BACKGROUND:Viruses, among them parvovirus B19 and other small, nonenveloped viruses, may be present in human blood and may contaminate plasma-derived therapeutics. Efficient inactivation or removal of such viruses, especially parvoviruses, represents a current problem and corresponding technologies are under investigation. In this report, such a technology is described. STUDY DESIGN AND METHODS:A recently developed pasteurization of human apolipoprotein A-I (apoA-I), which is performed at 60 degrees C for 10 hours in the presence of guanidine hydrochloride (GdnHCl), was validated by using a series of model viruses, including members of the families parvoviridae and picornaviridae. The model viruses were spiked into the apoA-I- and GdnHCl-containing solutions, and virus inactivation was evaluated by infectivity assays in cell cultures. The mechanism of virus inactivation was studied by virus sedimentation analysis using the picornavirus model. RESULTS:All viruses tested were inactivated to levels below the limit of detection, although different inactivation kinetics were obtained for the different viruses. The mechanism of virus inactivation by this pasteurization was disassembly of the virus particles into single proteins or small noninfectious viral subunits. CONCLUSION:The pasteurization validated in this report has the potential to inactivate a wide range of transfusion-relevant viruses including parvoviruses and picornaviruses.

Background: IgG preparations have rarely transmitted infectious diseases; however, because such transmission has occurred a few times, manufacturers are required to present experimental proof that their specific production process removes and/or inactivates viruses that may be present in the starting material.Study Design and Methods: The kinetics of virus inactivation mediated by pepsin treatment at pH 4 during the production of intravenous immunoglobulin was assessed with spiking experiments using human immunodeficiency virus, bovine viral diarrhea virus, Semliki Forest virus, and pseudorabies virus. The influence of various factors on the rate of virus inactivation also was studied by modifying the composition of the IgG solutions with respect to IgG, sucrose, and NaCl content.Results: Virus inactivation at 37 degrees C was extremely rapid and resulted in a complete loss of infectivity within 5 minutes to 1 hour. Inactivation was much slower at lower temperatures. Furthermore, inactivation was dependent on the solute composition. Increasing the sucrose content from 0 to 15 percent reduced the rate of inactivation of pseudorabies virus but did not affect the rate of inactivation of Semliki Forest virus. in contrast, increasing the NaCl content from 0 to 150 mM resulted in a reduction in the rate of inactivation of Semliki Forest virus, whereas the rate of inactivation of pseudorabies virus remained unaffected. Moreover, increasing the IgG concentration from 0 to 10 percent resulted in an increased rate of inactivation of pseudorabies virus but a decreased rate of inactivation of Semliki Forest virus.Conclusion: Inactivation of viruses by pepsin treatment at pH 4 essentially is temperature-dependent, and the reaction rate is selectively influenced by the solute composition of the IgG solution. This has to be taken into account when safety data for different products are compared.

Abstract The ability to quantify low copy numbers of a specific nucleic acid target in limited amounts of sample is important in both basic and applied medical research. The combination of PCR and TGGE (temperature gradient gel electrophoresis) provides an exact and reliable system for such quantification (1).

Stimulation of secretion in exocrine cells by agonists involving cAMP as second messenger is associated with the phosphorylation of a specific membrane‐associated 22.4‐kDa protein (protein III) (Jahn). Here it is shown by subcellular fractionation of rat parotid gland lobules that protein III is associated with the endoplasmic reticulum. The submicrosomal fractions containing protein III, also contain the ATP‐dependent microsomal calcium pump activity. Protein III in microsomal subfractions can be phosphorylated in vitro with catalytic subunit from cAMP‐dependent protein kinase. Phosphorylated protein III contains exclusively P‐serine. Protein III can be removed from ER‐membranes with acid chloroform—methanol or Triton X‐114, but not by high salt wash indicating that it is tightly associated with the membranes. Protein III is smaller than phospholamban and, in contrast to phospholamban, resistant to heating in SDS. A relationship between phosphorylation of protein III and microsomal calcium sequestration is discussed.

ATP‐dependent calcium uptake was studied in isolated guinea pig parotid gland microsomes. The apparent K m for free Ca 2+ was 0.41 μM, the apparent K m for ATP·Mg 2− 0.23 mM. The pH optimum was 6.8–7.0. Subfractionation of the microsomes revealed that the highest specific uptake activity resided in a rather dense fraction of the endoplasmic reticulum. The calcium uptake/ATPase stoichiometry was determined in the absence of exogenous magnesium in the submicrosomal fractions. It ranged from 1–2. It is concluded that in vivo the stoichiometry is the same as in sarcoplasmic reticulum, namely 2.