Pitfalls in diagnosing human poxvirus infections.
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Adult↗
Biomedical subjects
Andreas Nitsche
Publications and source records attributed to Andreas Nitsche.
A new quantitative real-time reverse transcriptase PCR assay and melting curve analysis for detection and genotyping of Ljungan virus strains.
Ljungan virus (LV), a new member of the Picornaviridae, recently isolated from vole species in both Sweden and the USA, is suspected to be pathogenic for humans as an aetiological agent in myocarditis, diabetes, neurological disease and perinatal disease. This study describes for the first time an RT-PCR assay that can identify and quantify LV infection in various tissue sample types using primers and two different minor-groove-binder probes targeting the 5'-untranslated region of the LV genome. The assay, evaluated using control samples derived from various virus cultures and rodent tissues, allows precise quantification of viral load over six orders of magnitude (10(1) to 10(6) viral copies per assay) for all known strains of LV with high sensitivity and specificity. Furthermore, a melting curve analysis (MCA) was developed using two amplicon-specific hybridisation probes that allows rapid genotyping of different LV strains. These new methods provide useful tools to investigate the putative role of LV as a pathogen in both rodents and humans.
Animals↗
Quantitative expression analysis of HHV-6 cell receptor CD46 on cells of human cord blood, peripheral blood and G-CSF mobilised leukapheresis cells.
Human herpesvirus-6 (HHV-6) can infect blood cells and thereby may inhibit hematopoietic stem and progenitor cell expansion and differentiation. In this context, it has been discussed if early progenitor cells can be infected by HHV-6. CD46 was identified as one possible cellular surface receptor for HHV-6. The study presented here had been done to get insight into the susceptibility of various leukocyte subpopulations to HHV-6 (including early hematopoietic progenitors) by determining the amount of CD46 molecules expressed on their surfaces. Human cord blood cells, peripheral blood cells and G-CSF mobilised progenitor cells were analysed by flow cytometry. CD46 molecule number per cell was determined and compared to calibration beads conjugated with known ratio of PE per bead. Highest CD46 expression was detected on B- lymphocytes, whereas T-lymphocytes only showed about half of the amount found on B cells. Hematopoietic progenitors also carried CD46 at intermediate levels. Unexpectedly, CD46 expression on progenitors from G-CSF mobilised leukapheresis products was approximately 20% of that found on comparable cells from untreated cord blood. In conclusion, hematopoietic progenitor cells express CD46 on their surface, thereby fulfilling a basic requirement for the susceptibility of HHV-6 infection.
Adult↗
Real-time PCR detection of parapoxvirus DNA.
BACKGROUND: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. METHODS: A minor groove binder-based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. RESULTS: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7-6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. CONCLUSION: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.
Animals↗
Detection of infectious poxvirus particles.
To enable rapid and reliable detection of poxviruses in clinical and environmental specimens, a diagnostic approach was developed to detect <3 PFU of infectious poxvirus particles in <5 hours. This approach involved virus culture combined with real-time reverse transcription-polymerase chain reaction detection of 2 viral genes expressed immediately after infection.
Cell Line, Tumor↗
Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region.
Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription. First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels. Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells. Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia.
Cell Line, Tumor↗
Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?
Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and >or=7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.
Aged↗
Development of a PCR-based assay for detection, quantification, and genotyping of human adenoviruses.
BACKGROUND: Adenoviruses (AdVs) can cause serious disease in immunosuppressed patients, particularly those undergoing allogeneic stem cell transplantation. A method for virus quantification in clinical specimens is essential for monitoring patient adenoviral loads and evaluating new therapeutic approaches. METHODS: We developed a PCR-based assay that combines detection and genotyping of human AdVs, targeting a highly conserved region of the adenoviral genome coding for the DNA polymerase (AdV DPol PCR). We tested the diagnostic applicability of this PCR-based assay by analyzing 159 clinical specimens from children with respiratory disease and comparing the results with those obtained by nested PCR analysis. RESULTS: The PCR assay detected all currently known AdV serotypes, with a detection limit of approximately 10 genome equivalents per reaction for 49 of 51 serotypes. No cross-reactivity to human DNA or other DNA viruses was observed. In addition, genotyping of PCR-positive samples was achieved within minutes by fluorescence curve melting analysis in a LightCycler instrument using 6 pairs of hybridization probes, each specific for a single AdV species. Results for clinical specimens were in good concordance with those obtained by nested PCR. CONCLUSION: The presented assay is a suitable tool for the detection and genotyping of human AdVs in clinical samples.
Adenoviruses, Human↗
Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections.
Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, beta-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.
Cell Line↗
High prevalence of cardiotropic viruses in myocardial tissue from explanted hearts of heart transplant recipients and heart donors: a 3-year retrospective study from a German patients' pool.
BACKGROUND: The prevalence of some cardiotropic viruses in virus-associated inflammatory cardiac disease remains controversial. The aim of this study was to examine myocardial tissue samples from explanted hearts of heart transplant recipients and heart donors for nucleic acids of myocardiotropic viruses and to observe the potential risk of viral-induced post-transplantation complications in recipients of cardiac allografts or heart valve homografts. METHODS: Myocardial tissue samples were analyzed by polymerase chain reaction (PCR) for enteroviruses, adenoviruses, human cytomegalovirus (HCMV), parvovirus B19 (PVB19), and influenza viruses. The results were compared with serologic and histopathologic findings. RESULTS: PCR analysis of 449 myocardial tissue samples from explanted hearts indicated infection in 34 (47%) of 73 heart transplant recipients and 48 (60%) of 80 donors. The prevalence of virus infection in donors aged over 65 years was significantly higher than in heart transplant recipients (p = 0.005) or donors aged under 65 years (p = 0.02). The most frequently detected viruses were enteroviruses (group B coxsackievirus) and adenoviruses. HCMV and PVB19 were found less frequently. All samples were negative for influenza viruses. Although the serologic findings and PCR results for different viruses were discordant in 4% to 27% of cases, PCR and histopathologic findings were highly correlated (88%). CONCLUSIONS: The frequent detection of viral genome sequences in myocardial tissue of both heart transplant recipients and heart donors suggests a significant risk for graft-transmitted viral infection in cardiac and heart valve transplant recipients.
Adenoviridae↗
Detection of vaccinia virus DNA on the LightCycler by fluorescence melting curve analysis.
After eradication of variola virus the worldwide vaccination program was stopped to avoid the severe complications observed in a small fraction of vaccinees. Hence, there is at least one non-vaccinated generation in the human population that is immunologically naive with respect to variola virus infections. The possibility of a deliberate release of variola virus by bioterrorist attacks has led to the resumption of vaccination of hospital employees and military personnel with vaccinia virus in certain parts of the world. However, the appearance of a single confirmed smallpox case worldwide would result in vaccination of possible contact persons with vaccinia virus. Therefore, reliable confirmation of vaccinia virus in patients presenting with smallpox-like syndromes is required. A vaccinia virus-specific single nucleotide polymorphism was identified in the gene B8R coding for a vaccinia virus IFNgamma receptor. Based on this polymorphism, the LightCycler real-time PCR assay detects vaccinia virus DNA in a linear range from 10(6) to 10 genome equivalents and discriminates vaccinia virus from other orthopoxviruses by fluorescence melting curve analysis (DeltaT = 9 degrees C). While the assay amplifies generically DNA of all orthopoxviruses tested, amplification curves are only displayed for vaccinia virus strains including strains formerly used for vaccination. In addition, an internal amplification control is described that allows reliable interpretation of results.
Base Sequence↗
Infections with human herpesvirus 6 variant B delay platelet engraftment after allogeneic haematopoietic stem cell transplantation.
The clinical significance of human herpesvirus (HHV-6) infections after allogeneic stem cell transplantation (SCT) remains controversial. We analysed cryoconserved plasma samples from 82 patients after allogeneic SCT by quantitative polymerase chain reaction for HHV-6 variants A and B. Platelet engraftment was delayed in patients with HHV-6B infections but not with HHV-6A infections detected before day +28. In multivariate analysis early HHV-6B infections and the type of conditioning were associated with platelet engraftment. In conclusion, the two variants of HHV-6 should be studied separately; early infections with HHV-6B may contribute to delayed platelet engraftment after allogeneic SCT.
Adolescent↗
Guideline to reference gene selection for quantitative real-time PCR.
Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and beta-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.
Base Sequence↗
Analysing myocardial tissue from explanted hearts of heart transplant recipients and multi-organ donors for the presence of parvovirus B19 DNA.
BACKGROUND: Parvovirus B19 (PVB19) is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes, dependent on the haematological and/or immunological status of the host. Reports of PVB19 infection as a causative agent of paediatric or adult inflammatory cardiac diseases, or of cardiac transplant rejection are rare. OBJECTIVES: To identify PVB19 and other cardiotropic viruses in the myocardium of heart transplant (HTx) recipients and multi-organ donors (MOD). Furthermore, to assess the prevalence of cardiotropic viral infection in inflammatory heart disease. STUDY DESIGN: Heart tissue samples from 110 explants were analysed for PVB19 using primers and a 5'-nuclease probe designed to amplify a 160-basepair PCR product from the VP1/NS1 gene region. Samples tested included those obtained from patients undergoing HTx or from MODs. The findings were correlated with clinical course, histologic analysis and serologic testing. Confirmation of the positive PCR-results was done by sequencing and in situ hybridisation. RESULTS: The new assay described here allows precise quantitation of viral load over 7 orders of magnitude (10(6) to 10(0)IU/assay). Measurable amounts of parvoviral genomes were detected in 4/56 (7%) explanted HTx-hearts and in 5/54 (9%) explanted MOD-hearts. CONCLUSIONS: The newly developed real-time PCR is a rapid, sensitive and specific method to detect PVB19 infection in heart tissue. It will be a useful tool to address important questions regarding viral infections transmitted by transplantation, acute infections, relapses and complications involving late or chronic rejection.
Adolescent↗
Inhibition of cord blood cell expansion by human herpesvirus 6 in vitro.
To elucidate the role of human herpesvirus 6 (HHV-6) in hematopoiesis, the influence of HHV-6A and HHV-6B on the in vitro expansion and differentiation of cord blood (CB) progenitor cells was investigated in liquid culture. Nonfractionated CB mononuclear cells (CB-MNC) or MACS-enriched CD34+ CB cells were seeded in liquid culture under conditions allowing maximal expansion of nucleated cells. Cells were either incubated with HHV-6A- or HHV-6B-containing cell culture supernatants or a virus-free control. After 7, 14, and 21 days, cells were analyzed for growth by cell count, for differentiation by flow cytometry, and for HHV-6 infection by antigen detection or PCR. Expansion of CB-MNC was significantly inhibited by HHV-6A and HHV-6B for a period of 3 weeks, including reduced proportions of CD34+ and CD33+ cells in HHV-6-treated cultures on day 7. In contrast, when starting with enriched CD34+ cells, the expansion was only affected by HHV-6A. Inhibition of CD34 and CD33 cell development was less pronounced in these cultures compared to CB-MNC cultures. However, HHV-6 antigen and DNA was detectable in these cultures. In conclusion, although HHV-6A inhibited expansion of CD34 progenitor cells, HHV-6B inhibited growth of immature CB cells only in interaction with nonfractionated CB-MNC.
Antigens, CD34↗
Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.
Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.
Base Sequence↗
First international quality assurance study on the rapid detection of viral agents of bioterrorism.
We have conducted an international quality assurance study of filovirus, Lassa virus, and orthopox virus PCR with 24 participants. Of the participating laboratories, 45.8 and 66.7% detected virus in all plasma samples, which contained > or = 5,000 and > or = 100,000 copies per ml, respectively. Sensitivity levels were not significantly different between viruses. False-negative results were attributable to a lack of sensitivity.
Bioterrorism↗
SARS coronavirus detection.
We developed a set of three real-time reverse transcription-polymerase chain reaction (PCR) assays that amplify three different regions of the SARS-associated coronavirus (SARS-CoV), can be run in parallel or in a single tube, and can detect <10 genome equivalents of SARS-CoV. The assays consider all currently available SARS-CoV sequences and are optimized for two prominent real-time PCR platforms.
Humans↗