Development of an improved method of detection of infectious parvovirus B19

Abstract

Background Parvovirus B19, the only known pathogenic human parvovirus is the aetiologic agent of erythema infectiosum, transient aplastic crisis, pure red cell aplasia, and hydrops fetalis. Transmission is either by respiratory secretions or, as it can be present at high titre in plasma, by blood and blood products. B19 is only cultured with difficulty in vitro, and there is no readily available assay for detecting B19 infectivity or neutralizing antibodies. Objectives In this study, we evaluated different methods to detect viral infection for the purpose of developing automated methods for large-scale testing of viral infectivity, development of neutralizing antibody and viral inactivation assays. Study design Different cell lines were evaluated for their ability to support B19 infection and assays tested for sensitivity and ease of performing. A high-throughput assay was validated by determining infectious virus in blood pools and for determining neutralizing antibody in sera. Results B19 protein production was detected by immunofluorescence (IF) staining and increased viral DNA production by dot blot hybridization and quantitative PCR. The detection of RNA transcripts by RT-PCR assay and quantitative RT-PCR (qRT-PCR) was used as an indirect marker for infection. Of the cell lines tested, the subclone UT7/Epo-S1 showed the greatest sensitivity to B19 infection, with detection of viral transcripts by qRT-PCR the preferred assay. The assays were validated by experiments to determine the infectious titre of sera from acutely infected humans, to evaluate the presence of infectious virus in human donor plasma pools and to measure neutralizing antibodies.