Growth and Distribution of Bacteria in Contaminated Whole Blood and Derived Blood Components.

Introduction: Bacterial contamination of blood products presumably occurs mainly during blood collection, starting from low initial concentrations of 10-100 colony-forming units (CFUs) per bag. As little is known about bacterial growth behavior and distribution in stored whole blood (WB) and WB-derived blood products, this study aims to provide data on this subject. Methods: WB units were inoculated with transfusion-relevant bacterial species (Acinetobacter baumannii, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas fluorescens, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus dysgalactiae, Streptococcus pyogenes, Yersinia enterocolitica; n = 12 for each species), stored for 22-24 h at room temperature, and then centrifuged for separation into plasma, red blood cells (RBCs), and buffy coats (BCs). The latter were pooled with 3 random donor BCs and one unit of PAS-E each to yield plasma-reduced platelet concentrates (PCs). Samples for bacterial colony counting were collected after WB storage and immediately after blood component production. Sterility testing in PCs (n = 12 for each species) was performed by bacterial culture after 7 days of storage. Results: Bacterial growth in WB varied remarkably between donations and species. Streptococcus species produced the highest titers in WB, whereas Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas fluorescens did not multiply. Centrifugation resulted in preferential accumulation of bacteria in BCs, with titers of up to 3.5 × 103 CFU/mL in BCs and up to ≤0.9 × 103 CFU/mL in BC-derived PCs. Overall, 72/144 PCs (50%) tested positive for bacteria after storage. Sterility test results were species-dependent, ranging from 12 of 12 PCs tested positive for Streptococcus pyogenes to 1 of 12 PCs positive for Escherichia coli. Bacterial contamination of RBC and plasma units was much less common and was associated with higher initial bacterial counts in the parent WB units. Conclusions: Bacterial growth in WB is species-dependent and varies greatly between donations. Preferential accumulation of bacteria in BCs during manufacturing is a critical determinant of the contamination risk of BC-derived pooled PCs.

Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices.

BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.

New ultraviolet C light-based method for pathogen inactivation of red blood cell units.

BACKGROUND: Pathogen inactivation (PI) technologies for platelet concentrates and plasma are steadily becoming more established, but new PI treatment options for red blood cells (RBCs), the most commonly used blood component, still need to be developed. We present a novel approach to inactivating pathogens in RBC units employing ultraviolet C (UVC) light. METHODS: Whole blood-derived leukoreduced RBCs suspended in PAGGS-C, a third generation additive solution, served as test samples, and RBCs in PAGGS-C or SAG-M as controls. Vigorous agitation and hematocrit reduction by diluting the RBCs with additional additive solution during illumination ensured that UVC light penetrated and inactivated the nine bacteria and eight virus species tested. Bacterial and viral infectivity assays and in vitro analyses were performed to evaluate the system's PI capacity and to measure the RBC quality, metabolic, functional, and blood group serological parameters of UVC-treated versus untreated RBCs during 36-day storage. RESULTS: UVC treatment of RBCs in the PAGGS-C additive solution did not alter RBC antigen expression, but significantly influenced some in vitro parameters. Compared to controls, hemolysis was higher in UVC-treated RBC units, but was still below 0.8% at 36 days of storage. Extracellular potassium increased early after PI treatment and reached ≤70 mmol/L by the end of storage. UVC-treated RBC units had higher glucose and 2,3-diphosphoglycerate levels than controls. CONCLUSION: As UVC irradiation efficiently reduces the infectivity of relevant bacteria and viruses while maintaining the quality of RBCs, the proposed method offers a new approach for PI of RBC concentrates.

Xeno-Free In Vitro Cultivation and Osteogenic Differentiation of hAD-MSCs on Resorbable 3D Printed RESOMER®.

The development of alloplastic resorbable materials can revolutionize the field of implantation technology in regenerative medicine. Additional opportunities to colonize the three-dimensionally (3D) printed constructs with the patient's own cells prior to implantation can improve the regeneration process but requires optimization of cultivation protocols. Human platelet lysate (hPL) has already proven to be a suitable replacement for fetal calf serum (FCS) in 2D and 3D cell cultures. In this study, we investigated the in vitro biocompatibility of the printed RESOMER® Filament LG D1.75 materials as well as the osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultivated on 3D printed constructs under the influence of different medium supplements (FCS, human serum (HS) and hPL). Additionally, the in vitro degradation of the material was studied over six months. We demonstrated that LG D1.75 is biocompatible and has no in vitro cytotoxic effects on hMSCs. Furthermore, hMSCs grown on the constructs could be differentiated into osteoblasts, especially supported by supplementation with hPL. Over six months under physiological in vitro conditions, a distinct degradation was observed, which, however, had no influence on the biocompatibility of the material. Thus, the overall suitability of the material LG D1.75 to produce 3D printed, resorbable bone implants and the promising use of hPL in the xeno-free cultivation of human MSCs on such implants for autologous transplantation have been demonstrated.

Digital droplet polymerase chain reaction to monitor ultraviolet C treatment of single-donor and buffy coat platelet units.

BACKGROUND: UVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology. STUDY DESIGN AND METHODS: Samples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out-of-specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross-validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed. RESULTS: Apheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single-nucleotide polymorphisms in the primer- and probe-binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra- and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment. CONCLUSION: The ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC-treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.

Hepatitis E virus is effectively inactivated in platelet concentrates by ultraviolet C light.

BACKGROUND AND OBJECTIVES: As previous investigations have shown, THERAFLEX UV-Platelets, a UVC-based pathogen inactivation (PI) system, is effective against non-enveloped transfusion-relevant viruses such as hepatitis A virus (HAV), which are insensitive to most PI treatments for blood products. This study investigated the PI efficacy of THERAFLEX UV-Platelets against HEV in platelet concentrates (PCs). MATERIALS AND METHODS: Buffy coat-derived PCs in additive solution were spiked with cell culture-derived HEV and treated with the THERAFLEX UV-Platelets system using various doses of UVC (0·05, 0·10, 0·15 and 0·20 (standard) J/cm2 ). Titres of infectious virus in pre- and post-treatment samples were determined using a large-volume plating assay to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets dose-dependently inactivated HEV in PCs. The standard UVC dose inactivated the virus to below the limit of detection, corresponding to a mean log reduction of greater than 3·5. CONCLUSION: Our study demonstrates that the THERAFLEX UV-Platelets system effectively inactivates HEV in PCs.

Inactivation of three emerging viruses - severe acute respiratory syndrome coronavirus, Crimean-Congo haemorrhagic fever virus and Nipah virus - in platelet concentrates by ultraviolet C light and in plasma by methylene blue plus visible light.

BACKGROUND: Emerging viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Crimean-Congo haemorrhagic fever virus (CCHFV) and Nipah virus (NiV) have been identified to pose a potential threat to transfusion safety. In this study, the ability of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate these viruses in platelet concentrates and plasma, respectively, was investigated. MATERIALS AND METHODS: Blood products were spiked with SARS-CoV, CCHFV or NiV, and then treated with increasing doses of UVC light (THERAFLEX UV-Platelets) or with methylene blue (MB) plus increasing doses of visible light (MB/light; THERAFLEX MB-Plasma). Samples were taken before and after treatment with each illumination dose and tested for residual infectivity. RESULTS: Treatment with half to three-fourths of the full UVC dose (0·2 J/cm2 ) reduced the infectivity of SARS-CoV (≥3·4 log), CCHFV (≥2·2 log) and NiV (≥4·3 log) to the limit of detection (LOD) in platelet concentrates, and treatment with MB and a fourth of the full light dose (120 J/cm2 ) decreased that of SARS-CoV (≥3·1 log), CCHFV (≥3·2 log) and NiV (≥2·7 log) to the LOD in plasma. CONCLUSION: Our study demonstrates that both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce the infectivity of SARS-CoV, CCHFV and NiV in platelet concentrates and plasma, respectively.

Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems.

In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media.

Gelatin-Methacryloyl (GelMA) Formulated with Human Platelet Lysate Supports Mesenchymal Stem Cell Proliferation and Differentiation and Enhances the Hydrogel's Mechanical Properties.

Three-dimensional (3D) cell culture is a major focus of current research, since cultivation under physiological conditions provides more reliable information about in vivo cell behavior. 3D cell cultures are used in basic research to better understand intercellular and cell-matrix interactions. However, 3D cell culture plays an increasingly important role in the in vitro testing of bioactive substances and tissue engineering. Gelatin-methacryloyl (GelMA) hydrogels of different degrees of functionalization (DoFs) are a versatile tool for 3D cell culture and related applications such as bioprinting. Human platelet lysate (hPL) has already demonstrated positive effects on 2D cell cultures of different cell types and has proven a valuable alternative to fetal calf serum (FCS). Traditionally, all hydrogels are formulated using buffers. In this study, we supplemented GelMA hydrogels of different DoF with hPL during adipose tissue-derived mesenchymal stem cell (AD-MSCs) encapsulation. We studied the effect of hPL supplementation on the spreading, proliferation, and osteogenic differentiation of AD-MSCs. In addition, the influence of hPL on hydrogel properties was also investigated. We demonstrate that the addition of hPL enhanced AD-MSC spreading, proliferation, and osteogenic differentiation in a concentration-dependent manner. Moreover, the addition of hPL also increased GelMA viscosity and stiffness.

Bacterial inactivation of platelet concentrates with the THERAFLEX UV-Platelets pathogen inactivation system.

BACKGROUND: The THERAFLEX UV-Platelets system (Maco Pharma) uses ultraviolet C (UVC) light for pathogen inactivation (PI) of platelet concentrates (PCs) without any additional photoactive compound. The aim of the study was to systematically investigate bacterial inactivation with this system under conditions of intended use. STUDY DESIGN AND METHODS: The robustness of the system was evaluated by assessing its capacity to inactivate high concentrations of different bacterial species in accordance with World Health Organization guidelines. The optimal use of the PI system was explored in time-to-treatment experiments by testing its ability to sterilize PCs contaminated with low levels of bacteria on the day of manufacture (target concentration, 100 colony-forming units/unit). The bacteria panel used for spiking experiments in this study included the World Health Organization International Repository Platelet Transfusion Relevant Reference Strains (n = 14), commercially available strains (n = 13), and in-house clinical isolates (n = 2). RESULTS: Mean log reduction factors after UVC treatment ranged from 3.1 to 7.5 and varied between different strains of the same species. All PCs (n = 12/species) spiked with up to 200 colony-forming units/bag remained sterile until the end of storage when UVC treated 6 hours after spiking. UVC treatment 8 hours after spiking resulted in single breakthrough contaminations with the fast-growing species Escherichia coli and Streptococcus pyogenes. CONCLUSION: The UVC-based THERAFLEX UV-Platelets system efficiently inactivates transfusion-relevant bacterial species in PCs. The comprehensive data from this study may provide a valuable basis for the optimal use of this UVC-based PI system.

Ultraviolet C light efficiently inactivates nonenveloped hepatitis A virus and feline calicivirus in platelet concentrates.

BACKGROUND: Nonenveloped transfusion-transmissible viruses such as hepatitis A virus (HAV) and hepatitis E virus (HEV) are resistant to many of the common virus inactivation procedures for blood products. This study investigated the pathogen inactivation (PI) efficacy of the THERAFLEX UV-Platelets system against two nonenveloped viruses: HAV and feline calicivirus (FCV), in platelet concentrates (PCs). STUDY DESIGN AND METHODS: PCs in additive solution were spiked with high titers of cell culture-derived HAV and FCV, and treated with ultraviolet C at various doses. Pre- and posttreatment samples were taken and the level of viral infectivity determined at each dose. For some samples, large-volume plating was performed to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets reduced HAV titers in PCs to the limit of detection, resulting in a virus reduction factor of greater than 4.2 log steps, and reduced FCV infectivity in PCs by 3.0 ± 0.2 log steps. CONCLUSIONS: THERAFLEX UV-Platelets effectively inactivates HAV and FCV in platelet units.

Inactivation of Ebola virus and Middle East respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene blue plus visible light, respectively.

BACKGROUND: Ebola virus (EBOV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have been identified as potential threats to blood safety. This study investigated the efficacy of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate EBOV and MERS-CoV in platelet concentrates (PCs) and plasma, respectively. STUDY DESIGN AND METHODS: PCs and plasma were spiked with high titers of cell culture-derived EBOV and MERS-CoV, treated with various light doses of ultraviolet C (UVC; THERAFLEX UV-Platelets) or methylene blue (MB) plus visible light (MB/light; THERAFLEX MB-Plasma), and assessed for residual viral infectivity. RESULTS: UVC reduced EBOV (≥4.5 log) and MERS-CoV (≥3.7 log) infectivity in PCs to the limit of detection, and MB/light decreased EBOV (≥4.6 log) and MERS-CoV (≥3.3 log) titers in plasma to nondetectable levels. CONCLUSIONS: Both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce EBOV and MERS-CoV infectivity in platelets and plasma, respectively.

Timing of gamma irradiation and blood donor sex influences in vitro characteristics of red blood cells.

BACKGROUND: There are few studies investigating the effect of irradiation on red blood cells (RBCs) during storage. This study analyzed changes in in vitro quality of RBCs irradiated at several points during storage with the aim of providing evidence to support current maximum pre- and postirradiation storage limits. STUDY DESIGN AND METHODS: Each of seven participating centers produced four pools of 7 standard RBC units (SAGM, AS-3, or PAGGSM), which were then split back into 7 units. All units in a pool were from sex-matched blood donors. Every week during 6 weeks of refrigerated storage, 1 unit was irradiated, while 1 unit was not irradiated (control). Units were tested weekly for biochemical variables, morphology, and mechanical fragility. RESULTS: The earlier during storage that units were irradiated, the higher the hemolysis and K+ at end of storage. Irrespective of the timing of irradiation, there was a rapid increase in extracellular K+ , followed by a more gradual increase in hemolysis. ATP levels decreased faster in irradiated units and were reduced below accepted values if irradiated early. Irradiated female RBCs had an absolute lower hemolysis and K+ level compared to male RBCs at all time points. CONCLUSIONS: The method of blood component manufacturing determined the absolute levels of hemolysis and potassium in irradiated and nonirradiated units, but did not influence the effect that timing of irradiation had on the in vitro quality characteristics. This study provides support for the current Council of Europe guidelines on the time limitations for the irradiation of RBCs.

Mitochondrial DNA multiplex real-time polymerase chain reaction inhibition assay for quality control of pathogen inactivation by ultraviolet C light in platelet concentrates.

BACKGROUND: Several ultraviolet (UV) light-based pathogen inactivation (PI) technologies for platelet (PLT) products have been developed or are under development. Upon implementation of PI technologies, quality control measures are required to ensure consistent efficiency of the treatment process. Previous reports showed that amotosalen/UVA and riboflavin/UV-based PI technologies induce modifications of the PLT-derived mitochondrial DNA (mtDNA) that can be detected by polymerase chain reaction (PCR) inhibition assays. In this study, we sought to establish a PCR inhibition assay to document the impact of ultraviolet C (UVC) treatment with the THERAFLEX UV-Platelets system on the mitochondrial genome in PLT concentrates (PCs). STUDY DESIGN AND METHODS: A multiplex real-time PCR inhibition assay with simultaneous short-amplicon (143 bp) and long-amplicon (794 bp) amplification was developed to detect mtDNA modifications in PLTs after UVC treatment. Assay performance was tested in UVC-treated and untreated, plasma-reduced pooled PCs, and apheresis PCs and challenged using PCs manufactured for a clinical trial under routine-like conditions. RESULTS: UVC illumination of PLTs resulted in dose-dependent inhibition of mtDNA amplification for the larger amplicon. Amplification of the shorter amplicon was not affected by UVC treatment. Evaluation of 283 blinded apheresis and pooled PLT samples from routine-like PC production resulted in prediction of UVC treatment status with 100% accuracy. CONCLUSION: The proposed dual-amplicon size real-time mtDNA PCR assay effectively detects nucleic acid damage induced by UVC illumination of PLTs and could be useful as an informative indicator of PI quality of the THERAFLEX UV-Platelets system.

Calcium spirulan derived from Spirulina platensis inhibits herpes simplex virus 1 attachment to human keratinocytes and protects against herpes labialis.

BACKGROUND: Chronic infections with herpes simplex virus (HSV) type 1 are highly prevalent in populations worldwide and cause recurrent oral lesions in up to 40% of infected subjects. OBJECTIVE: We investigated the antiviral activity of a defined Spirulina platensis microalga extract and of purified calcium spirulan (Ca-SP), a sulfated polysaccharide contained therein. METHODS: The inhibitory effects of HSV-1 were assessed by using a plaque reduction assay and quantitative PCR in a susceptible mammalian epithelial cell line and confirmed in human keratinocytes. Time-of-addition and attachment experiments and fluorescence detection of the HSV-1 tegument protein VP16 were used to analyze the mechanism of HSV-1 inhibition. Effects of Ca-SP on Kaposi sarcoma-associated herpesvirus/human herpes virus 8 replication and uptake of the ORF45 tegument protein were tested in human retinal pigment epithelial cells. In an observational trial the prophylactic effects of topically applied Ca-SP were compared with those of systemic and topical nucleoside analogues in 198 volunteers with recurrent herpes labialis receiving permanent lip makeup. RESULTS: Ca-SP inhibited HSV-1 infection in vitro with a potency at least comparable to that of acyclovir by blocking viral attachment and penetration into host cells. Ca-SP also inhibited entry of Kaposi sarcoma-associated herpesvirus/human herpes virus 8. In the clinical model of herpes exacerbation, the prophylactic effect of a Ca-SP and microalgae extract containing cream was superior to that of acyclovir cream. CONCLUSION: These data indicate a potential clinical use of Ca-SP containing Spirulina species extract for the prophylactic treatment of herpes labialis and suggest possible activity of Ca-SP against infections caused by other herpesviruses.

Viral inhibition of BAK promotes murine cytomegalovirus dissemination to salivary glands.

Apoptosis induction is an important host defense mechanism to control viral infection, which is antagonized by viral proteins. Murine cytomegalovirus m41.1 encodes a viral inhibitor of BAK oligomerization (vIBO) that blocks the mitochondrial apoptosis mediator BAK. However, its importance for viral fitness in vivo has not been investigated. Here, we show that an m41.1-deficient virus attains reduced titers in salivary glands of wild-type but not Bak1(-/-) mice, indicating a requirement of BAK inhibition for optimal dissemination in vivo.

Live or let die: manipulation of cellular suicide programs by murine cytomegalovirus.

Cytomegaloviruses (CMVs) are large double-stranded DNA viruses that replicate slowly and cause life-long persisting infections in their hosts. To achieve this, the CMVs had to evolve numerous countermeasures against innate and adaptive immune responses. Induction of programmed cell death is one important host defense mechanism against intracellular pathogens such as viruses. For a multicellular organism, it is advantageous to let infected cells die in order to thwart viral replication and dissemination. For a virus, by contrast, it is better to inhibit cell death and keep infected cells alive until the viral replication cycle has been completed. As a matter of fact, the CMVs encode a number of proteins devoted to interfering with different forms of programmed cell death: apoptosis and necroptosis. In this review, we summarize the known functions of the four best characterized cell death inhibitors of murine cytomegalovirus (MCMV), which are encoded by open reading frames, M36, m38.5, m41.1, and M45. The viral proteins interact with key molecules within different cell death pathways, namely caspase-8, Bax, Bak, and RIP1/RIP3. In addition, we discuss which events during MCMV infection might trigger apoptosis or necrosis and how MCMV's countermeasures compare to those of other herpesviruses. Since both, MCMV and its natural host, are amenable to genetic manipulation, the mouse model for CMV infection provides a particularly suitable system to study mechanisms of cell death induction and inhibition.

Mutations in the M112/M113-coding region facilitate murine cytomegalovirus replication in human cells.

Cytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host but are highly species specific. For instance, human cytomegalovirus (HCMV) does not replicate in mouse cells, and human cells are not permissive for murine cytomegalovirus (MCMV) infection. However, the underlying molecular mechanisms are so far poorly understood. In the present study we isolated and characterized a spontaneously occurring MCMV mutant that has gained the capacity to replicate rapidly and to high titers in human cells. Compared to the parental wild-type (wt) virus, this mutant formed larger nuclear replication compartments and replicated viral DNA more efficiently. It also disrupted promyelocytic leukemia (PML) protein nuclear domains with greater efficiency but caused less apoptosis than did wt MCMV. Sequence analysis of the mutant virus genome revealed mutations in the M112/M113-coding region. This region is homologous to the HCMV UL112-113 region and encodes the viral early 1 (E1) proteins, which are known to play an important role in viral DNA replication. By introducing the M112/M113 mutations into wt MCMV, we demonstrated that they are sufficient to facilitate MCMV replication in human cells and are, at least in part, responsible for the efficient replication capability of the spontaneously adapted virus. However, additional mutations probably contribute as well. These results reveal a previously unrecognized role of the viral E1 proteins in regulating viral replication in different cells and provide new insights into the mechanisms of the species specificity of cytomegaloviruses.

Generation and characterization of genetic reassortants between Puumala and Prospect Hill hantavirus in vitro.

Hantaviruses belong to the family Bunyaviridae characterized by tri-segmented RNA genomes. Depending on the hantavirus species, infection can lead to hantavirus cardiopulmonary or haemorrhagic fever with renal syndrome. In vitro studies suggest that pathogenic hantaviruses evade induction of innate antiviral responses, and this ability might determine the virulence in humans. Since reverse genetic systems are not available, in vitro reassortment is currently the only way to culture defined hantavirus variants. Here, we demonstrate for the first time the generation of a reassortant between a pathogenic Old World and a non-pathogenic New World hantavirus in vitro. The reassortant contained the glycoprotein coding M-segment derived from the pathogenic Puumala virus (PUUV) and the other genomic segments coding for the nucleocapsid protein and RNA-dependent RNA-polymerase from Prospect Hill virus (PHV), which is taken as non-pathogenic in humans. Exchange of the M-segment was confirmed by sequencing and virus neutralization test with PUUV-specific sera. Functional analysis of the reassortant and parental viruses revealed characteristic growth kinetics and innate immune responses as determined by expression analyses for lambda interferon and MxA, and by interferon-stimulated response element reporter gene studies. Consistent with previous studies with other pathogenic hantaviruses, PUUV elicited reduced innate responses if compared with PHV. In all these functional assays the reassortant revealed PHV-like phenotypes. Thus, neither the PUUV M-segment nor entry via specific M-segment directed pathways modulated the virus type-specific innate responses. Moreover, the data imply that this approach might be an option for production of attenuated viruses that could be used as vaccines against pathogenic hantaviruses.

Defective particles can lead to underestimated antibody titers in virus neutralization tests.

OBJECTIVE: Stocks of RNA viruses often contain divergent mixtures of infectious and defective particles (DPs). Since the surface of both particles share the same immunoreactivity, differing ratios of these particles in stocks used for virus neutralization tests might alter the results. Here the impact of such off-target effects were measured. METHODS: To determine the relative content of DPs in 3 stocks with high, medium and low titers of infectious virions, the amounts of nucleocapsid protein and viral RNA were quantified by Western blot and quantitative RT-PCR. Thereafter, stocks with different amount of DPs were used to determine the titer of neutralizing antibodies in serum from a convalescent Puumala virus-infected patient by a focus reduction neutralization test. RESULTS: The relative amount of DPs was at least 5-fold higher in the stock with the lowest titer compared to the stock with the highest. Stocks with lower levels of DPs led to higher antibody titers compared to the analysis performed with stocks containing higher levels of DPs. CONCLUSION: These results imply that DPs in virus stocks should be considered when performing virus neutralization tests to quantify neutralizing antibodies.