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