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
Panel Dates No B19 pos 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
Prevalence of variant B19
* Only detected in those born before 1973
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
B19V Transcription Map
Alignment of 7.5 kDa region of different parvovirus B19 isolates
T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC B19T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVBAUAT GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC NC_000883.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028255.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249436T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249435T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249433T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249432T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249429T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249428T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249427T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249426T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249425T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249424T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249422T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028247.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028244.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028241.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028238.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028237.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028234.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028232.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028229.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF236880T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC ERTHRU1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF161226T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF161225T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF161224T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF113323T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF162273T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X599T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X565T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X562T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X560T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X557T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X545T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X530T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X528T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19NSVPT GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AB030694T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AB030693T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AB030673T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVBPROT GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGA PVB19X578- GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X571- GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X568- - - AGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X573T GCAGAT GCCCT CCACCCAAACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028227.1T GCAGAT GCCCT CCACCCAAACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AY028225.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT GT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC AF161223T GCAGAT GCCCT CCACCCAGACCT CCAAGCCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC EB1249423- - - - - AT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC S63987.1T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCAAT T GT CACAGACACCAGT AT CAGCAGCAGGGGT GGT GAAAGCT T T GAAGAACT CAGT GAAAGCAGC PVB19X549- - - AGAT GCCCACCACCCAGACCT CCAAACCACCCCAAT T GT CACAGAGACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC PVB19X534T GCAGAT GCCCT CCACCCAGACCT CCAAACCGT CCCCAT T GT CGCAGACACCAGT GT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC HER249434T GCAGAT GCCCT CCACCCAGACCT CCAAACCGT CCCCAT T GT CGCAGACACCAGT GT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC HER249431T GCAGAT GCCCT CCACCCAGACCT CCAAACCACCCCCAT T GT CCCAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC HER249430T GCAGAT GCCCT CCACCCAGAT CT CCAAACCACCCCCAT T GT CCCAGACACCAGT AT CAGCAGCAGT GGT GGT GAAAGCT CT GAAGAACT CAGT GAAAGCAGC V9_c22-1
MfeI site
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
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 G2G3
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
+ 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 11 of 4 NTP added to mixIf incorporated PPi produced
Step 2Sulphurlyase converts PPi to ATPLuciferase converts ATP to light
Apyrase degrades dNTPs and ADP
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
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
B19 in Brazil
50 IgM samples
29 PCR positive
Range of concentrations: 102 -1010 ge/mL
All B19 genotype 1 (3 point mutations)
69 bone marrow samples PCR analysis only
12 B19 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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