strains dfr2072 and dfr2736 were isolated from 2 patients originating from senegal and cte d'ivoire , respectively . the patients were living in france when chronic delta hepatitis was diagnosed ; however , because no risk factors were identified , each patient was suspected to have been infected during childhood in africa . full - length hdv genome sequences from isolates dfr2072 and dfr2736 were characterized to determine their genetic affiliation . hdv rna extraction and cdna synthesis were performed as previously described ( 5 ) , and 4 overlapping regions of the genome were amplified ( table 1 ) . amplicons were sequenced bidirectionally with the bigdye terminator v3.1 sequencing kit ( applied biosystems , courtabuf , france ) . * name of the amplified region and position on the genome ( according to wang et al . ) . s , forward primer ; as , reverse primer . in a first approach , the complete dfr2072 and dfr2736 sequences were aligned with 41 complete genome sequences gathering all of the 7 hdv clades , plus sequence dfr644 . the sequence alignment was generated in 2 ways : 1 with clustalx using a gap - opening penalty ( gop ) of 15 and a gap - extension penalty ( gep ) of 6.66 and 1 with the soap program ( http://evol-linux1.ulb.ac.be/ueg/soap/ ) ( gop from 12 to 17 in steps of 0.5 ; gep from 6 to 8 in steps of 1 ) . phylogenetic analyses were performed with paup*4.010 ( sinauer associates , inc . , sunderland , ma , usa ) from a soap sequence alignment that excluded 833 unstable characters . neighbor - joining ( nj ) distance and maximum parsimony ( mp ) analyses were performed . the robustness of the topologic features was determined by bootstrap methods ( 10 replicates for nj and mp ) . a bayesian approach ( 11 ) was also used on the data matrix : 5,000 trees were initially built by using the mrbayes program , version 3.0 4 , from 210 generations , and the first 250 trees were burned . parameters specified during mrbayes analysis ( table 2 ) were also imported into paup*4.0 10 for a maximum likelihood ( ml ) analysis using the general time reversible model with a gamma distribution . * each substitution rate is expressed as compared with the g / t substitution rate . an 89.4% similarity score was observed between dfr644 , dfr2072 , and dfr2736 , which allowed these 3 sequences to be grouped inside the same genotype . by contrast , the similarity obtained among dfr644 , dfr2072 , and dfr2736 and the sequences representative of hdv-1 to hdv-7 was < 76.4% ( figure 1 ) . on the phylogenetic tree built from the ml data ( figure 2 ) , isolates dfr644 , dfr2072 , and dfr2736 appeared as a monophyletic group , with bootstrap values of 100 ( nj and mp ) and a posterior probability value of 100 ( mrbayes ) . percent similarity between hepatitis delta virus ( hdv ) genotypes calculated from complete and small hepatitis delta ( shd ) nucleotide sequences . above the oblique line are represented scores of similarity obtained from alignment and comparison of 49 shd nucleotide sequences including 13 hdv-1 sequences , 7 hdv-2 , 7 hdv-3 , 6 hdv-4 , 6 hdv-5 , 4 hdv-6 , 3 hdv-7 , and 3 hdv-8 . below the oblique line are represented scores of similarity obtained from alignment and comparison of 44 complete nucleotide sequences including 13 hdv-1 sequences , 7 hdv-2 , 4 hdv-3 , 6 hdv-4 , 6 hdv-5 , 3 hdv-6 , 2 hdv-7 , and 3 hdv-8 . maximum likelihood trees inferred from hepatitis delta virus ( hdv ) nucleotide sequences . left panel : maximum - likelihood phylogram obtained from the small hepatitis delta antigen dataset . right panel : maximum - likelihood phylogram obtained from the full - length hdv genome dataset . bootstrap values ( 10 replicates ) obtained for neighbor - joining and maximum parsimony are indicated above the branches ; posterior probabilities ( inferred from 510 trees generated from mrbayes application ) are indicated below the branches . the accession numbers of the sequences used were af209859 , af209859 ; cagliari , x85253 ; china , x77627 ; dfr45 , ax741164 ; dfr47 , ax741149 ; dfr48 , ax741164 ; dfr73 , ax741154 ; dfr644 , ax741169 ; dfr910 , ax741159 ; dfr1843 , aj583885 ; dfr2005 ( guinea - bissau ) , am183331 ; dfr2020 , aj583887 ; dfr2072 ( senegal ) , am183330 ; dfr2139 ( central african republic ) , am183332 ; dfr2158 ( cameroon ) , am183333 ; dfr2600 ( togo ) , am183326 ; dfr2627 ( nigeria ) , am183329 ; dfr2703 ( senegal ) , am183328 ; dfr2736 ( cte d'ivoire ) , am183327 ; ethiopia , u81989 ; hdv - iran , ay633627 ; italy , x04451 ; japan , x60193 ; l215 , ab088679 ; lebanon , m84917 ; miyako , af309420 ; miyako-36 , ab118845 ; miyako-37 , ab118846 ; nagasaki-2 , ab118849 ; nauru , m58629 ; peru-1 , l22063 ; somalia , u81988 ; taiwan , m92448 ; taiwan-3 , u19598 ; taiwan - tw-2b , af018077 ; tokyo , ab118847 ; tw2476 , af104264 ; tw2667 , af104263 ; us-1 , d01075 ; us-2 , l22066 ; vnzd8349 , ab037948 ; vnzd8375 , ab037947 ; vnzd8624 , ab037949 ; yakut-26 , aj309879 ; and yakut-62 , aj309880 . because of claims that the shd protein trans - complements the corresponding hdv type more efficiently ( 12 ) , we compared the shd coding nucleotide sequences of dfr644 , dfr2072 , and dfr2736 with 46 sequences , by using the same phylogenetic approaches ( table 2 ) . analysis of the shd genes confirmed the results obtained with the full - length sequences , showing 93.8% similarity between dfr644 , dfr2072 , and dfr2736 versus only 70.8%82.9% when compared with sequences of the other genotypes ( figure 1 ) . bootstrap values of 100 ( nj and mp ) and posterior probability values of 100 ( mrbayes ) were obtained and are represented on the phylogenetic tree built from the ml parameters ( figure 2 ) . taken together , these results fulfill the recommendations for the designation of a major clade ( i.e. , > 3 distinct isolates repeatedly showing high scores of similarity and high bootstrap values ) . in this study , an eighth hdv clade ( hdv-8 ) was identified from 3 complete sequences obtained from strains isolated from patients of african origin . isolate dfr644 , originating from congo - brazzaville , was initially described by radjef et al . and tentatively affiliated with hdv-7 ( bootstrap value 84 , posterior probability value 97 ) , despite a similarity of only 77.8% with the other hdv-7 sequences ( 1 ) . isolates dfr2072 and dfr2736 presented similarity of 89.4% with dfr644 and only 76.4% with hdv-7 sequences . thus , an additional lineage was individualized , bringing the number of hdv clades with a probable african origin to 4 . since 1999 , a total of 468 hdv isolates collected in france were analyzed in our laboratory for phylogenetic characterization of the r0 region ( defined in table 1 ) . of these , 98 isolates ( 21% ) were affiliated with hdv-5 ( 15.2% ) , hdv-6 ( 1.7% ) , hdv-7 ( 3.0% ) , or hdv-8 ( 1.1% ) ( paul dny , unpub data ) . by contrast , all patients of european origin were specifically infected by hdv-1 isolates . to date , no evidence exists that hdv-5 , -6 , -7 or -8 circulates among native populations in france . nevertheless , epidemiologic studies in africa should be carried out to specify the prevalence and geographic distribution of all hdv clades . if the african origin of hdv-5 , -6 , -7 and -8 viruses is confirmed , detection of these clades in france among local populations would reveal an emerging process that should be anticipated in epidemiologic surveys . thus , the molecular assays used for diagnostic purposes should rely on primers and probes defined in the most conserved regions of the hdv genome to avoid false - negative results ( 5,14,15 ) . in conclusion , the deltavirus genus includes at least 8 major clades , with specific geographic distribution . future development of molecular assays for diagnosis of delta hepatitis should take into account this high genetic variability . the relationship between hdv diversity and pathogenesis has previously been suggested ( 7,9 ) but remains to be clarified by taking into account the extension of the diversity . treatment of chronic delta hepatitis , which relies on long - term administration of high doses of interferon - alpha , is not very effective ( 16 ) . it is not known whether some hdv genotypes might be more susceptible to therapy than others , as has been described for chronic hepatitis c ( 17 ) . thus , the clinical effect of hdv diversity , in terms of severity of disease and response to therapy , remains to be determined .
hepatitis delta virus is the only representative of the deltavirus genus , which consists of 7 differentiated major clades . in this study , 
 an eighth clade was identified from 3 distinct strains . 
 deltavirus genetic variability should be considered for diagnostic purposes . 
 clinical consequences of the diversity have yet to be evaluated .