Drug resistance of clinical varicella-zoster virus strains confirmed by recombinant thymidine kinase expression and by targeted resistance mutagenesis of a cloned wild-type isolate.

In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (Δaa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results.

Varicella-zoster virus open reading frame 48 encodes an active nuclease.

Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity.

ProTides of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide as potential anti-tubercular and anti-viral agents.

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.

In vitro-selected drug-resistant varicella-zoster virus mutants in the thymidine kinase and DNA polymerase genes yield novel phenotype-genotype associations and highlight differences between antiherpesvirus drugs.

Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACV(r)) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the "palm domain" of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Studying protein complexes by the yeast two-hybrid system.

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

The alphaherpesvirus US3/ORF66 protein kinases direct phosphorylation of the nuclear matrix protein matrin 3.

The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.

Hyperphosphorylation of histone deacetylase 2 by alphaherpesvirus US3 kinases.

A serine/threonine (S/T) kinase encoded by the US3 gene of herpes simplex virus type 1 (HSV-1) is conserved in varicella-zoster virus (VZV) and pseudorabies virus (PRV). Expression of US3 kinase in cells transformed with US3 expression plasmids or infected with each virus results in hyperphosphorylation of histone deacetylase 2 (HDAC2). Mapping studies revealed that each US3 kinase phosphorylates HDAC2 at the same unique conserved Ser residue in its C terminus. HDAC2 was also hyperphosphorylated in cells infected with PRV lacking US3 kinase, indicating that hyperphosphorylation of HDAC2 by PRV occurs in a US3-independent manner. Specific chemical inhibition of class I HDAC activity increases the plaquing efficiency of VZV and PRV lacking US3 or its enzymatic activity, whereas only minimal effects are observed with wild-type viruses, suggesting that VZV and PRV US3 kinase activities target HDACs to reduce viral genome silencing and allow efficient viral replication. However, no effect was observed for wild-type or US3 null HSV-1. Thus, we have demonstrated that while HDAC2 is a conserved target of alphaherpesvirus US3 kinases, the functional significance of these events is virus specific.

VZV ORF47 serine protein kinase and its viral substrates.

ORF47, a serine protein kinase of varicella-zoster virus (VZV) and homolog of herpes simplex virus UL13, is an interesting modulator of VZV pathogenesis. This chapter summarizes research showing that ORF47 protein kinase activity, by virtue of phosphorylation of or binding to various viral substrates, regulates VZV proteins during all phases of viral infection and has a pronounced effect on the trafficking of gE, the predominant VZV glycoprotein, which in turn is critical for cell-to-cell spread of the virus. Casein kinase II, an ubiquitous cellular protein kinase, recognizes a similar but less stringent phosphorylation consensus sequence and can partially compensate for lack of ORF47 activity in VZV-infected cells. Differences between the phosphorylation consensus sites of the viral and cellular kinases are outlined in detail.

Varicella-zoster virus open reading frame 66 protein kinase and its relationship to alphaherpesvirus US3 kinases.

The varicella-zoster virus (VZV) open reading frame (ORF) 66 encodes a basophilic kinase orthologous to the US3 protein kinases found in all alphaherpesviruses. This review summarizes current information on the ORF66 kinase, and outlines apparent differences from other US3 kinases, as well as some of the conserved functions. One critical difference is the VZV ORF66 kinase targeting of the major regulatory VZV IE62 protein to control its nuclear import and assembly into the VZV virion, which is so far unprecedented in the alphaherpesviruses. However, ORF66 targets some cellular targets which are also targeted by US3 kinases of other herpesviruses, including the histone deacetylase-1 and 2 proteins, pathways that lead to changes in actin dynamics, and the targeting of substrates of protein kinase A, including the nuclear matrix protein matrin 3.

Histone deacetylases 1 and 2 are phosphorylated at novel sites during varicella-zoster virus infection.

ORF66p, a virion-associated varicella-zoster virus (VZV) protein, is a member of a conserved Alphaherpesvirinae kinase family with homology to herpes simplex virus US3 kinase. Expression of ORF66p in cells infected with VZV or an adenovirus expressing only ORF66p results in hyperphosphorylation of histone deacetylase 1 (HDAC1) and HDAC2. Mapping studies reveal that phosphorylation is at a unique conserved Ser residue in the C terminus of both HDACs. This modification requires an active kinase domain in ORF66p, as neither protein is phosphorylated in cells infected with VZV lacking kinase activity. However, hyperphosphorylation appears to occur indirectly, as within the context of in vitro kinase reactions, purified ORF66p phosphorylates a peptide derived from ORF62p, a known substrate, but does not phosphorylate HDAC. These results support a model where ORF66p is necessary but not sufficient to effect hyperphosphorylation of HDAC1 and HDAC2.

Viral ribonucleotide reductase attenuates the anti-herpes activity of acyclovir in contrast to amenamevir.

Amenamevir is a helicase-primase inhibitor of herpes simplex virus (HSV) and varicella-zoster virus (VZV) and is used for the treatment of herpes zoster in Japan. The half maximal effective concentrations (EC50s) of acyclovir and sorivudine for VZV increased as the time of treatment was delayed from 6 to 18 h after infection, while those of amenamevir and foscarnet were not affected. Susceptibility of infected cells at 0 and 18 h after infection was examined with four anti-herpes drugs, and the fold increases in EC50 for acyclovir, sorivudine, amenamevir, and foscarnet were 13.1, 6.3, 1.3, and 1.0, respectively. The increase in the EC50s for acyclovir in the late phase of infection in VZV and HSV was abolished by hydroxyurea, a ribonucleotide reductase (RR) inhibitor. The common mechanism affecting antiviral activities of acyclovir to HSV and VZV was examined in HSV-infected cells. The amount of HSV DNA in cells treated with amenamevir at 10 x EC50 was similar at 0 and 12 h but less than that in cells treated with acyclovir at 10 x EC50. dGTP, produced through viral RR, peaked at 4 h and decreased thereafter as it was consumed by viral DNA synthesis. Because acyclovir and amenamevir inhibited viral DNA synthesis, thus making dGTP unnecessary, dGTP was significantly more abundant in the presence of acyclovir and amenamevir than in untreated, infected cells. Abundant dGTP supplied by RR may compete with acyclovir triphosphate and attenuate its antiviral activity. In contrast, abundant dGTP did not influence the inhibitory action of amenamevir on viral helicase-primase activity. ATP was significantly decreased at 12 h after infection and significantly more abundant in untreated infected cells compared to cells treated with acyclovir and amenamevir. The anti-herpetic activity of amenamevir was not affected by the replication cycle of VZV and HSV, indicating the suitability of amenamevir for the treatment of herpes zoster and suppressive therapy for genital herpes.

Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells.

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.

Synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxamides with broad-spectrum human herpesvirus polymerase inhibition.

A versatile synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxylate esters has been developed which has lead to the identification of a new series of non-nucleoside inhibitors of human herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

Titration of cell-associated varicella-zoster virus with the MV9G reporter cell line for antiviral studies.

Titration of the cell-associated virus (CAV) of varicella-zoster virus (VZV) is essential for antiviral studies. A VZV reporter cell line, MV9G, generated in our previous study expresses firefly luciferase upon CAV infection in a dose-dependent manner, suggesting that use of the cell line for titration is feasible. In this study, MeWo cells infected with VZV vaccine Oka (vOka) strain or with clinical isolates obtained from patients with varicella or zoster were used as CAV. A co-culture of MV9G cells with the virus-infected MeWo cells were set up and optimized for titration of CAV. Luciferase activities of MV9G cells measured as relative light units (RLUs) of chemiluminescence correlated well (r > 0.9, p < 0.05) both with quantities of viral DNAs measured by TaqMan PCR and with numbers of viral foci detected by immunostaining with a monoclonal antibody against VZV IE62. In addition, the usefulness of MV9G for antiviral studies was exemplified by treatment of the VZV-infected cells with various concentrations of acyclovir. Thus, the reporter cell-based titration of CAV by measuring the induced RLUs may be a reliable way to estimate viral foci and viral DNAs.

Synthesis and preliminary evaluation of 18F- or 11C-labeled bicyclic nucleoside analogues as potential probes for imaging varicella-zoster virus thymidine kinase gene expression using positron emission tomography.

Two radiolabeled bicyclic nucleoside analogues (BCNAs) were synthesized, namely 3-(2'-deoxy-beta-d-ribofuranosyl)-6-(3-[18F]fluoroethoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([18F]-2) and 3-(2'-deoxy-beta-d-ribofuranosyl)-6-(3-[11C]methoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([11C]-3), and evaluated as PET reporter probes for varicella-zoster virus thymidine kinase (VZV-tk) gene expression imaging in brain. [18F]-2 and [11C]-3 were synthesized starting from phenol precursor 1. The phenol precursor 1 was converted to stable as well as to radiolabeled compounds 2 and 3 using (19/18)FCH(2)CH(2)Br or (12/11)CH(3)I as alkylating agent. In vitro evaluation of [18F]-2 and [11C]-3 in 293T cells showed a 4.5 and 53-fold higher uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. However, biodistribution studies in mice demonstrated low uptake of these tracers in the brain. RP-HPLC analysis of plasma and urine samples of mice injected with [11C]-3 revealed that this tracer is very stable in vivo. These data warrant further evaluation of these tracers as noninvasive imaging agents for VZV infection and VZV-tk reporter gene expression in vivo.

A p-[18F]fluoroethoxyphenyl bicyclic nucleoside analogue as a potential positron emission tomography imaging agent for varicella-zoster virus thymidine kinase gene expression.

We recently reported a new positron emission tomography (PET) reporter gene, namely, varicella-zoster virus thymidine kinase (VZV-tk) in combination, with carbon-11 or fluorine-18 labeled m-alkoxyphenyl bicyclic nucleoside analogues (BCNAs) as PET reporter probes. We now report the synthesis and evaluation of p-alkoxyphenyl-BCNA tracers ([11C]-4 and [18F]-5), which are found to be superior to the m-alkoxyphenyl-BCNA tracers. In particular, the fluorine-18 labeled tracer ([18F]-5, IC50 of 5 is 4.2 microM) shows a higher accumulation in VZV-tk expressing cells than the previously reported m-methoxyphenyl BCNA. [11C]-4 and [18F]-5 were synthesized by heating the phenol precursor 3 with 11CH 3I and 18FCH 2CH 2Br, respectively, as alkylating agents. In vitro evaluation of [11C]-4 and [18F]-5 in 293T cells showed about 14- and 54-fold higher uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. LC-MS analysis confirmed the formation of monophosphate derivative of 5 upon catalysis by VZV TK. In vivo studies of this new reporter gene/probe system are in progress.

5-alkynyl analogs of arabinouridine and 2'-deoxyuridine: cytostatic activity against herpes simplex virus and varicella-zoster thymidine kinase gene-transfected cells.

A group of arabinouridines (TMSEAU, EAU, IEAU-TA) and 2'-deoxyuridines (TMSEDU, EDU, IEDU) having a variety of substituents at the uracil C-5 position (trimethylsilylethynyl, TMSE; ethynyl, E; or iodoethynyl, IE), and the sugar C-2' position (2'-arabino OH in arabinouridine, AU; or 2'-deoxyribo H in 2'-deoxyuridine, DU) were prepared to acquire antiviral structure-activity relationships. A broad-spectrum viral panel screen showed that these 5-alkynylarabino/deoxy-uridines exhibit moderate anti-HSV-1 activity, with no difference in potency between arabinouridines and 2'-deoxyuridines. The 2'-deoxyuridines TMSEDU, EDU, and IEDU, unlike the arabinouridines, exhibited potent antiviral activity against cytomegalovirus, but they were also highly cytostatic. The abilities of the 5-alkynylarabino/deoxy-uridines to inhibit nontransfected (wild-type or thymidine kinase-deficient, tk-) and viral gene transfected (HSV-1, HSV-2, or VZV thymidine kinase-positive, tk+) FM3A and OST (osteosarcoma) cells were determined. This group of 5-alkynylarabino/deoxy-uridines showed an enhanced ability to inhibit cells transfected with a viral thymidine kinase gene (HSV-1tk+, HSV-2tk+, VZVtk+) relative to wild-type or thymidine kinase-deficient (tk-) cells.

Genetic polymorphism of thymidine kinase (TK) and DNA polymerase (pol) of clinical varicella-zoster virus (VZV) isolates collected over three decades.

BACKGROUND: Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. OBJECTIVES: To examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. STUDY DESIGN: The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. RESULTS: In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. CONCLUSIONS: Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations.

Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy.

Profile of anti-herpetic action of ASP2151 (amenamevir) as a helicase-primase inhibitor.

The antiherpetic drugs acyclovir (ACV, valaciclovir) and penciclovir (famciclovir) are phosphorylated by viral thymidine kinase and terminate DNA synthesis. ASP2151 (amenamevir) and foscavir (PFA) directly inhibit viral helicase-primase and DNA polymerase, respectively, and inhibit replication of herpes simplex virus (HSV) and varicella-zoster virus. ACV, ASP2151, and PFA all inhibit HSV with a different mechanism of action and as a consequence, the kinetics of viral DNA accumulation and progeny virus production differ. This study focused on how viral DNA synthesis and its related events in the replication cycle would influence anti-HSV action of ACV, ASP2151, and PFA. ASP2151 suppressed HSV replication more efficiently than ACV at 10 × 50% effective concentration of plaque formation (EC50), when treatments were started 0-24 h after infection. ASP2151 and PFA were more potent than ACV in suppressing viral DNA synthesis and infectious virus production when they were added up to 3 h following infection. The virus replicated in the presence of ACV was compared for the ratios of HSV DNA copy number to infectivity with that without ACV and infectivity of ACV-treated virus was less efficient than that without ACV-treatment. The EC50 of infected cells in the time course after infection was preserved in PFA, limited in ASP2151, and much increased for ACV, indicating that viral DNA synthesis had little effect on antiviral action of PFA and ASP2151 but reduced the susceptibility of ACV. ASP2151 showed a preferable profile as an anti-herpetic agent with a better pharmacokinetic profile than ACV.