4D-π-1A type ß-substituted ZnII-porphyrins: ideal green sensitizers for building-integrated photovoltaics.

Two novel green ß-substituted ZnII-porphyrins, G1 and G2, based on a 4D-π-1A type substitution pattern have been synthesized. Their enhanced push-pull character, by reduction of H-L energy gaps, promotes broadening and red-shifting of absorption bands. The effective synthetic pathway and the remarkable spectroscopic properties make G2 ideal for BIPV application.

Non-contiguous finished genome sequence and description of Streptococcus varani sp. nov.

Strain FF10(T) (= CSUR P1489 = DSM 100884) was isolated from the oral cavity of a lizard (Varanus niloticus) in Dakar, Senegal. Here we used a polyphasic study including phenotypic and genomic analyses to describe the strain FF10(T). Results support strain FF10(T) being a Gram-positive coccus, facultative anaerobic bacterium, catalase-negative, non-motile and non-spore forming. The sequenced genome counts 2.46 Mb with one chromosome but no plasmid. It exhibits a G+C content of 40.4% and contains 2471 protein-coding and 45 RNA genes. On the basis of these data, we propose the creation of Streptococcus varani sp. nov.

The rediscovery of smallpox.

Smallpox is an infectious disease that is unique to humans, caused by a poxvirus. It is one of the most lethal of diseases; the virus variant Variola major has a mortality rate of 30%. People surviving this disease have life-long consequences, but also assured immunity. Historically, smallpox was recognized early in human populations. This led to prevention attempts--variolation, quarantine, and the isolation of infected subjects--until Jenner's discovery of the first steps of vaccination in the 18th century. After vaccination campaigns throughout the 19th and 20th centuries, the WHO declared the eradication of smallpox in 1980. With the development of microscopy techniques, the structural characterization of the virus began in the early 20th century. In 1990, the genomes of different smallpox viruses were determined; viruses could be classified in order to investigate their origin, diffusion, and evolution. To study the evolution and possible re-emergence of this viral pathogen, however, researchers can only use viral genomes collected during the 20th century. Cases of smallpox in ancient periods are sometimes well documented, so palaeomicrobiology and, more precisely, the study of ancient smallpox viral strains could be an exceptional opportunity. The analysis of poxvirus fragmented genomes could give new insights into the genetic evolution of the poxvirus. Recently, small fragments of the poxvirus genome were detected. With the genetic information obtained, a new phylogeny of smallpox virus was described. The interest in conducting studies on ancient strains is discussed, in order to explore the natural history of this disease.

Classification of TTV and related viruses (anelloviruses).

Ten years after the identification of the first partial sequences of Torque teno virus (TTV), more than 200 full-length related genomes have been characterized in humans and in several animal species. As suspected in the earlier stages of their description, a considerable genetic variability characterizes TTV and related viruses, the current members of the floating genus Anellovirous. Since information related to anelloviruses diversity is in constant evolution, the challenge in their taxonomic classification is to take into account all pertinent parameters, along with the taxonomic situation of other viruses having circular single-stranded DNA genomes. Past, present and future phylogenetic and taxonomic considerations are exposed.

[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.

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.

Pig anelloviruses are highly prevalent in swine herds in France.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular analysis of HCV type 1 to 5 envelope gene: application to investigations of posttransfusion transmission of HCV.

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.