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ABSTRACT: Rapid alternative methods are required to evaluate easily acyclovir (ACV) sensitivity of clinical herpes simplex virus (HSV) isolates. The objective of this study was to screen 54 ACV-sensitive and 41 ACV-resistant clinical HSV-1 isolates, well characterized by phenotypic and genotypic methods, for the phosphorylation activity of the viral thymidine kinase (TK) using a commercially available and modified non-radioactive DiviTum(®) test on the basis of an indirect enzyme linked immunosorbent assay. The ACV-sensitive HSV-1 isolates had high TK activity values between 31.5±6.4 DiviTum(®) Units per liter (DU/L) and 487.4±60.1 DU/L. The mean activity of all ACV-sensitive isolates was calculated as 212.3±15.7 DU/L. By contrast, the mean activity of all ACV-resistant HSV-1 isolates was significantly lower at 5.5±1.3 DU/L. Out of the 41 ACV-resistant HSV-1 isolates, 38 had no or very low phosphorylation activities of the viral TK between 0 DU/L and 9.3±3.2 DU/L. The remaining three ACV-resistant viral isolates had TK activities between 44.6±5.1 DU/L and 80.9±13.3 DU/L. In conclusion, the modified DiviTum(®) test can be used to screen HSV-1 isolates for their sensitivity to ACV. Acyclovir-sensitive HSV-1 isolates show TK activities >30 DU/L and ACV-resistant isolates have activity values <10 DU/L. However, single ACV-resistant HSV-1 isolates can have TK activity values >30 DU/L. These strains are most likely ACV-resistant TK-altered mutants, but no evidence was provided for an alteration of the TK.
Journal of virological methods 12/2012; · 2.13 Impact Factor
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ABSTRACT: BACKGROUND: There is agreement that the infectivity assay with the duck hepatitis B virus (DHBV) is a suitable surrogate test to validate disinfectants for hepatitis B virucidal activity. However, since this test is not widely used, information is necessary whether disinfectants with limited virucidal activity also inactivate DHBV. In general, disinfectants with limited virucidal activity are used for skin and sensitive surfaces while agents with full activity are more aggressive. The present study compares the activity of five different biocides against DHBV and the classical test virus for limited virucidal activity, the vaccinia virus strain Lister Elstree (VACV) or the modified vaccinia Ankara strain (MVA). METHODS: Virucidal assay was performed as suspension test according to the German DVV/RKI guideline. Duck hepatitis B virus obtained from congenitally infected Peking ducks was propagated in primary duck embryonic hepatocytes and was detected by indirect immunofluorescent antigen staining. RESULTS: The DHBV was inactivated by the use of 40% ethanol within 1-min and 30% isopropanol within 2-min exposure. In comparison, 40% ethanol within 2-min and 40% isopropanol within 1-min exposure were effective against VACV/MVA. These alcohols only have limited virucidal activity, while the following agents have full activity. 0.01% peracetic acid inactivated DHBV within 2 min and a concentration of 0.005% had virucidal efficacy against VACV/MVA within 1 min. After 2-min exposure, 0.05% glutardialdehyde showed a comparable activity against DHBV and VACV/MVA. This is also the case for 0.7% formaldehyde after a contact time of 30 min. CONCLUSIONS: Duck hepatitis B virus is at least as sensitive to limited virucidal activity as VACV/MVA. Peracetic acid is less effective against DHBV, while the alcohols are less effective against VACV/MVA. It can be expected that in absence of more direct tests the results may be extrapolated to HBV.
BMC Infectious Diseases 10/2012; 12(1):276. · 3.12 Impact Factor
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ABSTRACT: 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.
Antiviral research 08/2012; 96(2):105-107. · 3.61 Impact Factor
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ABSTRACT: Commercial serologic assays for varicella-zoster virus (VZV), which enable reliable determination of VZV immune status and are amenable to automation, are needed. The present study compares the automated performance of the VZV whole-cell enzyme-linked immunosorbent assay (ELISA) Enzygnost anti-VZV/IgG, the Euroimmun anti-VZV ELISA (IgG) based on highly purified viral proteins, and the VZV glycoprotein (gp)-based Serion ELISA Classic VZV IgG. The fluorescent-antibody-to-membrane-antibody (FAMA) test was used as a reference. A total of 638 serum samples from VZV-negative children, blood donors, varicella vaccinees, and bone marrow transplant recipients were included. The Enzygnost anti-VZV/IgG and the Serion ELISA Classic VZV IgG showed sensitivities of 99.6% and 99.2%, respectively, and the Euroimmun anti-VZV ELISA (IgG) had a significantly lower sensitivity of 90.5%. Specificity was calculated as 100% for both the Euroimmun anti-VZV ELISA (IgG) and for the Enzygnost anti-VZV/IgG, and the Serion ELISA Classic VZV IgG had a significantly lower specificity of 89.4%. Quantitative results of all ELISAs correlated well, but there was a poor quantitative correlation between the ELISAs and FAMA. In conclusion, this study does not show any superiority of a gp- and a protein-based ELISA compared to a whole-cell ELISA for the automated detection of VZV-specific IgG. The automated performance of the Enzygnost anti-VZV/IgG assay correlated best with the FAMA reference assay.
Clinical and vaccine immunology: CVI 06/2012; 19(8):1261-8. · 2.37 Impact Factor
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ABSTRACT: The objective of this study was to genotype 375 clinical herpes simplex virus type 1 (HSV-1) isolates collected from the German Reference Laboratory of HSV and VZV between 1973 and 2010. The method is based on the amplification and the restriction fragment length polymorphism analysis of the glycoprotein G (gG) and gI. 45.1% of isolates were classified as genotype A, 28.5% as B, and 4.3% as C. 22.1% presented different cleavage patterns for gG and gI suggesting intergenic recombinants A/B in 7.7%, A/C in 0.5%, B/A in 9.3%, B/C in 1.9%, C/A in 1.6%, and C/B in 0.5% of isolates. Two isolates from 1982 and 2010 presented atypical gI cleavage pattern consistent with novel intragenic recombination between genotypes A and C. There were no significant differences of the prevalence of genotypes A, B as well as the recombinants A/B, B/A dependent on the age/gender of patients and the time period in which the strains were isolated. Likewise, there were no significant differences in the distribution of the genotypes A and B as etiological agents of eczema herpeticum, herpes labialis, herpes genitalis, and herpetic gingivostomatitis. The number of recombinants was not different significantly in the groups of the distinct herpetic diseases. In conclusion, the study confirms the high prevalence of recombinants in clinical HSV-1 strains. HSV-1 infections result in clinical manifestations which are independent of the gG/gI genotype and recombinants are not associated with special herpetic diseases.
Journal of Medical Virology 04/2012; 84(4):651-6. · 2.82 Impact Factor
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ABSTRACT: In the present study, antibody response to seasonal influenza vaccination and to the adjuvanted one-shot influenza A H1N1 vaccine (Pandemrix(®)) was investigated in 57 hemodialysis (HD) patients and 48 renal transplant (RT) recipients. Specific antibodies were measured by hemagglutination inhibition (HI) test using a pandemic H1N1 strain and a seasonal H3N2 virus. HI titers of ≥1:40 were considered as protective. Hemodialysis patients showed seroprotection against pandemic H1N1 in 35.1%, against seasonal influenza in 36.8% and against both in 14.0%. In comparison, renal transplant recipients developed protective antibody titers against the pandemic H1N1 virus in 47.9%, against the seasonal H3N2 strain in 31.3% and against both in 18.8%. HD patients and renal transplant recipients younger than 60 years developed protective antibody response to the pandemic influenza H1N1 vaccine in 50.0% of the HD patients and 55.2% of the RT recipients and against seasonal influenza in 45.0/20.7% (HD/RT) of the cases. Patients aged ≥60 years showed seroprotection against pandemic influenza in 27.0/36.8% (HD/RT) and against seasonal influenza in 32.4/47.4% (HD/RT). Side effects were reported in only four patients. In hemodialysis patients and renal transplant recipients, vaccination against pandemic H1N1 and seasonal influenza is well tolerated. However, more than a half of these patients did not develop seroprotective antibody levels. Thus, new vaccines and altered vaccination regimes are likely necessary to achieve relevant antibody levels in these patient groups.
Medical Microbiology and Immunology 02/2012; 201(3):297-302. · 3.83 Impact Factor
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ABSTRACT: Herpes simplex virus type 2 (HSV-2) can cause radiculo-myelitis as a neurological manifestation. We report a case of ongoing HSV-2 DNA positivity in the cerebrospinal fluid (CSF) of at least eight weeks under antiviral therapy with acyclovir in a highly immunocompromised hemato-oncologic patient with HSV-2-associated radiculitis. Upon admission, the patient presented with pain, leg paresis, and urinary incontinence, as well as pleocytosis in the CSF. Quantitative real-time PCR of the CSF at day 3 after admission revealed HSV-2 with a concentration of 2.0×10(5) copies/ml and treatment with acyclovir intravenously and prednisolone by mouth was started. Clinical symptoms resolved almost completely after approximately 3 weeks of antiviral therapy. However, CSF samples of day 12, 19, 26, 33, 39, 48 and 54 after admission showed a slow decline of HSV-2 DNA concentrations. HSV-2 DNA was still detectable (1.6×10(4) copies/ml) at day 54 after admission. Genotypic resistance testing showed, as far as available, no mutations indicative for acyclovir resistance. Since an increasing specific antibody index for HSV was observed, we speculate that the prolonged detectability of HSV-2 DNA in the CSF might not necessarily indicate ongoing viral replication but neutralized virus. Other hypotheses and the consequences on treatment are discussed. To our knowledge this is the first report about the long-term viral load kinetics of HSV-2 in the CSF of a patient with radiculitis under antiviral therapy, highlighting the need for further studies on HSV DNA kinetics in the CSF and their significance for an appropriate antiviral treatment.
Antiviral therapy 01/2012; 17(1):125-8. · 3.16 Impact Factor
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ABSTRACT: Varicella-zoster virus (VZV), the cause of varicella and zoster, is divided into five major clades and four provisional clades, the latter of which have been rarely reported worldwide to date. We present a varicella outbreak by the provisional clade VI within an Indian couple in Germany returning from a trip to Amsterdam. To the best of our knowledge, this is the first case of varicella by the VZV clade VI described in Germany, but the disease was acquired in The Netherlands.
Case Reports in Medicine 01/2012; 2012:838241.
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ABSTRACT: Genotyping of 21 varicella-zoster virus (VZV) strains using a scattered single nucleotide polymorphism (SNP) method revealed ambiguous SNPs and two nontypeable isolates. For a further genetic characterization, the genomes of all strains were sequenced using the 454 technology. Almost-complete genome sequences were assembled, and most remaining gaps were closed with Sanger sequencing. Phylogenetic analysis of 42 genomes revealed five established and two novel VZV genotypes, provisionally termed VIII and IX. Genotypes VIII and IX are distinct from the previously reported provisional genotypes VI and VII as judged from the SNP pattern. The alignments showed evidence of ancient recombination events in the phylogeny of clade 4 and recent recombinations within single strains: 3/2005 (clade 1), 11 and 405/2007 (clade 3), 8 and DR (clade 4), CA123 and 413/2000 (clade 5), and strains of the novel genotypes VIII and IX. Bayesian tree inference of the thymidine kinase and the polymerase genes of the VZV clades and other varicelloviruses revealed that VZV radiation began some 110,000 years ago, which correlates with the out-of-Africa dispersal of modern humans. The split of ancestral clades 2/4 and 1/3/5/VIII/IX shows the greatest node height.
Journal of Virology 11/2011; 86(3):1608-22. · 5.40 Impact Factor
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ABSTRACT: 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.
Antiviral research 06/2011; 90(3):242-7. · 3.61 Impact Factor
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Journal der Deutschen Dermatologischen Gesellschaft 06/2011; 9(6):444-7. · 1.47 Impact Factor
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ABSTRACT: The thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are important targets for genotypic determination of HSV resistance to antiviral drugs. The knowledge of gene polymorphism is an absolute requirement for the correct interpretation of genotypic findings.
In this study, the natural polymorphism of TK and DNA pol genes was examined by DNA sequencing in 56 HSV-1 and 12 HSV-2 strains sensitive to acyclovir.
In 56 HSV-1 strains, 26 different non-synonymous polymorphism-associated mutations were detected in the TK gene. To our knowledge, 8 of them have never been reported in the literature. In the TK gene of 12 HSV-2 strains, 6 polymorphism-related non-synonymous mutations were observed, whereas there was 1 novel mutation in 1 strain. The DNA pol gene of 53 HSV-1 isolates contained 47 distinct polymorphism-associated amino acid substitutions and 11 substitutions were found in the DNA pol gene of 12 HSV-2 strains. Altogether, 31 novel substitutions could be identified in the DNA pol gene of HSV-1 and 3 in HSV-2 strains. In these strains, any resistance to foscarnet was excluded.
The 43 novel non-synonymous mutations enrich the knowledge about the natural genetic polymorphism of TK and DNA pol in clinical HSV strains. The findings have to be considered for genotypic analysis of HSV in case of clinical resistance.
Antiviral therapy 01/2011; 16(7):989-97. · 3.16 Impact Factor
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Andreas Sauerbrei,
Kathrin Bohn,
Albert Heim,
Jörg Hofmann,
Benedikt Weissbrich,
Paul Schnitzler,
Dieter Hoffmann,
Roland Zell,
Gerhard Jahn,
Peter Wutzler,
Klaus Hamprecht
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ABSTRACT: Studies to verify correlations between phenotypes and genotypes of herpes simplex virus (HSV) are an important tool to establish a database of resistance-associated mutations.
In this study, 32 acyclovir (ACV)-resistant clinical HSV-1 and 4 ACV-resistant clinical HSV-2 isolates were examined in parallel by both phenotypic and genotypic resistance testing. Additionally, five non-viable HSV-1 strains and two non-viable HSV-2 strains with clinical resistance were included in genotypic resistance analysis.
All ACV-resistant HSV isolates showed cross-resistance to brivudin and penciclovir, and were sensitive to foscarnet and cidofovir. Acyclovir resistance was assigned to frameshift and single non-synonymous mutations of the thymidine kinase (TK) gene in 32 out of 37 HSV-1 strains and in 4 out of 6 HSV-2 strains. In three HSV-1 isolates, there were resistance-associated amino acid substitutions of the DNA polymerase (pol). Six substitutions in the TK and two in the DNA pol gene could not be attributed without doubt to either ACV resistance or natural gene polymorphism. Altogether, 10 resistance-related mutations in the TK and 1 in the DNA pol gene have not been reported previously.
The novel non-synonymous mutations found in this study enrich the knowledge about the genetic alterations of TK and DNA pol genes in ACV-resistant clinical HSV strains. Together with data from the literature, the findings justify the generation of a HSV database that contains resistance mutations associated with ACV resistance phenotype.
Antiviral therapy 01/2011; 16(8):1297-308. · 3.16 Impact Factor
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ABSTRACT: Varicella has the highest contagiosity index of all viral diseases. We report an endemic outbreak of varicella among 4 Indian students in Magdeburg in November and December 2008. An initially severe course was observed in three of these patients with a negative vaccination status. Large vesicular skin lesions with a diameter of up to 8 mm were found in all patients. Molecular genetic tests revealed African/Indian clade 5 in 2 patients, although the European clades (i.e., clade 1 and 3) are the most common in Germany, accounting for 85 %. All patients recovered without any complications after administration of intravenous aciclovir at a dosage of 10 mg per kg body weight. Although isolated cases of varicella are not notifiable according to the German Protection against Infection Act, endemic outbreaks must be reported to the appropriate health surveillance authorities.
Journal der Deutschen Dermatologischen Gesellschaft 12/2010; 9(6):444-7. · 1.47 Impact Factor
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ABSTRACT: Eighty-six varicella-zoster virus (VZV) strains of clades 1, 3 and 5, isolated from varicella and zoster patients in Germany, were analyzed by sequencing the glycoprotein E gene. Four novel non-synonymous and 10 novel synonymous mutations were detected. Of these, two synonymous (C513T, C885T) and two non-synonymous mutations (T485G, C524T) were located within the coding regions of e1 and c1. The profile of single-nucleotide polymorphisms was found to be significantly associated with the VZV clades 1, 3 and 5.
Archives of Virology 11/2010; 156(3):505-9. · 2.11 Impact Factor
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ABSTRACT: We report the case of a 25-year-old lung and liver transplant recipient who developed respiratory failure. High levels of Epstein-Barr virus (EBV) genome copies were detectable in respiratory tract specimens, while the search for various other viral, bacterial or fungal pathogens remained empty. Post-transplant lymphoproliferative disease was excluded. Due to the rapid progression of respiratory insufficiency, a re-transplantation of the lung was performed. EBV-encoded small RNAs could be demonstrated by in situ hybridization within pneumocytes and lymphocytes of the explanted lung tissue. The clinical situation improved soon after re-transplantation, and the EBV load detected in the lower respiratory tract decreased significantly.
Medical Microbiology and Immunology 11/2010; 199(4):317-22. · 3.83 Impact Factor
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ABSTRACT: Influenza is associated with substantial morbidity and mortality in pregnant women and neonates, but few countries offer annual influenza vaccination with the inactivated vaccine to all women who are, or intend to become, pregnant.
To provide seroepidemiological information on influenza A and B antibodies in pregnant women and their offspring in Germany.
Anti-influenza antibodies were determined using commercially available enzyme-linked immunosorbent assays (ELISA) on serum obtained from 209 women and their newborns at delivery.
The prevalence of antibodies against influenza A virus was 93.8% [89.6-96.6%] in the mothers and 96.7% [93.2-98.6%] in the newborns. The prevalence of antibodies against influenza B virus was 42.1% [35.3-49.1%] in the mothers and 78.5% [72.3-83.8%] in their newborns, which was a significant difference. The antibody concentrations against both influenza A and influenza B viruses were significantly lower in mother than in their newborns.
Because of active placental transport of IgG antibodies, neonates have higher prevalence and/or concentrations of influenza A and B virus-specific antibodies induced by natural infections than their mothers. Considering these serological findings, especially the lower prevalence of maternal antibody against influenza B virus, annual influenza vaccination may improve the protection of pregnant women and their offspring against influenza.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 08/2009; 46(2):161-4. · 3.12 Impact Factor
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ABSTRACT: Rapid and reliable detection of varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and -2 (HSV-2) is of clinical significance in immunocompromised patients and patients with infections of the central nervous system. This paper describes the detection of VZV and HSV using the commercially available Affigene VZV and Affigene HSV 1/2 tracer kits in comparison to "in-house" polymerase chain reaction (PCR) assays. For sample preparation, Qiagen (Hilden, Germany) and Affigene (Cepheid AB, Bromma, Sweden) DNA extraction kits were used. 175 samples were analyzed for VZV and 352 samples for HSV-1 and -2. Generally more positive results were obtained using the Affigene assays compared to the "in-house" methods independent of the DNA preparation method used. There were significant differences in sensitivity between the Affigene HSV 1/2 tracer and the "in-house" PCR assays for the detection of both HSV-1 and -2 in cerebrospinal fluid and vesicle/skin swabs. The Affigene HSV 1/2 and VZV tracers are very sensitive assays for detection of VZV and HSV. A wide variety of clinical samples can be examined in combination with either the Qiagen or the Affigene DNA extraction kits for preparation.
Journal of virological methods 06/2009; 160(1-2):193-6. · 2.13 Impact Factor
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ABSTRACT: The novel RIDASCREEN varicella-zoster virus (VZV) IgG enzyme immunoassay (EIA) based on viral envelope glycoproteins was evaluated using the fluorescent antibody to membrane antigen test as a reference. For the detection of VZV immunity in latently infected persons, the sensitivity of the RIDASCREEN assay was 94-100%. Thus, the novel EIA had comparable sensitivity as the SERION enzyme-linked immunosorbent assay (ELISA) classic VZV IgG and higher sensitivity than the ELISA Enzygnost Anti VZV/IgG. Sensitivity of the RIDASCREEN EIA for the determination of immunity following vaccination was 57-89% and not different from the sensitivity of the SERION and the Enzygnost assay. While the specificity of the RIDASCREEN EIA was calculated as 88-98%, SERION and Enzygnost ELISAs had 100% specificity. In conclusion, the novel RIDASCREEN EIA can be recommended for detection of VZV immunity after natural infection taking account of the reduced specificity. For determination of immunity post vaccination, a different approach for the interpretation of borderline results should be considered.
Clinical laboratory 02/2009; 55(1-2):1-7. · 0.90 Impact Factor
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ABSTRACT: The objective of this study was to provide seroepidemiological information on influenza A and B antibodies in children and adolescents. Viral immunoglobulin G antibodies were determined retrospectively using enzyme-linked immunosorbent assays in a group of 1,111 children and adolescents. Sera (809) from healthy adult blood donors served as controls. In children, the prevalence of antibodies against influenza A was 82.0% and against influenza B 9.6%, whereas in adults the prevalence of antibodies against influenza A was calculated as 99.4% and against influenza B 56.7%. After decline of maternal antibodies during the first year of life, there was an increase of prevalence of influenza A antibodies up to 100% by the age of 12 years. In contrast, only 1-2% of children up to 9 years had influenza B antibodies increasing to 25% by the age of 18 years and to 70% among adults aged 30 years. Children aged 0-6 years had significantly lower concentrations and >12-15-year-old adolescents had significantly higher concentrations of antibodies against influenza A than adults. For all age groups of children and adolescents, significantly lower antibody concentrations against influenza B were measured in comparison to the blood donor control group. In conclusion, the annual influenza vaccination in children and adolescents may improve considerably the protection against influenza virus infection occurring during epidemics.
Medical Microbiology and Immunology 02/2009; 198(2):93-101. · 3.83 Impact Factor
