Unité dOncologie Virale, Département du SIDA et des Rétrovirus, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France1
Cancer Research Center, Moscow, Russia2
Central Dermato-Venerological Institute, Moscow, Russia3
Author for correspondence: Antoine Gessain. Fax +33 1 40 61 34 65. e-mail agessain{at}pasteur.fr
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Abstract |
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Main text |
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We report here the molecular characterization, with subtyping of both K1 and K14.1/K15 genomic regions, of seven new HHV-8 strains from Russian patients with classical KS. The main clinical and epidemiological features of the patients are summarized in Table 1. All seven patients (four males, three females; mean age of 74 years) were HIV seronegative and suffered from cutaneous lesions mainly located on the legs. They all lived in and originated from the Moscow area, except for one patient (case 78/48), an 82 year old female who originated from the Chita province in eastern Siberia. DNA was extracted from cutaneous tumour biopsies. The complete 870 bp ORF K1 coding region, from nucleotide position 105 to 974 of the prototype sequence (Russo et al., 1996
), was amplified directly from these DNA samples as a 1073 bp PCR product, as previously described (Fouchard et al., 2000
), using a single step PCR. The 870 bp sequence was performed after TA cloning of the PCR product (Invitrogen), using ABI 377 Perkin Elmer apparatus (Eurogentec). Three clones were sequenced on both strands from four amplified samples, while only one clone was sequenced from the three other samples.
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Phylogenetic studies were performed using the seven new sequences and all the other complete K1 gene sequences (33) available in GenBank. Since tree building algorithms rely on different assumptions, we used two different methods, neighbour joining and DNA maximum parsimony, to increase the reliability of the derived tree topologies. As seen in Fig. 1, the four main already known clades (A, B, C, D) were clearly identified on the basis of consistent topological associations, and high bootstrap values, in the two phylogenetic analyses. Furthermore, the location of the seven new HHV-8 strains from Russia, with six of them belonging to the A subtype and one to the C subtype, were identical in the two analyses. Moreover, quite similar results were obtained by performing phylogenetic analyses on the nucleotide or on the amino acid sequences (Fig. 1
).
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Sequence analyses of strain 76/42 indicate that this new Russian strain possesses an unusual deletion of 57 nucleotides in the VR2 region. This deletion is located at position 186204 of the amino acid sequence of the K1 protein and so has only a four amino acid overlap with a five amino acid deletion specific for the C subtype (amino acids 201205). This peculiar strain also possesses all the specific mutations of the A4 molecular subgroup, including the four amino acid deletion characteristic of the A4 subgroup and located downstream of the large deletion, at position 207210.
Comparison with all the 160 complete or partial K1 sequences either available in GenBank or in tables of the previously quoted papers indicate that none of these sequences possesses the deletion found in the 76/42 Russian strain. The GK18 strain originating from a patient from Greece with classical KS possesses an amino acid deletion located at position 197206 in the VR2 fragment which overlaps four amino acids with the deletion of the 76/42 strain, but this GK18 isolate was otherwise a typical C subtype. Such data suggest that this region of the VR2 fragment, which contains neither glycosylation sites nor cysteine residues, may be prone to genomic rearrangements.
In four samples, we analysed the complete K1 sequences of three different clones, one obtained in a first PCR, the two others in a second PCR performed 4 months later. The genetic variability within the three clones was very low for three patients (02 changes both at the nucleotide and amino acid level), while for one patient, the variability was slightly higher (25 changes at both nucleotide and amino acid level). These data thus indicate that the intra-strain K1 variability in a given patient, at a given time, is very low at least for a KS tumour sample (range from 0 to 0·6% nucleotide difference). Furthermore, a phylogenetic analysis was performed using the three clones from these patients. The location of the sequences was identical to when only one clone was analysed (data not shown).
Molecular subtype characterization of the right hand side (RHS) of the KSHV/HHV-8 genome was determined as previously described (Poole et al., 1999 ). The extreme RHS genomic region comprises K14.1 and K15 genes. The latter codes for an integral membrane protein related to the LMP2 latency protein of EpsteinBarr virus. Two different PCR strategies were used, a single PCR using a pair of primers unique to the M subtype of ORF K15, and a triple primer PCR set of ORF K14.1 covering the divergent junction of the two subtypes of ORF K15 genes. Specific amplification of ORF K15 subtype M gave a positive result for samples 74/8T, 76/42, 79/55 and BC-1 (subtype M control) and a negative result for 75/10T, 77/47, 78/48, 80/56 and BCBL-1 (subtype P control) (data not shown). The second strategy gave a 450 bp product specific for subtype M for samples 74/8T, 76/42, 79/55 and BC-1 and a 362 bp product specific for the P subtype for the other samples, 75/10T, 77/47, 78/48, 80/56 and BCBL-1 (Fig. 2
). Thus, a perfect correlation was obtained between the two strategies used to characterize the M or P subtypes of the RHS genomic region of the seven new Russian HHV-8 strains.
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Acknowledgments |
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Footnotes |
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References |
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Received 12 November 1999;
accepted 4 February 2000.