Valacyclovir or valganciclovir for cytomegalovirus prophylaxis: A randomized controlled trial in adult and pediatric kidney transplant recipients.

BACKGROUND: Valganciclovir (valG), a cytomegalovirus (CMV) prophylactic agent, has dose-limiting side effects. The tolerability and effectiveness of valacyclovir (valA) as CMV prophylaxis is unknown. METHODS: We conducted a randomized, open-label, single-center trial of valA versus valG for all posttransplant CMV prophylaxis in adult and pediatric kidney recipients. Participants were randomly assigned to receive valA or valG. Primary endpoints were the incidence of CMV viremia and side-effect related drug reduction with secondary assessment of incidence of EBV viremia. RESULTS: Of the 137 sequential kidney transplant recipients enrolled, 26 % were positive and negative for CMV antibody in donor and recipient respectively. The incidence of CMV viremia (4 of 71 [6 %]; 8 of 67 [12 %] P = 0.23), time to viremia (P = 0.16) and area under CMV viral load time curve (P = 0.19) were not significantly different. ValG participants were significantly more likely to require side-effect related dose reduction (15/71 [21 %] versus 1/66 [2 %] P = 0.0003). Leukopenia was the most common reason for valG dose reduction and granulocyte-colony stimulating factor was utilized for leukopenia recovery more frequently (25 % in valG vs 5 % in valA: P = 0.0007). Incidence of EBV viremia was not significantly different. CONCLUSIONS: ValA has significantly less dose-limiting side effects than valG. In our study population, a significant increase in CMV viremia was not observed, in adults and children after kidney transplant, compared to valG. TRIAL REGISTRATION NUMBER: NCT01329185.

Study of tissue engineered vascularised oral mucosa-like structures based on ACVM-0.25% HLC-I scaffold in vitro and in vivo.

PURPOSE: To explore the feasibility of constructing tissue-engineered vascularised oral mucosa-like structures with rabbit ACVM-0.25% HLC-I scaffold and human gingival fibroblasts (HGFs), human gingival epithelial cells (HGECs) and vascular endothelial-like cells (VEC-like cells). METHOD: Haematoxylin and Eosin (H&E) staining, immunohistochemical, immunofluorescence, 5-ethynyl-2'-deoxyuridine (EdU) staining and scanning electron microscope (SEM) were performed to detect the growth status of cells on the scaffold complex. After the scaffold complex implanted into nude mice for 28 days, tissues were harvested to observe the cell viability and morphology by the same method as above. Additionally, biomechanical experiments were used to assess the stability of composite scaffold. RESULTS: Immunofluorescence and Immunohistochemistry showed positive expression of Vimentin, S100A4 and CK, and the induced VEC-like cells had the ability to form tubule-like structures. In vitro observation results showed that HGFs, HGECs and VEC-like had good compatibility with ACVM-0.25% HLC-I and could be layered and grow in the scaffold. After implanted, the mice had no immune rejection and no obvious scar repair on the body surface. The biomfechanical test results showed that the composite scaffold has strong stability. CONCLUSION: The tissue-engineered vascularised complexes constructed by HGFs, HGECs, VEC-like cells and ACVM-0.25% HLC-I has good biocompatibility and considerable strength.

Virtual screening, ADME/T, and binding free energy analysis of anti-viral, anti-protease, and anti-infectious compounds against NSP10/NSP16 methyltransferase and main protease of SARS CoV-2.

Recently, a pathogen has been identified as a novel coronavirus (SARS-CoV-2) and found to trigger novel pneumonia (COVID-19) in human beings and some other mammals. The uncontrolled release of cytokines is seen from the primary stages of symptoms to last acute respiratory distress syndrome (ARDS). Thus, it is necessary to find out safe and effective drugs against this deadly coronavirus as soon as possible. Here, we downloaded the three-dimensional model of NSP10/NSP16 methyltransferase (PDB-ID: 6w6l) and main protease (PDB-ID: 6lu7) of COVID-19. Using these molecular models, we performed virtual screening with our anti-viral, inti-infectious, and anti-protease compounds, which are attractive therapeutics to prevent infection of the COVID-19. We found that top screened compound binds with protein molecules with good dock score with the help of hydrophobic interactions and hydrogen bonding. We observed that protease complexed with Cyclocytidine hydrochloride (anti-viral and anti-cancer), Trifluridine (anti-viral), Adonitol, and Meropenem (anti-bacterial), and Penciclovir (anti-viral) bound with a good docking score ranging from -6.8 to -5.1 (Kcal/mol). Further, NSP10/NSP16 methyltransferase complexed with Telbivudine, Oxytetracycline dihydrate (anti-viral), Methylgallate (anti-malarial), 2-deoxyglucose and Daphnetin (anti-cancer) from the docking score of -7.0 to -5.7 (Kcal/mol). In conclusion, the selected compounds may be used as a novel therapeutic agent to combat this deadly pandemic disease, SARS-CoV-2 infection, but needs further experimental research.HighlightsNSP10/NSP16 methyltransferase and main protease complex of SARS CoV-2 bind with selected drugs.NSP10/NSP16 methyltransferase and protease interacted with drugs by hydrophobic interactions.Compounds show good DG binging free energy with protein complexes.Ligands were found to follow the Lipinski rule of five.

Tricyclic Derivative of Acyclovir and Its Esters in Relation to the Esters of Acyclovir Enzymatic Stability: Enzymatic Stability Study.

The 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-6-(4-methoxyphenyl)-9-oxo-5H-imidazo[1,2-a]-purine (6-(4-MeOPh)-TACV) was selected to assess the enzymatic stability of the tricyclic acyclovir derivatives from the imidazo[1,2-a]-purine group. The parent compound and its esters (acetyl, isobutyryl, pivaloyl, nicotinic, ethoxycarbonyl) were subjected to kinetic studies and compared with the stability of analogous acyclovir (ACV) esters. The enzymatic hydrolysis was observed in vitro in a medium of 80% human plasma in the absence and presence of porcine liver esterase (PLE). The tests were carried out at 37 °C. To determine the kinetic parameters (kobs., t0.5) of the observed reaction, the validated HPLC-UV method in the reversed phase was used. The HPLC-MS/MS method was used to identify the degradation products under the tested conditions. In summary, it was found that 6-(4-MeOPh)-TACV esters are more susceptible to esterase metabolism than ACV esters. It was confirmed by HPLC-MS/MS that in the plasma, the main product of their hydrolysis is 6-(4-MeOPh)-TACV and not ACV, which confirms that their antiviral activity observed in vitro does not result from ring degradation.

Antiviral effect of Chinese herbal prescription JieZe-1 on adhesion and penetration of VK2/E6E7 with herpes simplex viruses type 2.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese Herbal Prescription JieZe-1(JZ-1), added and subtracted from Yihuang Decoction, a famous formula in the 12th year of Kangxi in Qing Dynasty, has a clear effect on Genital Herpes (GH) and no obvious adverse reactions occur clinically. JZ-1 also has preventive and therapeutic effects on Trichomonas vaginitis, Candida albicans vaginitis and GH in vitro and in vivo experiments. AIM OF STUDY: The effect and mechanism of JZ-1 on anti-herpes simplex virus type 2(HSV-2) in vitro focusing on adhesion and penetration stages were investigated. MATERIALS AND METHODS: A model of HSV-2 infection of VK2/E6E7 was developed. In order to explore JZ-1's anti-HSV-2 effect in vitro, cell morphology, ultrastructural pathology, cell viability and expression of viral glycoprotein D (gD) were assessed at 6 h, 12 h, 18 h, and 24 h of JZ-1 treatment. Then we measured the exact time required for adhesion and penetration of HSV-2 into VK2/E6E7 among a series of times at room temperature and under temperature control techniques. We treated VK2/E6E7 with JZ-1, penciclovir, or berberine and explored the mechanism of JZ-1 in blocking HSV-2 adhesion and penetration of host cells by assessing the cell ultrastructural pathology, viability, viral proteins gB, gD, VP16, ICP5, and ICP4 and host cell proteins HVEM, Nectin-1, and Nectin-2. RESULTS: HSV-2 can fully adhere and penetrate into VK/E6E7 within 5 mins at room temperature while it takes 60mins under temperature control techniques. JZ-1 and penciclovir showed significant anti-HSV-2 effects, with improved host cell morphologies and increased host cell viabilities observed after treatment for 24 h. The anti-HSV-2 effect of JZ-1 can be detected after treatment for 6 h while that of penciclovir was not obvious until treatment for 12 h. JZ-1 showed distinct effect on HSV-2 adhesion and penetration stages by significantly reducing the expression of viral proteins gB, gD, VP16, ICP5, and ICP4, improving cell morphology and increasing cell viability. However, these effects were not exerted via downregulated expression of membrane fusion-related proteins such as HVEM, Nectin-1, or Nectin-2. The specific anti-HSV-2 mechanism of JZ-1 need to be further explored. CONCLUSION: The anti-HSV-2 effect of JZ-1 was superior to that of penciclovir and berberine in vitro, and was mainly mediated by enhancing host cell defense and blocking adhesion and penetration of HSV-2.

Synthesis and anti-HSV activity of tricyclic penciclovir and hydroxybutylguanine derivatives.

A series of tricyclic penciclovir (PCV) and hydroxybutylguanine (HBG) derivatives have been prepared with enhanced lipophilicity following an efficient synthetic route. All the novel tricyclic derivatives were evaluated for inhibitory activity against herpes simplex virus 1 and 2 (HSV-1, HSV-2) and thymidine kinase deficient (ACV resistant) HSV-1. The tricyclic HBG derivatives were devoid of inhibitory activity however several of the tricyclic PCV derivatives showed promising antiviral activity, in particular 9g (R = 4-MeO-C6H4) displayed good inhibitory activity (HSV-1 EC50 1.5 µM, HSV-2 EC50 0.8 µM) and retained inhibitory activity in HSV-1 TK- cells (EC50 0.8 µM). Computational docking experiments supported the biological data observed and this preliminary study provides useful data for further development of tricyclic acyclic nucleoside derivatives with improved lipophilicity and retention of activity in HSV-1 TK deficient strains. Also, the new tricyclic derivatives were evaluated against a broad range of other DNA and RNA viruses, but were found to be inactive at subtoxic concentrations. In addition, weak to moderate cytostatic effect was observed for the new compounds.

Pilot Study of the Safety and Tolerability of a Subconjunctival Penciclovir Implant in Cats Experimentally Infected with Herpesvirus.

PURPOSE: To assess safety and tolerability of a subconjunctival penciclovir implant in cats infected with feline herpesvirus type 1 (FHV-1). METHODS: Subconjunctival blank (n = 4 cats) or penciclovir-impregnated (n = 6) silicone implants were placed bilaterally in 10 normal, FHV-1-naive cats 7-8 days before viral inoculation. Outcomes included disease score, FHV-1 serology, conjunctival viral load, Schirmer tear tests (STT), tear film break-up times (TFBUTs), conjunctival histology, goblet cell density (GCD), body weight, tear and plasma penciclovir concentration, and corneal ulcer evaluation. RESULTS: Both groups had similar clinical and histologic disease scores, STT values, TFBUTs, GCD, FHV-1 titers, viral loads, and body weight changes. No ocular or systemic signs of toxicity were noted. Tear penciclovir concentration varied widely among cats and across time points. Tear penciclovir concentrations exceeded the lowest published half maximal inhibitory concentration (IC50) in 5/6 treated cats. Plasma penciclovir concentrations remained below 10 ng/mL. Cats with higher tear penciclovir concentrations at inoculation and/or time of peak disease had fewer corneal ulcers than cats in which tear penciclovir concentrations were inconsistent, low, or unrecordable. CONCLUSIONS: Subconjunctival blank and penciclovir-impregnated implants were well tolerated at the ocular surface and not associated with systemic toxicity, adverse effect, or appreciable plasma penciclovir concentrations. Tear penciclovir concentrations >IC50 were sometimes achieved, especially during burst release soon after implant placement. Further study is necessary to determine efficacy of locally delivered penciclovir when penciclovir concentration is consistently maintained above IC50. This will be especially useful in patients unable to receive systemic therapy.

Synthesis of Chiral Acyclic Nucleosides by Sharpless Asymmetric Dihydroxylation: Access to Cidofovir and Buciclovir.

An efficient method to construct chiral acyclic nucleosides via Sharpless asymmetric dihydroxylation of N-allylpyrimidines or N-alkenylpurines is reported. A range of chiral acyclic nucleosides with two adjacent hydroxyl groups present on the side chains could be produced in good yields (up to 97% yield) and excellent enantioselectivities (90-99% ee). The synthetic utility of the reaction was demonstrated by the catalytic asymmetric synthesis of ( S)-Cidofovir and ( R)-Buciclovir.

An atypical presentation of herpes simplex virus infection in Harlequin ichthyosis.

We present an atypical presentation of herpes simplex virus infection in a patient with Harlequin ichthyosis, which we attribute to abnormal cornification.

Evaluation of the antiviral activities of Bacharis dracunculifolia and quercetin on Equid herpesvirus 1 in a murine model.

Equid herpesvirus 1 (EHV-1) is a pathogen of high economic importance in equine breeding operations around the world. EHV-1 infection causes respiratory, neurologic and reproductive disease. The absence of an efficient therapy has caught the attention of the scientific community and the therapeutic activities of natural products with its antivirals effects might be effective for the disease's treatment. Herein it was evaluated the prophylactic and therapeutic potential of quercetin and ethanolic extracts of Bacharis dracunculifolia formulations compared to Penciclovir® in an in vivo EHV-1 infection model. Six to seven-week-old female C57BL/6 mice were randomly organized into fifteen groups with six animals each. Ex-1 represents the treatment post-challenge groups to assess morbidity, mortality and weight variation. Ex-2 represents the animals that received treatment for 5 days post-challenge for lesion evaluation. In Ex-3 animals were treated prior to viral challenge to assess morbidity, mortality and weight variation. All mice in the treatment groups were challenged by intranasal inoculation of 3.0 × 105 TCID50 EHV-1. The quercetin and B. dracunculifolia treatment decreased morbimortality in post-challenge treatment (Ex-1) and EHV-1 related lesions (Ex-2). Treatment prior to viral challenge (Ex-3) did not show any significant results. Based on the results of the present study, both tested formulations are promising antiviral agents for the treatment of EHV-1 infection.

Antiviral Drugs Against Alphaherpesvirus.

The discovery of acyclovir and penciclovir has led to the development of a successful systemic therapy for treating herpes simplex virus infection and varicella-zoster virus infection, and the orally available prodrugs, valacyclovir and famciclovir, have improved antiviral treatment compliance. Acyclovir and penciclovir are phosphorylated by viral thymidine kinase and are incorporated into the DNA chain by viral DNA polymerase, resulting in chain termination. Helicase-primase plays an initial step in DNA synthesis to separate the double strand into two single strands (replication fork) and is a new target of antiviral therapy. The helicase-primase inhibitors (HPIs) pritelivir and amenamevir have novel mechanisms of action, drug resistance properties, pharmacokinetic characteristics, and clinical efficacy for treating genital herpes. The clinical study of amenamevir in herpes zoster has been completed, and amenamevir has been submitted for approval for treating herpes zoster in Japan. The clinical use of HPIs will be the beginning of a new era of anti-herpes therapy.

Anxiety, depression, and hopelessness in patients before and after treatment for peripheral facial paralysis.

We conducted a prospective study to investigate the effectiveness of pharmacologic treatment on alleviating facial paralysis, as well as the anxiety and depression that are associated with it. Our study population was made up of 105 patients-59 men and 46 women, aged 18 to 60 years (mean: 38.2)-who had acute idiopathic peripheral facial paralysis. Before treatment, paralysis was classified as House-Brackmann grade II or III in 44 patients (41.9%) and grade IV to VI in the remaining 61 (58.1%). After treatment, 73 patients (69.5%) improved to grade I, 29 (27.6%) were at grade II or III, and only 3 (2.9%) remained at grade IV or higher. Mean scores on the Beck anxiety inventory, the Beck depression inventory, and the Beck hopelessness scale were 20.30, 19.75, and 7.57, respectively, before treatment and 5.72, 5.68, and 2.85 afterward; the difference in all three measures was statistically significant (p < 0.001). We found no correlation between the degree of facial paralysis and anxiety levels (r = 0.094, p = 0.338) or depression levels (r = 0.181, p = 0.064). Clinicians should consider asking patients with peripheral facial paralysis about their feelings of anxiety, depression, and hopelessness and refer them for a psychiatric consultation if necessary.

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of valacyclovir and its metabolite acyclovir in mouse and human plasma.

It is challenging to conduct a pharmacokinetic (PK) study on mice due to the limited amount of plasma one can obtain, which is also true for some clinical studies. Here, we developed and validated a simple, sensitive and robust LC-MS/MS method for measuring the prodrug valacyclovir (VACV) and its metabolite acyclovir (ACV) in mouse and human plasma. This assay utilized an acetonitrile protein precipitation method with isotope-labeled internal standards (IS) and enabled precise and accurate quantification of VACV and ACV in 10 µL plasma samples with a nine-min gradient. The analytes were separated on a Waters Atlantis T3 C18 column. The precursor-product ion transitions for VACV (m/z 325.2 > 152.1), ACV (m/z 226.2 > 152.1), VACV-D4 (m/z 329.2 > 152.1, IS) and ACV-D4 (m/z 230.2 > 152.1, IS) were detected in a multiple reaction monitoring (MRM) positive ion mode using an API4000 LC-MS/MS system. The lower limit of quantification (LLOQ) was 2 nM for both VACV and ACV. The linear range was validated over the concentration ranges of 2-200 nM and 200-5000 nM for both compounds. The matrix effect and stability of VACV and ACV were also evaluated. This assay was successfully applied to a PK study in mice.

Fracaso renal agudo y neurotoxicidad severa tras sobredosis accidental por valaciclovir en población geriátrica: a propósito de un caso / Acute renal failure and severe neurotoxicity after unintentional overdose of valacyclovir in a geriatric population: A case report

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Establishment of a novel microscale thermophoresis ligand-binding assay for characterization of SLC solute carriers using oligopeptide transporter PepT1 (SLC15 family) as a model system.

INTRODUCTION: Membrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purification tasks even more challenging compared to soluble proteins. This issue also holds true for conventional ligand-binding assays (LBAs) which usually require high-quality, pure and concentrated protein samples. Herein, we report a novel binding assay strategy to overcome these issues, taking advantage of a unique combination of yeast expression and microscale thermophoresis (MST). Following yeast overexpression of SLC15A1/PepT1 ortholog from moss Physcomitrella patens, PepTPp, which exhibits remarkable similarity to human PepT1, the approach was validated using dipeptide glycylsarcosine (Gly-Sar) and antiviral prodrug valacyclovir as test substrates. METHOD: The originality of our approach is based on the comparative analysis of solubilized total membrane preparations with or without expression of the SLC target of interest, using a yeast strain (S. cerevisiae), in which the corresponding endogenous SLC homolog is depleted. MST is a recently developed technique that takes advantage of the properties of biomolecules in solution to migrate along a temperature gradient. Importantly, this migration is affected by substrate binding. It is being monitored by fluorescence using labelled SLC molecules in the presence of different ligand concentrations. RESULTS: We herein report a novel MST/yeast-based method to characterize binding of ligands to SLCs without the need for a prior SLC-purification step. For validation purposes, we used a close eukaryotic homolog of the human H+-coupled oligopeptide transporter PepT1 (SLC15A1) that mediates uptake of di-tripeptides and peptide-like drugs as a test model. This approach allowed the successful confirmation of the binding of Gly-Sar at the mM range and revealed for the first time the KD of the antiviral prodrug valacyclovir to the PepT1 homolog at around 50 µM. DISCUSSION: This novel LBA approach is independent of protein purification. It is suitable for drug discovery as it is upscalable to high throughput compound screening. It works well for SLC transporters which are underrepresented targets due to their difficulties to study them. Moreover, this approach could make a significant contribution toward "deorphanization" of SLCs, revealing their transport substrates.

[Cerebral infarction related to varicella zoster virus vasculopathy].

A 14-year-old girl developed transient disturbance of consciousness, dysarthria, and clumsiness of the right upper limb 4 months after herpes zoster ophthalmicus. Brain MRI showed acute cerebral infarction in the left middle cerebral artery (MCA) territory. CT angiography demonstrated mild stenosis in the top of the left internal carotid artery and the proximal side of the MCA. Cerebrospinal fluid (CSF) examination showed slightly mononuclear pleocytosis (6/µl). Titer of the anti-varicella zoster virus (VZV) IgG antibodies in CSF was increased, and gadolinium-enhanced brain MRI (T1-weighted imaging) revealed enhancement of the vessel walls at the stenotic lesions. Based on the diagnosis of VZV vasculopathy, methylprednisolone and valacicrovir were administered, followed by acyclovir, in addition to antithrombotic therapy using aspirin and warfarin. After these treatment, her right upper clumsiness was resolved and gadolinium-enhancement of the vessel walls was disappeared on MRI. VZV vasculopathy may cause ischemic stroke in young patients, especially in children. A careful history-taking about herpes is necessary to detect the disease as a potential cause in young stroke patients.

[Topical ozone therapy: An innovative solution to patients with herpes zoster].

OBJECTIVE: To observe the clinical efficacy and safety of topical ozone therapy for patients with herpes zoster by reflectance confocal microscopy (RCM).
 Methods: A total of 60 patients with herpes zoster were divided into a control group and an ozone treatment group (n=30). In the control group, patients took oral valacyclovir tablets or granules (0.3 g per day, three times a day) and they were subjected to local weak laser irradiation treatment plus topical 2% mupirocin ointment twice a day. In the ozone group, the treatment is same as the control group except mupirocin ointment was replaced with topical ozone treatment (hydrotherapy every day plus ozonated oil twice a day). The clinical symptoms, discoid cell and adverse reactions were observed and taken records at day 0, 3, 7 and 14. Statistical analysis was performed to compare the clinical efficacy between the 2 groups. 
 Results: On the seventh day of treatment, the discoid cells of the ozone group disappeared, and the difference between the control group and the ozone group was statistically significant (P<0.05). The difference of decreased percentage of pain scores at each time point between the 2 groups was statistically significant (P<0.05). The clinical efficacy was 100% in the ozone group and 86.7% in the control group, with significant difference between the 2 groups (P<0.05).
 Conclusion: Topical ozone therapy in patients with herpes zoster is helpful in relieving pain, shortening the course as well as improving the clinical efficacy without obvious adverse reactions. It is worth to be popularized.

Management of Recurrent Vestibular Neuritis in a Patient Treated for Rheumatoid Arthritis.

PURPOSE: This clinical report is presented to describe how results of vestibular function testing were considered along with other medical history to develop a management plan that was ultimately successful. METHOD: The patient underwent audio-vestibular assessment including comprehensive audiogram, videonystagmography, cervical vestibular evoked myogenic potential, and postural stability testing. RESULTS: Results from initial testing were most consistent with uncompensated peripheral vestibular dysfunction affecting the right superior vestibular nerve. These results, considered along with history and symptoms, supported vestibular neuritis. After a second vertigo event, we became concerned about the potential temporal association between the patient's rheumatoid arthritis treatment and symptom onset. It is established that treatment for rheumatoid arthritis can exacerbate latent viral issues, but this has not specifically been reported for vestibular neuritis. There are reports in the literature in which patients successfully used viral suppressant medication to decrease viral activity while they were able to continue benefiting from immunosuppressive therapy. We hypothesized that, if the current patient's vestibular neuritis events were related to her treatment for rheumatoid arthritis, she may also benefit from use of viral suppressant medication while continuing her otherwise successful immunosuppressive intervention. CONCLUSIONS: Patients treated with biologic disease-modifying antirheumatic drugs are more susceptible to viral issues, and this may include vestibular neuritis. For the current case, identifying this possibility and recommending viral suppressant medication allowed her to continue with successful treatment of rheumatoid arthritis while avoiding additional vertigo events.

Relevance of non-synonymous thymidine kinase mutations for antiviral resistance of recombinant herpes simplex virus type 2 strains.

Therapy or prophylaxis of herpes simplex virus type 2 (HSV-2) infections with the nucleoside analog aciclovir (ACV) can lead to the emergence of drug-resistant HSV-2 strains, particularly in immunocompromised patients. In this context, multiple amino acid (aa) changes can accumulate in the ACV-converting viral thymidine kinase (TK) which hampers sequence-based diagnostics significantly. In this study, the so far unknown or still doubted relevance of several individual aa changes for drug resistance in HSV-2 was clarified. For this purpose, ten recombinant fluorescent HSV-2 strains differing in the respective aa within their TK were constructed using the bacterial artificial chromosome (BAC) pHSV2(MS)Lox. Similar TK expression levels and similar replication behavior patterns were demonstrated for the mutants as compared to the unmodified BAC-derived HSV-2 strain. Subsequently, the resulting strains were tested for their susceptibility to ACV as well as penciclovir (PCV) in parallel to a modified cytopathic effect (CPE) inhibition assay and by determining the relative fluorescence intensity (quantified using units, RFU) as a measure for the viral replication capacity. While aa changes Y53N and R221H conferred ACV resistance with cross-resistance to PCV, the aa changes G25A, G39E, T131M, Y133F, G150D, A157T, R248W, and L342W maintained a susceptible phenotype against both antivirals. The CPE inhibition assay and the measurement of relative fluorescence intensity yielded comparable results for the phenotypic testing of recombinant viruses. The latter test showed some technical advantages. In conclusion, the significance of single aa changes in HSV-2 TK on ACV/PCV resistance was clarified by the construction and phenotypic testing of recombinant viral strains. This was facilitated by the fluorescence based method.