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Kinetics of all resistant PP (a), MR (b) and 06 (c) strains vs. virus control. Corresponding non-resistant parental virus control under non-selective conditions. Numbers indicate peak titers of selected time points. Plaque titrations were performed four times for each time point in all figures
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The treatment of varicella-zoster virus (VZV) reactivation is based on nucleoside analogues acyclovir (ACV) and bromevinyldeoxyuridine (BVdU) and a phosphonic acid derivative (PFA). Drug-resistant mutants of 3 wild-type (WT) VZV strains were obtained by exposure of human retinal pigment epithelial (hRPE) cells inoculated with cell-free WT virus at...
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... shown in Fig. 2b indicate peak values of MR-BVdU and MR-PFA at 96 hpi. Solely, MR-ACV exhibits peak values at 120 hpi. Progression of the MR- ACV curve indicates a slow increase and rather low but stable titers unlike MR-PFA, whose overall titers are even higher compared to the parental control and present a rather fast increase and also a decline in titers right after peak values were ...
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... shown in Fig. 2c, kinetics of strain 06 differed from kinetics of the other two strains. Even the parental strain demonstrated an extended peak of virus growth between 48 and 96 hpi. Resistant mutant titers generally turned out lower at all time points of investigation. After an initial strong increase in viral titers (about 100-fold) between 24 and 48 hpi, the titers remain on a similar level, except for a peak at 144 hpi for 06-ACV. A decrease in overall titers of the resistant strains compared to parental virus control titers exists for 06 and PP strains but shows no statistical (Fig. 2a). Com- pared to all other kinetics, only the PP-BVdU curve pre- sented a slow rise, a rather constant progress, and a slow decline. Even 168 hpi a titer of 1 9 10 5 PFU/ml was measured. The parental control titer measured at the same point of time revealed a decrease to 4 9 10 3 PFU/ml. Microscopy pictures taken 168 hpi from PP-VC as well as from PP-BVdU showed a nearly complete detachment of the PP-VC cell monolayer, whereas PP-BVdU still pre- sented a confluent cell monolayer merely permeated with multiple viral plaques (data not ...
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... shown in Fig. 2c, kinetics of strain 06 differed from kinetics of the other two strains. Even the parental strain demonstrated an extended peak of virus growth between 48 and 96 hpi. Resistant mutant titers generally turned out lower at all time points of investigation. After an initial strong increase in viral titers (about 100-fold) between 24 and 48 hpi, the titers remain on a similar level, except for a peak at 144 hpi for 06-ACV. A decrease in overall titers of the resistant strains compared to parental virus control titers exists for 06 and PP strains but shows no statistical (Fig. 2a). Com- pared to all other kinetics, only the PP-BVdU curve pre- sented a slow rise, a rather constant progress, and a slow decline. Even 168 hpi a titer of 1 9 10 5 PFU/ml was measured. The parental control titer measured at the same point of time revealed a decrease to 4 9 10 3 PFU/ml. Microscopy pictures taken 168 hpi from PP-VC as well as from PP-BVdU showed a nearly complete detachment of the PP-VC cell monolayer, whereas PP-BVdU still pre- sented a confluent cell monolayer merely permeated with multiple viral plaques (data not ...
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... parental strains PP, MR, and 06 demonstrated peak titers at 72 hpi (1,625,000 PFU/ml), 72 hpi (44,000 PFU/ml), and 96 hpi (162,500 PFU/ml), respectively (Fig. 2a-c
(Fig. 2a-c). The resistant isolates partly revealed a different behav- ior. Titers of PP-BVdU showed a distinct shift of the peak values of 334,000 PFU/ml to 144 hpi, respectively. The overall increase in titers progressed much slower, and interestingly, titers showed certain stability without declining even after 168 hpi. This fact counts especially for PP-PFA whose titers even progressed until 168 hpi and never declined. However, kinetics of PP-ACV showed no difference to PP-VC kinetics (Fig. 2a). Overall titers of resistant strains were generally lower compared to parental control titers without being statistically significant (P \ ...
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... parental strains PP, MR, and 06 demonstrated peak titers at 72 hpi (1,625,000 PFU/ml), 72 hpi (44,000 PFU/ml), and 96 hpi (162,500 PFU/ml), respectively (Fig. 2a-c
(Fig. 2a-c). The resistant isolates partly revealed a different behav- ior. Titers of PP-BVdU showed a distinct shift of the peak values of 334,000 PFU/ml to 144 hpi, respectively. The overall increase in titers progressed much slower, and interestingly, titers showed certain stability without declining even after 168 hpi. This fact counts especially for PP-PFA whose titers even progressed until 168 hpi and never declined. However, kinetics of PP-ACV showed no difference to PP-VC kinetics (Fig. 2a). Overall titers of resistant strains were generally lower compared to parental control titers without being statistically significant (P \ ...
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... parental strains PP, MR, and 06 demonstrated peak titers at 72 hpi (1,625,000 PFU/ml), 72 hpi (44,000 PFU/ml), and 96 hpi (162,500 PFU/ml), respectively (Fig. 2a-c
(Fig. 2a-c). The resistant isolates partly revealed a different behav- ior. Titers of PP-BVdU showed a distinct shift of the peak values of 334,000 PFU/ml to 144 hpi, respectively. The overall increase in titers progressed much slower, and interestingly, titers showed certain stability without declining even after 168 hpi. This fact counts especially for PP-PFA whose titers even progressed until 168 hpi and never declined. However, kinetics of PP-ACV showed no difference to PP-VC kinetics (Fig. 2a). Overall titers of resistant strains were generally lower compared to parental control titers without being statistically significant (P \ ...
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... mutations do not necessarily have to occur within active domains of an enzyme for causing relevant phenotypical changes such as resistance versus antiviral substances [21][22][23][24]. Boivin et al. [21] sequenced 11 clinical VZV isolates of AIDS patients who were unsuccessfully treated with ACV because of prolonged or recurring HZ for more than 3 months. Exchanges within active domains of the vTK occured in one isolate at position 24, and exchanges 38 and 54 were located close to the ATP- binding site (Fig. 4). The eight remaining isolates showed aa substitutions between positions 171 and 308 [21]. vTK activity, measured with the help of enzymatic turnover of radioactive-labeled thymidine nucleosides, was completely abolished in all isolates [21]. These observations coincide with our sequence data in ACV-resistant mutants. Even though aa substitutions found at positions 256 (Thr ? Met), 479 (Asn ? Ile), and 601 (Arg ? Met) (Table 3 and Fig. 4) were located outside of the active domain of the vTK, the post-adaptational IC 50 of the resistant isolates MR-ACV, 06-ACV, and PP-ACV exhibited a distinct rise beyond the tenfold proving Mutations found in our resistant isolates are indicated in red resistance toward ACV. Isolate 06-ACV even tolerated ACV concentrations of more than 4,000 lM although the selection process was terminated at a final concentration of 1,200 lM ACV. These findings suggest a complete loss of the functional activity of the vTK although the aa substi- tution is located peripheral to the active ATP-binding site at position 601 (Arg ? Met). Substitutions in positions 62 (Ile ? Phe) and 85 (Leu ? Pro) within the vTK were found in isolates PP-BVdU and 06-BVdU, whereas the substitution (Leu ? Met) at position 92 was detected in isolate MR-PFA. All three substitutions are located close to the enzymatic active site of the vTK, comparable to sites of mutations artificially induced by El Omari et al. [25], thereby suggesting a derogation of the phosphoryla- tion activity of the enzyme. The adaptation of isolates PP-BVdU, 06-BVdU, and MR-PFA lead to mutations potentially influencing the conformation of the active domain of the vTK resulting in changes in kinetics. Whereas 06-VC peaks at 96 hpi, 06-BVdU shows an initial titer increase between 24 and 48 hpi followed by a plateau, where titer values remain on a similar level with the highest value of 9,700 PFU/ml at 120 hpi. Overall titers of 06-BVdU remain substantially lower, and the curve progression shows no fast increase, high peak, and fast decrease as 06-VC does (Fig. 2c). These findings suggest an impaired function of the vTK in 06-BVdU. In contrast, MR-PFA behaves differently. The course of the curve progression is quite similar to the virus control MR-VC as titers rise quickly and no plateau is observed. The titer peak value is even higher compared to the virus control. After an initial strong increase in the virus titer (of about 100-fold) between 24 and 48 hpi, the titers remain on a similar level, except for a peak at 144 hpi for 06-ACV. In contrast to vTK-deficient mutants who lose their pathogenicity and present retarded growth rates, vTK-modified mutants still replicate efficiently and show unaltered pathogenicity [26]. This could be a reason, why isolates MR-PFA and 06-BVdU present such a different phenotypical behavior. As isolates PP-PFA and 06-PFA also possess mutations within the nucleoside-binding domain (region III) of vDNA pol, they were investigated for cross-resistance versus nucleoside analogues BVdU and ACV. Analysis revealed increased IC 50 values above the tenfold threshold for ACV of isolates PP-PFA and 06-PFA but also for MR-PFA. In contrast, IC 50 of BVdU was identical for all PFA-resistant strains to the corresponding controls (data not shown). Therefore, a cross-resistance could only be determined for PFA-resistant isolates vs. ACV. IC 50 values of PFA of ACV-resistant isolates remained within therapeutical ran- ges not indicating resistance (Fig. 1b) ...
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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 prodru...
Citations
... The thymidine kinase deficient strains may have reduced pathogenicity, however those with altered specificity may retain pathogenicity and neurovirulence (Darby et al., 1981). Work is ongoing to characterize the virulence and resistance phenotypes of specific mutations in acyclovir resistant strains (Bleymehl et al., 2011;Frobert et al., 2005;Schubert et al., 2014). This will enable the use of genotypic testing of acyclovir resistant isolates to guide rational clinical practice (Piret and Boivin, 2011;2016). ...
Many clinically used antiviral drugs are nucleoside or nucleotide analogue drugs, which have a unique mechanism of action that requires intracellular phosphorylation. This dependence on intracellular activation presents novel challenges for the discovery and development of nucleoside/nucleotide analogue drugs. Contrary to many small molecule drug development programs that rely on plasma pharmacokinetics and systemic exposures, the precise mechanisms that result in efficacious intracellular nucleoside triphosphate concentrations must be understood in the process of nucleoside/nucleotide drug development. The importance is highlighted here, using the following as case studies: the herpes treatment acyclovir, the cytomegalovirus therapy ganciclovir, and human immunodeficiency virus (HIV) treatments based on tenofovir, which are also in use for HIV prophylaxis. For each drug, the specificity of metabolism that results in its activation in different cells or tissues is discussed, and the implications explored. Acyclovir's dependence on a viral enzyme for activation provides selective pressure for resistance mutations. Ganciclovir is also dependent on a viral enzyme for activation, and suicide gene therapy capitalizes on that for a novel oncology treatment. The tissue of most relevance for tenofovir activation depends on its use as treatment or as prophylaxis, and the pharmacogenomics and drug-drug interactions in those tissues must be considered. Finally, differential metabolism of different tenofovir prodrugs and its effects on toxicity risk are explored. Taken together, these examples highlight the importance of understanding tissue specific metabolism for optimal use of nucleoside/nucleotide drugs in the clinic. Significance Statement Nucleoside and nucleotide analogue drugs are cornerstones in current antiviral therapy and prevention efforts that require intracellular phosphorylation for activity. Understanding their cell and tissue specific metabolism enables their rational, precision use for maximum efficacy.
... In the corresponding conserved TK gene regions (aa 48-53, 183-189), only two substitutions (E48G, E48L) conferring resistance to ACV have been reported yet [7,22]. By contrast, out of the corresponding conserved centers of DNA pol, the region III (aa 769-809) contains seven known resistance-associated substitutions (A773V, N779S, I804T, G805C, R806S, M808V, L809S) [17,[23][24][25] and the region VI (aa 736-755) no substitution related to any resistance. Additionally, the novel DNA pol substitution T417I of this study is close to the conserved region IV (aa 418-460). ...
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.
... This study included seven VZV strains from patients with zoster and one obtained from a patient with encephalitis (5) ( Table 1). The phenotypes of three strains with the following TK substitutions were described previously: first, the amino acid exchange Q303stop, observed in two patients with AIDS developing persistent zoster under ACV treatment (17,18); second, the T256M substitution found after in vitro adaption to ACV (19); and third, the W225R substitution in one strain cultivated from a patient specimen (5). Suspected clinical ACV resistance related to the substitutions L73I, A163stop, N334stop, and the deletion of nucleotides (nt) 19 to 223 (deletion of amino acids [aa] 7 to 74 [⌬aa 7 to 74]) was not verified (5). ...
... In all but one TK mutant virus, the resistance phenotype was clearly defined. While the W225R (5), T256M (19), and Q303stop (17,18) substitutions were confirmed to be related to resistance, A163stop, N334stop, and ⌬aa 7 to 74 were identified as the results of novel resistance mutations. The hitherto unclear substitution L73I led to reduced TK activity, which was considered a sensitive phenotype. ...
In this study, approaches were developed to examine the phenotype of non-viable clinical VZV strains with amino acid (aa) substitutions in the thymidine kinase (TK, ORF36) and/or DNA polymerase (Pol, ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing aa substitutions described as known or suspected cause of antiviral resistance were analyzed by measuring the TK activity applying a modified commercial enzyme immunoassay. To examine the effect of these TK and several Pol substitutions on the replication of recombinant virus strains, the method of en-passant mutagenesis was used. Targeted mutations within the ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluores-cent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the aa substitutions with unknown function in TK, Q303stop, N334stop, A163stop, and aa Δ7-74, were associated with resistance against acyclovir (ACV), penciclovir, or brivudin, whereas L73I and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activity of recombinant TK proteins represents a useful tool to evaluate the significance of aa substitutions for antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of non-synonymous mutations in a VZV bacterial artificial chromosome using en-passant mutagenesis led to reliable phenotypic results.
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... As with HSV-1/2, acyclovir prophylaxis is largely successful for preventing VZV reactivated infections and routine surveillance for VZV infections is not the norm in transplant or other types of immunocompromised patients -rather, they are dealt with when they are suspected, usually with high dose therapy with acyclovir or one of its related compounds. However, as with HSV-1/2, although not routinely tested, VZV drug resistance to acyclovir does occur also (Bleymehl et al., 2011;Sauerbrei et al., 2011) and co-existing wild-type and resistant viral populations may be present in different compartments (e.g. plasma versus CSF versus skin) within a single immunocompromised host (Brink et al., 2011). ...
With the modern sequencing technologies and the capability to sequence large viral genomes virtually overnight, full-genome sequencing of large DNA viruses and correlating any sequence variation to specific clinical manifestations is now eminently feasible - but is it worth doing, i.e. can such large-scale viral genomic analyses be clinically useful? A few clinical conditions, such as organ and bone marrow transplant recipients, require the regular diagnostic sampling and testing for certain viruses over an extended period of time. Such frequent sampling leads to the routine archiving of multiple samples from the same patient over an extended time period and it is the purpose of this mini-review to explore whether such routinely collected samples can be utilized for viral sequencing studies to produce clinically useful outcomes. Specific examples of this include the monitoring of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) levels in transplant recipients, as rising levels of these viruses in these patients can cause serious and sometimes fatal disease during the post-transplant period when their immune system is gradually recovering from the transplant process. Other herpesviruses, such as herpes simplex virus (HSV) and varicella zoster virus (VZV), may also reactivate and cause disease in post-transplant and other types of immunompromised patients, though the specific complications may differ between patients. Although the natural mutation rate for these human herpes DNA viruses are very low, all of these viruses are capable of developing specific drug resistance within a few weeks of therapy, demonstrating that these viruses do have the ability to adapt rapidly to their local environment if the need arises. Specific problems with analyzing these large DNA virus genomes include the selection of the appropriate gene target (unless a full-genome analysis is the aim), and how to deal with overlapping and inverted reading frames, which are discussed in this mini-review.
... A DNA polymerase mutation of unknown significance (S689N, GenBank accession number JQ745674) was found in patient 6 after treatment with ACV, FOS, and CDV. This mutation is outside functional domains of the viral DNA polymerase, but adjacent mutations at codon 684 [26] and 692 [27] have been associated with FOS resistance. No resistance-associated mutations were found in the DNA polymerase gene in samples from the 5 patients with tk mutations. ...
Background:
Varicella zoster virus (VZV) infections are a relevant cause of morbidity and mortality in hematological patients and especially in hematopoietic stem cell transplant (HSCT) recipients. The present study aimed to investigate the prevalence and clinical significance of viral persistence and antiviral resistance by systematically analyzing all episodes of VZV diagnosed in our laboratory in pediatric and adult hematological patients between 2007 and 2010.
Methods:
Patient charts were reviewed to document patient and disease characteristics. VZV loads were determined in all available clinical samples from the day of diagnosis and thereafter. Persistent VZV infection was defined as a VZV infection that lasted at least 7 days. Analysis of resistance was performed in all patients with persistent VZV infection by sequence analysis of viral thymidine kinase and DNA polymerase genes.
Results:
In total, 89 episodes occurred in 87 patients, of whom 65 were recipients of an allogeneic HSCT. Follow-up samples were available in 54 episodes. Persistent VZV was demonstrated in 32 of these episodes (59%). Complications occurred in 16 of the persistent episodes (50%) vs 2 of 22 nonpersistent episodes (9%). Mutations possibly associated with resistance were found in 27% of patients with persistent VZV, including patients with treatment-unresponsive dermatomal zoster that progressed to severe retinal or cerebral infection.
Conclusions:
In hematological patients, VZV-related complications occur frequently, especially in persistent infections. Antiviral resistance is a relevant factor in persistent infections and needs to be investigated in various affected body sites, especially when clinical suspicion of treatment failure arises.
... However, the replicative capacity of these strains could not be assessed because of the failure to isolate the viruses in cell culture. Since the substitution T256M is located in the conserved gene region and has been reported recently to be associated phenotypically with resistance to ACV (Bleymehl et al., 2011), its relation to resistance seems to be also verified. By contrast, the significance of the substitution L73I for resistance has to be confirmed either by correlation between phenotype and genotype or by site-directed mutagenesis experiments. ...
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.
This study was designed to uncover the antiviral compounds contained in Duchesneae Indicae. Two non-sulfated acidic heteropolysaccharides (DIP30 and DIP60) with anti-varicella-zoster virus (anti-VZV) activity were purified and investigated with a combination of DEAE C-52 ion-exchange chromatography, Sphacryl S-300 gel chromatography, high performance size exclusion chromatography, pre-column derivation with 1-phenyl-3-methyl-5-pyrazolone (PMP) method, infrared (IR) spectroscopy, (1)H and (13)C nuclear magnetic resonance (NMR). Results indicated that molecular weight (MW) of DIP30 and DIP60 are 1.98×10(3) kDa and 34 kDa, respectively. Both DIP30 and DIP60 have a backbone of β-conformation and consist of more than four kinds of monosaccharides. The anti-VZV 50% effective concentrations (EC50) of DIP30 and DIP60 are 265.2±35.4μg/ml and 325.6±42μg/ml, respectively, suggesting that DIP30 and DIP60 could be explored as novel antiviral agents.
A look back is done to some clinical and basic research activities recently published in medical microbiology and immunology. The review covers clinical experiences and in vitro experiments to understand the emergency, pathogenicity, epidemic spread, and vaccine-based prevention of avian and swine-origin flu. Some new developments and concepts in diagnosis, (molecular) epidemiology, and therapy of AIDS, viral hepatitis C, and herpesvirus-associated diseases are outlined. Regulation of immune system has been discussed in a special issue 2010 including some aspects of CNS affections (measles). Mycobacterial infection and its prevention by modern recombinant vaccines have reached new interest, as well as new concepts of vaccination and prophylaxis against several other bacteria. Adaptation to host niches enables immune escape (example brucella) and determines virulence (example N. meningitidis). Chlamydia pneumoniae, previously considered to trigger atherosclerosis, is hypothetically associated to Alzheimer disease, while CMV, another putative trigger of atherosclerosis, gains evidence of oncomodulation in CNS tumor diseases. In terms of globalization, exotic virus infections are increasingly imported from southern countries.
