micelles of gel. Diffusion studies showed that liposomes were trapped within these aggregates and were hardly able to diffuse across a polycarbonate membrane. In addition, although the liposomes were shown to be highly stable in vitro, the formation of these aggregates destabilized their membrane resulting in a premature release of foscarnet from liposomes. The efficacy of both gel formulations was higher than that of solutions of free or liposomal foscarnet suggesting that the gel formulation is a suitable matrix for the delivery of drugs. Thus, strategies aimed at reducing the interaction of liposomes with the gel could be a convenient approach to improve the efficacy of liposome-encapsulated drug over the free drug.

Evaluation of antioxidant healing formulations in topical therapy of experimental cutaneous and genital herpes simplex virus infections

A genital HSV-2 infection of guinea pigs and a cutaneous HSV-1 infection in mice were used to examine the ability of antioxidant components CRT 1 CRT was known by the internal project name of Triad during these investigations. 1 to reduce lesion development, duration, and severity. CRT is a patented antioxidant formulation developed by Warner-Lambert Worldwide Consumer Healthcare R. and D. CRT contains three components that work synergistically: vitamin E, sodium pyruvate and membrane stabilizing fatty acids. The MS strain of HSV-2 was utilized for intravaginal inoculation of guinea pigs with approximately 1.2×10 5 plaque forming units. The guinea pigs were treated on the external genital skin four times daily for 10 days beginning 48 h post viral inoculation. This study was designed to optimize the CRT formulations that would be used to quantify the synergistic effect of the CRT components. SKH-1 male hairless mice were inoculated with 1×10 7 HSV-1 (McIntyre strain) on the dorsal surface of the mouse and treated with CRT formulations starting on the afternoon of the day of infection, and treated for the following 14 days. In the guinea pig model, the CRT formula that contained all three CRT components, worked synergistically to reduce lesion development, duration and severity scores significantly compared to vehicle control or acyclovir. Acyclovir was the only compound that reduced viral titers, but in contrast to CRT, acyclovir did not reduce lesion development, duration or severity. The quantitative effect of the three CRT components was demonstrated in the mouse model.

The relationship of basal cell layer drug concentration (C*) to the antiviral efficacy: A rigorous evaluation of the C* concept for predicting the entire range of topical efficacy of acyclovir formulations in the treatment of cutaneous HSV-1 infections in hairless mice: Samir C. Mehta, Mohsen I. Afouna, Abdel-Halim Ghanem, William I. Higuchi, Earl R. Kern a and Hamed El-Shattawy b.

The relationship of basal cell layer drug concentration (C*) to the antiviral efficacy: A rigorous evaluation of the C* concept for predicting the entire range of topical efficacy of acyclovir formulations in the treatment of cutaneous HSV-1 infections in hairless mice: Samir C. Mehta, Mohsen I. Afouna, Abdel-Halim Ghanem, William I. Higuchi, Earl R. Kern a and Hamed El-Shattawy b.

Comparative effects of famciclovir and valaciclovir on cutaneous and neurological HSV-1 infection in normal and immunocompromised mice: A.M. Thackray and H.J. Field, University of Cambridge, Madingley Road, Cambridge, CB3 OES, UK

Comparative effects of famciclovir and valaciclovir on cutaneous and neurological HSV-1 infection in normal and immunocompromised mice: A.M. Thackray and H.J. Field, University of Cambridge, Madingley Road, Cambridge, CB3 OES, UK

Significance of high skin fluxes produced by acyclovir topical formulations for achieving therapeutic levels of drug at the target site in the treatment of cutaneous HSV-1 infections in hairless mice: A closer look at the C* concept: Phenil J. Patel1, Abdel-Halim Ghanem 1, William I. Higuchi 1, Jer Hsu 1, V. Srinivasan 2 and Earl R. Kern 3. 1Dept. of

Significance of high skin fluxes produced by acyclovir topical formulations for achieving therapeutic levels of drug at the target site in the treatment of cutaneous HSV-1 infections in hairless mice: A closer look at the C* concept: Phenil J. Patel1, Abdel-Halim Ghanem 1, William I. Higuchi 1, Jer Hsu 1, V. Srinivasan 2 and Earl R. Kern 3. 1Dept. of

Synthesis, Oral Bioavailability and in vivo Activity of Acetal Derivatives of the Selective Antiherpesvirus Agent 9-(3-hydroxypropoxy) Guanine (BRL 44385)

Acyclic acetal derivatives of the selective antiherpesvirus agent 9-(3-hydroxypropoxy) guanine (BRL44385) and of its 2-aminopurine congener (BRL46720) have been prepared and evaluated in mice for oral delivery of BRL 44385. Guanine derivatives (6 a-c) were prepared via Mitsunobu condensation of an alcohol with a 9-hydroxy-6-methoxypurine (Harnden and Wyatt, 1990). Synthesis of derivatives of 2-aminopurine (10 a-d) was achieved by hydrogenolysis of 9-alkoxy-6-chloropurines, which were obtained either by reaction of an alkoxyamine with 4,6-dichloro-2,5-diformamidopyrimidine and subsequent ring closure or by Mitsunobu condensation of an alcohol with a 6-chloro-9-hydroxypurine. Following oral administration, 2-amino-9-[3-(iso-propoxymethyl)propoxy]-purine (10b, BRL 55792) was very well absorbed and provided high and prolonged concentrations of BRL44385 in the blood. In a cutaneous HSV-1 infection in the ear pinna of mice, orally dosed BRL 55792 was at least 3-fold more potent than both BRL44385 and Acyclovir in reduction of lesion severity.

Estimation of skin target site acyclovir concentrations following controlled (trans)dermal drug delivery in topical and systemic treatment of cutaneous HSV-1 infections in hairless mice.

The use of controlled transdermal delivery of acyclovir (ACV) in the treatment of cutaneous herpes simplex virus type 1 infections in hairless mice was investigated. Using an in vivo animal model (A. Gonsho, et al. Int. J. Pharm. 65:183-194 (1990)) made it possible to quantify both, the topical and the systemic antiviral efficacy of ACV transdermal patches as a function of the drug delivery rate of the patches. Drug delivery rates required to attain systemic efficacy were found to be higher than the rates required to attain the same magnitude of topical efficacy. The ACV concentrations in the basal cell layer of the epidermis for 50% topical efficacy and 50% systemic efficacy were estimated. The basal epidermis layer was considered to be the site of antiviral drug activity (skin target site). Systemic plasma levels were obtained from pharmacokinetic studies and were used to estimate the ACV concentration achieved systemically in the basal epidermis layer. A computational model for drug permeation across skin was employed to estimate the ACV concentration achieved topically in the basal epidermis layer. Equal topical and systemic efficacies were found to correspond to equal drug concentrations at the site of antiviral activity. The length of the effective diffusion pathway of drug molecules in the dermis prior to entering the blood circulation was assumed to be approximately equal to 1/20 of the anatomical dermis thickness because of dermis vascularization.

An application of the [formula omitted] concept in predicting the topical efficacy of finite dose acyclovir in the treatment of cutaneous HSV-1 infections in hairless mice

This study has examined C ∗ (and its temporal pattern) as a parameter for predicting topical antiviral efficacy of acyclovir (ACV) formulations in a recently developed hairless mouse model for the treatment of HSV-1 cutaneous infections. C ∗ referred to here is the free drug concentration at the target site, which is believed to be the basal cell layer of the epidermis. Two different topical ACV formulations, one available commercially (Zovirax 5% ointment) and the other prepared in our laboratories (3% ACV suspension) were investigated in a finite dose multiple dosing regimen (twice a day application) to simulate the clinical situation. C ∗ was calculated from in vitro flux data and information obtained from previous baseline studies. In vitro ACV flux determinations involved an in vivo-in vitro experimental design that was believed to approximate closely the actual in vivo antiviral treatment protocol. For the in vivo antiviral efficacy studies, a 1-day delayed (after virus inoculation) 4-day treatment protocol was implemented and the animals wore a Velcro jacket to protect the formulations from being removed during the drug application period. With Zovirax 5% ointment given 3 mg every 12 h, our results showed a 0% topical efficacy which was consistent with the C ∗ predictions. In the case of the laboratory prepared 3% ACV suspension, a dose of 2 μl was given every 12 h and our results showed a modest level (33–44%) of topical effectiveness, which was somewhat higher than predicted (0–25%) from C ∗ considerations. Also, in the efficacy studies (but not in the in vitro flux studies) with the laboratory prepared formulation, there were instances (20–33%) of severe skin irritation/damage noted at the end (day 5) of the experiments. Taking this into consideration, it is suggested that these studies represent a good correlation between predictions (C ∗) based on the in vitro data and the in vivo antiviral efficacy and validate the predictive value of this approach.