1. Identification, persistence and serological cross-reactivity of B19 genotype 2 Kati Hokynar, MSc Haartman-institute, Dept. of virology University of Helsinki Finland

2. Identification of B19 type 2 in skin (Hokynar et al. 2002, Virology) B19 Au genome 1 5 1 1 2 p 3 r p 5 r p 6 f p r i m 8 f 393 1066 N S o r e v N S i r e v N S o f w d N S i f w d 436 1998 V P 2 o r e V P 2 i r e V P 2 o f w V P 2 i f w 389 1660 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. Hokynar et al, Virology 2002; Nguyen et al, Virology 2002; Servant et al, J Virol 2002. Sequence variability

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 x x x Liver x x x Tonsil x1, 2 and 3 (5 DNA copies each) RealArt Parvo B19 LC PCR 1, 3 (1.5 and 15 copies, resp.) Genotype 1 PCR x x x2 (1.5 DNA copies) Nested K71- PCR x x1, 2 and 3 (15 DNA copies each) Nested VP1- PCR x1, 2 and 3 (5 DNAcopies each) Non-nested VP1-PCR SerumSynovium Skin Genotypes detectable (sensitivity) PCR

11. Prevalence of virus types 1-3 in solid human tissues and sera 16483 (136) 0% (0) 17% (28) Serum 7736% (28)31 (24)32% (25) Liver 22082% (181)1% (3)16% (36) Tonsil 8657% (49)8% (7)35% (30) Synovia 14052% (73)17% (24)31% (43) Skin NegativeVirus type 2Virus type 1Tissue Virus type 3 was absent from all the tissues and sera studied

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

13. Conclusion I Virus type 1 Birth year range 1923-1996 (mean 1965, SD 14) 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. 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 p6-promoter activities

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

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