Genotype analysis of anti-B19 IgM positive sera from Brazil

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Genotype analysis of anti-B19 IgM positive sera
from Brazil
Kevin E Brown Immunisation and Diagnosis
Unit Virus Reference Department Centre for
Infections
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Prevalence of variant B19
Panel Dates No B19 pos Variant Variant
A HIV patients France 1992-1997 21 1 1 Type 3 5 Servant, J. Virol 2002
B Fetal hydrops France 1995-1997 73 16 0 0
D B19 Ag positive France 1972-1999 87 87 1 Type 3 1
E B19-like symptoms France 1999-2001 633 88 9 Type 2 3 10
C B19-like symptoms USA 270 204 0 0
Blood donors UK 1999-2001 1000 9 0/4 Candotti, J.Virol 2004
Blood donors Ghana 1999-2001 1000 13 12/12 Type 3 100
B19-like symptoms Brazil 2003-2005 69 12 7 6 type 3 1 type 2 58 Sanabani, J. Clin Micro 2006
Tissues Finland 523 190 58 Type 2 11 Norja, PNAS 2006
Only detected in those born before 1973
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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

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B19V Transcription Map
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Alignment of 7.5 kDa region of different
parvovirus B19 isolates
MfeI site
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NS1/7.5 PCR
Samples tested V9 PCR ve MfeI site Not B19
Serum samples (1998-2001) 149 29 28 0
Bone marrow (1998-2001) 18 4 4 0
B19 dotblot pos or equiv (1991-1998) 58 53 51 1
Danish plasma pools (2,000 donors each) 62 40 40 0
Nguyen et al, Virology 2002
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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)

G1
G2
G3
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Light cycler vs in house

• Detect V9 and A6 sequences
• Sensitivity of assay
• lt 1ge/uL
• BUT
• ?Confirmation of low positives
• ? Sequence/genotype information

Artus LC Artus LC
- Total
NS1/7.5 29 5 34
NS1/7.5 - 0 55 55
Total 29 60 89
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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

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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
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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

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Pyrosequencing results
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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

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B19 in Brazil

• 50 IgM samples
• 29 PCR positive
• Range of concentrations 102 -1010 ge/mL
• All B19 genotype 1 (3 point mutations)

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• 69 bone marrow samples PCR analysis only
• 12 B19 positive 7/12 variants

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Acknowledgements

• VRD
• Stuart Beard
• Jayshi Ghandhi
• Members of IDU
• Angie Lackenby
• Cath Arnold
• Brazil
• Marilda Siqueira
• Solange Oliveira