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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