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Viral RNA (viral + rna)
Selected AbstractsLateral transmission of equine arteritis virus among Lipizzaner stallions in South AfricaEQUINE VETERINARY JOURNAL, Issue 6 2003A. J. GUTHRIE Summary Reasons for performing study:A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. Objectives: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. Methods: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF5-specific primers, subjected to sequence and phylogenetic analysis. Results: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stal lions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from be dding or fo mites contaminated with semen. Conclusions: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. Potential relevance: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV-infected semen. [source] Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplificationJOURNAL OF FISH DISEASES, Issue 4 2006P J Millard Abstract A new method for the molecular detection of the fish pathogens, infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV), is described. By employing molecular padlock probe (MPP) technology combined with rolling circle amplification (RCA) and hyperbranching (Hbr), it is possible to detect RNA target sequence from these viruses at levels comparable with those detected by the polymerase chain reaction (PCR), but without prior reverse transcription. The use of MPP technology combined with RCA and Hbr for the detection of IHNV and ISAV in fish exhibited selectivity comparable with that of PCR while potentially reducing the time and cost required for analysis. The method described was used to detect as few as 104 DNA oligonucleotide targets and was sequence-specific at the single base level. Viral RNA could be detected directly, either alone or in the presence of non-viral RNA from fish tissue. This technology is applicable for detecting a variety of microbes, in addition to IHNV and ISAV, and is ideal for further integration into a biosensor platform for on-site diagnosis of pathogen infection in fish. [source] Characterization of hepatitis A virus isolates from subgenotypes IA and IB in Rio de Janeiro, Brazil,JOURNAL OF MEDICAL VIROLOGY, Issue 1 2002Vanessa S. de Paula Abstract Hepatitis A virus (HAV) isolates from around the world have been classified into seven genotypes (I,VII). Most human strains belong to genotype I, which has been divided into two subgenotypes, A and B. South America has provided a small number of strains studied at the genome level. In the present study, IgM anti-HAV antibodies were detected in 116 out of 250 (46%) serum samples collected from consecutive patients with acute hepatitis referred to the Brazilian Reference Center for Viral Hepatitis, Rio de Janeiro. Viral RNA were extracted from all 250 samples and submitted to a reverse transcription-polymerase chain reaction (RT-PCR) assay designed to amplify a genome segment in the VP1/2A junction region. HAV RNA was detected in 54/116 (47%) and 17/134 (13%) IgM anti-HAV-positive and -negative sera, respectively. In addition, HAV RNA was detected in 17/35 (49%) IgM anti-HAV-positive sera that had been collected at a day care center where cases of acute hepatitis were being observed for 3 months. Nucleotide sequences (168 bp) of PCR products were determined for 30 HAV isolates. Phylogenetic analysis showed that 21 belonged to subgenotype IB, while 9 were of subgenotype IA. Interestingly, a concomitant circulation of isolates from subgenotypes IA and IB was observed in the day care center. J. Med. Virol. 66:22,27, 2002. © 2002 Wiley-Liss, Inc. [source] Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in FinlandJOURNAL OF MEDICAL VIROLOGY, Issue 1 2001Xiuqi Han Abstract Approximately 20 cases of tick-borne encephalitis (TBE) occur annually in Finland. The known endemic areas are situated mainly in the archipelago and coastal regions of Finland, with highest incidence in Åland islands. Ixodes ricinus panels collected in 1996,1997 from two endemic areas were screened for the presence of RNA. Two distinct RT-PCR methods were applied, and were shown to have an approximate detection limit of 10 focus forming doses (FFD)/100 ,l. One out of 20 pools (a total of 139 ticks) from Helsinki Isosaari Island and one out of 48 pools (a total of 450 ticks) from Åland were positive with both methods, whereas the remaining pools were negative. The observed overall frequency (0.34%) in ticks in endemic areas of Finland, was similar to the low incidence found by virus isolation in mice in the 1960s (0.5%). Viral RNA was detectable in a diluted sample representing 0.005% of a positive pool of ten nymphs suggesting that the viral RNA load within an infected tick pool was approximately equivalent to 20,000,200,000 FFD. Sequence analysis did not show geographical clustering of the Finnish strains, suggesting an independent emergence of different TBE foci from the south. TBE virus RNA positive ticks were not found in I. ricinus panels consisting of 130 pools (726 ticks) from Helsinki city parks or 41 pools (197 ticks) from Võrmsi Island in Estonia. J. Med. Virol. 64:21,28, 2001. © 2001 Wiley-Liss, Inc. [source] Hepatitis C virus infection among drug injectors in St Petersburg, Russia: social and molecular epidemiology of an endemic infectionADDICTION, Issue 11 2009Elijah Paintsil ABSTRACT Aims To understand the epidemiology and transmission patterns of hepatitis C virus (HCV), the predominant blood borne-pathogen infecting injection drug users (IDUs), in a part of the former Soviet Union. Design Cross-sectional respondent-driven sample of IDUs. Setting St Petersburg, Russia. Participants A total of 387 IDUs were recruited in late 2005 and throughout 2006. Measurements Participants were surveyed to collect demographic, medical and both general and dyad-specific drug injection and sexual behaviors. A blood sample was collected to detect antibodies to hepatitis C and to amplify viral RNA for molecular analysis. The molecular data, including genotypes, were analyzed spatially and linkage patterns were compared to the social linkages obtained by respondent-driven sampling (RDS) for chains of respondents and among the injection dyads. Findings HCV infection was all but ubiquitous: 94.6% of IDUs were HCV-seropositive. Among the 209 viral sequences amplified, genotype 3a predominated (n = 119, 56.9%), followed by 1b (n = 61, 29.2%) and 1a (n = 25, 11.9%). There was no significant clustering of genotypes spatially. Neither genotypes nor closely related sequences were clustered within RDS chains. Analysis of HCV sequences from dyads failed to find associations of genotype or sequence homology within pairs. Conclusions Genotyping reveals that there have been at least five unique introductions of HCV genotypes into the IDU community in St Petersburg. Analysis of prevalent infections does not appear to correlate with the social networks of IDUs, suggesting that simple approaches to link these networks to prevalent infections, rather than incident transmission, will not prove meaningful. On a more positive note, the majority of IDUs are infected with 3a genotype that is associated with sustained virological response to antiviral therapy. [source] Quantitation of reduced glutathione and cysteine in human immunodeficiency virus-infected patientsELECTROPHORESIS, Issue 10-11 2004Elena Sbrana Abstract Plasma viral load (VL) values and CD4+ cell count are employed clinically for initiation of therapy in the treatment of patients infected with human immunodeficiency virus (HIV), as previous clinical studies have shown a marked prevalence of acquired immunodeficiency sydrome (AIDS) development in seropositive individuals with VL values over 30,000 copies/mL. Many studies have shown that reduced glutathione (GSH) and cysteine (Cys) deficiency play an important role in the infection. We have developed capillary zone electrophoresis (CZE)-based assays and have used them to investigate the relationship between plasma and intracellular thiol levels and HIV-1 viremia in plasma. Blood samples from healthy volunteers and seropositive patients undergoing different antiretroviral regimes were analyzed in the study. The VL assay was based on CZE-UV detection of viral RNA at 260 nm. Determination of endogenous reduced Cys and GSH was achieved by CZE-UV detection of their mercurial complexes at 200 nm. We found that a decrease in GSH and Cys levels may be associated with disease progress. In fact, reduced GSH and Cys levels appear progressively reduced with increasing VL. [source] Unexpected roles for DEAD-box protein 3 in viral RNA sensing pathwaysEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2010Orla Mulhern Abstract Detection of viral nucleic acid within infected cells is essential to an effective anti-viral response. The retinoic acid-inducible gene-I-like receptors (RLR) form part of the virus detection repertoire and are critically important in sensing viral RNA in the cytoplasm. Efforts continue to define the signalling components downstream of RLR that are required to induce type I IFN (IFN-, and promoter stimulator-1) after viral infection. One surprising finding was that the Asp-Glu-Ala-Asp box helicase DEAD/H Box 3 (DDX3), known for some time to have a number of roles in cellular RNA regulation in the nucleus, has a role in the RLR cytoplasmic signalling pathway involved in promoter stimulator-1 induction. In this issue of the European Journal of Immunology, an article reports an additional distinct positive role for DDX3 in the RLR RNA sensing pathway. This further emphasises the importance of DDX3 in anti-viral immunity, and is consistent with the idea that viruses target DDX3 for immune evasion. [source] A new and efficient method for inhibition of RNA viruses by DNA interferenceFEBS JOURNAL, Issue 16 2009Monika Nowak We report here a new method for inhibition of RNA viruses induced by dsDNA. We demonstrated that both long dsDNA molecules and short interfering DNA with a sequence complementary to that of viral RNA inhibited tobacco mosaic virus expression and prevented virus spread. Also, the expression of the HIV-1 gp41 gene in HeLa cells was inhibited by complementary short interfering DNA. We showed that Dicer processed dsDNA, which suggests activation of the cellular machinery involved in silencing of RNA. For the silencing of viral RNA effected with dsDNA, we coined the term DNA interference technology. [source] Structure of the HIV-1 Rev response element alone and in complex with regulator of virion (Rev) studied by atomic force microscopyFEBS JOURNAL, Issue 15 2009Jesper Pallesen The interaction of multiple HIV-1 regulator of virion (Rev) proteins with the viral RNA target, the Rev response element (RRE), is critical for nuclear export of incompletely spliced and unspliced viral RNA, and for the onset of the late phase in the viral replication cycle. The heterogeneity of the Rev,RRE complex has made it difficult to study using conventional structural methods. In the present study, atomic force microscopy is applied to directly visualize the tertiary structure of the RRE RNA alone and in complex with Rev proteins. The appearance of the RRE is compatible with the earlier proposed RRE secondary structure in dimensions and overall shape, including a stalk and a head interpreted as stem I, and stem-loops II,V in the secondary structure model, respectively. Atomic force microscopy imaging of the Rev,RRE complex revealed an increased height of the structure both in the stalk and head regions, which is in accordance with a binding model in which Rev binding to a high affinity site in stem IIB triggers oligomerization of Rev proteins through cooperative binding along stem I in RRE. The present study demonstrates that atomic force microscopy comprises a useful technique to study complex biological structures of nucleic acids at high resolution. [source] HCV RNA-dependent RNA polymerase replicates in vitro the 3, terminal region of the minus-strand viral RNA more efficiently than the 3, terminal region of the plus RNAFEBS JOURNAL, Issue 22 2001Sandrine Reigadas The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3, end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3, UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3, end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3, end of the minus-strand RNA: (a) the presence of a C residue as the 3, terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3, end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B. [source] HIV-1 proviral resistance mutations: usefulness in clinical practiceHIV MEDICINE, Issue 8 2010B Kabamba-Mukadi Objectives Transmitted HIV strains may harbour drug resistance mutations. HIV-1 drug resistance mutations are currently detected in plasma viral RNA. HIV-1 proviral DNA could be an alternative marker, as it persists in infected cells. Methods This was a prospective study assessing the prevalence and persistence of HIV-1 drug resistance mutations in DNA from CD4 cells before and after protease inhibitor (PI)- or nonnucleoside reverse transcriptase inhibitor (NNRTI)-based therapy initiation in 69 drug-naïve patients. Results Before therapy, 90 and 66% of detected mutations were present in CD4 cells and plasma, respectively. We detected seven key mutations, and four of these (M184M/V, M184M/I, K103K/N and M46M/I) were only found in the cells. When treatment was started, 40 patients were followed; the mutations detected at the naïve stage remained present for at least 1 year. Under successful treatment, new key mutations emerged in CD4 cells (M184I, M184M/I and Y188Y/H). Conclusions The proportion of mutations detected in the DNA was statistically significantly higher than that detected in standard RNA genotyping, and these mutations persisted for at least 1 year irrespective of therapy. The pre-existence of resistance mutations did not jeopardise treatment outcome when the drug concerned was not included in the regimen. Analysis of HIV-1 DNA could be useful in chronic infections or when switching therapy in patients with undetectable viraemia. [source] Seroprevalence of hepatitis C in patients with type 2 diabetes mellitus and non-diabetic on haemodialysisINTERNATIONAL JOURNAL OF CLINICAL PRACTICE, Issue 6 2006S. Ocak Summary Type 2 diabetes mellitus (DM) has emerged as the commonest cause of end-stage renal disease. Haemodialysis (HD) treatment constitutes a high-risk environment for the transmission of hepatitis C virus (HCV). The aim of this study was to establish a potential relationship between type 2 DM and HCV infection in HD patients. Of the 267 HD patients, 67 (25.1%) had type 2 DM and 200 (74.9%) were with diverse aetiology for end-stage renal disease. The serum markers of HCV infection were tested by a second-generation enzyme-linked immunosorbent assay test for antibodies and by qualitative reverse-transcription polymerase chain reaction technique for viral RNA. The overall prevalence of anti-HCV antibodies and HCV RNA was found to be 12.7% (34/267) and 10.1% (27/267), respectively. Patients with type 2 DM were found to have a higher HCV prevalence compared with non-diabetic patients [20.8% (14/67) vs. 10% (20/200)] (p < 0.05). The mean period on dialysis of anti-HCV-positive patients with type 2 DM was shorter than that observed for anti-HCV-positive non-diabetic patients (43.9 ± 9.8 months vs. 59.7 ± 28.4 months) (p < 0.05). This study has shown that although the period on dialysis of diabetic patients are shorter than non-diabetic patients, the prevalence of HCV in HD patients with type 2 DM is higher than that detected in non-diabetic HD patients. [source] Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virusJOURNAL OF FISH DISEASES, Issue 2 2002-Maganja, D Barli Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase,polymerase chain reaction (RT,PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G-gene sequences were used for IHNV. For the detection of IPNV the VP2-coding region was selected for RT,PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT,PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT,PCR, a semi-nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT,PCR. Because of the possibility of template carry-over contamination, a closed one step RT,PCR method was tested. This technically simplified approach was then combined with the PCR,enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR,ELISA method for the detection of RT,PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work. [source] Development of RNA interference (RNAi) as potential antiviral strategy against enterovirus 70JOURNAL OF MEDICAL VIROLOGY, Issue 6 2008Eng Lee Tan Abstract Enterovirus 70 (EV70) is recognized as the main causative agent of acute hemorrhagic conjunctivitis (AHC), a highly contagious viral infection of the eye. Currently, there is no available treatment for EV70 infections. In this study, we developed a potential intervention strategy using RNA interference (RNAi) against EV70 infection in an in vitro system. Two synthetic 19-mer siRNAs, si-3D1 and si-3D2, were designed to target the 3Dpol region of the EV70 genome. Significant dosage dependent inhibition of EV70 in rhabdomyosarcoma cell line, as shown by reduction of viral RNA and VP1 production, was observed. Both siRNAs prevented EV70 replication in RD cells when transfected into these cells 48 hr prior to virus infection. Introduction of these siRNAs into RD cells 1,3 hr after infection with EV70 reduced production of viral RNA by approximately 60%. Thus, RNAi is a promising strategy to prevent EV70 infections and may have therapeutic potential. J. Med. Virol. 80:1025,1032, 2008. © 2008 Wiley-Liss, Inc. [source] Human parechovirus 1 infections in young children,no association with type 1 diabetesJOURNAL OF MEDICAL VIROLOGY, Issue 4 2007Sisko Tauriainen Abstract The epidemiology, transmission and clinical symptoms of human parechoviruses [HPeV, classified earlier as enteroviruses; echovirus 22 (HPeV1) and echovirus 23 (HPeV2)] remain poorly characterized. Enteroviruses and one parechovirus species, the Ljungan virus, have been associated with type 1 diabetes in humans and rodents. The occurrence of human parechovirus 1 (HPeV1) infections in young children and their possible association with type 1 diabetes was evaluated. The prospective birth cohort study comprised 221 Finnish children carrying genetic type 1 diabetes susceptibility and who were observed from birth. Thirty-four children developed multiple diabetes-associated autoantibodies, and 18 children progressed to clinical type 1 diabetes during the follow-up. HPeV1 infections were diagnosed by measuring neutralizing antibodies from the follow-up sera taken every 3,12 months. In addition, viral RNA was analysed by RT-PCR from stool samples taken every month from six of the participants. HPeV1 infections were found to occur early in childhood. The median age of infection was 18 months and 20% of the children had had an infection by the age of 1 year. The number of infections started to increase from the age of 6 months and most children had their first infection by 36 months. Nearly all (99%) mothers were HPeV1 antibody positive. No difference was found in infection frequency between boys and girls, nor between prediabetic, diabetic and control subjects. Most infections (87%) occurred during autumn, winter and spring. J. Med. Virol. 79:457,462, 2007. © 2007 Wiley-Liss, Inc. [source] Lack of susceptibility of Chacma baboons (Papio ursinus orientalis) to hepatitis C virus infectionJOURNAL OF MEDICAL VIROLOGY, Issue 4 2002N.P. Sithebe Abstract The main reason to ascertain whether baboons are susceptible to infection with hepatitis C virus (HCV) is the need to replace chimpanzees, which are endangered, as an animal model for undertaking research into the biology and host,virus interactions of HCV, and for developing a vaccine against this virus. A second reason is that baboons are a possible source of xenografts for human liver transplantation. We inoculated serum containing HCV into four Chacma baboons and monitored them for 52 weeks for evidence of infection. Serum was tested for antibody to HCV, HCV RNA, and aminotransferase concentrations at 2-week intervals for 26 weeks and thereafter at 4-week intervals. Liver tissue was examined at 28 and 52 weeks for histopathological changes and viral RNA, and at 52 weeks for viral particles using electron microscopy. Reverse transcription-polymerase chain reaction assay was used to detect HCV RNA, and the results were confirmed by Southern hybridization. Serum aminotransferase concentrations remained within the normal range and liver histology was normal during the follow-up period. Passive transmission of anti-HCV to the baboons was observed during the first 4 weeks. HCV RNA was not detectable in any serum or liver sample and electron microscopy failed to reveal viral particles in liver tissue. In conclusion, we did not find Chacma baboons to be susceptible to infection with HCV, although we cannot deny that in an immunosuppressed liver transplant recipient, infection of a baboon xenograft might occur. Another animal model for HCV infection must be sought. J. Med. Virol. 66:468,471, 2002. © 2002 Wiley-Liss, Inc. [source] Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritisJOURNAL OF MEDICAL VIROLOGY, Issue 3 2002D.S. Subekti Abstract Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis. J. Med. Virol. 66:400-406, 2002. © 2002 Wiley-Liss, Inc. [source] Suppression of proinflammatory cytokines and induction of IL-10 in human monocytes after coxsackievirus B3 infectionJOURNAL OF MEDICAL VIROLOGY, Issue 4 2001P. Hofmann Abstract Coxsackievirus B3 (CVB3) causes acute and chronic myocarditis, which is accompanied by an intense mononuclear leukocyte infiltration. Because myocardial tissue damage may either result from viral infections or from a dysregulated immune response, the susceptibility of human monocytes and macrophages to CVB3 was examined in this study with regard to virus replication, virus persistence, and release of cytokines. Monocytes were infected by CVB3 as shown by the intracellular appearance of plus- and minus-strand viral RNA, which was also capable of persisting for more than 10 days. Fresh monocytes were not permissive for full virus replication whereas monocyte-derived macrophages yielded a low amount of new viruses, which led to cell death. Although CVB3 infection induced the mRNA for the proinflammatory cytokines tumor necrosis factor-alpha (TNF-,), interleukin (IL)-1, and IL-6, only little cytokine production occurred. When infected monocytes were stimulated in addition by lipopolysaccharides (LPS), cytokine production was partially suppressed. In striking contrast, IL-10 expression was strongly and persistently induced by CVB3 on the mRNA and the protein level. These data show a dysregulated cytokine response in CVB3-exposed human monocytes and macrophages, which is characterized by a suppression of proinflammatory cytokines and a dominance of IL-10. This viral strategy may aid CVB3, causing chronic myocardiopathy. J. Med. Virol. 64:487,498, 2001. © 2001 Wiley-Liss, Inc. [source] Oral candidiasis as a clinical marker related to viral load, CD4 lymphocyte count and CD4 lymphocyte percentage in HIV-infected patientsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 1 2002J. Campo Abstract Background:, High viral load is currently considered to be one of the main indicators of the progression of HIV-induced immunodepression, but few studies have analysed its relationship to the presence of oral candidiasis (OC). The aim of this cross-sectional study is to analyse the relationship between viral load, total CD4 lymphocyte count, and percentage of CD4 lymphocytes to the occurrence of OC. Methods:, The present cross-sectional study included 156 HIV-infected patients seen at a clinic for sexually transmitted diseases and HIV. We assesed the presence or absence of OC, and microbiological samples were obtained from the palatine mucosa and dorsal tongue for a smear stained with KOH (potassium hydroxide) and culture on Sabouraud's dextrose agar in all patients. Viral load was determined by quantification of viral RNA in peripheral blood with a minimum detectable level of 500 RNA copies/ml. CD4+ counts/CD4+ percentage were categorized as <200/<14%, 200,499/14,28%, and >500/>29%, and HIV viral loads were categorized as <500, 500,10,000, >10,000 copies/ml. Results:, Thirty-eight percent (37.8%) of the patients had OC. Patients with CD4+ lymphocyte counts below 200×106/l and CD4+ percentages below 14% showed a significantly higher frequency of OC (57.9% and 48.0%, respectively). Patients with a viral load over 10,000 copies/ml also had OC more frequently (44.8%). In the multiple logistic regression analysis, OC showed a statistically significant association with high viral load [>10,000 vs <500, odds ratio (OR)=11.4], low percentage of CD4+ lymphocytes (<14% vs >28%, OR=5), and injection drug use (IDU vs heterosexual transmission, OR=10.2). In HIV-infected patients, high viral load was associated with more frequent OC, regardless of CD4+ lymphocyte level. Conclusions:, These findings suggest that oral candidiasis could be a useful clinical marker of patients with high viral load. In view of these results, emphasis should be placed on the importance of systematic examination of the oral cavity in all medical follow-up examinations of HIV-infected patients. [source] Optimization of RT,PCR for the Detection of Bean leaf roll virus in Plant Hosts and Insect VectorsJOURNAL OF PHYTOPATHOLOGY, Issue 2 2005V. Ortiz Abstract The detection of luteoviruses by reverse transcription polymerase chain reaction (RT,PCR) depends on the adequate quality and quantity of extracted viral nucleic acids. We have optimized the detection of Bean leaf roll virus (BLRV) using selective precipitation by LiCl of viral RNA from a small quantity of infected plant tissues and insect vectors. The optimal template for PCR was 15 ,l of RT reaction mixture. BLRV was detected in different plant hosts and aphid vectors and Aphis fabae, previously considered to be a non-vector of BLRV, was found to acquire the virus from infected plants. [source] Presence of HCV-RNA after ultracentrifugation of serum samples during the follow-up of chronic hepatitis C patients with a sustained virological response may predict reactivation of hepatitis C virus infectionALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 5 2009I. CASTILLO Summary Background, Concentration of viral particles by ultracentrifugation of serum prior to PCR allows detection of hepatitis C virus (HCV) RNA in patients with undetectable viral RNA by conventional PCR assays. Aim, To analyse if HCV-RNA is detected after serum ultracentrifugation in chronic hepatitis C patients with a sustained virological response to antiviral therapy (defined as serum HCV-RNA negativity by conventional assays 6 months after the end of therapy). Methods, HCV-RNA was tested using real-time PCR in ultracentrifuged sera collected during the post-treatment follow-up (mean: 42 ± 27 months) in 57 sustained virological responders (SVR). Results, After serum ultracentrifugation, HCV-RNA was detected on at least one occasion during the follow-up in 29/57 (51%) SVR. Thirteen (23%) of these 57 SVR suffered a reactivation 18 ± 8 months after the end of therapy (reappearance of serum HCV-RNA detectable by conventional assays). Among reactivated patients, 11/13 (85%) had HCV-RNA in ultracentrifuged serum samples (detectable 10 ± 5 months before reactivation), while HCV-RNA was positive after ultracentrifugation in 18/44 (41%) long-term SVR (P = 0.01). Persistence of detectable HCV-RNA after serum ultracentrifugation was associated with reactivation (P = 0.001). Conclusions, Serum ultracentrifugation prior to PCR allows detection of HCV-RNA in SVR and its persistence may predict late reactivation. [source] Suppression of hepatitis C virus replication by protein kinase C-related kinase 2 inhibitors that block phosphorylation of viral RNA polymeraseJOURNAL OF VIRAL HEPATITIS, Issue 10 2009S.-J. Kim Summary., Hepatitis C virus (HCV) infection is a serious threat to human health worldwide. In spite of the continued search for specific and effective anti-HCV therapies, the rapid emergence of drug-resistance variants has been hampering the development of anti-HCV drugs designed to target viral enzymes. Targeting host factors has therefore emerged as an alternative strategy offering the potential to circumvent the ever-present complication of drug resistance. We previously identified protein kinase C-related kinase 2 (PRK2) as a cellular kinase that phosphorylates the HCV RNA-dependent RNA polymerase (RdRp). Here, we report the anti-HCV activity of HA1077, also known as fasudil, and Y27632, which blocks HCV RdRp phosphorylation by suppressing PRK2 activation. Treatment of a Huh7 cell line, stably expressing a genotype 1b HCV subgenomic replicon RNA, with 20 ,m each of HA1077 and Y27632 reduced the HCV RNA level by 55% and 30%, respectively. A combination of the inhibitors with 100 IU/mL interferon , (IFN-,) significantly potentiated the anti-HCV drug activities resulting in approximately a 2-log10 viral RNA reduction. We also found that IFN-, does not activate PRK2 as well as its upstream kinase PDK1 in HCV-replicating cells. Furthermore, treatment of HCV-infected cells with 20 ,m each of HA1077 and Y27632 reduced the levels of intracellular viral RNA by 70% and 92%, respectively. Taken together, the results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation. [source] Discovery of the hepatitis C virusLIVER INTERNATIONAL, Issue 2009Michael Houghton Abstract After nearly 6 years of intensive investigations between 1982 and 1988 in my laboratory at Chiron corporation, in which numerous molecular biological methods were used to investigate the viral aetiology of parenterally transmitted non-A, non-B viral hepatitis (NANBH), a single cDNA clone (5-1-1) was isolated that was shown to be derived from a new flavi-like virus, termed the hepatitis C virus (HCV). After screening hundreds of millions of bacterial cDNA clones derived from different liver and plasma samples obtained from experimentally infected chimpanzees, a single HCV clone was eventually isolated using a novel, blind immunoscreening method in which antibodies derived from a clinically diagnosed NANBH patient were used to identify a cDNA clone encoding an immunodominant epitope within HCV nonstructural protein 4. Its viral origin was demonstrated by its specific hybridization to a large single-stranded RNA molecule of ,10 000 nucleotides found only in NANBH-infected samples that shared distant sequence identity with flaviviruses. Further, HCV clone 5-1-1 was shown to be extrachromosomal and to encode an antigen eliciting antibody seroconversion only in NANBH-infected chimpanzees and humans. Subsequent work demonstrated that HCV was the principal cause of parenterally transmitted NANBH around the world, with an estimated 170 million global carriers and that blood screening tests detecting circulating HCV antibodies and viral RNA could effectively eradicate the transmission of transfusion-associated NANBH. Key viral-encoded enzymes essential to its life cycle are now the targets of vigorous, ongoing drug development activities, and the feasibility of successful vaccination strategies has been demonstrated using the valuable chimpanzee model, without which any progress on HCV would not have been possible. My colleagues and coworkers who made essential contributions to the discovery of HCV were George Kuo, who had his own laboratory at Chiron and who provided intellectual and practical input, Dan Bradley of the Centers for Disease Control and Prevention, who provided a large supply of well-characterized chimpanzee samples and knowledge of the NANBH field, and Qui-Lim Choo, in my own laboratory, who provided many years of outstandingly dedicated and precise molecular biology expertise. [source] Single nucleotide insertion in the 5,-untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfectionLIVER INTERNATIONAL, Issue 1 2002Consolato Sergi Abstract: Hepatitis C virus (HCV) infection is an important etiology in patients undergoing orthotopic liver transplantation (OLT) world-wide. Antiviral therapy-related clearance of HCV RNA may occur both in patients with chronic HCV infection and in transplanted patients for HCV-related liver cirrhosis, but the role of the 5,-untranslated region (UTR) of HCV containing the internal ribosome entry site (IRES), which directs the translation of the viral open reading frame has not hitherto been evaluated. We studied the 5,-UTR in an HCV-infected recipient of a liver graft that showed spontaneous clearance of HCV RNA during an acute hepatitis B virus (HBV) superinfection. Sequencing of the 5,-UTR of HCV showed a nucleotide A insertion at position 193 of the IRES. [source] Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2bLIVER TRANSPLANTATION, Issue 4 2010Elizabeth B. Haagsma Hepatitis E virus (HEV) infections are known to run a self-limiting course. Recently, chronic hepatitis E has been described in immunosuppressed patients after solid-organ transplantation. Besides the general recommendation to lower the immunosuppressive medication in these patients, there is currently no specific treatment. We here describe the successful use of pegylated interferon alpha-2b in the treatment of 2 liver transplant recipients who suffered a chronic HEV infection for 9 years (case A) or 9 months (case B). After 4 weeks of therapy, a 2-log decrease (case A) and a 3-log decrease (case B) in the viral load were observed. In case A, who received treatment for 1 year, serum viral RNA became undetectable from week 20 onward, and serum liver enzymes normalized completely. In case B, interferon was discontinued at week 16 because of a lack of a further decline in the viral load. However, 4 weeks after the cessation of therapy, viral RNA was no longer detectable in the serum, and this was probably related to a further decline in the immunosuppressive medication. Liver tests normalized completely. In both cases, no relapse has been noted so far. We conclude that pegylated interferon alpha-2b may be useful in the treatment of chronic HEV infections in patients in whom the reduction of the immunosuppressive medication alone is not sufficient. Liver Transpl , 2010. © 2010 AASLD. [source] Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming unitsMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2009Nina Jonsson ABSTRACT Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID50), methods that are laborious, time-consuming and take on average 3,7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID50/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID50). The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID50 and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID50 or PFU for each enterovirus serotype included was determined. [source] A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem-tip culturesPLANT PATHOLOGY, Issue 1 2000A sensitive method was used to detect bamboo mosaic virus (BaMV) and its associated satellite RNA (satBaMV) by 32P- and digoxigenin (Dig)-labelled probes synthesized from cDNA clones of BaMV genomic (L probe) and satBaMV (S probe) RNA. Both the 32P- and Dig-labelled L and S probes could detect as little as 490 pg of BaMV viral RNA by slot- and dot-blot hybridization. In infected leaf extracts, 32P-labelled L and S probes detected virus at 25-fold higher dilutions than Dig-labelled probes, which were also successfully used to detect BaMV infection in plants derived from meristem-tip culture. However, immunoassays failed to detect BaMV in meristem culture. By dot-blot hybridization assays, 25% of the seedlings were shown to be virus-free. These results suggest that a highly sensitive method for the detection of BaMV infection is required for the establishment of BaMV-free cultures. Meristem-tip culture also provides an efficient method for obtaining virus-free bamboo plants. [source] Two Subgroups of Stapes Fixation: Otosclerosis and Pseudo-Otosclerosis,THE LARYNGOSCOPE, Issue 11 2005Tamás Karosi MD Abstract Hypothesis: Stapes ankylosis is a disease with variable histopathology and can be caused by otosclerosis or pseudo-otosclerosis. Viral pathogenesis of otosclerosis could be established only by correlative analysis: histologic examination of the stapes footplate and reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of the viral RNA. Background: Presence of the RNA genome of measles virus was demonstrated in the footplates of clinically otosclerotic patients by RT-PCR, and also viral proteins were detected by immunohistochemistry. Methods: Nucleic acids were extracted from ankylotic stapes footplates of clinically stapes fixation patients (n = 104). Measles virus genomic nucleoprotein (NP) RNA was amplified by seminested RT-PCR. Amplification results were correlated to postoperative histologic and audiologic findings. Results: Measles virus RNA was detectable only in histologically otosclerotic stapes footplates (n = 67). Histology for virus negative footplates (n = 37) excluded otosclerosis. Virus negative stapes footplates showed nonotosclerotic, degenerative disorders. Conclusions: Stapes ankylosis is a heterogeneous disease causing conductive hearing loss with different etiologies. Nonotosclerotic stapes fixations could be established as pseudo-otosclerosis and may belong to nonspecific, degenerative disorders with variable and noncharacteristic histopathology. Otosclerosis is an inflammatory disease caused by persisting measles virus infection of the otic cap-sule. [source] Retroviral vector production under serum deprivation: The role of lipidsBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009A.F. Rodrigues Abstract The use of retroviral vectors for gene therapy applications demands high titer preparations and stringent quality standards. However, the manufacturing of these vectors still represents a highly challenging task due to the low productivity of the cell lines and reduced stability of the vector infectivity, particularly under serum-free conditions. With the objective of understanding the major limitations of retroviral vector production under serum deprivation, a thorough study of viral production kinetics, vector characterization and cell growth and metabolic behavior was conducted, for 293 FLEX 18 and Te Fly Ga 18 producer cell lines using different serum concentrations. The reduction of serum supplementation in the culture medium resulted in pronounced decreases in cell productivity of infectious vector, up to ninefold in 293 FLEX 18 cells and sevenfold in Te Fly Ga 18 cells. Total particles productivity was maintained, as assessed by measuring viral RNA; therefore, the decrease in infectious vector production could be attributed to higher defective particles output. The absence of the serum lipid fraction was found to be the major cause for this decrease in cell viral productivity. The use of delipidated serum confirmed the requirement of serum lipids, particularly cholesterol, as its supplementation not only allowed the total recovery of viral titers as well as additional production increments in both cell lines when comparing with the standard 10% (v/v) FBS supplementation. This work identified lower production ratios of infectious particles/total particles as the main restraint of retroviral vector production under serum deprivation; this is of the utmost importance concerning the clinical efficacy of the viral preparations. Lipids were confirmed as the key serum component correlated with the production of infective retroviral vectors and this knowledge can be used to efficiently design medium supplementation strategies for serum-free production. Biotechnol. Bioeng. 2009; 104: 1171,1181. © 2009 Wiley Periodicals, Inc. [source] Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirusCELLULAR MICROBIOLOGY, Issue 6 2010Mustafa Ulasli Summary Coronaviruses (CoV) are enveloped positive-strand RNA viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. The origin, the protein composition and the function of these structures are not well established. To shed further light on these structures, we have performed a time-course experiment in which the mouse hepatitis virus (MHV)-induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)-electron microscopy. With our approach we were able to confirm the appearance of 6, previously reported, membranous structures during the course of a complete infection cycle. These structures include the well-characterized double-membrane vesicles (DMVs), convoluted membranes (CMs) and virions but also the more enigmatic large virion-containing vacuoles (LVCVs), tubular bodies (TBs) and cubic membrane structures (CMSs). We have characterized the LVCVs, TBs and CMSs, and found that the CoV-induced structures appear in a strict order. By combining these data with quantitative analyses on viral RNA, protein synthesis and virion release, this study generates an integrated molecular and ultrastructural overview of CoV infection. In particular, it provides insights in the role of each CoV-induced structure and reveals that LVCVs are ERGIC/Golgi compartments that expand to accommodate an increasing production of viral particles. [source] | ||||||||||||||||