1. GRIFOLS PLASMA: genotype 2 vB19 sample
Marta José, Instituto Grifols S.A., Barcelona, SPAIN
SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

2. Characterisation of a genotype 2 vB19 plasma sample: SAMPLE IDENTIFICATION
During the validation of our current vB19 PCR method, this plasma sample was analysed by two different PCR method with the following results:
An In house method, that we know that only detects the genotype 1 of vB19: NEGATIVE
Our in house method, that it was designed to identify the 3 vB19 genotypes: POSITIVE
Due to the discrepant results, we decide:
Quantify the sample
Genotype the sample
SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

3. Characterisation of a genotype 2 vB19 plasma sample: METHODS
PCR method
The sample was analysed using an in-house quantitative PCR method using primers located in the VP1 capsid structural protein region.
The PCR method is validated according to current guidelines for the following parameters: linearity, range, accuracy, precision, detection limit, quantitation limit, specificity and robustness.
The quantitation limit was established in 2.97 log10 IU/ml.
The method detects genotypes 1, 2 and 3 with the same efficacy.
Genotyping
Genotyping was carried out by a phylogenetic analysis as described by Candiotti et al (J. Virol 204, 76(22): ). To do this, a 1550 bp fragment of B19 genome (NS1-VP1 region) was sequenced.
SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

4. Characterisation of a genotype 2 vB19 plasma sample: RESULTS
The plasma sample is positive for vB19 DNA and negative for HCV RNA, HIV-1 RNA, HAV RNA, HBV DNA. It is also negative for antibodies anti-HCV and anti-HIV 1/2 and for HBsAg.
The titre was established in 1.90x107 IU/mL (7.3 log10 IU/mL) of vB19 DNA.
The sample is negative for antibodies anti-vB19 IgG and anti-vB19 IgM using commercial ELISAs (Biotrin).
Phylogenetic tree
AB030694
M24682
AF162273 NC
Z70528
M13178
Z70560
AF113323
AY386330
Isolate Sanquin
Isolate Sanquin
AY064476
AY064475
Isolate Sanquin
Sample Grifols
ref sample NIBSC
AY044266
AY083234
HER249437 NC
AX003421
100 77 50 69 80 98 56 70 51 32 63 53 37
0,01
B19 gt 1
B19 gt 2
B19 gt 3
By sequencing a 1550 bp fragment of B19 genome (NS1-VP1 region) the sample was identified like genotype 2 according to the following phylogenetic tree.
SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007