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Biomedical subjects

P de Micco

Publications and source records attributed to P de Micco.

At least 19 recordsLinked to original sources
PubMed2006

Use of the PSA enhancer core element to modulate the expression of prostate- and non-prostate-specific basal promoters in a lentiviral vector context.

Composite promoters combining the prostate-specific antigen (PSA) enhancer core element with promoter elements derived from gene coding for human prostate-specific transglutaminase gene, prostate-specific membrane antigen gene, prostate-specific antigen, rat probasin or phosphoglycerate kinase were characterized for their ability to specifically express the enhanced green fluorescent protein (EGFP) gene in prostate versus non-prostate cancer cell lines when transferred with a human immunodeficiency virus-1-based lentiviral vector. By themselves minimal proximal promoter elements were found to inefficiently promote relevant tissue-specific expression; in all the vectors tested, addition of the PSA enhancer core element markedly improved EGFP expression in LnCaP, a cancer prostate cell line used as a model for prostate cancer. The composite promoter was inactive in HuH7, a hepatocarcinoma cell line used as a model of neighboring non-prostate cancer cells. Among the promoters tested, the combination of the PSA enhancer and the rat probasin promoter showed both high specificity and a strong EGFP expression. Neither a high viral input nor the presence of the cPPT/CTS sequence affected composite promoter behavior. Our data suggest that composite prostate-specific promoters constructed by combining key elements from various promoters can improve and/or confer tissue specific expression in a lentiviral vector context.

Cell Line, Tumor↗
PubMed2006

Distribution of killer-cell immunoglobulin-like receptor (KIR) in Comoros and Southeast France.

Killer-cell immunoglobulin-like receptors (KIRs) expressed by natural killer cells are cell surface molecules able to recognize groups of HLA class I alleles. The number and distribution of KIR genes vary among individuals and populations. The aim of this study is to analyse the KIR gene content in a Comorian population in order to investigate genetic relationships with other populations and to reconstruct past migration events. The Comorian population consisted of 54 unrelated immigrants living in France and a control population consisted of 38 individuals from Southeast France. We investigated the presence or absence of 15 KIR genes, two pseudogenes expressed and non-expressed forms of KIR2DL5 and the two major subtype full-length and deleted forms of KIR2DS4. All individuals were typed positive for the framework genes, i.e. KIR2DL4, KIR3DL2 and KIR3DL3, and the two pseudogenes KIR3DP1 and KIR2DP1. The frequencies of full-length KIR2DS4 (*00101/00102/002) were lower in the French population (F = 29%) than in the Comorian population (F = 72%) (P(c) < 0.05). No significant differences were found for other KIR genes. A total of 11 genotypes were identified in the Southeast French population and 22 genotypes in the Comorian population. The most common genotype (2DL1, 2DL3, 2DL4, 3DL1, 3DL2, 3DL3 and 2DS4) accounted for 41% in the Comorian population and 34% in the Southeast French population. Principal component analysis using KIR gene data from 20 populations was performed to determine genetic differences and relations between populations. The Comorian population exhibited closest kinship with Africans and Asians. As KIR gene content is heterogeneous among ethnic groups, it can probably be used to assess the genetic relationships among populations from different geographic areas.

Comoros↗
PubMed2003

Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe.

Little is known about the genetic relationships between European and other Old-World strains of West Nile virus (WNV) and persistence of WNV North of Mediterranean. We characterized the complete genomes of three WNV strains from France (horse-2000), Tunisia (human-1997) and Kenya (mosquito-1998), and the envelope, NS3 and NS5 genes of the Koutango virus. Phylogenetic analyses including all available full-length sequences showed that: (1) Koutango virus is a distant variant of WNV; (2) the three characterized strains belong to lineage 1, clade 1a; (3) the Tunisian strain roots the lineage of viruses introduced in North America. We established that currently available partial envelope sequences do not generate reliable phylogenies. Accordingly, establishing a large WNV sequence database is pivotal for the understanding of spatial and temporal epidemiology of this virus. For rapid completion of that purpose, colinearized E-NS3-NS5 gene sequences were shown to constitute a valuable surrogate for complete sequences.

Africa↗
PubMed2002

Genus Coltivirus (family Reoviridae): genomic and morphologic characterization of Old World and New World viruses.

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.

Americas↗
PubMed2002

Multivariate analysis of determinants of bacterial contamination of whole-blood donations.

BACKGROUND AND OBJECTIVES: Introduction of bacteria into blood components at the collection stage seems to be a frequent occurrence. We therefore assessed determinants of bacterial contamination of whole-blood donations to gain insight into contamination mechanisms and direct prevention. MATERIALS AND METHODS: A cross-sectional study was carried out on donors accepted for whole-blood donation in four French blood banks. Each blood bank used its own two-stage procedure for phlebotomy site preparation. Contamination was identified by culturing two 15-ml samples (collected aseptically at the outset of donation) in a BacT/Alert 240 system. Determinants were assessed by logistic regression analysis. RESULTS: Bacterial contamination, mainly by skin flora, occurred in 76 (2.2%) out of 3385 donations. Significant determinants were as follows: the blood bank (odds ratio [OR] range = 3.0-5.6, P < 0.001); lack of repetition of scrub (OR = 2.7, P = 0.032); and donor age > 35 years (OR = 1.8, P = 0.036). CONCLUSION: Systematic scrub repetition should be implemented to reduce bacterial contamination by skin flora at the collection stage. Further research is required to clarify the role of different antiseptic agents and of donor age.

Adult↗
PubMed2002

Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus.

Tamana bat virus (TABV, isolated from the bat Pteronotus parnellii) is currently classified as a tentative species in the genus FLAVIVIRUS: We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genus Flavivirus were identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the family FLAVIVIRIDAE: However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the family Flaviviridae, TABV should be assigned to a new genus.

Amino Acid Sequence↗
PubMed2001

Sequence characterization of Ndelle virus genome segments 1, 5, 7, 8, and 10: evidence for reassignment to the genus Orthoreovirus, family Reoviridae.

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.

Genome, Viral↗
PubMed2001

[Methodology for the development of a program for following and maintaining the competency of human resources].

The progressive introduction of a management program for the maintenance and assessment of staff competence has also focussed attention on the human factor, a major consideration in risk management and quality control. This article has examined the relevant tools and practical means of application, and proposes a methodology combining a methodical analysis of processes with the determination of the minimal knowledge required for participation in the practical and theoretical training programs that provide a means of objective evaluation. The results obtained in terms of technical, organizational and cultural impact have also been analyzed.

Blood Banks↗
PubMed2001

Comparison of systems performance for TT virus detection using PCR primer sets located in non-coding and coding regions of the viral genome.

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.

3' Untranslated Regions↗
PubMed2001

Evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome sequences.

Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The 'Recombination Detection Program', bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5' and 3' genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.

Base Sequence↗
PubMed2001

Hepatitis C virus RNA detection by in situ RT-PCR in formalin-fixed paraffin-embedded liver tissue. Comparison with serum and tissue results.

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.

Formaldehyde↗
PubMed2000

Complete genomic sequence of a dengue type 2 virus from the French West Indies.

Severe forms of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome, were not prominent in the Americas until the epidemic of Cuba in 1981. Since that time, they have spread to other countries in Central and South America, correlating with the spread of dengue type 2 viruses related to Southeast Asian strains. We report here the complete genomic sequence of a dengue type 2 virus isolated during the epidemic in La Martinique in 1998. This constitutes the first complete genetic characterization of a dengue virus strain from French West Indies, and also the first molecular identification in this region of a dengue 2 strain phylogenetically related to the emerging American type 2 dengue viruses.

Amino Acid Substitution↗
PubMed2000

Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae).

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.

Animals↗
PubMed2000

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae).

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.

Animals↗
PubMed2000

Complete sequences of two highly divergent european isolates of TT virus.

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.

Circoviridae↗
PubMed2000

Strategies for the sequence determination of viral dsRNA genomes.

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.

Animals↗
PubMed2000

TT virus: a study of molecular epidemiology and transmission of genotypes 1, 2 and 3.

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.

Blood Donors↗