First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS).

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

Snapshot of the Phylogenetic Relationships among Avian Poxviruses Circulating in Portugal between 2017 and 2023.

Avipoxvirus (APV), a linear dsDNA virus belonging to the subfamily Chordopoxvirinae of the family Poxviridae, infects more than 278 species of domestic and wild birds. It is responsible for causing avian pox disease, characterized by its cutaneous and diphtheric forms. With a high transmission capacity, it can cause high economic losses and damage to the ecosystem. Several diagnostic methods are available, and bird vaccination can be an effective preventive measure. Ten APV-positive samples were analyzed to update the molecular characterization and phylogenetic analysis of viruses isolated in Portugal between 2017 and 2023. A P4b gene fragment was amplified using a PCR, and the nucleotide sequence of the amplicons was determined using Sanger sequencing. The sequences obtained were aligned using ClustalW, and a maximum likelihood phylogenetic tree was constructed. With this study, it was possible to verify that the analyzed sequences are distributed in subclades A1, A2, B1, and B3. Since some of them are quite similar to others from different countries and obtained in different years, it is possible to conclude that there have been several viral introductions in Portugal. Finally, it was possible to successfully update the data on Avipoxviruses in Portugal.

A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices.

Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.

Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner.

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.

Loss of endothelial membrane KIT ligand affects systemic KIT ligand levels but not bone marrow hematopoietic stem cells.

A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.

TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity.

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.

Highlights of ionospheric investigations at Comandante Ferraz Brazilian Antarctic Station.

The ionospheric investigations have improved our understanding of the space weather role in the upper atmosphere conditions, particularly at higher latitudes where the geospace phenomena print their signatures. The simultaneous observations using multi-instruments have improved our knowledge of the coupling processes inside the ionosphere, and their connection with the magnetosphere and neutral atmosphere processes under the space weather phenomena. The ionosphere probing at EACF started on 1986 using an analogical very low frequency (VLF) system, and after the year 2004 using digital VLF system, global navigation satellite system (GNSS), riometers and Canadian digital ionosonde (CADI). This paper presents the different radio techniques that have been used at Brazilian Antarctic Station Comandante Ferraz (EACF) to characterize the ionospheric conditions, and the highlights of the studies using multi-instrument observations performed in the last few decades.

West Nile virus transmission potential in Portugal.

It is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country's adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966-2020), explored mosquito (2016-2019) and land type distributions (1992-2019), and used climate data (1981-2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.

Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures.

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.

Manipulating niche composition limits damage to haematopoietic stem cells during Plasmodium infection.

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and-coupled with reactive oxygen species quenching-enables partial rescuing of HSC function.

Single-cell lineage tracing approaches in hematology research: technical considerations.

The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

Divide and Rule: Mitochondrial Fission Regulates Quiescence in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) remain quiescent to preserve long-term integrity. In this issue of Cell Stem Cell, Hinge et al. (2020) and Liang et al. (2020) demonstrate that HSCs achieve this by regulating mitochondrial fission and lysosomal activity, suppressing glucose uptake, and maintaining healthy punctate mitochondria with low metabolic activity.

Biological implications of clonal hematopoiesis.

Adult hematological malignancies, such as acute myeloid leukemia, are thought to arise through the gradual acquisition of oncogenic mutations within long-lived hematopoietic stem cells (HSCs). Genomic analysis of peripheral blood DNA has recently identified leukemia-associated genetic mutations within otherwise healthy individuals, an observation that is strongly associated with age. These genetic mutations are often found at high frequency, suggesting dominance of a mutant HSC clone. Expansion of clones carrying other mutations not associated with leukemia or larger chromosomal deletions was also observed. This clinical observation has been termed clonal hematopoiesis, a condition associated with increased risk of both hematological malignancy and cardiovascular disease. Here, we discuss the identification of clonal hematopoiesis and its implications on human health, based on the May 2019 International Society for Experimental Hematology New Investigator Committee Webinar.

Development and validation of a real-time PCR for the detection and quantification of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is a spherical and non-enveloped virus belonging to the genus Circovirus of the Circoviridae family with a single stranded circular DNA genome. This virus, already detected worldwide, has been associated to several diseases and was implicated as the etiological agent of a disease named postweaning multisystemic wasting syndrome. Several methods have been described for the detection of PCV2, being real-time PCR the most simple and reliable. As far as we know, all the real-time PCR systems described until now are based on ORF2 gene, that exhibit the highest variability. This paper reports the development and validation of a real-time PCR targeted to ORF1 and based on a TaqMan probe for the detection of porcine circovirus type 2 DNA in swine samples. Due to the lack of PCV1 samples, the ability of the test to discriminate between PCV1 and PCV2 positive samples was evaluated in silico. Estimations of 100% specificity and 100% sensitivity were obtained based on the qPCR results with panel of 81 swine samples (known PCV2-positive (n = 50); known PCV2-negative (n = 17); samples positive to other common swine viral pathogens (n = 13) and one sample from a BFDV-positive parrot (n = 1)). Intra- and inter-assay coefficients of variation obtained with three positive samples of different viral charges in five replicates or in five independent assays were below the acceptance threshold. The limit of detection determined with a recombinant plasmid containing the amplicon, led to conclude that this assay can detect at least three plasmid copies.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.