The establishment of an infectivity assay for human parvovirus B19 to investigate the efficacy of protocols for the inactiviation of pathogens from plasma products

Chaput, Cécile Claire (2005). The establishment of an infectivity assay for human parvovirus B19 to investigate the efficacy of protocols for the inactiviation of pathogens from plasma products. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fe6b

Abstract

Erythrovirus B19 is the only parvovirus known to cause disease in humans and represents a concern in transfusion medicine since several blood products were shown to contain B19 despite various inactivation steps and some products have even transmitted viral infection. The aim of the study was to establish a reliable and reproducible infectivity assay that could be used to evaluate the efficiency of viral removal/inactivation methods. This assay was based on the detection of B 19-specific mRNA transcripts from susceptible cells inoculated with virus: the human leukaemic cell line UT-7/EPO-S1. B19 DNA concentrations were also determined using an optimised quantitative real-time PCR assay. Both assays were used to assess the efficacy of one virus removal method, nanofiltration, and four virus inactivation techniques, dry heating treatment, supercritical fluids, INACTINE™ system and Helinx® technology. Nanofiltration of a spiked solution of 0.5% albumin with 15N and 20N filters resulted in the removal of more than logio 5 and logio 6 of infectious B19, respectively. Dry heat treatment of factor VIE showed a reduction of infectivity greater than log₁₀ 2 after 24 hours and below the limit of detection of the assay after 72 hours. The treatment of plasma with the SuperFluids™ system removed more than log₁₀ 5 infectious viruses. Two chemicals methods used novel small molecules which target and crosslink nucleic acids: PEN 110 (INACTINE™ system) and Psoralen S-59 (Helinx® technology). Treatment of red cells with PEN 110 showed a reduction of infectivity below the detection limit while platelet concentrates treated with S-59 resulted in a reduction greater than log₁₀ 5. Although chemical methods can be used on cells which do not have a nucleus (red cells and platelets), they also introduce potential toxicity. In contrast, physical methods of removal and inactivation are less toxic but would damage red cells and platelets. Overall, this research work allowed the establishment of a reproducible infectivity assay that was successfully used to perform preliminary studies on the efficacy of various viral removal/inactivation methods.