What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole.

UNLABELLED: The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species. IMPORTANCE: We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent.

Stiff matrix induces switch to pure ß-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular ß-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive ß-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-ß-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and ß-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express ß-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only ß-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of ß-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure ß-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation.

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks.

A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients' heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 106 CMs is expected. Moreover, to 'uncouple' cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation.

Blood-generating heart-forming organoids recapitulate co-development of the human haematopoietic system and the embryonic heart.

Despite the biomedical importance of haematopoietic stem cells and haematopoietic progenitor cells, their in vitro stabilization in a developmental context has not been achieved due to limited knowledge of signals and markers specifying the multiple haematopoietic waves as well as ethically restricted access to the human embryo. Thus, an in vitro approach resembling aspects of haematopoietic development in the context of neighbouring tissues is of interest. Our established human pluripotent stem cell-derived heart-forming organoids (HFOs) recapitulate aspects of heart, vasculature and foregut co-development. Modulating HFO differentiation, we here report the generation of blood-generating HFOs. While maintaining a functional ventricular-like heart anlagen, blood-generating HFOs comprise a mesenchyme-embedded haemogenic endothelial layer encompassing multiple haematopoietic derivatives and haematopoietic progenitor cells with erythro-myeloid and lymphoid potential, reflecting aspects of primitive and definitive haematopoiesis. The model enables the morphologically structured co-development of cardiac, endothelial and multipotent haematopoietic tissues equivalent to the intra-embryonic haematopoietic region in vivo, promoting research on haematopoiesis in vitro.

Protein-free media for cardiac differentiation of hPSCs in 2000 mL suspension culture.

BACKGROUND: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). METHODS AND RESULTS: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. CONCLUSIONS: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.

Transient stabilization of human cardiovascular progenitor cells from human pluripotent stem cells in vitro reflects stage-specific heart development in vivo.

AIMS: Understanding the molecular identity of human pluripotent stem cell (hPSC)-derived cardiac progenitors and mechanisms controlling their proliferation and differentiation is valuable for developmental biology and regenerative medicine. METHODS AND RESULTS: Here, we show that chemical modulation of histone acetyl transferases (by IQ-1) and WNT (by CHIR99021) synergistically enables the transient and reversible block of directed cardiac differentiation progression on hPSCs. The resulting stabilized cardiovascular progenitors (SCPs) are characterized by ISL1pos/KI-67pos/NKX2-5neg expression. In the presence of the chemical inhibitors, SCPs maintain a proliferation quiescent state. Upon small molecules, removal SCPs resume proliferation and concomitant NKX2-5 up-regulation triggers cell-autonomous differentiation into cardiomyocytes. Directed differentiation of SCPs into the endothelial and smooth muscle lineages confirms their full developmental potential typical of bona fide cardiovascular progenitors. Single-cell RNA-sequencing-based transcriptional profiling of our in vitro generated human SCPs notably reflects the dynamic cellular composition of E8.25-E9.25 posterior second heart field of mouse hearts, hallmarked by nuclear receptor sub-family 2 group F member 2 expression. Investigating molecular mechanisms of SCP stabilization, we found that the cell-autonomously regulated retinoic acid and BMP signalling is governing SCP transition from quiescence towards proliferation and cell-autonomous differentiation, reminiscent of a niche-like behaviour. CONCLUSION: The chemically defined and reversible nature of our stabilization approach provides an unprecedented opportunity to dissect mechanisms of cardiovascular progenitors' specification and reveal their cellular and molecular properties.

High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling.

To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

Human heart-forming organoids recapitulate early heart and foregut development.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

Patchy Occurrence of Cowpox Virus in Voles from Germany.

Cowpox virus (CPXV), genus Orthopoxvirus, family Poxviridae, is a zoonotic pathogen in Eurasian wild rodents. High seroprevalences have been reported previously for vole and murine species in Europe. In contrast, viral DNA was only rarely detected, and very few reservoir-derived CPXV isolates exist. In this study, CPXV DNA and CPXV-reactive antibodies were monitored in wild small mammals for 5 years in four German federal states. Screening of liver tissues of 3966 animals by CPXV real-time PCR (qPCR) revealed five voles of two species positive for CPXV DNA. Two positive bank voles (Myodes glareolus) and two positive common voles (Microtus arvalis) originated from two plots in Baden-Wuerttemberg. One positive bank vole originated from Mecklenburg-Western Pomerania. None of the small mammals from Thuringia and North Rhine-Westphalia was positive in the qPCR. CPXV antigen-based indirect immunofluorescence assays of 654 highly diluted chest cavity fluid samples detected two bank voles and two common voles from the same sites in Baden-Wuerttemberg to be highly seroreactive. Five animals were CPXV DNA positive, and four other animals were orthopoxvirus seropositive. Our study indicates both a very low prevalence and a patchy occurrence of CPXV in common and bank voles and absence in other rodent and shrew species in Germany. The multiple detection of infected voles at one site in Baden-Wuerttemberg and continued detection in a region of Mecklenburg-Western Pomerania classify these regions as potential endemic foci.

Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of NaV1.5 sodium channels.

Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit NaV1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-NaV1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells' immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27-42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-NaV1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs' relevance for investigating channelopathies in a dish.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.

Paracrine mechanisms in early differentiation of human pluripotent stem cells: Insights from a mathematical model.

With their capability to self-renew and differentiate into derivatives of all three germ layers, human pluripotent stem cells (hPSCs) offer a unique model to study aspects of human development in vitro. Directed differentiation towards mesendodermal lineages is a complex process, involving transition through a primitive streak (PS)-like stage. We have recently shown PS-like patterning from hPSCs into definitive endoderm, cardiac as well as presomitic mesoderm by only modulating the bulk cell density and the concentration of the GSK3 inhibitor CHIR99021, a potent activator of the WNT pathway. The patterning process is modulated by a complex paracrine network, whose identity and mechanistic consequences are poorly understood. To study the underlying dynamics, we here applied mathematical modeling based on ordinary differential equations. We compared time-course data of early hPSC differentiation to increasingly complex model structures with incremental numbers of paracrine factors. Model simulations suggest at least three paracrine factors being required to recapitulate the experimentally observed differentiation kinetics. Feedback mechanisms from both undifferentiated and differentiated cells turned out to be crucial. Evidence from double knock-down experiments and secreted protein enrichment allowed us to hypothesize on the identity of two of the three predicted factors. From a practical perspective, the mathematical model predicts optimal settings for directing lineage-specific differentiation. This opens new avenues for rational stem cell bioprocessing in more advanced culture systems, e.g. in perfusion-fed bioreactors enabling cell therapies.

Differential Expression of Cholinergic System Components in Human Induced Pluripotent Stem Cells, Bone Marrow-Derived Multipotent Stromal Cells, and Induced Pluripotent Stem Cell-Derived Multipotent Stromal Cells.

The components of the cholinergic system are evolutionary very old and conserved molecules that are expressed in typical spatiotemporal patterns. They are involved in signaling in the nervous system, whereas their functions in nonneuronal tissues are hardly understood. Stem cells present an attractive cellular system to address functional issues. This study therefore compared human induced pluripotent stem cells (iPSCs; from cord blood endothelial cells), mesenchymal stromal cells derived from iPSCs (iPSC-MSCs), and bone marrow-derived MSCs (BM-MSCs) from up to 33 different human donors with respect to gene expressions of components of the cholinergic system. The status of cells was identified and characterized by the detection of cell surface antigens using flow cytometry. Acetylcholinesterase expression in iPSCs declined during their differentiation into MSCs and was comparably low in BM-MSCs. Butyrylcholinesterase was present in iPSCs, increased upon transition from the three-dimensional embryoid body phase into monolayer culture, and declined upon further differentiation into iPSC-MSCs. In BM-MSCs a notable butyrylcholinesterase expression could be detected in only four donors, but was elusive in other patient-derived samples. Different nicotinic acetylcholine receptor subunits were preferentially expressed in iPSCs and during early differentiation into iPSC-MSCs, low expression was detected in iPS-MSCs and in BM-MSCs. The m2 and m3 variants of muscarinic acetylcholine receptors were detected in all stem cell populations. In BM-MSCs, these gene expressions varied between donors. Together, these data reveal the differential expression of cholinergic signaling system components in stem cells from specific sources and suggest the utility of our approach to establish informative biomarkers.

Laser bioprinting of human induced pluripotent stem cells-the effect of printing and biomaterials on cell survival, pluripotency, and differentiation.

Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient's own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells' behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

Classification of Cowpox Viruses into Several Distinct Clades and Identification of a Novel Lineage.

Cowpox virus (CPXV) was considered as uniform species within the genus Orthopoxvirus (OPV). Previous phylogenetic analysis indicated that CPXV is polyphyletic and isolates may cluster into different clades with two of these clades showing genetic similarities to either variola (VARV) or vaccinia viruses (VACV). Further analyses were initiated to assess both the genetic diversity and the evolutionary background of circulating CPXVs. Here we report the full-length sequences of 20 CPXV strains isolated from different animal species and humans in Germany. A phylogenetic analysis of altogether 83 full-length OPV genomes confirmed the polyphyletic character of the species CPXV and suggested at least four different clades. The German isolates from this study mainly clustered into two CPXV-like clades, and VARV- and VACV-like strains were not observed. A single strain, isolated from a cotton-top tamarin, clustered distantly from all other CPXVs and might represent a novel and unique evolutionary lineage. The classification of CPXV strains into clades roughly followed their geographic origin, with the highest clade diversity so far observed for Germany. Furthermore, we found evidence for recombination between OPV clades without significant disruption of the observed clustering. In conclusion, this analysis markedly expands the number of available CPXV full-length sequences and confirms the co-circulation of several CPXV clades in Germany, and provides the first data about a new evolutionary CPXV lineage.

Experimental Cowpox Virus (CPXV) Infections of Bank Voles: Exceptional Clinical Resistance and Variable Reservoir Competence.

Cowpox virus (CPXV) is a zoonotic virus and endemic in wild rodent populations in Eurasia. Serological surveys in Europe have reported high prevalence in different vole and mouse species. Here, we report on experimental CPXV infections of bank voles (Myodes glareolus) from different evolutionary lineages with a spectrum of CPXV strains. All bank voles, independently of lineage, sex and age, were resistant to clinical signs following CPXV inoculation, and no virus shedding was detected in nasal or buccal swabs. In-contact control animals became only rarely infected. However, depending on the CPXV strain used, inoculated animals seroconverted and viral DNA could be detected preferentially in the upper respiratory tract. The highest antibody titers and virus DNA loads in the lungs were detected after inoculation with two strains from Britain and Finland. We conclude from our experiments that the role of bank voles as an efficient and exclusive CPXV reservoir seems questionable, and that CPXV may be maintained in most regions by other hosts, including other vole species. Further investigations are needed to identify factors that allow and modulate CPXV maintenance in bank voles and other potential reservoirs, which may also influence spill-over infections to accidental hosts.

EBIO Does Not Induce Cardiomyogenesis in Human Pluripotent Stem Cells but Modulates Cardiac Subtype Enrichment by Lineage-Selective Survival.

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.

Fatal Cowpox Virus Infection in an Aborted Foal.

The article describes the isolation of a cowpox virus (CPXV) isolate originating from a horse. The skin of a foal, aborted in the third trimester, displayed numerous cutaneous papules. The histological examination showed A-type inclusion bodies within the lesion, typical for CPXV infections. This suspicion was confirmed by real-time PCR where various organs were analyzed. From skin samples, virus isolation was successfully performed. Afterwards, the whole genome of this new isolate "CPXV Amadeus" was sequenced by next-generation technology. Phylogenetic analysis clearly showed that "CPXV Amadeus" belongs to the "CPXV-like 1" clade. To our opinion, the study provides important additional information on rare accidental CPXV infections. From the natural hosts, the voles, species such as rats, cats, or different zoo animals are occasionally infected, but until now only two horse cases are described. In addition, there are new insights toward congenital CPXV infections.