Hepatitis A virus in relation to contamination of factor VIII

Abstract

Outbreaks of hepatitis A amongst hemophiliacs receiving solvent detergent treated factor VIII in Europe and the USA have recently caused concern to blood product manufacturers and control agencies. In this study, the use of terminal dry heat treatment to inactivate HAV (strains HM175A.2 and HM175/18f) in spiked factor VIII was assessed using tissue culture based HAV assays, including a novel cytopathic microtitre plate assay, and a quantitative RT-PCR assay. The infectivity of HAV was quickly destroyed by heating to 80 °C. However, it was not until samples were heated to 100 °C for 8 hr that PCR positivity was lost. The use of nucleic acid amplification techniques in blood transfusion virology is increasing due to the lack of suitable cell culture assays for viruses such as HCV and parvovirus B19. This study shows that ability to detect viral genome in blood products does not necessarily imply the presence of infectious virus after the use of virucidal treatments. The relationship between PCR positivity and infectivity of HAV was further assessed in a time course study of cytopathic HAV HM175A.2 replication in BS-C-1 cells. Titres of released HAV RNA were similar to that of infectious virus with a ratio of 2 genome equivalents per infectious unit, whereas 19 genome equivalents per infectious unit were found associated with the cell. The infectious titre of contaminating HAV in blood products prior to viral inactivation was similar to that of HAV RNA. Comparison of infectious titres with genome copies of HAV during the time course demonstrated that little viral RNA was available for replication in the early stages of infection due to efficient encapsidation. The uptake and replication of the less cytopathic strain, HM175/18f, was slower than that of HM175A.2, however similar RNA titres were reached. The ability of HAV to cause cytopathology may be related to rate of replication. The attenuation of HM175A.2 and HM175/18f was studied in a tamarin model. HAV HM175/18f showed a degree of attenuation compared to wild type HM175 and HM175A.2 appeared to be completely attenuated. There was no difference in time of seroconversion between strains. HAV HM175A.2 may prove to be a suitable vaccine strain.