Use of Single-Domain Antibodies Against the NSm Protein for the Detection of Cells Infected by Rift Valley Fever Virus.

Single-domain antibodies, referred to as VHH (variable heavy chains of heavy chain-only antibodies) or in their commercial name as nanobodies, are potent tools for the detection of target proteins in biological samples. They have the advantage of being highly stable, specific, and sensitive, with affinities reaching the nanomolar range. We utilized this tool to develop a rapid detection method that discriminates cells infected with Rift Valley fever virus (RVFV), based on the intracellular detection of the viral nonstructural NSm protein localized on the outer membrane of mitochondria. Here we describe how NSm-specific VHHs have been produced, cloned, and characterized, highlighting their value in RVFV research and diagnosis. This work may also raise interest in other potential applications such as antiviral therapy.

Intranasal Exposure to Rift Valley Fever Virus Live-Attenuated Strains Leads to High Mortality Rate in Immunocompetent Mice.

Rift Valley fever virus (RVFV) is a pathogenic arthropod-borne virus that can cause serious illness in both ruminants and humans. The virus can be transmitted by an arthropod bite or contact with contaminated fluids or tissues. Two live-attenuated veterinary vaccines-the Smithburn (SB) and Clone 13 (Cl.13)-are currently used during epizootic events in Africa. However, their residual pathogenicity (i.e., SB) or potential of reversion (i.e., Cl.13) causes important adverse effects, strongly limiting their use in the field. In this study, we infected immunocompetent mice with SB or Cl.13 by a subcutaneous or an intranasal inoculation. Interestingly, we found that, unlike the subcutaneous infection, the intranasal inoculation led to a high mortality rate. In addition, we detected high titers and viral N antigen levels in the brain of both the SB- and Cl.13-infected mice. Overall, we unveil a clear correlation between the pathogenicity and the route of administration of both SB and Cl.13, with the intranasal inoculation leading to a stronger neurovirulence and higher mortality rate than the subcutaneous infection.

Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins.

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.

The ring finger protein 213 gene (Rnf213) contributes to Rift Valley fever resistance in mice.

Rift Valley fever (RVF) is an emerging viral zoonosis that primarily affects ruminants and humans. We have previously shown that wild-derived MBT/Pas mice are highly susceptible to RVF virus and that part of this phenotype is controlled by a locus located on distal Chromosome 11. Using congenic strains, we narrowed down the critical interval to a 530 kb region containing five protein-coding genes among which Rnf213 emerged as a potential candidate. We generated Rnf213-deficient mice by CRISPR/CAS9 on the C57BL/6 J background and showed that they were significantly more susceptible to RVF than control mice, with an average survival time post-infection reduced from 7 to 4 days. The human RNF213 gene had been associated with the cerebrovascular Moyamoya disease (MMD or MYMY) but the inactivation of this gene in the mouse resulted only in mild anomalies of the neovascularization. This study provides the first evidence that the Rnf213 gene may also impact the resistance to infectious diseases such as RVF.

Identification of Umbre Orthobunyavirus as a Novel Zoonotic Virus Responsible for Lethal Encephalitis in 2 French Patients with Hypogammaglobulinemia.

BACKGROUND: Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients. METHODS: Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis. RESULTS: Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals. CONCLUSIONS: Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe.

The NSs Protein Encoded by the Virulent Strain of Rift Valley Fever Virus Targets the Expression of Abl2 and the Actin Cytoskeleton of the Host, Affecting Cell Mobility, Cell Shape, and Cell-Cell Adhesion.

Rift Valley fever virus (RVFV) is a highly pathogenic zoonotic arbovirus endemic in many African countries and the Arabian Peninsula. Animal infections cause high rates of mortality and abortion among sheep, goats, and cattle. In humans, an estimated 1 to 2% of RVFV infections result in severe disease (encephalitis, hepatitis, or retinitis) with a high rate of lethality when associated with hemorrhagic fever. The RVFV NSs protein, which is the main virulence factor, counteracts the host innate antiviral response to favor viral replication and spread. However, the mechanisms underlying RVFV-induced cytopathic effects and the role of NSs in these alterations remain for the most part unknown. In this work, we have analyzed the effects of NSs expression on the actin cytoskeleton while conducting infections with the NSs-expressing virulent (ZH548) and attenuated (MP12) strains of RVFV and the non-NSs-expressing avirulent (ZH548ΔNSs) strain, as well as after the ectopic expression of NSs. In macrophages, fibroblasts, and hepatocytes, NSs expression prevented the upregulation of Abl2 (a major regulator of the actin cytoskeleton) expression otherwise induced by avirulent infections and identified here as part of the antiviral response. The presence of NSs was also linked to an increased mobility of ZH548-infected cells compared to ZH548ΔNSs-infected fibroblasts and to strong changes in cell morphology in nonmigrating hepatocytes, with reduction of lamellipodia, cell spreading, and dissolution of adherens junctions reminiscent of the ZH548-induced cytopathic effects observed in vivo Finally, we show evidence of the presence of NSs within long actin-rich structures associated with NSs dissemination from NSs-expressing toward non-NSs-expressing cells.IMPORTANCE Rift Valley fever virus (RVFV) is a dangerous human and animal pathogen that was ranked by the World Health Organization in 2018 as among the eight pathogens of most concern for being likely to cause wide epidemics in the near future and for which there are no, or insufficient, countermeasures. The focus of this work is to address the question of the mechanisms underlying RVFV-induced cytopathic effects that participate in RVFV pathogenicity. We demonstrate here that RVFV targets cell adhesion and the actin cytoskeleton at the transcriptional and cellular level, affecting cell mobility and inducing cell shape collapse, along with distortion of cell-cell adhesion. All these effects may participate in RVFV-induced pathogenicity, facilitate virulent RVFV dissemination, and thus constitute interesting potential targets for future development of antiviral therapeutic strategies that, in the case of RVFV, as with several other emerging arboviruses, are presently lacking.

Comparison of dengue case classification schemes and evaluation of biological changes in different dengue clinical patterns in a longitudinal follow-up of hospitalized children in Cambodia.

BACKGROUND: The World Health Organization (WHO) proposed guidelines on dengue clinical classification in 1997 and more recently in 2009 for the clinical management of patients. The WHO 1997 classification defines three categories of dengue infection according to severity: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Alternative WHO 2009 guidelines provide a cross-sectional classification aiming to discriminate dengue fever from dengue with warning signs (DWWS) and severe dengue (SD). The primary objective of this study was to perform a comparison of two dengue classifications. The secondary objective was to describe the changes of hematological and biochemical parameters occurring in patients presenting with different degrees of severity during the course of the disease, since progression to more severe clinical forms is unpredictable. METHODOLOGY/PRINCIPAL FINDINGS: We performed a prospective, monocentric, cross-sectional study of hospitalized children in Cambodia, aged from 2 to 15 years old with severe and non-severe dengue. We enrolled 243 patients with acute dengue-like illness: 71.2% were dengue infections confirmed using quantitative reverse transcription PCR or NS1 antigen capture ELISA, of which 87.2% and 9.0% of DF cases were respectively classified DWWS and SD, and 35.9% of DHF were designated SD using an adapted WHO 2009 classification for SD case definition. Systematic use of ultrasound at patient admission was crucial for detecting plasma leakage. No difference was observed in the concentration of secreted NS1 protein between different dengue severity groups. Lipid profiles were different between DWWS and SD at admission, characterized by a decrease in total cholesterol, HDL cholesterol, and LDL cholesterol, in SD. CONCLUSIONS/SIGNIFICANCE: Our results show discrepancies between the two classifications, including misclassification of severe dengue cases as mild cases by the WHO 1997 classification. Using an adapted WHO 2009 classification, SD more precisely defines the group of patients requiring careful clinical care at a given time during hospitalization.

NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice.

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.

Genetic dissection of Rift Valley fever pathogenesis: Rvfs2 locus on mouse chromosome 11 enables survival to early-onset hepatitis.

Infection of mice with Rift Valley fever virus (RVFV) reproduces major pathological features of severe human disease, notably the early-onset hepatitis and delayed-onset encephalitis. We previously reported that the Rvfs2 locus from the susceptible MBT/Pas strain reduces survival time after RVFV infection. Here, we used BALB/cByJ (BALB) mice congenic for Rvfs2 (C.MBT-Rvfs2) to investigate the pathophysiological mechanisms impacted by Rvfs2. Clinical, biochemical and histopathological features indicated similar liver damage in BALB and C.MBT-Rvfs2 mice until day 5 after infection. However, while C.MBT-Rvfs2 mice succumbed from acute liver injury, most BALB mice recovered and died later of encephalitis. Hepatocytes of BALB infected liver proliferated actively on day 6, promoting organ regeneration and recovery from liver damage. By comparison with C.MBT-Rvfs2, BALB mice had up to 100-fold lower production of infectious virions in the peripheral blood and liver, strongly decreased RVFV protein in liver and reduced viral replication in primary cultured hepatocytes, suggesting that the BALB Rvfs2 haplotype limits RVFV pathogenicity through decreased virus replication. Moreover, bone marrow chimera experiments showed that both hematopoietic and non-hematopoietic cells are required for the protective effect of the BALB Rvfs2 haplotype. Altogether, these results indicate that Rvfs2 controls critical events which allow survival to RVFV-induced hepatitis.

Genetic Diversity of Collaborative Cross Mice Controls Viral Replication, Clinical Severity, and Brain Pathology Induced by Zika Virus Infection, Independently of Oas1b.

The explosive spread of Zika virus (ZIKV) has been associated with major variations in severe disease and congenital afflictions among infected populations, suggesting an influence of host genes. We investigated how genome-wide variants could impact susceptibility to ZIKV infection in mice. We first describe that the susceptibility of Ifnar1-knockout mice is largely influenced by their genetic background. We then show that Collaborative Cross (CC) mice, which exhibit a broad genetic diversity, in which the type I interferon receptor (IFNAR) was blocked by an anti-IFNAR antibody expressed phenotypes ranging from complete resistance to severe symptoms and death, with large variations in the peak and the rate of decrease in the plasma viral load, in the brain viral load, in brain histopathology, and in the viral replication rate in infected cells. The differences in susceptibility to ZIKV between CC strains correlated with the differences in susceptibility to dengue and West Nile viruses between the strains. We identified highly susceptible and resistant mouse strains as new models to investigate the mechanisms of human ZIKV disease and other flavivirus infections. Genetic analyses revealed that phenotypic variations are driven by multiple genes with small effects, reflecting the complexity of ZIKV disease susceptibility in the human population. Notably, our results rule out the possibility of a role of the Oas1b gene in the susceptibility to ZIKV. Altogether, the findings of this study emphasize the role of host genes in the pathogeny of ZIKV infection and lay the foundation for further genetic and mechanistic studies.IMPORTANCE In recent outbreaks, ZIKV has infected millions of people and induced rare but potentially severe complications, including Guillain-Barré syndrome and encephalitis in adults. While several viral sequence variants were proposed to enhance the pathogenicity of ZIKV, the influence of host genetic variants in mediating the clinical heterogeneity remains mostly unexplored. We addressed this question using a mouse panel which models the genetic diversity of the human population and a ZIKV strain from a recent clinical isolate. Through a combination of in vitro and in vivo approaches, we demonstrate that multiple host genetic variants determine viral replication in infected cells and the clinical severity, the kinetics of blood viral load, and brain pathology in mice. We describe new mouse models expressing high degrees of susceptibility or resistance to ZIKV and to other flaviviruses. These models will facilitate the identification and mechanistic characterization of host genes that influence ZIKV pathogenesis.

Landscape genetic analyses of Cervus elaphus and Sus scrofa: comparative study and analytical developments.

Red deer and wild boar are two major game species whose populations are managed and live in areas impacted by human activities. Measuring and understanding the impact of landscape features on individual movements and spatial patterns of genetic variability in these species is thus of importance for managers. A large number of individuals sampled across Wallonia (Belgium) for both species have been genotyped using microsatellite markers (respectively > 1700 and > 1200 genotyped individuals) and some individuals have also been followed using a capture-mark-recapture (CMR) protocol. The combined data set represents an unprecedented opportunity to study and compare the environmental factors impacting the interconnectivity of these large mammals. The present study describes and uses a landscape genetic workflow to compare spatial patterns of genetic variability and the impact of environmental factors on genetic differentiation. For the latter analyses, we investigate the correlation between genetic and environmental distances (pairwise approach) and also between local genetic dissimilarity and environmental conditions (point approach). Preliminary analyses of CMR data confirm that motorways act as significant barriers to dispersal. However, analyses performed with the pairwise approach do not highlight any evidence of an impact of motorways on genetic differentiation, which is presumably due to their recent establishment. Complementary analyses performed with the point approach reveal that low altitude tends to be associated with higher genetic dissimilarity. From a methodological point of view, the present workflow illustrates the complementary application of both pairwise and point approaches, as well as univariate and multivariate analyses.

Stress-induced unfolded protein response contributes to Zika virus-associated microcephaly.

Accumulating evidence support a causal link between Zika virus (ZIKV) infection during gestation and congenital microcephaly. However, the mechanism of ZIKV-associated microcephaly remains unclear. We combined analyses of ZIKV-infected human fetuses, cultured human neural stem cells and mouse embryos to understand how ZIKV induces microcephaly. We show that ZIKV triggers endoplasmic reticulum stress and unfolded protein response in the cerebral cortex of infected postmortem human fetuses as well as in cultured human neural stem cells. After intracerebral and intraplacental inoculation of ZIKV in mouse embryos, we show that it triggers endoplasmic reticulum stress in embryonic brains in vivo. This perturbs a physiological unfolded protein response within cortical progenitors that controls neurogenesis. Thus, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex, whereupon sustained endoplasmic reticulum stress leads to apoptosis. Furthermore, we demonstrate that administration of pharmacological inhibitors of unfolded protein response counteracts these pathophysiological mechanisms and prevents microcephaly in ZIKV-infected mouse embryos. Such defects are specific to ZIKV, as they are not observed upon intraplacental injection of other related flaviviruses in mice.

Using genetic tools to estimate the prevalence of non-native red deer (Cervus elaphus) in a Western European population.

Game species like the red deer have been subjected to anthropogenic impacts for centuries. Translocations are often carried out-sometimes illegally-not only for sporting purposes, but also to increase trophy quality, reduce inbreeding, or mitigate bottlenecks after excessive persecution. Apart from the blurring of large-scale genetic structure, translocations without adequate quarantine measure risk introducing pathogens into potentially immunologically naïve populations. It is therefore important to understand the frequency of clandestine translocations. Identification of non-autochthonous animals and their potential origin is often difficult and, in red deer, has been hampered by the lack of large-scale genotypic datasets for comparison. In the present study, we make use of a recently published European-wide microsatellite dataset to detect and quantify the presence of non-autochthonous red deer in a large population sample (n = 1,780) from Central Europe (Belgium). Using factorial correspondence analysis, assignment tests and Bayesian clustering algorithms we arrive at an estimate of 3.7% non-autochthonous animals (or their descendants). Some of these animals were assigned to a nearby French population and may have immigrated into Belgium naturally, but the large majority must have been introduced by humans. Our analysis pointed to the British Isles and Germany/Poland as the potential origin of many introduced deer, regions known to have been source populations for translocations in Europe and beyond. We found evidence for recreational hunters using carcasses from farmed deer to fulfill mandatory hunting quotas. Our study is the first to quantify the extent of human-mediated introductions in a European game species at such a large scale with large and representative sample sizes.

Innate Immune Basis for Rift Valley Fever Susceptibility in Mouse Models.

Rift Valley fever virus (RVFV) leads to varied clinical manifestations in animals and in humans that range from moderate fever to fatal illness, suggesting that host immune responses are important determinants of the disease severity. We investigated the immune basis for the extreme susceptibility of MBT/Pas mice that die with mild to acute hepatitis by day 3 post-infection compared to more resistant BALB/cByJ mice that survive up to a week longer. Lower levels of neutrophils observed in the bone marrow and blood of infected MBT/Pas mice are unlikely to be causative of increased RVFV susceptibility as constitutive neutropenia in specific mutant mice did not change survival outcome. However, whereas MBT/Pas mice mounted an earlier inflammatory response accompanied by higher amounts of interferon (IFN)-α in the serum compared to BALB/cByJ mice, they failed to prevent high viral antigen load. Several immunological alterations were uncovered in infected MBT/Pas mice compared to BALB/cByJ mice, including low levels of leukocytes that expressed type I IFN receptor subunit 1 (IFNAR1) in the blood, spleen and liver, delayed leukocyte activation and decreased percentage of IFN-γ-producing leukocytes in the blood. These observations are consistent with the complex mode of inheritance of RVFV susceptibility in genetic studies.

Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses.

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment.

Genetic Structure and Effective Population Sizes in European Red Deer (Cervus elaphus) at a Continental Scale: Insights from Microsatellite DNA.

We analyzed more than 600 red deer (Cervus elaphus) from large parts of its European distribution range at 13 microsatellite loci, presenting the first continent-wide study of this species using nuclear markers. Populations were clearly differentiated (overall F ST = 0.166, Jost's D est = 0.385), and the BAPS clustering algorithm yielded mainly geographically limited and adjacent genetic units. When forced into only 3 genetic clusters our data set produced a very similar geographic pattern as previously found in mtDNA phylogeographic studies: a western group from Iberia to central and parts of Eastern Europe, an eastern group from the Balkans to Eastern Europe, and a third group including the threatened relict populations from Sardinia and Mesola in Italy. This result was also confirmed by a multivariate approach to analyzing our data set, a discriminant analysis of principal components. Calculations of genetic diversity and effective population sizes (linkage disequilibrium approach) yielded the lowest results for Italian (Sardinia, Mesola; N e between 2 and 8) and Scandinavian red deer, in line with known bottlenecks in these populations. Our study is the first to present comparative nuclear genetic data in red deer across Europe and may serve as a baseline for future analyses of genetic diversity and structuring in this widespread ungulate.