Biomedical subjects
Publications and source records attributed to P Biagini.
We demonstrate for the first time that a putative third member of the genus Anellovirus (TTV/TTMV) is present in the blood of healthy persons (20% prevalence), and also in their PBMNC and saliva samples.
A survey of anelloviruses in swine herds from Britanny, France, is reported. By using PCR targeted to the conserved untranslated region, prevalences of 93 and 73 % were found among 15 herds and 33 animals, respectively. The lung was the organ found to be positive most frequently among the five organs tested from 32 animals. The highest identity levels of our nucleotide sequences were found with pig isolates from Japan and with an isolate from Tupaia belangeri. Interestingly, when aligning all available swine isolates from France and Japan, at least two phylogenetic groups were identified, each one containing clones from France and Japan. Some animals carried clones from both groups, demonstrating intra-individual variability. Despite the putative harmlessness of anelloviruses, the potential inoculum carried by pigs must be further evaluated as a sanitary threat.
We report a genomic and morphologic study of the European Eyach (EYA) virus (genus Coltivirus, family Reoviridae) and a comparative analysis with the American Colorado tick fever (CTF) virus (the type species of the genus). The previously established, but distant, antigenic relationship between these viruses was strengthened by genetic findings (presence of cognate genes, amino acid identity between 55 and 88%, similar conserved terminal motifs, suspected read-through phenomenon in segment 9 of both viruses) and by indistinguishable ultramicroscopic morphologies. Moreover, putative constitutive modifying enzyme activities were suspected to be carried out by homologous viral proteins (RNA-dependent RNA polymerase, methyl/guanylyl transferase, NTPase). These findings, together with the comparative analysis to genomes of southeast Asian isolates, support the recent classification of arboviruses with 12 segments of dsRNA within two distinct genera (genus Coltivirus and genus Seadornavirus) and raise interesting questions about the evolutionary origins of coltiviruses. The previously proposed hypothesis that EYA virus was derived from an ancestral virus introduced in Europe with the migration of lagomorphs from North-America, would imply a divergence date between American and European isolates of over 50 million years ago (MYA). This analysis allows for the first time to propose an evolutionary rate for virus dsRNA genomes which was found to be in the order of 10(-8) to 10(-9) mutations/nt/year, a rate similar to that of dsDNA genomes.
The full-length nucleotide sequences of genome segments 1, 5, 7, 8 and 10 from Ndelle virus (NDEV) have been characterized. Comparison of the deduced protein amino acid sequences with those of other member viruses of the family Reoviridae demonstrates that NDEV was originally assigned incorrectly to the genus Orbivirus (aa identity values of <20%). In contrast, high levels of amino acid identity were found with members of the species Mammalian orthoreovirus (MRV); for example, amino acid identity in gamma3(Pol) is between 91 and 97%. These findings, together with previous antigenic analyses, provide evidence that NDEV should be reclassified as a new serotype (designated MRV-4) within the Mammalian orthoreovirus species.
BACKGROUND: The most recent polymerase chain reaction (PCR) detection protocols for the TT virus (TTV) permit one to identify the presence of viral DNA in the serum of a majority of healthy individuals, in the absence of any particular risk factor. This is in contrast with previous epidemiological studies that reported a higher prevalence of TTV infection in populations such as haemodialysis patients (HD), haemophiliacs, intravenous drug users or diabetics. OBJECTIVES: To show that these discrepant results were due to the different sensitivity (number of viral copies detected) of the detection protocols used in initial and more recent epidemiological studies. STUDY DESIGN AND RESULTS: We designed a standardised primary PCR assay that detects only viraemia >5x10(3) to 5x10(4) copies/ml for genotypes 1, 2 and 3, and compared the results of this test with those of a nested PCR assay which is 100-fold more sensitive. Viraemia >5x10(3) to 5x10(4) copies/ml were statistically more frequent in HD patients (54.3%), diabetics (54.7%), and HIV-infected patients with CD4 cells <200/mm(3) (69%) than in blood donors (37%) or HIV-infected patients with CD4 cells >500/mm(3) (33%). CONCLUSIONS: These data suggest a possible relationship between the prevalence of elevated viral loads and the level of immunocompetence of the populations studied, and therefore that of an immune control of TTV viraemia. This corroborates previous findings showing that the stimulation of the immune system by an interferon treatment was able to clear TTV viraemia.
BACKGROUND: the heterogeneity of the TT virus (TTV) DNA prevalence values reported from comparable human cohorts suggests that diagnostic PCR protocols still require to be optimized. OBJECTIVES: to design TTV PCR primer sets with low genotype restriction and to compare their performances with commonly used amplification systems. STUDY DESIGN: we compared full length TTV genomic sequences and identified conserved nucleotide patterns in the 5' and 3' non-coding regions of the viral genome. This permitted to design two new primer sets usable for the PCR amplification of the most divergent human isolates of TTV described to date. The performances of these amplification systems were compared with those of three other PCR systems earlier used for prevalence studies. RESULTS: the primer systems P5Bx and P3Bx exhibited higher PCR scores than the other systems tested; 14 to 34% improvement values were obtained, and divergent positive results of earlier described PCR systems were confirmed systematically by our new detection assays. CONCLUSIONS: an optimized detection of TT virus DNA is a pre-requisite for the accurate epidemiological survey of viral infection and for the realization of phylogenetic studies. Such PCR systems with low genotype restriction will be helpful in the future for a better knowledge of natural history of TT virus infection.
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The purpose of this study was to localize HCV RNA in formalin-fixed paraffin-embedded liver biopsies of 15 patients with chronic hepatitis C using in situ RT-PCR method. The results were compared to serum and tissue extract analysis of HCV RNA. HCV RNA was detected in 80% of the sera tested, in 40% of the corresponding hepatic tissue extract and in 60% of the tissue sections tested by in situ RT-PCR. Compared to the serum positive cases, 67% of the cases were positive with in situ RT-PCR and 41% were positive with tissue extract detection. 50% of the cases in situ RT-PCR positive were also positive with tissue extract detection. These results underlined the complementarity of the different methods of viral detection for the precise diagnosis of hepatitis C.
The genome segments 1, 2, and 3 of the grass carp reovirus (GCRV), a tentative species assigned to genus Aquareovirus, family Reoviridae, were sequenced. The respective segments 1, 2, and 3 were 3949, 3877, and 3702 nucleotides long. Conserved motifs 5' (GUUAUUU) and 3' (UUCAUC) were found at the ends of each segment. Each segment contains a single ORF and the negative strand does not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VP1 might represent the viral guanylyl/methyl transferase (involved in the capping process of RNA transcripts) and VP3 the NTPase/helicase (involved in the transcription and capping of viral RNAs). The highest amino acid identities (26-41%) were found with orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses, and orthoreoviruses. It should also permit to precise the taxonomic status of these different viruses.
The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5' end (SACUUUUGY) and at the 3' end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.
TT virus is a virus distantly related to the Circoviridae family. We report here the complete genome characterization of two European human isolates (T3PB and TUPB) using a new and simple protocol for sequencing GC-rich genomic regions. Sequence analysis confirmed the existence of two major ORFs, of a CAV-like VP2 motif in ORF2 and of potential stem-loop structures in non-coding regions. Phylogenetic analyses based on complete genomic sequences of human isolates suggested that three different lineages exist at least. The first lineage includes genotypes 1, 2, and 3, and two other lineages include viruses related to the Japanese SANBAN and to the North American TUS01 isolates respectively. Sequence comparison made it possible to assign strain T3PB to genotype 3, and strain TUPB to the TUS01 group. Consequently, this study reports the first full-length sequence of a genotype 3 isolate and demonstrates that viruses belonging to the TUS01 lineage are present in the Old Word.
The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.
The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.
BACKGROUND: TT virus (TTV) is a recently discovered virus, which is not related to any other known virus infecting humans. OBJECTIVES: To investigate: (i) the world-wide distribution of the three major TTV genotypes; and (ii) the possible routes of viral transmission. STUDY DESIGN: (i) The phylogenetic distribution of 494 TTV isolates originating from 31 countries was analysed, using partial ORF1 sequences. (ii) Faeces samples (n=22) and saliva samples (n=72) from French individuals were tested for the presence of TTV DNA. (iii) Viral titres in paired serum and saliva samples were compared. RESULTS: (i) Genotypes 1, 2 and 3 were distributed world-wide, with a high proportion of type 1 in Asia (71%) and no type 3 identified in Africa to date. In the USA, 77% of isolates were grouped in four clusters only (genetic distances <10%). This was also the case of 76% of French isolates, 76% of Japanese isolates, and 89% of Hong Kong isolates. (ii) TTV DNA was detected in 18% of faeces samples and 68% of saliva samples tested. (iii) Viral titre in saliva samples was 100-1000 times higher than that of the corresponding serum. CONCLUSIONS: (i) The observed epidemiological distribution of TTV isolates is compatible with an ancient dissemination of viral ancestors belonging to the different genotypes and a slow genetic evolution in sedentary populations. (ii) Besides the possible transmission of TTV by the parental and oral-faecal routes, the high titre of TTV DNA observed in saliva raises the hypothesis of the viral transmission by saliva droplets. This route of transmission could explain the high degree of exposure to viral infection observed in the general population.
BACKGROUND: The purpose of this study was to determine the prevalence of TT virus (TTV) infection in voluntary blood donors in Southeastern France. STUDY DESIGN AND METHODS: The sera of 289 blood donors were tested for the presence of TTV DNA by two PCR systems detecting genes located in the 5' UTR (primer set A [Set A]) and the open reading frame (ORF2) (primer set B [Set B]) of the viral genome. A randomized sample of 40 blood donors was also tested by a nested-PCR system in the ORF1 by use of primer set C (Set C). Donors were questioned for possible risk factors for virus transmission. RESULTS: In the entire population studied, 30.8 percent of blood donors tested positive with both Sets A and B, and 70.6 percent with at least one set. In the sample tested with three sets of primers, 27.5 percent of blood donors were positive in testing with all PCR systems and 80 percent with at least one system. The specificity of TTV DNA amplification was confirmed by sequencing 10 PCR products obtained with each set of primers. Statistical analysis revealed that the prevalence of TTV reactivity increased with age. CONCLUSION: The high prevalence of TTV reactivity and the absence of a pathologic condition or risk factors obviously associated with the infection in blood donors suggest that there is no need for systematic detection of TTV infection before blood donation. Further studies are required to determine if TTV isolates can be responsible for a pathologic condition in humans after blood transfusion.
BACKGROUND: Until 1990, HCV infection was common in transfused patients, resulting in more than 200,000 cases of posttransfusion hepatitis C in France alone. A molecular method that permits the investigation of posttransfusion hepatitis C infections is presented. STUDY DESIGN AND METHODS: Viral sequences in the envelope region of HCV were obtained for 12 pairs of blood recipients and their respective blood donors. The HCV strains studied belonged to types 1 (subtypes 1a and 1b), 2, 3, 4, and 5. Genetic distances and mutation rates were determined, and sequences were submitted to phylogenetic analysis along with sequences retrieved from nucleotide databases. RESULTS: Pairwise distances in the donor-recipient pairs were found to be less than 0.05 mutation per site, which corresponds to a mutation rate ranging from 0.6 x 10(-3) to 2.1 x 10(-3) per site per year. Sequences obtained from the 12 donor-recipient pairs clustered in 12 monophyletic nests. CONCLUSION: The genetic analysis of the envelope region of HCV can be used for the forensic evaluation of virus transmission. It permits the refutation of a link between blood transfusion and HCV transmission, rather than proof of the existence of such a link.
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