Identification, persistence and serological

cross-reactivity ofB19 genotype 2

Kati Hokynar, MScHaartman-institute, Dept. of virology

University of HelsinkiFinland

Identification of B19 type 2 in skin

(Hokynar et al. 2002, Virology)

B19 Au genome

1

51

12

p3r

p5r

p6f

pri

m8f

393

1066

NS

ore

v

NS

irev

NS

ofw

d

NS

ifw

d

436

1998

VP

2ore

VP

2ir

e

VP

2ofw

VP

2ifw

389

1660

Skin and sera from patients with B19-nonrelated skin lesions or healthy members of hospital staff (N=35) were examined for B19 DNA with three nested PCRs

Results

B19 VP1-DNA in skin: 14/20 seropositives 0/15 seronegatives

Only 5/14 VP1-positive samples were also positive for NS1- and VP2-DNA

The remaining 9 samples showed poorly reproducible amplicons or weak hybridisation signals

DNA Protein

non-CDS CDS NS1 VP1/2 VP2 uVP1 NS1 VP1/2 VP2

LaLi vs Au 26,5 10,8 12,9 9 10,9 4,6 6 2,1 1,1

LaLi vs V9 17,2 8,6 7,6 9,5 10,1 8,2 3 2,7 1,3

V9 vs Au 30,7 12,5 13,4 11,7 13 8,5 5,8 3,1 1,4

SEQUENCE DIVERGENCE (%)

Near-full-length genome was sequenced from 2 dermal DNA isolates

DNA sequence of isolate LaLi differed extensively from all the previously reported erythroviruses including the variant V9.

Hokynar et al, Virology 2002; Nguyen et al, Virology 2002; Servant et al, J Virol 2002.

Sequence variability

B19 type 2 - specific nested PCR (K71-PCR)

Skin samples contained B19 DNA 25% prototypic 45% type 2

From synovium, only 1/31 samples gave a weak PCR signal.

RealArt Parvo B19 LC PCR (Artus)

-detected and differentiated (plasmid constructs of) all three B19 genotypes

(Hokynar et al. 2004, J Clin Microbiol)

Do the virus types 2 and 3 differ from virus type 1 in biology – or in epidemiology?

Of 140 000 plasma donations collected during 2002-2003,none contained B19 types 2 or 3

Tissues and Sera

Synovia + skin: from healthy adults with joint trauma, n=86

Skin: from patients with B19 unrelated dermatological disorders or from healthy laboratory staff, n=54

Tonsils: from patients with tonsillitis or tonsillar hypertrophy, n=220

Liver: collected for diagnostic purposes in Germany, n=77

Altogether 523 solid tissue samples

Sera: collected in 1983-1997 for virus diagnosis n=1640

PCRs used

x

x

x

Liver

x

x

x

Tonsil

x1, 2 and 3(5 DNA copies each)

RealArt Parvo B19 LC PCR

1, 3(1.5 and 15 copies, resp.)

Genotype 1 PCR

x x x2

(1.5 DNA copies)

Nested K71-PCR

x x1, 2 and 3(15 DNA copies each)

Nested VP1-PCR

x1, 2 and 3

(5 DNAcopies each) Non-nested VP1-PCR

SerumSynovium Skin Genotypes

detectable

(sensitivity)

PCR

Prevalence of virus types 1-3 in solid human tissues and sera

16483 (136)0% (0)17% (28)Serum

7736% (28)31 (24)32% (25)Liver

22082% (181)1% (3)16% (36)Tonsil

8657% (49)8% (7)35% (30)Synovia

14052% (73)17% (24)31% (43)Skin

NegativeVirus type 2Virus type 1Tissue

Virus type 3 was absent from all the tissues and sera studied

Norja et al, PNAS 2006;103:7450-3

B19 types in tissue by birth year

Conclusion IVirus type 1

Birth year range 1923-1996 (mean 1965, SD 14)

Virus type 2

Birth year range 1935-1972 (mean 1945, SD 10)

The new variant (virus type 2) is ”older” in occurence than the prototype virus (type 1); Both circulated in Northern Europe in equal frequency up to the 1950’s, after which virus type 2 disappeared from wide circulation.

Biological relations of B19 types 1-3

At the nucleotide level, the most striking sequence difference between the three B19 types occur within the p6 promoter→The promoter activities of the three virus types were measured in several B19 permissive and non-permissive cell-lines

NS1 acts as a transcription transactivator and, at the amino acid level, is the most divergent protein among the three B19 types→The effect of the NS1 of B19 type 1 on promoters of B19 types 1-3 was measured

All B19 types have been associated with anemia or aplastic crisis indicating erythroid tropism→The ability of B19 types 1-3 to infect B19 type-1 permissive cells was examined

The p6 promoter activities of the three B19 types were of similar strength in all cell types studied; strongest activity was observed in cell lines permissive for B19 prototype

Type 1 NS1 protein enhanced the activity of all three promoters equally

p6-promoter activities

InfectivityTwo B19-permissive cell lines were infected with B19 type 1, 2 or 3 containing plasma / sera

SPR3 (type 1), BTS, FinlandIM-81 (type 2), Baxter, AustriaD91.1 (type 3), Dr. Garbarg-Chenon, France

All three genotypes were able to infect these cells

IgG cross-reactivity of B19 types 1 and 2

Recombinant VLPs, composed either of VP2 protein alone or VP1 and VP2 together, of B19 type 1 and type 2 were produced in a baculovirus system.

Four EIAs, using as antigen VP2 or VP1/2 antigens of either virus type, were set up.

Sera from three groups of subjects were examined:

Sera from subjects carrying in tissue B19 type 1 DNA (n=24),B19 type 2 DNA (n=25)B19 IgG negative subjects (n=13)

100 nm

*) One sample showed borderline reactivity. **) The overall reactivity was equally low for both genotypes.

100 % IgG cross-reactivity between B19 types 1 and 2

VP2 VP1/2 VP2** VP1/2Sera EIA absorbance

B19 type 1 -carriers mean 2,650 2,698 1,038 2,189n=24 range 0.867 - 3.838 0.837 - 3.756 0.830 - 1.616 0.822 - 3.032

B19 type 2 -carriers mean 2,588 2,613 1,049 2,142n=25 range 0.451 - 3.343 0.456 - 3.539 0.134* - 1.358 0.351 - 2.686

B19 IgG-negatives mean 0,034 0,041 0,045 0,039n=13 SD 0,028 0,029 0,04 0,036

cut-off (mean + 3 SD) 0,119 0,128 0,166 0,146

ANTIGENTYPE 1 TYPE 2

Conclusion II

Despite their sequence and epidemiological differences, the three B19 types are highly similar variants of the same species

B19 types 1-3 constitute a single serotype

Acknowledgements

Dept. of Virology, University of Helsinki Maria Söderlund-Venermo, Klaus Hedman

Päivi Norja, Anna Ekman, Kalle Kantola, Laura Kakkola, Heidi Bonden, Anne Lahtinen, Simo Miettinen, Lea Hedman, Jaakko Lommi, Renwei Chen, Olli Vapalahti, Antti Vaheri,

CollaborationAnna-Maria Eis-Hübinger, Irja Davidkin, Beate Schneider, Hans-Peter Fischer, Rene Tolba, Matthias Gessner, Claudia Aberham, Antoine Garbarg-Chenon, Harri Laitinen, Eiji Morita, Kazuo Sugamura, Eiji Miyagawa, Frederic Morinet, Doris Morgan, Susanne Modrow

Clinical collaboratorsLeena-Maija Aaltonen, Annamari Ranki, Esa Partio, Olli Kiviluoto, Tomi Leivo