1. Genotype analysis of anti-B19 IgM positive sera from Brazil Kevin E Brown Immunisation and Diagnosis Unit Virus Reference Department Centre for Infections

2. PanelDatesNoB19 posVariant A HIV patients France 1992-19972111 Type 3 5%Servant, J. Virol 2002 B Fetal hydrops France 1995-1997731600 D B19 Ag positive France 1972-199987 1 Type 3 1% E B19-like symptoms France 1999-2001633889 Type 2 & 3 10% C B19-like symptoms USA 27020400 Blood donors UK 1999-2001100090/4Candotti, J.Virol 2004 Blood donors Ghana 1999-200110001312/12 Type 3 100% B19-like symptoms Brazil 2003-200569127 6 type 3 1 type 2 58%Sanabani, J. Clin Micro 2006 Tissues Finland 52319058* Type 2 11%Norja, PNAS 2006 Prevalence of variant B19 * Only detected in those born before 1973

3. Parvovirus B19 in Brazil Part of study of rash like illness in Brazil Samples tested for measles, rubella, dengue and B19 Preliminary/feasibility study 50 B19 IgM positive 5 from 10 different regions

4. B19V Transcription Map

5. Alignment of 7.5 kDa region of different parvovirus B19 isolates MfeI site

6. NS1/7.5 PCR Samples# testedV9 PCR +veMfeI site Not B19 Serum samples (1998-2001) 14929280 Bone marrow (1998-2001) 18440 B19 dotblot pos or equiv (1991-1998) 5853511 Danish plasma pools (2,000 donors each) 6240 0 Nguyen et al, Virology 2002

7. NS1/7.5 qPCR Modified PCR Quantitect SYBR Green (Qiagen) Light cycler machine 4 step cycling conditions: 95C for 15 min 45 cycles of: 94C for 15s 55C for 20s 72C for 20s 78C for 5s (data acquisition) G1G2G3

8. Light cycler vs in house Detect V9 and A6 sequences Sensitivity of assay < 1ge/uL BUT ?Confirmation of low positives ? Sequence/genotype information Artus LC +-Total NS1/ 7.5 +29534 -055 Total296089

9. Pyrosequencing Sequencing by synthesis Detection of pyrophophate Fast and reliable Ideal for short-read sequencing or mutation/SNP analysis Requirement ss DNA Use biotinylated primer

10. Principle of Method Step 1 1 of 4 NTP added to mix If incorporated PPi produced Step 2 Sulphurlyase converts PPi to ATP Luciferase converts ATP to light Apyrase degrades dNTPs and ADP

11. B19 pyrosequencing SYBR green qPCR Biotinylated reverse primer PCR products saved ssDNA by binding to streptavidin beads DNA incubated with forward primer, polymerase, sulphurylase, luciferase and apyrase Sequential addition of dNTP Record light emmision with PyroMark ID (Biotage) Identify sequence with Identifire software

12. Pyrosequencing results

13. Validation of pyrosequence approach 2 variants identified Both correctly identified by pyrosequencing No. tested 57 routine samples (since Dec 2006) All genotype 1 1 additional sample identified – suspected as variants

14. B19 in Brazil 50 IgM samples 29 PCR positive Range of concentrations: 10 2 -10 10 ge/mL All B19 genotype 1 (3 point mutations)

15. 69 bone marrow samples PCR analysis only 12 B19 positive; 7/12 variants

16. Acknowledgements VRD: Stuart Beard Jayshi Ghandhi Members of IDU Angie Lackenby Cath Arnold Brazil: Marilda Siqueira Solange Oliveira