P. De Micco

Classification of hepatitis C virus variants in six major types based on analysis of the envelope 1 and nonstructural 5B genome regions and complete polyprotein sequences.

The phylogenetic status of recently described isolates of hepatitis C virus (HCV) from Vietnam, Thailand and Indonesia (previously classified as types 7, 8, 9, 10 and 11) was re-analysed by the neighbour-joining method instead of the unweighted pair-group method with arithmetic mean (UPGMA) that was first used by the discoverers of these strains. The analysis of complete amino acid sequences and of nucleotide sequences of the envelope 1 (672 nt) and nonstructural 5B (1092 nt) genomic regions permitted the re-assignment of the type 7, 8, 9 and 1 1 isolates to type 6, and that of type 10 strains to type 3. Finally, this study made possible the classification of the previously described HCV strains (including these South-East Asian isolates) in six major types and at least 30 subtypes. It confirms that analysis of the E 1 and NS5B genomic regions using the neighbour-joining method is a reliable tool for the assignment of most new isolates.

Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns

The influence of the DNA concentration was tested using two different primers and nine DNA samples. Major modifications in the DNA banding pattern were apparent between successive dilutions. Such differences could be explained by concomitant changes in three different molecular conditions: the presence of perfect priming sites, the amplification of rare sites and the existence of mismatch annealing events. At low DNA concentrations (less than 1 pg/microliter), molecular events occurred at random and had a direct consequence on the reproducibility of RAPD profiles. At the appropriate DNA concentration (between 100 ng/microliters and 10 pg/microliters), reproducibility was adequate at a given concentration, but RAPD profiles differed from one dilution to another. These observations demonstrate the usefulness of the bis-benzimide method for quantification of DNA extracts.

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.

COMPARATIVE SEQUENCE ANALYSIS OF AMERICAN, EUROPEAN AND ASIAN ISOLATES OF VIRUSES IN THE GENUS COLTIVIRUS

In this study, the basis for the classification of virus isolates grouped within the genus Coltivirus, family Reoviridae, is discussed. Sequences of dsRNA segments from American (segments 9-12), European (segment 12) and Asian (segments 7-12) isolates were characterized and polythetic criteria were defined for their taxonomic classification. These criteria (including sequence analysis) permitted the different species to be distinguished and classified into two groups. In both groups, subgroups were defined according to the degree of homology between the genomic sequences. American and European isolates are classified within group A, which includes subgroups A1 (Colorado tick fever virus species) and A2 (Eyach virus species). Asian isolates are classified in group B, which includes subgroups B1 (JKT-7075 virus species) and B2 (JKT-6423 virus species). The proteins encoded by the sequenced genomic segments were analysed. This allowed the identification of dsRNA binding domains in the proteins encoded by segment 8 of subgroup B1 isolates and segment 12 of subgroup B2 isolates. A conserved pattern of amino acids in segment 7 of group B isolates matched sequences found in the catalytic domains of protein kinases.

Identification and strain differentiation of Mycobacterium species on the basis of DNA 16S–23S spacer region polymorphism

Amplification of the region separating the genes coding for the two rRNA species 16S and 23S was performed to identify 56 mycobacterial strains, belonging to eleven species: Mycobacterium tuberculosis, M. avium, M. kansasii, M. gordonae, M. abscessus, M. fortuitum, M. xenopi, M. bovis, M. bovis/BCG, M. africanum and M. intracellulare. Reproducible amplification patterns were obtained with most species with the exception of M. kansasii which showed heterogeneity, confirming the existence of a genetically distinct subspecies within this species. In addition, we used the amplified products as target DNA for restriction endonuclease digestion and RAPD (randomly amplified polymorphic DNA) analysis to compare strains of M. abscessus, M. tuberculosis and M. avium. The discriminatory power of these two typing methods was higher than when whole genomic DNA is used as target. Our results demonstrate that the two-step approach to identification and typing on the basis of the hypervariability of 16S-23S spacer region is reliable, rapid and simple, and consequently could be an epidemiological tool in clinical laboratories.

Detection of JC Virus by Polymerase Chain Reaction and Colorimetric DNA Hybridization Assay

A procedure for rapid detection of JC virus (JCV) using the polymerase chain reaction is described. The procedure was tested in eight HIV-1-seropositive patients with progressive multifocal leukoencephalopathy. One-step DNA extraction using a chelating resin was carried out on clinical samples of cerebrospinal fluid (CSF), urine and brain tissue. After amplification, PCR products were detected by a DNA hybridization method. Microplates were coated with a specific probe and hybridized PCR products were revealed by a commercial colorimetric immunoassay. Using this procedure JC virus DNA was detected in all CSF specimens from patients with progressive multifocal leukoencephalopathy. This sensitive and rapid (24 h) procedure could greatly facilitate use of the DNA probe assay for detection of JC virus in clinical laboratories.

PCR analysis of hepatitis B virus DNA in paraffin-embedded liver tissue from patients with chronic liver disease.

We described a nested polymerase chain reaction protocol to detect hepatitis B viral DNA in paraffin-embedded liver tissue and tried to determine whether this virus was associated with non-B chronic liver disease. Fifty-five samples were obtained from 28 patients with B, and 27 patients with non-B chronic liver disease (35 cirrhosis, 4 hepatocellular carcinoma and 16 chronic hepatitis). The two sets of primers amplify a sequence located in a conserved polymerase/surface region of the viral genome. Reaction products were analysed using a nonisotopic hybridization method. None of the 27 (0%) seronegative samples and 20 of the 28 (71%) seropositive specimens were positive for hepatitis B virus DNA. There were 4 false negatives in which beta-globin PCR was positive. Although its sensitivity is reduced in formalin-fixed paraffin-embedded tissue, nested PCR allows rapid detection of HBV DNA sequences and can be a useful tool if no frozen tissue is available.

High prevalence of human T-lymphotropic virus (HTLV) infection in pregnant women in southern France

Screening of blood and organ donations for human T-lymphotropie viruses (HTLV-I/II) has been obligatory in France since July 1991. It underscores the increasing concern of the French public health authorities for HTLV-I and HTLV-II dissemination [1]. Among HTLV-I transmission routes, vertical mother-to-child transmission is estimated at 25% and is essentially through maternal breast-feeding [2, 3]. The possibility of prevention through breast-feeding abstention led us to evaluate HTLV-I/II seroprevalence in women at the end of their pregnancy. Previous data in France have only concerned a highrisk pregnant women population in Paris (0.66%) [4]. In an attempt to improve our knowledge of HTLV dissemination in our cosmopolitan region [5], we have focused this prospective study on a general population of pregnant women. Between April and November 1992, blood samples from pregnant women in two public maternity wards in Marseille were collected. A short questionnaire with possible risk-factors was established for each subject. With their agreement, data on age, region of origin and sexual behaviour were recorded. Detection of HTLV-I/II antibodies was performed by enzyme immunoassay (EIA) (Vironostika HTLV-I, Organon Teknika Corp., Durham, NC, USA) and confirmed by Western blot (WB) using the manufacturers positivity criteria (HTLV blot 2.3; Diagnostic Biotechnology Ltd., Singapore and Biotech Laboratories, Rockville, MD, USA). Seropositive women were then assayed by DNA amplification (primers SK43/SK44, SK54/SK55 and SK58/SK59) to discriminate HTLV type I from type II [6]. All sera were also tested for HIV-1 and HIV-2 by two EIA tests (Genelavia mixt; Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France and Wellcozyme HIV recombinant; Murex, Dartford, UK). A total of 932 women has been tested (mean age: 26.8 years, HIV-1 seropositive subjects: 1.3%). Ten subjects (1.1%) were positive for HTLV-I/II antibodies with EIA test. After WB, three (30%) were negative, three (30%) were confirmed positive by WB and PCR and four (40%) had an undetermined WB and were PCR negative (Table 1). The differentiation by PCR identified two HTLV-1 and one HTLV-II. All three women presented a risk factor identified by the questionnaire: a region of origin endemic for HTLV. None of them (1 from Comoro Islands aged 16, 1 from Ghana aged 22, and 1 from French West Indies aged 26) presented an HIVseropositivity associated with HTLV nor did any have clinical signs of HTLV-I infection. This study presents a seroprevalence of 0.32% of HTLV-I/II in a general population of pregnant women from two public hospital maternity wards in Marseilte. Although this prevalence rate is low, it is significantly higher (a hundred times more) than those observed in the blood donor population in Marseille (0.37 %0) (G. Cotte, personal communication) or in France (0.39 %o) [7] as previously shown by Courtois [4]. It also demonstrates that the principal variable associated with HTLV-I/II seropositivity is region of origin [8]. Even if these three cases probably correspond to an old acquisition of HTLV in high-risk endemic areas before coming to France, they represent a potential origin for viral dissemination in our region. This serological survey could