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GRIFOLS PLASMA: genotype 2 vB19 sample

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.

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GRIFOLS PLASMA: genotype 2 vB19 sample

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  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): 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

  4. AB030694 56 Phylogenetic tree M24682 Phylogenetic tree 70 AF162273 32 NC 000883 98 Z70528 63 B19 gt 1 M13178 Z70560 53 100 AF113323 37 AY386330 51 Isolate 90332 Sanquin Isolate 163429 Sanquin 80 AY064476 100 77 AY064475 B19 gt 2 Isolate 207458 Sanquin 100 Sample 420028819 Grifols ref sample NIBSC 69 AY044266 50 B19 gt 3 AY083234 HER249437 100 NC 004295 100 AX003421 0,01 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). • By sequencing a 1550bp 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

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