[Anelloviruses (TTV and variants): state of the art 10years after their discovery]. / Les anellovirus (TTV et variants): données actuelles dix ans après leur découverte.

Ten years after their discovery, anelloviruses combine some characteristics making them particularly intriguing. In support of their extreme genetic diversity and high prevalence in various populations, their natural history is still poorly understood along with their implication in human health. These viruses have been identified in blood and blood-derived products, and are probably remarkable examples of co-existence and co-evolution in their various hosts. This article presents epidemiological and molecular characterizing this new viral family.

Overview of revised measures to prevent malaria transmission by blood transfusion in France.

Plasmodial transmission by blood donation is rare in non-endemic countries, but a very serious complication of blood transfusion. The French national blood service (Etablissement Français du Sang and Centre de Transfusion sanguine des Armees) intended to revise the measures to strengthen blood safety with regard to Plasmodiae as transmissible pathogens. To limit the risk of transmission during infusion, serious additive measures have been taken for more than a decade in France, which is the European country with the highest rate of exposure to imported plasmodial infections or malaria. These measures were revised and strengthened after the occurrence of a lethal transfusion-transmitted infection in 2002, but did not prevent another occurrence in 2006. This report examines the weaknesses of the systems and aims at emphasizing the safety measures already taken and addresses issues to best respond to that risk.

Inactivation of a European strain of West Nile virus in single- donor platelet concentrate using the INTERCEPT blood system.

BACKGROUND AND OBJECTIVE: In order to prevent West Nile virus (WNV) contaminations by transfusion, the French National Blood Service decided to evaluate the INTERCEPT Blood System's efficiency on a European strain. MATERIALS AND METHODS: Culture supernatant of WNV was used to infect six platelets concentrates. Viral titre was determined by plaque reduction neutralization test before and after viral inactivation using the INTERCEPT Blood System. RESULTS: In all assays, the absence of plaque forming unit was observed after viral inactivation. The log reduction observed ranged between > 5.1 logs to > 5.2 logs. CONCLUSION: INTERCEPT Blood System is a commercially viral inactivation method potentially useful in order to prevent WNV transmission by blood products in France during re-emerging outbreaks.

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.

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.

Identification of a third member of the Anellovirus genus ("small anellovirus") in French blood donors.

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.

[West Nile virus (WNV): generalities and implications for blood transfusion]. / Le virus West Nile: généralités et implications en transfusion sanguine.

West Nile virus (WNV) is an arbovirus (genus Flavivirus, Family Flaviviridae, transmitted to humans by mosquito bite. In most cases (80%), human infection remains asymptomatic. Severe central nervous system complications (encephalitis and meningoencephalitis) are rare. In the Old World, the virus circulation has been demonstrated in Asia, Australia, Africa, Middle East and Europe. Several outbreaks in humans have been described. Following its introduction into North America in 1999, WN virus has been responsible of a large number of human cases in USA and Canada. For the first time, viral transmission by blood products was clearly demonstrated in USA in 2002. In France, the presence of virus has been reported in the Southeastern departments since 1962. In 2003, the occurrence of humans cases at specific geographical foci urged the French National Blood Agency (etablissement francais du sang) to take preventive measures for evaluating the virus transmission risks.

Immunohistochemical detection of C-100 hepatitis C virus antigen in formaldehyde-fixed paraffin-embedded liver tissue. Correlation with serum, tissue and in situ RT-PCR results.

We localized HCV C-100 protein in liver biopsies of 15 patients with chronic hepatitis C using immunohistochemistry. The results were compared to serum, tissue extract analysis of HCV RNA and in situ RT-PCR described in a previous study. HCV was detected in 80% of the sera tested, in 40% of the tissue extracts and in 80% and 60% of the tissue sections tested by immunohistochemistry and in situ RT-PCR respectively. Compared to the serum positive cases, 83% and 67% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR and 41% were positive with tissue extract detection. Compared to the tissue extract positive cases, 25% and 50% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR. Finally, 75% of the cases positive by immunohistochemistry were also positive by in situ RT-PCR. These results underline the complementarity of the different methods for the precise diagnosis of hepatitis C.

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.

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.

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.

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.

Complete coding sequence of the Alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in Saudi Arabia.

To date, tick-borne flaviviruses responsible for hemorrhagic fever in humans have been isolated in Siberia (Omsk hemorrhagic fever virus), India (Kyasanur Forest disease virus, KFDV), and in Saudi Arabia (Alkhurma virus, ALKV). Prior to this study, only partial coding sequences of these severe pathogens had been determined. We report here the complete coding sequence of ALK virus, which was determined to be 10,248 nucleotides (nt) long, and to encode a single 3,416 amino acid polyprotein. Independent analyses of the complete polyprotein and the envelope protein provided genetic and phylogenetic evidence that ALKV belongs to the tick-borne flavivirus group, within which it is most closely related to KFDV. Analysis of structural genes, genetic distances, and evolutionary relationship indicate that ALKV and KFDV derived from a common phylogenetic ancestor and constitute two genetic subtypes of the same virus species according to current genetic criteria of classification.

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.

Evaluation of disinfectant efficacy against hepatitis C virus using a RT-PCR-based method.

The methods traditionally used to evaluate the antiviral activity of antiseptics and disinfectants are based on cell cultures. However, such methods are not applicable to non-cultivable viruses such as hepatitis C (HCV). Therefore, in this case, virucidal activity is normally tested using surrogate viruses able to grow in cell culture. This paper describes a RT-PCR method for testing antiseptic/disinfectant activity against HCV, as a model for non-cultivable viruses. A chlorine-based agent used for skin and tissues, and a 2% glutaraldehyde solution used for endoscope disinfection, were the test materials. The results are discussed in the light of the use of these agents. The method is simple, fast and inexpensive, and could be used for tests on other viruses with minor modification.

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.

TT virus infection: prevalence of elevated viraemia and arguments for the immune control of viral load.

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.