Novel parvovirus and related variant in human plasma

Abstract

We report a novel parvovirus (PARV4) and related variants in pooled human plasma used in the manufacture of plasma-derived medical products. Viral DNA was detected by using highly selective polymerase chain reaction assays; 5% of pools tested positive, and amounts of DNA ranged from <500 copies/mL to >106 copies/mL plasma.

Figure 1

A) Specificity of primers for PARV4. Samples in lanes 1–5 were amplified by using primers directed to open reading frame 1 (ORF1) of PARV4. Template DNA in lane 1 was a plasmid subclone of the PARV4 ORF1 region. In lane 2, the template DNA was derived from parvovirus B19 International Standard (99/800, National Institute for Biological Standards and Control, South Mimms, UK) as representative of genotype 1 erythrovirus sequences; in lane 3, the template DNA was derived from a genotype 2 erythrovirus plasmid clone (A6; obtained from K. Brown, National Heart, Lung and Blood Institute, Bethesda, MD, USA); in lane 4, the template DNA was derived from a genotype 3 erythrovirus plasmid clone (D91.1; obtained from A. Garbarg-Chenon, Hôpital Trousseau, Paris, France). Template DNA in the erythrovirus samples (lanes 2–4) was adjusted to give ≈10^5.5 copies of each genotype per reaction. Lane 5, no template control. Polymerase chain reaction (PCR) products were analyzed on a 2.5% agarose gel alongside PCR Markers (M) (Promega, Madison, WI, USA). B) Screening manufacturing plasma samples for PARV4. Samples in lanes 1–6 were amplified by using primers directed to the ORF1 region of PARV4. Template DNA in lanes 1 and 2 consisted of 1 × 10² and 1 × 10³ copies of the ORF1 subclone of PARV4. In lane 3, the template DNA was derived from a plasma pool containing 3.9 × 10⁶ PARV4 genome copies/mL plasma; in lane 4, the template DNA was derived from a plasma pool containing <500 PARV4 genome copies/mL plasma; in lane 5, the template DNA was derived from a plasma pool that tested negative for PARV4 sequences. Lane 6, no template control. PCR products were analyzed on a 2.5% agarose gel alongside PCR Markers (M) (Promega).

Figure 2

hylogenetic analysis of a 178-bp sequence of ORF1 of PARV4 and PARV5 (GenBank accession no. DQ112361) with other members of the Parvoviridae subfamily. The alignment includes the members of the Erythrovirus genus (parvovirus B19 [5]) and related viruses such as V9 (6), D91.1 (7), and A6 (8), as well as the closely related viruses infecting the cynomolgus macaque (LTMPV) (9) and rhesus (RMPV) and pig-tailed macaques (PTMPV) (10). Two other viruses tentatively assigned to the group include a parvovirus isolated from chipmunks (11); BPV3, a novel bovine parvovirus (BPV3) (12); and porcine parvovirus 2 (PPV2) (13). Analysis was performed by using the program ClustalW (14).