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Acyclovir resistance of varicella-zoster virus (VZV) has been reported in rare cases of immunocompromised patients. In this study, the natural polymorphism of the thymidine kinase (TK) and DNA polymerase (pol) genes was examined in 51 clinical VZV isolates sensitive to acyclovir (ACV). In addition, 16 VZV strains with clinical resistance to ACV were analyzed. None of the ACV-sensitive strains of the clades 1, 3 and 5 showed gene polymorphism of the TK. By contrast, the DNA pol gene exhibited polymorphism-related substitutions as a function of the VZV clade. The novel substitutions M286I, E824Q, R984H and H1089Y were detected in strains of clades 3 and 5. In the TK gene of 7 VZV strains with clinical ACV resistance, the novel substitutions L73I, A163stop, W225R, T256M, N334stop and the deletion of nucleotides 19–223 were found to be associated most likely with resistance. In one strain showing the substitution W225R, ACV resistance could be confirmed by the viral phenotype. In the DNA pol gene, the novel amino acid substitutions T237K and A955T could be detected, but their significance remains unclear. In conclusion, the characterization of resistance using genetic analysis of the TK and DNA pol genes has to be considered the method of choice for the determination of VZV resistance to antiviral drugs. In a considerable number of patients with clinical ACV-resistant VZV infections, resistance cannot be verified by virological methods.

Brivudin [(E)-5-(2-bromovinyl)-2'-deoxyuridine] is a nucleoside analogue with a high and selective antiviral activity against varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1). The double-blind, randomized study presented here compared efficacy and safety of oral brivudin 1 x 125 mg and acyclovir 5 x 800 mg, both for 7 days, in 1227 immunocompetent patients with herpes zoster. Main results were as follows: brivudin was superior to acyclovir in accelerating the "time to last formation of new vesicles" (primary parameter; risk ratio(ITT): 1.13, P=0.014). Equivalent effects of brivudin and acyclovir were observed for the secondary parameters "time to first crust" (RR(ITT): 0.93, P=0.004), "time to full crusting" (risk ratio(ITT): 1.03, P0.001), and "time to loss of crusts" (RR(ITT): 0.95, P=0.002). The incidence of potentially treatment-related adverse events was similar under brivudin (7.7%) and acyclovir (10.0%). In conclusion, brivudin proved to be more effective than acyclovir in terminating vesicle formation, the parameter which reflects the end of viral replication, thus confirming, in the clinical setting, the greater in vitro antiviral activity of brivudin. Compared with acyclovir, brivudin provides a similar safety profile and a significant improvement in efficacy.

The in vitro antiviral activity of a new series of cycloSal-pro-nucleotides derived from the acyclic nucleoside analogues aciclovir and penciclovir against herpes simplex virus type 1 (HSV-1), thymidine kinase deficient (TK-) HSV-1, and Epstein-Barr virus (EBV) was evaluated. Using the XTT-based tetrazolium reduction assay EZ4U, the cycloSal derivatives were examined for their antiviral and cytotoxic effects in HSV-1 as well as HSV-1-TK--infected Vero cells. The anti-EBV activity was assessed by means of an EBV DNA hybridization assay using a digoxigenin-labeled probe specific for the Bam H1-W-fragment of the EBV genome and by measuring viral capsid antigen (VCA) expression in P3HR-1 cells by indirect immunofluorescence. Among the new cycloSal-phosphotriesters the three aciclovir monophosphates proved to be potent and selective inhibitors of HSV-1 replication, EBV DNA synthesis and EB-VCA expression. Of interest is the retention of activity of the aciclovir monophosphates in HSV-1-TK--infected cells. Particularly 3-methyl-cycloSal-aciclovir monophosphate retained the same effectiveness, as compared to the wild type virus strain. In contrast to the aciclovir pro-nucleotides the penciclovir cycloSal-phosphotriesters exhibited at best only a marginal antiviral effect on HSV and EBV replication.

The antiherpes virostatics acyclovir (ACV), valaciclovir (VACV), penciclovir (PCV), famciclovir (FCV) and ganciclovir (GCV), which belong to the group of purine acyclic nucleoside analogues, were tested for clastogenic and sister chromatid exchange (SCE)-inducing activity in Chinese hamster V79-E cells upon chronic application with and without a recovery period. ACV induced borderline effects in both cytogenetic assays, a dose-dependent reduction of the mitotic index and an increasing cell cycle delay. With VACV and PCV only a decrease of the mitotic index and an increase of cell cycle delay were observed. FCV was negative with respect to the four parameters studied, presumably due to the incapacity of the target cells of metabolizing FCV to PCV. GCV was a very potent genotoxin in both assays. It induced a statistically significant SCE response even in the range of the cytomegalovirus IC50 of < 10 μM. By variation of the experimental protocol it was shown that SCEs are induced in the second cell cycle following exposure to GCV but not in the first one. It is assumed that the drugs under study are metabolized to their respective triphosphates and then inhibit DNA replication as detected by decreasing mitotic index and increasing cell cycle delay. In the case of GCV it is suggested that GCV-TP is incorporated into the target cell DNA and that chromosomal aberrations and SCEs are secondary lesions due to repair processes at the substituted template.

A real-time quantitative PCR was developed to assess antiviral activity of molecules against human herpesvirus 8 (HHV-8) and the Epstein-Barr virus (EBV). The antiviral activity of the reference molecules acyclovir, ganciclovir, cidofovir, adefovir and brivudin, as assessed by this methodology, proved very similar to the activity as determined by a DNA-DNA hybridisation method.

The analysis of the viral thymidine kinase (TK) genotype is of rising significance for testing resistance of herpes simplex virus (HSV) to antivirals especially acyclovir. However, numerous of the described amino acid (aa) substitutions are diagnostically less conclusive because of the pronounced natural polymorphism of this gene. In this study, several aa substitutions in the TK sequence of HSV-1 with unclear significance for resistance were analyzed by expression of recombinant TK proteins and determination of enzymatic activity on the basis of an enzyme linked immunosorbent assay using bromodeoxyuridine (BrdU) as TK substrate. The recombinant TK wild-type protein resulted in high TK activity and TK mutant with stop of translation showed negative results. The recombinant TK proteins containing the aa substitutions R41H or V348I had high phosphorylation activities suggesting most likely natural gene polymorphisms. By contrast, the aa changes Y53H, L139V, R163H, L298A and L315S were accompanied by negative or weakly positive TK activities indicating resistance association. In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains.

In the flu season 2005/2006 amantadine-resistant human influenza A viruses (FLUAV) of subtype H3N2 circulated in Germany. This raises questions on the neuraminidase inhibitor (NAI) susceptibility of FLUAV. To get an answer, chemiluminescence-based neuraminidase inhibition assays were performed with 51 H1N1, H1N2, and H3N2 FLUAV isolated in Germany from 2001 to 2005/2006. According to the mean IC50 values (0.38–0.91 nM for oseltamivir and 0.76–1.13 nM for zanamivir) most H1N1 and H3N2 FLUAV were NAI-susceptible. But, about four times higher zanamivir concentrations were necessary to inhibit neuraminidase activity of H1N2 viruses. Two H1N1 isolates were less susceptible to both drugs in NA inhibition as well as virus yield reduction assays. Results from sequence analysis of viral hemagglutinin and neuraminidase genes and evolutionary analysis of N2 gene revealed (i) different subclades for N2 in H1N2 and H3N2 FLUAV that could explain the differences in zanamivir susceptibility among these viruses and (ii) specific amino acid substitutions in the neuraminidase segment of the two less NAI-susceptible H1N1 isolates. One H3N2 was isolate proved to be a mixture of a NA deletion mutant and full-length NA viruses.

Amino acid 1092 (AA1092) in capsid protein 1 of coxsackievirus B3 (CVB3) is located in close vicinity to the central phenoxy group of capsid binders (i.e. pleconaril). Whereas isoleucine is associated with drug susceptibility, leucine and methionine confer resistance to pleconaril. In the present study, novel analogues with different substitutions in the central phenoxy group were synthesized to study their influence on anti-CVB3 activity with the aim to overcome pleconaril resistance. Two [(biphenyloxy)propyl]isoxazoles and pleconaril were synthesized without methyl groups in the central phenoxy ring using Suzuki coupling reaction and tested for antiviral activity against the pleconaril-resistant CVB3 Nancy. Furthermore, pleconaril with 3-methyl, 3-methoxy, 3-bromine, 2,3-dimethyl in the central ring as well as the external rings in meta position were synthesized for structure–activity relationship analysis with CVB3 variants containing leucine, methionine or isoleucine at position 1092, other coxsackieviruses B (CVB) as well as several rhinoviruses. The results demonstrate a high impact of substituents in the central ring of capsid inhibitors for anti-enteroviral activity. Pleconaril resistance of CVB3 based on Leu1092 or Met1092 was overcome by unsubstituted analogues or by monosubstitution with 3-methyl as well as 3-bromine in the central phenyl. The 3-bromine derivative inhibited a broad spectrum of CVB and rhinoviruses.

As an intermediate host of avian and human influenza A viruses (FLUAV) pigs may play a potential role in interspecies virus transmission and reassortment of viral genes including those conferring antiviral drug resistance. Porcine FLUAV isolated in Germany between 1989 and 2001 contains mutations in the M2 gene inducing amantadine resistance. No data exist on neuraminidase inhibitor (NAI) susceptibility of these porcine FLUAV. We studied the antiviral activity of NAI against seven selected H3N2 FLUAV isolated from pigs in Germany between 1982 and 1999. All isolates were susceptible towards oseltamivir and zanamivir in neuraminidase enzyme-inhibition assays. Both compounds inhibited virus spreading and reduced the virus yields and plaque size at low concentrations. Higher concentrations were necessary to reduce the plaque number. Two isolates that differed in glycosylation pattern of viral hemagglutinin (HA) showed markedly reduced drug susceptibility in cell culture-based assays.

Because variola virus might be used as a pathogen in biological attacks, there is an urgent need to provide effective antiviral drugs for the treatment of orthopoxvirus infections. Thus, the aim of the present study was to test the antiviral activity of 3 pro-nucleotides of the acyclic nucleoside analogues aciclovir (ACV), 3 of penciclovir (PCV) and 38 of the cyclic nucleoside analogue brivudin (BVDU), on the basis of cycloSaligenyl-nucleoside monophosphate approach against vaccinia virus and cowpox virus in vitro. In further experiments, 13 synthetic humic acid-like polymers, so-called polyhydroxycarboxylates, were examined. Antiviral screening was performed by means of the plaque reduction assay and for quantification of the cytotoxicity of the test compounds the XTT-based tetrazolium reduction assay EZ4U was used. As result, three cycloSal-monophosphate derivatives of ACV proved to be potent inhibitors of both vaccinia virus and cowpox virus replication in vitro. Among the tested monophosphate derivatives of cycloSal-PCV and cycloSal-BVDU, selected substances showed a promising antiviral activity against vaccinia virus and cowpox virus. For the polyanionic compounds, no relevant antiviral activity was detected. In conclusion, by the delivery of nucleoside monophosphates from neutral, membrane-permeable prodrugs on the basis of the cycloSaligenyl-nucleotide concept, different ACV, PCV and BVDU derivatives can act as potent and selective inhibitors of orthopoxvirus replication. However, most of the cycloSal-monophosphate derivatives of BVDU had a higher cytotoxicity than their parent nucleosides.

This concerns a double-blind survey study on 608 herpes zoster patients treated with 1x 125 mg oral brivudin (n=309) or 5x 800 mg acyclovir (n=299), both for 7 days, during two prospective, randomised clinical herpes zoster trials. The survey aimed at evaluating the outcome of the two treatment regimens on postherpetic neuralgia (PHN). During a follow-up ranging from 8 to 17 months after start of treatment, former study participants aged >/=50 years were interviewed for the occurrence of PHN. Neither the investigators nor the patients were aware of which treatment the patients received during acute herpes zoster. The incidence of PHN, defined as zoster-associated pain occurring or persisting after rash healing was significantly lower in brivudin recipients (32.7%) than in acyclovir recipients (43.5%, P=0.006). Mean duration of PHN was similar with brivudin (173 days) and acyclovir (164 days, P=0.270). Despite some methodological disadvantages common to this type of study, the present survey provides for the first evidence that brivudin treatment during acute herpes zoster favourably affects the incidence of PHN in immunocompetent elderly herpes zoster patients.

The aim of the study was to establish a murine model for sensitive screening of potential compounds with in vitro anti-influenza A virus activity. The evaluation in this in vivo model is based on semi-quantitative detection of viral RNA using one-step reverse transcriptase polymerase chain reaction (RT-PCR). After intranasal infection of fully-conscious mice with influenza A virus, the viral load of the respiratory tract tissues was investigated. Peaks were observed in the nasopharynx between Days 1 and 4, in the trachea on Day 4, and in the lungs between Days 4 and 7 post infection. The elimination of virus correlated with the appearance of specific serum antibodies. After 4 days of treatment with zanamivir, trachea and lungs revealed negative RT-PCR results, whereas viral load in the nasopharynx was significantly reduced. In conclusion, the virus spread in the described murine model is similar to upper respiratory tract infection with influenza virus in human. Viral load measurement by semi-quantitative detection of viral RNA allows rapid and sensitive screening of potential compounds with in vitro anti-influenza A virus activity.

Two drug formulations of povidone-iodine (PVP-I)--an aqueous PVP-I solution (Betaisodona) and a liposomal PVP-I formulation--were tested for their virucidal activity and cytotoxicity in cell culture. As to the virudical activity against influenza A virus, herpes simplex virus type 1, adenovirus type 8 and human rhinovirus type 14, the liposomal formulation of PVP-I proved to be approximately as active as the aqueous one. Half maximum cytotoxic PVP-I concentrations were 0.01-0.07% for aqueous PVP-I and 0.03-0.27% for the liposomal PVP-I formulation (XTT reduction assay EZ4U). The detection of lactate dehydrogenase (LDH) release, DNA fragmentation (ELISA) and DNA strand breakage (TUNEL assay) after 24 h exposure of human embryonic lung fibroblasts to PVP-I revealed that necrosis predominates in cells treated with aqueous 0.08-0.32% PVP-I solutions, whereas apoptosis was the predominant type of cell death in cells treated with equivalent concentrations of liposomal PVP-I formulation. The favorable virucidal efficiency together with the preferred apoptotic route of cell death makes the liposomal PVP-I formulation a promising candidate for topical use in prevention and treatment of infections of the eye and the upper respiratory tract.

The 5-substituted 1-β-D-arabinofuranosyl (araU) analogues, (E)-5-(2-bromovinyl)-araU (BrVaraU) and 5-vinyl-araU (VaraU), which can be considered as structural analogues of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BrVUdR), are potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) replication in vitro. BrVaraU and VaraU have been compared with BrVUdR for their therapeutic effect on acute HSV-1 keratitis in rabbits. Both araU derivatives applied as 0.1% eyedrops suppressed the development of keratitis as monitored by the reduced number of herpes efflorescences. The healing effect of BrVaraU and VaraU was less pronounced than that of 0.1% BrVUdR eyedrops, the difference between BrVUdR and VaraU being statistically significant at the 10th day of treatment. As a further indication of the healing effect the number of corneae with opacities seen after cessation of drug treatment were 3.3, 7.4, 27.6 and 46.9% for the BrVUdR-BrVaraU-, VaraU- and placebo-treated eyes, respectively.

We investigated the influence of disodium phosphonic formate (PFA-Na 2 ) and trisodium thiophosphonic formate (TPFA-Na 3 ), in comparison with acyclovir (Zovirax®) and trisodium phosphonic formate (PFA-Na 3 ) (Triapten®) ointment, on the course of primary cutaneous herpes simplex virus infection in a guinea pig skin model. PFA-Na 2 at 3.0%, TPFA-Na 3 at 0.5% and PFA-Na 3 at 0.5% as well as Triapten ointment (2.0% PFA-Na 3 ) completely inhibited virus infection. Zovirax cream (5.0% acyclovir), applied five times (15 min., 4, 20, 24, and 28 h) after virus inoculation did not prevent virus infection. Similarly, application of Zovirax cream 5 times daily for 5 days did not prevent a vesicle formation following cutaneous herpes simplex virus infection of the guinea pig.

( E )-5-(2-Bromovinyl-2′-deoxyuridine (BrVUdR) showed strong antiviral activity against different laboratory strains and clinical isolates of herpes simplex virus type 1 (HSV-1) on primary rabbit testes (PRT) cells with a 50% inhibition of plaque formation (ID 50 ) at 0.01–0.02 μM. One laboratory strain (HSV-1-S), however, was completely refractory even at concentrations as high as 100 μM. In contrast, the ID 50 s for all herpes simplex virus type 2 (HSV-2) strains were about 10 2 – 10 3 times higher (8–25 μM) than for the HSV-1 strains. No toxicity in mice treated with 140 mg BrVUdR/kg/day for 14 days was observed, and successful treatments of herpes encephalitis in mice induced experimentally by intracerebral infection with one laboratory strain (HSV-1-Kupka) and one clinical isolate (HSV-1-64) were achieved. Treatment of encephalitis in mice induced by the strain HSV-1-S insensitive to BrVUdR in cell culture failed to be effective. Similar antibody titers against HSV-1 were found in surviving mice of the control and of the BrVUdR-treated groups.

The efficiency of (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil (BrVaraU, VaraU) as inhibitors of three herpes simplex virus type 1 (HSV-1) strains was assessed in comparison to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVUdR), 9-(2-hydroxyethoxymethyl)guanine (ACV), and trisodium phosphonoformate (Na3PFA) using a plaque assay in human embryonic lung fibroblast (HELF) cell cultures. The following order of decreasing activity was found: BrVaraU greater than VaraU greater than BrVU-dR greater than ACV much greater than Na3PFA. In HELF cell cultures, the selectivity indexes of VaraU and BrVaraU were 10 times higher than those of BrVUdR and ACV. Protection of mice from encephalitis and death due to intracerebral (i.c.) infection with a clinical HSV-1 isolate was nearly complete if mice were treated intraperitoneally (i.p.) with two daily doses of VaraU and BrVaraU (100 or 200 mg/kg per day) over a period of 5 or 10 days. The efficacy was similar to ACV, but, using a treatment schedule of three daily i.p. doses over 10 days, with equimolar amounts of the nucleoside analogs, VaraU and BrVaraU (140 and 180 mg/kg per day) were superior to ACV (130 mg/kg per day) (P less than 0.05).