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Identification, persistence and serological cross-reactivity of B19 genotype 2

Identification, persistence and serological cross-reactivity of B19 genotype 2. Kati Hokynar, MSc Haartman-institute, Dept. of virology University of Helsinki Finland. B19. Au genome. d. w. d. w. f. w. f. w. 8. f. o. f. i. f. m. o. 2. 2. i. f. i. P. P. S. S. 6. r.

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Identification, persistence and serological cross-reactivity of B19 genotype 2

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  1. Identification, persistence and serological cross-reactivity ofB19 genotype 2 Kati Hokynar, MSc Haartman-institute, Dept. of virology University of Helsinki Finland

  2. B19 Au genome d w d w f w f w 8 f o f i f m o 2 2 i f i P P S S 6 r N N V V p p 2 1 1 1 5 v v r r e e e e 5 3 r r r r i p p i o 2 o S 2 P S N P V N V 393 436 389 1066 1998 1660 Identification of B19 type 2 in skin (Hokynar et al. 2002, Virology) Skin and sera from patients with B19-nonrelated skin lesions or healthy members of hospital staff (N=35) were examined for B19 DNA with three nested PCRs

  3. Results B19 VP1-DNA in skin: 14/20 seropositives 0/15 seronegatives Only 5/14 VP1-positive samples were also positive for NS1- and VP2-DNA The remaining 9 samples showed poorly reproducible amplicons or weak hybridisation signals

  4. Near-full-length genome was sequenced from 2 dermal DNA isolates DNA sequence of isolate LaLi differed extensively from all the previously reported erythroviruses including the variant V9.

  5. Sequencevariability Hokynar et al, Virology 2002; Nguyenet al, Virology 2002; Servant et al, J Virol 2002.

  6. B19 type 2 - specific nested PCR (K71-PCR) Skin samples contained B19 DNA 25% prototypic 45% type 2 From synovium, only 1/31 samples gave a weak PCR signal.

  7. RealArt Parvo B19 LC PCR (Artus) -detected and differentiated (plasmid constructs of) all three B19 genotypes (Hokynar et al. 2004, J Clin Microbiol)

  8. Do the virus types 2 and 3 differ from virus type 1 in biology – or in epidemiology? Of 140 000 plasma donations collected during 2002-2003, none contained B19 types 2 or 3

  9. Tissues and Sera Synovia + skin: from healthy adults with joint trauma, n=86 Skin: from patients with B19 unrelated dermatological disorders or from healthy laboratory staff, n=54 Tonsils: from patients with tonsillitis or tonsillar hypertrophy, n=220 Liver: collected for diagnostic purposes in Germany, n=77 Altogether 523 solid tissue samples Sera: collected in 1983-1997 for virus diagnosis n=1640

  10. PCRs used PCR Genotypes detectable (sensitivity) Skin Synovium Tonsil Liver Serum Non-nested VP1-PCR 1, 2 and 3 (5 DNAcopies each) x x Nested VP1-PCR 1, 2 and 3 (15 DNA copies each) x x Nested K71-PCR 2 (1.5 DNA copies) x x x x x Genotype 1 PCR 1, 3 (1.5 and 15 copies, resp.) x RealArt Parvo B19 LC PCR 1, 2 and 3 (5 DNA copies each) x x x

  11. Tissue Virus type 1 Virus type 2 Negative Skin 31% (43) 17% (24) 52% (73) 140 Synovia 35% (30) 8% (7) 57% (49) 86 Tonsil 16% (36) 1% (3) 82% (181) 220 Liver 32% (25) 31 (24) 36% (28) 77 Serum 17% (28) 0% (0) 83 (136) 164 Prevalence of virus types 1-3 in solid human tissues and sera Virus type 3 was absent from all the tissues and sera studied

  12. B19 types in tissue by birth year Norja et al, PNAS 2006;103:7450-3

  13. Virus type 1 Birth year range 1923-1996 (mean 1965, SD 14) Conclusion I Virus type 2 Birth year range 1935-1972 (mean 1945, SD 10) The new variant (virus type 2) is ”older” in occurence than the prototype virus (type 1); Both circulated in Northern Europe in equal frequency up to the 1950’s, after which virus type 2 disappeared from wide circulation.

  14. Biological relations of B19 types 1-3 At the nucleotide level, the most striking sequence difference between the three B19 types occur within the p6 promoter →The promoter activities of the three virus types were measured in several B19 permissive and non-permissive cell-lines NS1 acts as a transcription transactivator and, at the amino acid level, is the most divergent protein among the three B19 types →The effect of the NS1 of B19 type 1 on promoters of B19 types 1-3 was measured All B19 types have been associated with anemia or aplastic crisis indicating erythroid tropism →The ability of B19 types 1-3 to infect B19 type-1 permissive cells was examined

  15. p6-promoter activities The p6 promoter activities of the three B19 types were of similar strength in all cell types studied; strongest activity was observed in cell lines permissive for B19 prototype Type 1 NS1 protein enhanced the activity of all three promoters equally

  16. Infectivity Two B19-permissive cell lines were infected with B19 type 1, 2 or 3 containing plasma / sera SPR3 (type 1), BTS, Finland IM-81 (type 2), Baxter, Austria D91.1 (type 3), Dr. Garbarg-Chenon, France All three genotypes were able to infect these cells

  17. IgG cross-reactivity of B19 types 1 and 2 Recombinant VLPs, composed either of VP2 protein alone or VP1 and VP2 together, of B19 type 1 and type 2 were produced in a baculovirus system. Four EIAs, using as antigen VP2 or VP1/2 antigens of either virus type, were set up. Sera from three groups of subjects were examined: Sera from subjects carrying in tissue B19 type 1 DNA (n=24), B19 type 2 DNA (n=25) B19 IgG negative subjects (n=13) 100 nm

  18. 100 % IgG cross-reactivity between B19 types 1 and 2 *) One sample showed borderline reactivity. **) The overall reactivity was equally low for both genotypes.

  19. Conclusion II • Despite their sequence and epidemiological differences, the three B19 types are highly similar variants of the same species • B19 types 1-3 constitute a single serotype

  20. Acknowledgements Dept. of Virology, University of Helsinki Maria Söderlund-Venermo, Klaus Hedman Päivi Norja, Anna Ekman, Kalle Kantola, Laura Kakkola, Heidi Bonden, Anne Lahtinen, Simo Miettinen, Lea Hedman, Jaakko Lommi, Renwei Chen, Olli Vapalahti, Antti Vaheri, Collaboration Anna-Maria Eis-Hübinger, Irja Davidkin, Beate Schneider, Hans-Peter Fischer, Rene Tolba, Matthias Gessner, Claudia Aberham, Antoine Garbarg-Chenon, Harri Laitinen, Eiji Morita, Kazuo Sugamura, Eiji Miyagawa, Frederic Morinet, Doris Morgan, Susanne Modrow Clinical collaborators Leena-Maija Aaltonen, Annamari Ranki, Esa Partio, Olli Kiviluoto, Tomi Leivo

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