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

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

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): 12469-12176). To do this, a 1550 bp fragment of B19 genome (NS1-VP1 region) was sequenced. SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

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 000883 Z70528 M13178 Z70560 AF113323 AY386330 Isolate 90332 Sanquin Isolate 163429 Sanquin AY064476 AY064475 Isolate 207458 Sanquin Sample 420028819 Grifols ref sample NIBSC AY044266 AY083234 HER249437 NC 004295 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