Inactivation of parvovirus B19 during STIM-4 vapor heat treatment of three coagulation factor concentrates.

BACKGROUND: To enhance the viral safety margins, nanofiltration has been widely integrated into the manufacturing process of plasma-derived medicinal products. Removal of smaller agents such as parvovirus B19 (B19V) by filtration, however, is typically less efficient. Because recent investigations have demonstrated that B19V may be more heat sensitive than animal parvoviruses, the potential B19V inactivation by a proprietary vapor heating procedure (STIM-4) as incorporated into the manufacturing processes of several nanofiltered coagulation factor concentrates was investigated. STUDY DESIGN AND METHODS: An infectivity assay based on quantitative reverse transcription-polymerase chain reaction (TaqMan, Applied Biosystems) detection of B19V mRNA after inoculation of a permissive cell line (UT7 Epo S1 cells) was used to investigate the virus inactivation capacity of the STIM-4 vapor heat treatment as used during the manufacture of nanofiltered second-generation Factor VIII inhibitor-bypassing activity (FEIBA), F IX complex, and FVII products. RESULTS: In contrast to animal parvoviruses, both B19V genotypes investigated, that is, 1 and 2, were shown to be surprisingly effectively inactivated by the STIM-4 vapor heat treatment process, with mean log reduction factors of 3.5 to 4.8, irrespective of the product intermediate tested. CONCLUSION: The newly demonstrated effective inactivation of B19V by vapor heating, in contrast to the earlier used animal parvoviruses, results in significant B19V safety margins for STIM-4-treated coagulation factor concentrates.

Neutralization of human parvovirus B19 by plasma and intravenous immunoglobulins.

BACKGROUND: Human parvovirus B19 (B19V) is a highly prevalent pathogen, and plasma pools for manufacturing of plasma-derived products have been shown to contain antibodies against B19V (B19V immunoglobulin G [IgG]). STUDY DESIGN AND METHODS: The megakaryoblastic cell line UT7/Epo-S1 can be infected with B19V Genotype 1 and as demonstrated here by immunocytochemistry, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) of B19V-specific mRNA, also with the more recently discovered Genotype 2. Based on B19V RT-PCR analysis of infected UT7/Epo-S1 cells, an infectivity assay was established and implemented for a B19V neutralization assay. To investigate the role of B19V neutralization in relation to B19V IgG titers, more than 1,000 manufacturing plasma pools were tested by enzyme-linked immunosorbent assay. RESULTS: Plasma pools were found to contain a mean B19V IgG titer of 33 +/- 9 IU per mL, with the lowest titer at 11 IU per mL. These 11 IU per mL B19V IgG neutralized 4.6 log B19V Genotype 1 and greater than 3.9 log Genotype 2 infectivity. Accordingly, a 10 percent intravenous immunoglobulin (IVIG) product prepared from such pools was found to contain an even higher B19V neutralization capacity. CONCLUSION: A high capacity of B19V Genotypes 1 and 2 neutralization was demonstrated in plasma pools for fractionation, an inherent feature based on the constantly high titer of B19V IgG in these pools. The neutralizing activity of B19V IgG was shown to be maintained in the 10 percent IVIG product tested.

Biological and immunological relations among human parvovirus B19 genotypes 1 to 3.

The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by approximately 10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1- or type 2-infected subjects (long-term immunity) was examined with homo- and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.

Characterization of Parvovirus B19 genotype 2 in KU812Ep6 cells.

An infectious parvovirus B19 (B19V) genotype 2 variant was identified as a high-titer contaminant in a human plasma donation. Genome analysis revealed a 138-bp insertion within the p6 promoter. The inserted sequence was represented by an additional 30 bp from the end of the inverted terminal repeat adjacent to a 108-bp element found also, in inverted orientation, at the extreme right end of the unique sequence of the genome. However, despite the profound variations in the promoter region, the pattern of gene expression and DNA replication did not differ between genotype 1 and genotype 2 in permissive erythroid KU812Ep6 cells. Capsid proteins of both genotypes differ in their amino acid sequences. However, equivalent kinetics of virus inactivation at 56 degrees C or pH 4 indicated a comparable physicochemical stability of virus capsids. Sera from six individuals infected by B19V genotype 1 were investigated on cross-neutralization of B19V genotype 2 in vitro. Similar neutralization of both B19V genotypes was observed in sera from three individuals, while the sera from three other individuals showed weaker cross-neutralization for genotype 2. In conclusion, the in vitro replication characteristics and physical stability of B19V capsids are very similar between human parvovirus B19 genotypes 1 and 2, and cross-neutralization indicates a close antigenic relation of genotypes 1 and 2.

Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA.

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.

GABA-ergic modulation of prefrontal spatio-temporal activation pattern during emotional processing: a combined fMRI/MEG study with placebo and lorazepam.

Various prefrontal cortical regions have been shown to be activated during emotional stimulation, whereas neurochemical mechanisms underlying emotional processing in the prefrontal cortex remain unclear. We therefore investigated the influence of the GABA-A potentiator lorazepam on prefrontal cortical emotional-motor spatio-temporal activation pattern in a combined functional magnetic resonance imaging/magnetoencephalography study. Lorazepam led to the reversal in orbito-frontal activation pattern, a shift of the early magnetic field dipole from the orbito-frontal to medial prefrontal cortex, and alterations in premotor/motor cortical function during negative and positive emotional stimulation. It is concluded that negative emotional processing in the orbito-frontal cortex may be modulated either directly or indirectly by GABA-A receptors. Such a modulation of orbito-frontal cortical emotional function by lorazepam has to be distinguished from its effects on cortical motor function as being independent from the kind of processing either emotional or nonemotional.