Efficient inactivation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in human apheresis platelet concentrates with amotosalen and ultraviolet A light.

OBJECTIVES: The detection of SARS-CoV-2 RNA in blood and platelet concentrates from asymptomatic donors, and the detection of viral particles on the surface and inside platelets during in vitro experiments, raised concerns over the potential risk for transfusion-transmitted-infection (TTI). The objective of this study was to assess the efficacy of the amotosalen/UVA pathogen reduction technology for SARS-CoV-2 in human platelet concentrates to mitigate such potential risk. MATERIAL AND METHODS: Five apheresis platelet units in 100% plasma were spiked with a clinical SARS-CoV-2 isolate followed by treatment with amotosalen/UVA (INTERCEPT Blood System), pre- and posttreatment samples were collected as well as untreated positive and negative controls. The infectious viral titer was assessed by plaque assay and the genomic titer by quantitative RT-PCR. To exclude the presence of infectious particles post-pathogen reduction treatment below the limit of detection, three consecutive rounds of passaging on permissive cell lines were conducted. RESULTS: SARS-CoV-2 in platelet concentrates was inactivated with amotosalen/UVA below the limit of detection with a mean log reduction of>3.31±0.23. During three consecutive rounds of passaging, no viral replication was detected. Pathogen reduction treatment also inhibited nucleic acid detection with a log reduction of>4.46±0.51 PFU equivalents. CONCLUSION: SARS-CoV-2 was efficiently inactivated in platelet concentrates by amotosalen/UVA treatment. These results are in line with previous inactivation data for SARS-CoV-2 in plasma as well as MERS-CoV and SARS-CoV-1 in platelets and plasma, demonstrating efficient inactivation of human coronaviruses.

Amotosalen and ultraviolet A light efficiently inactivate MERS-coronavirus in human platelet concentrates.

OBJECTIVE: This study aimed to assess the efficacy of the INTERCEPT™ Blood System [amotosalen/ultraviolet A (UVA) light] to reduce the risk of Middle East respiratory syndrome-Coronavirus (MERS-CoV) transmission by human platelet concentrates. BACKGROUND: Since 2012, more than 2425 MERS-CoV human cases have been reported in 27 countries. The infection causes acute respiratory disease, which was responsible for 838 deaths in these countries, mainly in Saudi Arabia. Viral genomic RNA was detected in whole blood, serum and plasma of infected patients, raising concerns of the safety of blood supplies, especially in endemic areas. METHODS: Four apheresis platelet units in 100% plasma were inoculated with a clinical MERS-CoV isolate. Spiked units were then treated with amotosalen/UVA to inactivate MERS-CoV. Infectious and genomic viral titres were quantified by plaque assay and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). Inactivated samples were successively passaged thrice on Vero E6 cells to exclude the presence of residual replication-competent viral particles in inactivated platelets. RESULTS: Complete inactivation of MERS-CoV in spiked platelet units was achieved by treatment with Amotosalen/UVA light with a mean log reduction of 4·48 ± 0·3. Passaging of the inactivated samples in Vero E6 showed no viral replication even after nine days of incubation and three passages. Viral genomic RNA titration in inactivated samples showed titres comparable to those in pre-treatment samples. CONCLUSION: Amotosalen and UVA light treatment of MERS-CoV-spiked platelet concentrates efficiently and completely inactivated MERS-CoV infectivity (>4 logs), suggesting that such treatment could minimise the risk of transfusion-related MERS-CoV transmission.

Cytomegaloviruses inhibit Bak- and Bax-mediated apoptosis with two separate viral proteins.

Apoptosis of infected cells can limit virus replication and serves as an innate defense mechanism against viral infections. Consequently, viruses delay apoptosis by expressing antiapoptotic proteins, many of which structurally resemble the cellular antiapoptotic protein Bcl-2. Like Bcl-2, the viral analogs inhibit apoptosis by preventing activation and/or oligomerization of the proapoptotic mitochondrial proteins Bax and Bak. Here we show that cytomegaloviruses (CMVs) have adopted a different strategy. They encode two separate mitochondrial proteins that lack obvious sequence similarities to Bcl-2-family proteins and specifically counteract either Bax or Bak. We identified a small mitochondrion-localized protein encoded by the murine CMV open reading frame (ORF) m41.1, which functions as a viral inhibitor of Bak oligomerization (vIBO). It blocks Bak-mediated cytochrome c release and Bak-dependent induction of apoptosis. It protects cells from cell death-inducing stimuli together with the previously identified Bax-specific inhibitor viral mitochondria-localized inhibitor of apoptosis (vMIA) (encoded by ORF m38.5). Similar vIBO proteins are encoded by CMVs of rats, and possibly by other CMVs as well. These results suggest a non-redundant function of Bax and Bak during viral infection, and a benefit for CMVs derived from the ability to inhibit Bak and Bax separately with two viral proteins.

Foamy virus--adenovirus hybrid vectors.

To confer adenovirus vectors (AdV), the feature of integration into the host cell genome hybrid vectors were characterized in vitro, which express vectors derived from the prototypic foamy virus (FV) in the backbone of a high-capacity AdV. FVs constitute a subfamily of retroviruses with a distinct replication pathway and no known pathogenicity. In the absence of envelope glycoprotein, the prototypic FV behaves like a retrotransposon, while it behaves like an exogenous retrovirus in its presence. Two principle types of vectors, which either allows the intracellular (HC-FAD-7) or, in addition, the extracellular (HC-FAD-2) pathway were constructed. In both chimeras the expression of the FV vector was controlled by the tetracycline-regulatable system. Hybrids were produced close to 10(10) infectious units/ml. By Southern blotting, the functionality of the hybrid vectors to generate host cell genomic integrants was shown. However, the efficiency of HC-FAD-7 to establish stable transgene expression was rather low, while around 70% of cells were stably transduced in secondary round following primary transduction with HC-FAD-2 at an MOI of 100. Given the benign characteristics of high-capacity adenovirus and FV vectors, hybrids based on HC-FAD-2 are probably suited for an in vivo application.

A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.

Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.