Philippe De Micco

Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae).

Full-length and partial genome sequences of four members of the genus Aquareovirus, family Reoviridae (Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus) were characterized. Based on sequence comparison, the unclassified Grass carp reovirus was shown to be a member of the species Aquareovirus C. The status of golden ide reovirus, another unclassified aquareovirus, was also examined. Sequence analysis showed that it did not belong to the species Aquareovirus A or C, but assessment of its relationship to the species Aquareovirus B, D, E and F was hampered by the absence of genetic data from these species. In agreement with previous reports of ultrastructural resemblance between aquareoviruses and orthoreoviruses, genetic analysis revealed homology in the genes of the two groups. This homology concerned eight of the 11 segments of the aquareovirus genome (amino acid identity 17-42%), and similar genetic organization was observed in two other segments. The conserved terminal sequences in the genomes of members of the two groups were also similar. These data are undoubtedly an indication of the common evolutionary origin of these viruses. This clear genetic relatedness between members of distinct genera is unique within the family Reoviridae. Such a genetic relationship is usually observed between members of a single genus. However, the current taxonomic classification of aquareoviruses and orthoreoviruses in two different genera is supported by a number of characteristics, including their distinct G+C contents, unequal numbers of genome segments, absence of an antigenic relationship, different cytopathic effects and specific econiches.

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.

Efficacy of a new collection procedure for preventing bacterial contamination of whole-blood donations.

BACKGROUND : Transfusion‐related bacterial contamination is a serious problem. The introduction of bacteria into donations at the collection stage seems frequent, despite well‐conducted phlebotomy site preparation. Additional preventive measures are required.

Complete sequence determination and genetic analysis of Banna virus and Kadipiro virus: proposal for assignment to a new genus (Seadornavirus) within the family Reoviridae.

Arboviruses with genomes composed of 12 segments of double-stranded (ds) RNA have previously been classified as members or probable members of the genus Coltivirus within the family REOVIRIDAE: A number of these viruses have been isolated in North America and Europe and are serologically and genetically related to Colorado tick fever virus, the Coltivirus type species. These isolates constitute subgroup A of the coltiviruses. The complete genome sequences are now presented of two Asian arboviruses, Kadipiro virus (KDV) and Banna virus (BAV), which are currently classified as subgroup B coltiviruses. Analysis of the viral protein sequences shows that all of the BAV genome segments have cognate genes in KDV. The functions of several of these proteins were also indicated by this analysis. Proteins with dsRNA-binding domains or with significant similarities to polymerases, methyltransferases, NTPases or protein kinases were identified. Comparisons of amino acid sequences of the conserved polymerase protein have shown that BAV and KDV are only very distantly related to the subgroup A coltiviruses. These data demonstrate a requirement for the subgroup B viruses to be reassigned to a separate new genus, for which the name Seadornavirus is proposed.

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.

Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates

The phylogenetic relationship between the complete genomic sequences of ten Japanese and one French isolate of TT virus-like mini virus (TLMV) was investigated. Analysis of the variability of the nucleotide sequences and the detection of signature patterns for overlapping genes suggested that ORFs 1 and 2 are probably functional. However, this was not the case for a putative third ORF, ORF3. Throughout the viral genome, several nucleotide or amino acid motifs that are conserved in circoviruses such as TT virus (TTV) and chicken anaemia virus were identified. Phylogenetic analysis distinguished three main groups of TLMV and allowed the identification of putative recombination breakpoints in the untranslated region. TLMV genomes were detected by PCR in the plasma of 38/50 French blood donors tested and were also identified in peripheral blood mononuclear cells, faeces and saliva. A phylogenetic study of 37 TLMV strains originating from France, Japan and Brazil showed that groupings were not related to geographical origin.

HLA-DRB1 alleles and Jka immunization

BACKGROUND: In transfusion medicine, anti‐Jka has been implicated in hemolytic transfusion reactions. Development of anti‐Jka after transfusion does not always occur after Jk(a–) patients receive at least 1 unit of Jk(a+) blood unit. This study was designed to identify HLA‐DRB1 alleles associated with predisposition to Jka immunization after blood transfusion or pregnancy.

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.

Variola Virus in a 300-Year-Old Siberian Mummy

This letter describes a distant lineage of the variola virus (the agent of smallpox) that was identified in a mummy found buried in the Siberian permafrost.

HLA-DRB1 polymorphism is associated with Kell immunisation

K immunisation is observed in some polytransfused patients and pregnant women but does not occur in all cases of K incompatibility. This study analysed the role of genetic background in this selective response to K antigen by investigating HLA‐DRB1 alleles associated with K immunisation in a southern European population. HLA‐DRB1 genotyping was performed by polymerase chain reaction sequence‐specific oligonucleotide/sequence‐specific primer procedures in 54 K immunised patients and 200 healthy controls. The frequency of HLA‐DRB1*11 was significantly higher in K immunised patients than healthy controls: 31 of 54 (57%) vs. 56 of 200 (28%) (Pc < 0·001). In the remaining K immunised HLA‐DRB1*11‐negative patients, the frequency of HLA‐DRB1*13 was increased: 14 of 23 (61%) vs. 49 of 144 in healthy controls (34%) (P < 0·02). The combined frequency of the two HLA‐DRB1 alleles (HLA‐DRB1*11 and HLA‐DRB1*13) was 83% in K immunised patients when compared with 52% in healthy controls (Pc < 0·001). K and k differ by a single amino acid T193 (M). The DRB1*11 and DRB1*13 alleles share a HLA‐DRB1 gene sequence containing S in position 13, D in 70 and A in 74, and coding for the P4 pocket within the HLA‐DR binding groove. This feature of the HLA‐DRB1 gene could be involved in the K peptide presentation through a polymorphism ligand specific for the T193 (M) of K. In conclusion, this study demonstrated a high frequency of HLA‐DRB1*11 or HLA‐DRB1*13 alleles in K immunised patients, which could be due to specific K peptide presentation by HLA‐DR molecules.