Home About us Contact
|
Home About us Contact | ||
|
|
||
Viral DNA (viral + dna)
Selected AbstractsA new strategy for studying In Vitro the drug susceptibility of clinical isolates of human hepatitis B virusHEPATOLOGY, Issue 4 2004David Durantel Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC50/IC90s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains. (Hepatology 2004;40:855,864). [source] Human herpesvirus 7-associated meningitis and optic neuritis in a patient after allogeneic stem cell transplantationJOURNAL OF MEDICAL VIROLOGY, Issue 3 2003Tetsushi Yoshikawa Abstract A 9-year-old boy who received allogeneic stem cell transplantation began to vomit from day 10 after transplantation. In addition to vomiting, the patient had a fever (from day 26) and severe headache (from day 34). His cerebrospinal fluid (CSF) (day 41) demonstrated pleocytosis with an absence of leukemic cells. Although the patient's symptoms were resolved with further supportive care, abrupt onset of bilateral decreased vision occurred at day 54. He was diagnosed with bilateral optic neuritis, due to the presence of disc edema and redness. Concomitant with the occurrence of aseptic meningitis, the human herpesvirus 7 (HHV-7) antibody titer increased significantly in this patient. Although neither HHV-6 nor cytomegalovirus (CMV) DNA was detected in CSF collected at day 41, HHV-7 DNA was detected in the sample. Viral DNA was not detected in CSF collected at day 93. J. Med. Virol. 70:440,443, 2003. © 2003 Wiley-Liss, Inc. [source] Viruses in periodontal disease , a reviewORAL DISEASES, Issue 4 2005I Cappuyns The purpose of this review was to evaluate the evidence supporting the hypothesis that viral infection plays a role in the development of periodontitis. An involvement in periodontal diseases has been suspected specifically for human immunodeficiency virus (HIV) and herpes viruses. An association has been demonstrated between HIV infection and some distinct forms of periodontal infection, i.e. necrotizing lesions. Furthermore, reports of increased prevalence and severity of chronic periodontitis in HIV-positive subjects suggests that HIV infection predispose to chronic periodontitis. Several studies, most of them from the same research group, have demonstrated an association of herpesviruses with periodontal disease. Viral DNA have been detected in gingival tissue, gingival cervicular fluid (GCF) and subgingival plaque from periodontaly diseased sites. In addition markers of herpesviral activation have been demonstrated in the GCF from periodontal lesions. Active human cytomegalovirus (HCMV) replication in periodontal sites may suggest that HCMV re-activation triggers periodontal disease activity. Concerns regarding sampling, methods and interpretation cast doubts on the role of viruses as causes of periodontal disease. [source] Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipientsCYTOPATHOLOGY, Issue 6 2008M. Koukoulaki Objective:, BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients. Patients and methods:, We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood. Results:, Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR. Conclusions:, Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation. [source] Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors, DNA-Sensors, and Enzyme BiosensorsELECTROANALYSIS, Issue 11 2003Eugenii Katz Abstract Impedance spectroscopy is a rapidly developing electrochemical technique for the characterization of biomaterial-functionalized electrodes and biocatalytic transformations at electrode surfaces, and specifically for the transduction of biosensing events at electrodes or field-effect transistor devices. The immobilization of biomaterials, e.g., enzymes, antigens/antibodies or DNA on electrodes or semiconductor surfaces alters the capacitance and interfacial electron transfer resistance of the conductive or semiconductive electrodes. Impedance spectroscopy allows analysis of interfacial changes originating from biorecognition events at electrode surfaces. Kinetics and mechanisms of electron transfer processes corresponding to biocatalytic reactions occurring at modified electrodes can be also derived from Faradaic impedance spectroscopy. Different immunosensors that use impedance measurements for the transduction of antigen-antibody complex formation on electronic transducers were developed. Similarly, DNA biosensors using impedance measurements as readout signals were developed. Amplified detection of the analyte DNA using Faradaic impedance spectroscopy was accomplished by the coupling of functionalized liposomes or by the association of biocatalytic conjugates to the sensing interface providing biocatalyzed precipitation of an insoluble product on the electrodes. The amplified detections of viral DNA and single-base mismatches in DNA were accomplished by similar methods. The changes of interfacial features of gate surfaces of field-effect transistors (FET) upon the formation of antigen-antibody complexes or assembly of protein arrays were probed by impedance measurements and specifically by transconductance measurements. Impedance spectroscopy was also applied to characterize enzyme-based biosensors. The reconstitution of apo-enzymes on cofactor-functionalized electrodes and the formation of cofactor-enzyme affinity complexes on electrodes were probed by Faradaic impedance spectroscopy. Also biocatalyzed reactions occurring on electrode surfaces were analyzed by impedance spectroscopy. The theoretical background of the different methods and their practical applications in analytical procedures were outlined in this article. [source] Detection of EHV-1 and EHV-4 in placental sections of naturally occurring EHV-1- and EHV-4-related abortions in the UK: use of the placenta in diagnosisEQUINE VETERINARY JOURNAL, Issue 5 2003S. GERST Summary Reasons for performing study: EHV-1 and EHV-4 abortion diagnosis is based upon detailed examination of the aborted fetus. However, in some cases, only the placenta is available for examination. Furthermore, the contribution of lesions in the placenta to pathogenesis and diagnosis of EHV-1 and EHV-4 abortion has been neglected. Objectives: To assess the utility of placental examination in equine herpesvirus-1 (EHV-1) and EHV-4 abortion diagnosis. Methods: Sections of allantochorion from 49 herpesvirus abortions were analysed by PCR, in situ hybridisation and immunostaining. Results: Virus-specific nested PCR confirmed the presence of viral DNA in 46 cases; 41 cases were EHV-1-positive and 5 EHV-4-positive. Microscopic changes were nonspecific. Examination of the PCR-positive sections of allantochorion revealed EHV-1 DNA by in situ hybridisation (ISH) in 21 cases and EHV-4 in 4 cases. In 2 samples, DNA of both viruses was present on PCR and ISH. Viral antigen was found by immunohistology in 15 cases. Regarding the localisation of virus in the placentae, both viral DNA and antigen of EHV-1 and EHV-4 were found in endothelial cells of chorionic villi and, occasionally, in trophoblast epithelium. In the stromal endothelium, only EHV-1 was found. Conclusions: The data indicate that examination of placentae is a useful diagnostic aid in EHV-1 and EHV-4 abortion diagnosis. Potential relevance: Virological examination of the placenta should be come standard practice in equine abortion investigations, particularly in those cases where the fetus is not available for examination. [source] Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UKJOURNAL OF FISH DISEASES, Issue 11 2007K R Jeffery Abstract Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14,15 to 19,21 °C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 °C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at ,70 °C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2. [source] Persistent parvovirus B19 infection detected by specific CD4+ T-cell responses in a patient with hepatitis and polyarthritisJOURNAL OF INTERNAL MEDICINE, Issue 3 2009G. Pongratz Abstract. We, here, report the case of a parvovirus B19 infection in an immunocompetent male patient presenting with acute hepatitis and polyarthritis. To follow the course of infection, we used a previously established enzyme-linked immunosorbent spot assay (ELISPOT) technique to detect CD4+ T cells specific for viral proteins. Even though symptoms of arthritis and hepatitis resolved in the immunocompetent individual within a few weeks, viral DNA in serum and CD4+ T cells specific for the viral protein VP1 unique region were still detectable more than 6 month after the onset of symptoms, thus pointing to a persistent state of infection. On the basis of this observation, we hypothesize that the intensity of liver involvement correlates with the likelihood of developing persistent parvovirus B19 infection. The described ELISPOT technique to detect virus-specific CD4+ T cells provides an excellent tool to analyse the state of parvovirus B19 infection for future studies to test this hypothesis. [source] Genotyping of the JC virus in urine samples of healthy Korean individualsJOURNAL OF MEDICAL VIROLOGY, Issue 2 2004Byung-Hoon Jeong Abstract A human polyomavirus, JC virus (JCV) is ubiquitous in humans and infects children asymptomatically. It persists in renal tissue and is excreted progeny in urine. DNAs from urine samples of 100 healthy Korean individuals were screened for the presence of JCV by polymerase chain reaction (PCR). Twenty of the samples were positive for JCV. JCV DNA was found in one individual (4%) in the 1,19-year group, two individuals (9%) in the 20,39-year group, ten individuals (38%) in the 40,59-year group, seven individuals (28%) in the over 60-year group. The prevalence of JC viral DNA was the highest in the 40,59-year-old Korean population. To investigate genotypes of JCV in Korea, the genotypes were determined by DNA sequence analysis of the regulatory region (333 bp) and the VT-intergenic region (656 bp) of DNA from the 20 JCV isolates. We have identified three distinctive JCV strains in the regulatory region and ten distinctive JCV strains in the VT-intergenic region of DNA from the 20 isolates. Based on restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis of the VT-intergenic region of JCV, two distinct subtypes, CY and type 2A (MY), were found to be prevalent in this Korean population. CY and type 2A of JCV were identified in 13 individuals (65%) and four individuals (20%), respectively. Interestingly, type 1, which was distributed mostly in Europe, was found in 3 (15%) isolates from healthy Korean individuals. J. Med. Virol. 72:281,289, 2004. © 2004 Wiley-Liss, Inc. [source] Use of a rodent model to show that varicella-zoster virus ORF61 is dispensable for establishment of latency,JOURNAL OF MEDICAL VIROLOGY, Issue S1 2003Hitoshi Sato Abstract Varicella-zoster virus (VZV) results in a latent infection in humans after primary infection. Latency has also been established in guinea pigs and rats after inoculation with the virus. It was found that infection of cotton rats with the Oka vaccine strain of VZV results in a latent infection. To begin to identify which genes are required for latency, we infected cotton rats with VZV strain Oka that is deleted for ORF61. ORF61 protein transactivates certain VZV promoters and enhances the infectivity of viral DNA in transient transfections. Deletion of ORF61 results in abnormal syncytia and impairs the growth of VZV in vitro. Inoculation of cotton rats with ORF61-deleted Oka virus resulted in latent VZV infection in the nervous system similar to that seen for animals infected with parental virus. Thus, the cotton rat can be used to study the ability of mutants in the Oka vaccine strain of VZV to establish latent infection. J. Med. Virol. 70:S79,S81, 2003. © 2003 Wiley-Liss, Inc. [source] Baculovirus expression of erythrovirus V9 capsids and screening by ELISA: Serologic cross-reactivity with erythrovirus B19JOURNAL OF MEDICAL VIROLOGY, Issue 2 2002Erik D. Heegaard Abstract Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (>,11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of ,23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96,97% homology). J. Med. Virol. 66:246,252, 2002. © 2002 Wiley-Liss, Inc. [source] Antibodies against human papillomavirus (HPV) type 16 and 18 E2, E6 and E7 proteins in sera: Correlation with presence of papillomavirus DNAJOURNAL OF MEDICAL VIROLOGY, Issue 4 2001Ricardo Rosales Abstract Human papillomavirus (HPV) infection is associated with cervical cancer. The E2 and E1 papillomavirus proteins are expressed at the early stage of infection and regulate DNA replication. The E2 protein activates and represses transcription from different HPVs promoters. At some stage when viral DNA gets integrated into the cellular genome, the E2 gene is disrupted or inactivated. This event leads to a derepression of the E6 and E7 viral oncogenes. These viral proteins are required normally for the maintenance of the malignant phenotype. Therefore, the E2, E6, and E7 proteins are present in all patients infected by papillomavirus. In this study, the association of antibody levels against E2, E6, and E7 proteins of HPV types 16, 18, and 6 was determined in relation to the presence of HPV DNA at the initial stages of HPV infection. Serum samples from 172 women with HPV infection, determined by Papanicolau (Pap) smears and colposcopy, were tested. Elevated antibody titers against E2 protein from the HPV 6 and HPV 16 were detected in 46.42 and 66.96% of the patients, respectively. Antibodies against the E7 and E6 proteins of HPV 16 were found in 51.78 and 36.60% of the patients, respectively. Antibodies against the E6 and E7 proteins of HPV 18 were 35 and 45%, respectively. A statistical difference was found for antibody titers against the E2, E6, and E7 proteins between patients with papillomavirus DNA and controls cases who had no cytological abnormalities and no HPV DNA. Sera titers were 1/500 for patients HPV positive and 1/50 for control individuals. Antibodies titers against E6 and E7 proteins were also examined in patients at 6 and 24 months after cryosurgery. In these patients, a slight decrease in the antibody level against the E2, E6, and E7 proteins was found. No correlation was found between age and number of sexual partners, with serum positivity to the E2, E6, and E7 papillomavirus proteins. These data suggest that antibodies against the E2, E6, and E7 proteins are good candidates for use as markers for monitoring cervical HPV infections. J. Med. Virol. 65:736,744, 2001. © 2001 Wiley-Liss, Inc. [source] Prevalence of oral herpes simplex virus reactivation in cancer patients: a comparison of different techniques of viral detectionJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 2 2009Milanko Djuric Background:, Oral reactivation of latent Herpes simplex virus (HSV) infection may easily occur in cancer patients. Virus reactivation can cause oral mucosa damage, worsen already existing lesions caused by stomatotoxic effect of cancer therapy and, whether symptomatic or asymptomatic, ample spreading and promote viral transmission. Methods:, Polymerase chain reaction (PCR), cell-culture and direct immunofluorescence have been used to determine the frequency of oral HSV reactivation in 60 patients undergoing chemotherapy for different malignancies. Results:, By means of PCR, the presence of viral DNA was detected in 71.7% of patients prior to chemotherapy and in 85.0% after chemotherapy. 33.3% of patients before and 40.0% after chemotherapy were viral-culture positive, while 3.3% of patients before and 11.7% after chemotherapy were positive as shown by direct immunofluorescence. No significant difference in HSV-1 reactivation was found before and after chemotherapy. In addition, no significant difference was found when comparing HSV-1 reactivation in patients with and without mucositis. HSV-2 was not detected in any of the patients. Conclusions:, Reactivation of latent HSV is exceptionally frequent in cancer patients. The results of this study suggest that virus reactivation occurs independently of cancer chemotherapy. The potential role of HSV reactivation in oral mucosa damage remains unclear. [source] A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV-associated oral hairy leukoplakia biopsiesJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 3 2000M. J. E. M. F. Mabruk Abstract: A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein,Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA. [source] Hepatitis B viral DNA is methylated in liver tissuesJOURNAL OF VIRAL HEPATITIS, Issue 2 2008P. Vivekanandan Summary., The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression. [source] Occult hepatitis B viral DNA in liver carcinomas from a region with a low prevalence of chronic hepatitis B infectionJOURNAL OF VIRAL HEPATITIS, Issue 4 2004R. Kannangai Summary., Occult hepatitis B is defined by the presence of hepatitis B viral (HBV) DNA in the serum or liver in persons lacking hepatitis B surface antigen (HBsAg) in the serum. A high prevalence of occult HBV has been reported in hepatocellular carcinoma (HCC) from Asia, but little information is available on the prevalence of occult HBV in HCC from regions with a low prevalence of typical chronic hepatitis B infection. In a retrospective study, 19 cases of primary liver cancer were investigated for the presence of occult HBV DNA by amplification of the surface, core, and X gene. In addition, HBV copy numbers were quantitated by real time polymerase chain reaction, genotyped, and samples tested for covalently closed circular HBV DNA, which is a marker of active viral replication. Occult HBV was found in three of 19 cases (16%). Genotyping was successful in two cases, both of which were genotype A. HBV DNA copy numbers were low, all less than 10 copies/,g liver DNA. No closed circular HBV DNA was detected. Thus, in this study occult HBV was of genotype A and was found in a low percentage of cases of HCC and was associated with low tissue HBV DNA copy numbers and no detectable evidence for viral replication. [source] Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissuesJOURNAL OF VIRAL HEPATITIS, Issue 2 2000Zhong The development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) frequently follows persistent HBV infection and may arise in individuals who are hepatitis B e antigen (HBeAg) negative, indicating the possible presence of precore/core mutants. It is unclear whether precore/core mutants are associated with tumour development or are selected for after chromosomal integration of the wild-type viral DNA. We studied the status and sequence variation of the precore/core region of HBV in 56 patients with HBV-associated HCC and in various corresponding non-tumour tissues by Southern blot analysis, polymerase chain reaction and direct sequencing. Southern blot showed that integrated HBV DNA existed in 43 of 56 HCC tissues. Sequence analysis revealed mutations in 65% of the HCC (26/40) and 45% (14/31) of the corresponding non-tumour tissues. The mutation at nucleotide (nt) 1896, known to prevent HBeAg synthesis, was detected in 40% (16/40) of the tumours and in 35.4% (11/31) of the non-tumour tissues. Other mutations were found at nt 1899 (eight of 40 in HCC; three of 31 in non-tumour tissues), nt 1898 (seven of 40 in HCC; two of 31 in non-tumour tissues), nt 1912 (seven of 40 in HCC; none of 31 in non-tumour tissues) and nt 1886 (three of 40 in HCC; none of 31 in non-tumour tissues). To determine whether this finding merely reflected the prevalence of such mutants in this geographical region, HBV DNA from the sera of patients (also in this region) with acute and chronic hepatitis were sequenced. The nt 1896 mutant was found in 5.6% (one of 18) of patients with acute hepatitis B and in 22.8% (nine of 35) of patients with chronic hepatitis B. However, the nt 1898 mutation was not found in any of these sera. The precore/core mutant was observed with increasing frequency from acute hepatitis to chronic hepatitis, non-tumour and HCC, and this difference in frequency was significant between HCC and acute hepatitis B groups (P < 0.01), suggesting that the precore/core mutant or hepatocytes harbouring this mutant may be under immune selection and that such mutations may facilitate integration and subsequent tumour development. [source] Human herpesvirus 6 infection in adult living related liver transplant recipientsLIVER TRANSPLANTATION, Issue 1 2008Masahiro Ohashi To analyze human herpesvirus 6 (HHV-6) infection in adult living related liver transplantation, we performed a virological analysis, including viral isolation, serological assay, and real-time polymerase chain reaction, of serially collected blood samples from 67 recipients. In addition, cytokine levels were measured to determine their role in viral reactivation. HHV-6 was isolated from only 4 recipients (6.0%), and viral DNA was detected in 15 (22.4%) of the 67 recipients. A significant increase in HHV-6 immunoglobulin G antibody titers was observed in 19 (28.4%) of the 67 recipients. Finally, 26 recipients (38.8%) had HHV-6 reactivation 2-6 weeks after transplantation. HHV-6 associated clinical features were analyzed in the 17 recipients presenting with either viremia or DNAemia. Two recipients with viremia and 3 recipients with DNAemia had unexplained fever at the time of viral infection. An increase in aminotransferase levels was observed in 2 recipients with viremia and 3 recipients with DNAemia. Recipients with liver cirrhosis caused by hepatitis B virus or hepatitis C virus infection as the underlying disease were more likely to have HHV-6 infection (P = 0.025). Mortality at the last follow-up in recipients with HHV-6 reactivation was significantly higher than in those without viral reactivation (P = 0.0118). Plasma interleukin-6 levels were significantly higher in the recipients with HHV-6 viremia than in the recipients without viremia at 4 weeks post-transplant (P = 0.0411). Moreover, tumor necrosis factor , levels were also higher in recipients with HHV-6 viremia (P < 0.0001) or reactivation (P = 0.0011) than in recipients without viremia or reactivation 4 weeks post-transplant. Liver Transpl, 2007. © 2007 AASLD. [source] Nasal CpG oligodeoxynucleotide administration induces a local inflammatory response in nonallergic individualsALLERGY, Issue 9 2009A. Månsson Background:, We have previously demonstrated the presence of toll-like receptor 9 in the nasal mucosa of both healthy and allergic individuals. CpG motifs, found in bacterial and viral DNA, elicit strong immunostimulatory effects via this receptor. CpG is known to skew the immune system towards a T helper 1 (Th1) profile, thereby suppressing Th2-driven allergic responses. This study was designed to examine the effects of CpG administration in the human nose. Methods:, Twenty subjects, of whom 10 suffered from seasonal allergic rhinitis (AR), were challenged intranasally with CpG outside pollen season. Symptom scores, nasal airway resistance (NAR), and nasal and pulmonary nitric oxide (NO) levels were assayed prior to challenge and 30 min, 6, 24 and 48 h post challenge. The presence of leukocytes and various cytokines were analyzed in nasal lavage (NAL) fluids before and after CpG exposure. Results:, Increased NAR, nasal NO production and secretion of interleukin (IL)-1,, IL-6, and IL-8 were seen after CpG exposure. Further analysis revealed that this inflammatory response was more marked in healthy subjects than among patients with AR, although a higher basal inflammatory response was recorded in the allergic group. In vitro experiments suggest that the effects induced by CpG are mediated by epithelial cells and neutrophils. Conclusion:, Nasal administration of CpG induces a local airway inflammation, more distinct among healthy than allergic individuals. The reduced responsiveness to CpG in allergic patients might be related to the ongoing minimal persistent inflammation. Results from cytokine analyses reflect the ability of CpG to induce a pro-inflammatory Th1-like immune response. [source] Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cellsTHE JOURNAL OF GENE MEDICINE, Issue 3 2009Min Liang Abstract Background Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed. Methods In the present study, we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors, based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction, we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector, developed in our laboratory, that allows targeted transduction to specific cell receptors via antibody recognition. Results Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. Conclusions Overall, the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. Copyright © 2009 John Wiley & Sons, Ltd. [source] N -acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entryTHE JOURNAL OF GENE MEDICINE, Issue 1 2002L. Jornot Abstract Background It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N -acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. Methods HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of ,-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24,h and 48,h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Results Pretreatment of cells with NAC prior to Ad infection enhanced ,-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in ,-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, ,-Gal activity was further enhanced, by 15-fold. Augmentation of ,-Gal activity was paralleled by an increase in ,-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Conclusion Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright © 2001 John Wiley & Sons, Ltd. [source] Postrenal Transplant Hemophagocytic Lymphohistiocytosis and Thrombotic Microangiopathy Associated with Parvovirus B19 InfectionAMERICAN JOURNAL OF TRANSPLANTATION, Issue 6 2008M. R. Ardalan Persistent anemia is a known consequence of Parvovirus B19 (B19) infection following renal transplantation. However, to date, no description of B19-related hemophagocytic lymphohistiocytosis (HLH) exists in renal transplant recipients. We report a 24-year-old male kidney recipient, who presented with fever, severe anemia and allograft dysfunction two years following transplantation. Hyperferritinemia, hypertriglyceridemia, elevated serum lactate dehydrogenase, pancytopenia and fragmented red blood cells on the peripheral blood were also noted. Bone marrow examination revealed giant pronormoblasts and frequent histiocytes with intracellular hematopoietic elements, consistent with HLH. Renal allograft biopsy revealed closure of the lumen of glomerular capillaries and thickening of the capillary walls compatible with thrombotic microangiopathy. The presence of anti-B19 IgM antibody and viral DNA in the patient's serum (detected by real-time PCR) confirmed an acute B19 infection. Following high-dose intravenous immunoglobulin therapy, the anemia gradually resolved and renal function improved. As far as we know, this is the first report of B19-associated HLH and thrombotic microangiopathy in a renal transplant recipient. [source] Detection of human herpesvirus-6 in cerebrospinal fluid of patients with encephalitis,ANNALS OF NEUROLOGY, Issue 3 2009Karen Yao MS Objective Virus infections are the most common causes of encephalitis, a syndrome characterized by acute inflammation of the brain. More than 150 different viruses have been implicated in the pathogenesis of encephalitis; however, because of limitations with diagnostic testing, causative factors of more than half of the cases remain unknown. Methods To investigate whether human herpesvirus-6 (HHV-6) is a causative agent of encephalitis, we examined for evidence of virus infection by determining the presence of viral sequence using polymerase chain reaction and assessed HHV-6 antibody reactivity in the cerebrospinal fluid of encephalitis patients with unknown cause. In a cohort study, we compared virus-specific antibody levels in cerebrospinal fluid samples of patients with encephalitis, relapsing-remitting multiple sclerosis, and other neurological diseases. Results Our results demonstrated increased levels of HHV-6 IgG, as well as IgM levels, in a subset of encephalitis patients compared with other neurological diseases. Moreover, cell-free viral DNA that is indicative of active infection was detected in 40% (14/35) of encephalitis patients, whereas no amplifiable viral sequence was found in either relapsing-remitting MS or other neurological diseases patients. In addition, a significant correlation between polymerase chain reaction detection and anti-HHV-6 antibody response was also demonstrated. Interpretation Collectively, these results suggested HHV-6 as a possible pathogen in a subset of encephalitis cases. Ann Neurol 2009;65:257,267 [source] Detection of JC virus DNA fragments but not proteins in normal brain tissueANNALS OF NEUROLOGY, Issue 4 2008Georgina Perez-Liz MD Objective Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the white matter affecting immunocompromised patients that results from the cytolytic destruction of glial cells by the human neurotropic JC virus (JCV). According to one model, during the course of immunosuppression, JCV departs from its latent state in the kidney and after entering the brain, productively infects and destroys oligodendrocytes. The goal of this study was to test the hypothesis that JCV may reside in a latent state in a specific region of the brains of immunocompetent (non-PML) individuals without any neurological conditions. Methods Gene amplification was performed together with immunohistochemistry to examine the presence of JCV DNA sequences and expression of its genome in five distinct regions of the brain from seven immunocompetent non-PML individuals. Results Although no viral proteins were expressed in any of these cases, fragments of the viral DNA were present in various regions of normal brain. Laser-capture microdissection showed the presence of JCV DNA in oligodendrocytes and astrocytes, but not in neurons. Interpretation The detection of fragments of viral DNA in non-PML brain suggests that JCV has full access to all regions of the brain in immunocompetent individuals. Thus, should the immune system become impaired, the passing and/or the resident virus may gain the opportunity to express its genome and initiate its lytic cycle in oligodendrocytes. The brain as a site of JCV latency is a possibility. Ann Neurol 2008; 64:379,387 [source] Methods for HPV detection in exfoliated cell and tissue specimensAPMIS, Issue 6-7 2010PETER J.F. SNIJDERS Snijders PJF, Heideman DAM, Meijer CJLM. Methods for HPV detection in exfoliated cell and tissue specimens. APMIS 2010; 118: 520,528. Given the causal involvement of high-risk human papillomaviruses (HPVs) in cervical cancer and a subset of squamous cell carcinomas of other anogenital regions as well as the oropharynx, much attention has been focused on the development and application of HPV detection assays. HPV detection assays are almost exclusively based on the detection of viral nucleic acids, mostly viral DNA. The HPV detection methods that are nowadays in use can broadly be subdivided into target amplification methods and signal amplification methods. In this review, several principles of various methodologies are explained and examples of some commonly used HPV detection assays are given. In addition, attention is paid to the use of HPV assays for detecting clinically meaningful HPV infections, i.e. infections related to (pre)cancerous lesions, e.g. cervical cancer screening purposes. For the latter, it is important that HPV tests are clinically validated according to validation strategies as outlined in guidelines. [source] Viral reactivation is not related to septic complications after major surgical resections,APMIS, Issue 4 2008T. VOGEL Anastomotic leakage and septic complications are the most important determinants of postoperative outcome after major surgical resections. Malignant diseases and surgical trauma can influence immune responses and the ability to react against infectious factors, such as bacteria and viruses. Comparable immune suppression can cause viral reactivation in transplantation and trauma patients. In this prospective study, patients who underwent major surgical resections for oesophageal or pancreatic cancer were investigated for the potential involvement of viral reactivation in the development of septic complications. 86 patients (40 oesophageal resections, 27 pancreatic resections, 19 surgical explorations) were included. Viral antigens, viral DNA, antibodies against viral structures (IgG, IgM, IgA) and, in part, viral cultivation were performed for CMV, EBV, HSV1, HSV2, HZV6 and VZV in serum, urine, sputum and swabs from buccal mucosa preoperatively and at postoperative days 1, 3 and 5. Test results were compared with the postoperative outcome (30-day morbidity, in-hospital mortality) and clinical scores (SOFA, TISS). For statistical analyses Student's t -tests and Chi2 -tests were used. The overall complication rate was 19.8% (30-day morbidity) with an in-hospital mortality of 1.2% (1/86 patients). Postoperatively, anti-CMV-IgG titres were significantly reduced (p<0.05) and remained suppressed in patients with septic complications. Anti-CMV-gB-IgG were also reduced, but showed considerable interindividual differences. Anti-CMV-IgA and -IgM did not show significant alterations in the postoperative course. In addition, direct viral detection methods did not support viral reactivation in patients in any of the investigated groups. The reduction of anti-CMV antibodies is likely caused by an immune suppression, specifically by reduced B-cell counts after major surgical interventions. Viral reactivation, however, did not occur in the early postoperative period as a specific risk for septic complications. [source] Generalized Wegener's granulomatosis in an immunocompetent adult after cytomegalovirus mononucleosis and bacterial urinary tract infectionARTHRITIS & RHEUMATISM, Issue 5 2009Stefania Varani Human cytomegalovirus (HCMV) is frequently detected in autoimmune diseases, but its role in such disorders is poorly understood. Herein we describe the case of a young woman who developed generalized Wegener's granulomatosis (WG) after HCMV mononucleosis and urinary tract infection. During mononucleosis, the patient had extraordinarily high plasma levels of proinflammatory cytokines such as interleukin-5 and lymphotoxin ,, autoantibodies, and a higher blood level of viral DNA than were found in other immunocompetent patients infected with HCMV or healthy controls. Active HCMV replication was detected after the onset of vasculitis, and HCMV genomes or antigens were found in blood, urine, and inflammatory lesions on the kidney. Thus, HCMV may have triggered or exacerbated inflammation and autoimmunity in this case of WG. [source] Clinical course of kidney transplant patients with acute rejection and BK virus replication following Campath therapyCLINICAL TRANSPLANTATION, Issue 3 2008Liise K. Kayler Abstract:, Background:, Kidney transplant recipients with active BK virus (BKV) replication are generally treated with reduction in immunosuppression to allow a successful immune response against the virus. Methods:, We inadvertently administered Campath to two patients with BKV viruria, and one patient with BKV nephropathy, since allograft biopsies showed severe tubulitis or intimal arteritis, and results of PCR and in situ hybridization were not available at the time of therapeutic intervention. Results:, Increased viral replication was observed, but not uniformly in all cases, and follow-up biopsies showed nephropathy in one additional case. Extra-renal dissemination did not occur. With subsequent reduction of immunosuppression or antiviral therapy, it was still possible to obtain clearance of viremia in all cases. Serum creatinine fell transiently after Campath in one patient; however, at one yr post-treatment all had increased levels over baseline. One graft was lost to persistent acute rejection that led to interstitial fibrosis and tubular atrophy. Conclusion:, These cases suggest that Campath treatment does not (i) irreversibly deplete cells believed to be important in mounting an immune response against BKV, or (ii) preclude subsequent eradication of viral DNA from the blood. [source] | ||||||||||||||||