Genotype analysis of anti-B 19 Ig. M positive sera from Brazil Kevin E Brown Immunisation and Diagnosis Unit Virus Reference Department Centre for Infections
Prevalence of variant B 19 Panel A Dates No B 19 pos HIV patients 1992 -1997 21 1 France B Fetal hydrops Variant 1 5% Type 3 1995 -1997 73 16 0 0 1972 -1999 87 87 1 Servant, J. Virol 2002 1% France D B 19 Ag positive France E B 19 -like symptoms Type 3 1999 -2001 633 88 France C 9 10% Type 2 & 3 B 19 -like symptoms 270 204 0 1999 -2001 1000 9 0/4 1999 -2001 1000 13 12/12 0 USA Blood donors Candotti, J. Virol 2004 UK Blood donors Ghana B 19 -like symptoms 100% Type 3 2003 -2005 69 12 Brazil 7 58% 6 type 3 J. Clin Micro 2006 1 type 2 Tissues Finland 523 190 58* Type 2 Sanabani, 11% Norja, PNAS 2006 * Only detected in those born before 1973
Parvovirus B 19 in Brazil Part of study of rash like illness in Brazil Samples tested for measles, rubella, dengue and B 19 Preliminary/feasibility study 50 B 19 Ig. M positive 5 from 10 different regions
B 19 V Transcription Map
Alignment of 7. 5 k. Da region of different parvovirus B 19 isolates Mfe. I site
NS 1/7. 5 PCR Samples # tested V 9 PCR +ve Mfe. I site Not B 19 149 29 28 0 18 4 4 0 B 19 dotblot pos or equiv (1991 -1998) 58 53 51 1 Danish plasma pools 62 40 40 0 Serum samples (1998 -2001) Bone marrow (1998 -2001) (2, 000 donors each) Nguyen et al, Virology 2002
NS 1/7. 5 q. PCR Modified PCR Quantitect SYBR Green (Qiagen) Light cycler machine 4 step cycling conditions: 95 C for 15 min 45 cycles of: 94 C for 15 s 55 C for 20 s 72 C for 20 s 78 C for 5 s (data acquisition) G 1 G 3 G 2
Light cycler vs in house Detect V 9 and A 6 sequences Artus LC Sensitivity of assay + NS 1/ 7. 5 BUT ? Confirmation of low positives ? Sequence/genotype information Total + < 1 ge/u. L - 29 5 34 - 0 55 55 Total 29 60 89
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
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 d. NTPs and ADP
B 19 pyrosequencing SYBR green q. PCR Biotinylated reverse primer PCR products saved ss. DNA by binding to streptavidin beads DNA incubated with forward primer, polymerase, sulphurylase, luciferase and apyrase Sequential addition of d. NTP Record light emmision with Pyro. Mark ID (Biotage) Identify sequence with Identifire software
Pyrosequencing results
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
B 19 in Brazil 50 Ig. M samples 29 PCR positive Range of concentrations: 102 -1010 ge/m. L All B 19 genotype 1 (3 point mutations)
69 bone marrow samples PCR analysis only 12 B 19 positive; 7/12 variants
Acknowledgements VRD: Stuart Beard Jayshi Ghandhi Members of IDU Angie Lackenby Cath Arnold Brazil: Marilda Siqueira Solange Oliveira
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