The tick-borne encephalitis virus (TBEV) is an important human pathogen that causes tick-borne encephalitis, a potentially fatal neurological disease. Human infections occur through tick bites or after the consumption of raw milk products from infected animals, causing alimentary outbreaks representing a significant public health problem in Slovakia. In the present study, a total of 1029 ruminant sera (from 672 sheep and 357 goats) from 18 localities, collected in Slovakia during 2017-2019 were initially screened for TBEV-specific antibodies by ELISA, and 98 (9.5 %) of them were confirmed as positive by the plaque reduction neutralization test or immunofluorescence assay. The differences in observed seroprevalence of 11.9 % (80/672) among sheep and of 5.0 % (18/ 357) among goats were significant. Tick screening was subsequently conducted near the surveyed farms where seropositive animals were identified. Overall, 2,534 ticks (2,528 Ixodes ricinus, 3 Dermacentor reticulatus, 2 Dermacentor marginatus, 1 Haemaphysalis concinna) from 7 collection sites were pooled and analysed by RT-qPCR, resulting in estimated prevalence of 2.86 % (ranging from 0.31 to 8.72 % at different sites). The estimated prevalence in adults was lower (2.15 %) than in nymphs (2.9 %). Positive ticks (all I. ricinus) and milk samples were then used for virus isolation. Three new cell culture isolates were prepared and sequenced. Obtained fulllength genome sequences revealed high genetic diversity and phylogenetic clustering with virus strains found across Europe. The seroprevalence of TBEV in farm animals is an effective tool for identifying areas of virus circulation and guiding more in-depth field investigations of local tick populations. This combined approach of serological and virological surveillance provides valuable data for assessing the risk of alimentary TBEV infections and characterizing local TBEV strains.

The increasing incidence of autism spectrum disorder (ASD) increases the urgency of establishing the mechanism of its development for effective prevention and treatment. ASD's etiology includes genetic predisposition and environmental triggers, both of which can play a role in the changed microbiota. Recent research has proved the impact of maternal microbiota on the neurodevelopment of the child. To investigate the co-play of genetic and microbiota factors in ASD development, we performed fecal microbiota transplantation (FMT) from children with ASD to female Shank3b+/- mice and studied the autism-like symptoms in the male Shank3b-/- and wild-type (WT) offspring. WT animals with prenatal exposure to ASD microbiota had delayed neurodevelopment and impaired food intake behavior, but also elevated plasma leptin concentration and body weight. Shank3b-/- mice after FMT ASD exhibited impaired learning and exacerbated anxiety-like behavior in adulthood. Interestingly, FMT ASD improved learning in adolescent Shank3b-/- mice. Prenatal exposure to ASD microbiota decreased the activity of hypocretin neurons of the lateral hypothalamic area in both genotypes. The combination of genetic predisposition and FMT ASD led to an increased colon permeability, evaluated by zonula occludens (ZO1, ZO3) and claudin factors. These results suggest the effect of parental FMT exposure on shaping offspring behavior in Shank3b-/- mice and the potential of microbiota in the modulation of ASD.

In this study, we aimed to elucidate the role of the gut microbiome in the multistage progression of colorectal cancer (CRC). We analyse the gut bacterial composition of three clinical groups: healthy individuals, patients with precancerous polyps (adenomas), and patients with cancerous tumours (carcinomas). Biopsy samples were profiled using the Illumina MiSeq platform. Significant differences in the composition of the gut microbiota emerged between the groups. The tumour-associated microbiome showed increased bacterial diversity and specific enrichments, including Verrucomicrobia (genus Akkermansia) and specific Bacteroidetes (genera Porphyromonas and Prevotella). In contrast, the healthy group exhibited a higher abundance of specific protobacteria (genus Microvirga). In particular, the adenoma group also showed elevated levels of specific bacteria compared to the control group. Our findings suggest a possible link between the gut microbiome and the progression of CRC, with variations observed at different stages of the disease. This opens avenues for further investigation of the potential of the gut microbiome as a biomarker and therapeutic target for prevention and management.

The diphyllobothriid tapeworm Dibothriocephalus dendriticus, one of the causative agents of the fish-borne zoonosis dibothriocephalosis, is mainly distributed in the Arctic/subarctic and temperate zones of the Northern Hemisphere (Europe, North America, and Asia), but also in the southern cone region of South America (Patagonia). The genetic structure and gene flow among 589 individuals of D. dendriticus, representing 20 populations, were studied using the mitochondrial cox1 gene as the first choice marker and 10 polymorphic nuclear microsatellite loci as a dominant molecular tool. The haplotype network of 30 cox1 haplotypes and Principal Coordinate Analysis/Structure analysis based on microsatellite data revealed close genetic relationships among populations within continents, namely northern and northwestern Europe (Norway, Finland, and UK/Scotland), North America (USA/Alaska, USA/Oregon, and Greenland), and South America (Argentina and Chile). The population from Iceland, located on the Mid-Atlantic Ridge between North America and Europe, was related to both Europe and North America. The mixed Nearctic and Palaearctic origin of D. dendriticus in Iceland was confirmed by a coalescent-based analysis of the microsatellite loci (in Migrate software). The most likely scenarios suggested that the Icelandic population is a genetic admixture of tapeworms from northwestern Europe and USA/Alaska. These findings corresponded with the distribution and migratory routes of piscivorous birds of the family Laridae, the main definitive hosts of D. dendriticus. The origin of an apparently non-native population of D. dendriticus in Patagonia was investigated in detail. The two most plausible hypotheses based on Migrate analysis represented different scenarios. One of them points to a European origin of the Patagonian population, the other to a North American origin. Future research with more extensive and a geographically broader sampling set is recommended to trace dispersal routes of D. dendriticus in Patagonia.

Due to the falling water level in the Aral Sea and Muynak Lake, the content of salts dissolved in the water has gradually increased, and toxic elements have been deposited at the lake’s bottom and subsequently washed into the Aral region by the river. Bacteria, archaea and fungi are crucial for the cycling of several important inorganic nutrients in soils. From 15 genera and 31 species of recovered microscopic filamentous fungi, a big group was melanized, of which most of them were also phytopathogenic. The second group consisted of keratinophilic species. Isolated bacteria mainly included members of the genera Arthrobacter, Bacillus, Massilia, Rhodococcus and Nocardiopsis. High-throughput sequencing analysis permitted a better view of the mycobiome and prokaryotic communities (comprising archaea). The cultivation and sequencing approaches were shown to be complementary. The aim of the work was to identify soil microorganisms, including the order Halobacteriales, and to discover the differences in species diversity depending on soil salinity and the presence of PTEs in soil.

Abstract Background: Severe alcohol-associated hepatitis (SAH) is the most serious phenotype of the alcohol-associated liver disease (ALD) spectrum, characterized by high short-term mortality. The only approved therapy with corticosteroids (CS) has modest and short-term survival benefits; moreover, new therapeutic alternatives are unmet need. Fecal microbiota transplantation (FMT) has gained attention as a potential therapeutic option for filling this gap. In this study, we aimed to determine the effect of FMT on mortality in patients with SAH beyond CS and search for factors predictive of outcome, which could be leveraged in a real-life predictive, preventive, and personalized medicine (3 PM) management of SAH. Methods: We conducted a prospective study (NCT04758806) in adult patients with SAH, non-responders (NR) to CS, or non-eligible (NE) for CS between January 2018 and August 2022. FMT intervention consisted of five 100 ml doses (prepared beforehand from 30g stool from an unrelated healthy donor and frozen at − 80°C) administered daily to the upper gastrointestinal (GI) tract. We evaluated the impact of FMT on short-term mortality as determined by the propensity score matching with the historical cohort derived from our RH7 registry (NCT04767945). Also, we scrutinized SAH-related prognostic factors and scores such as Model for end-stage liver disease (MELD), Maddrey Discriminant Function (MDF), Acute-on-chronic liver failure (ACLF), Liver Frailty Index (LFI), Hepatic venous-portal gradient (HVPG) and AAHS (Alcoholic Hepatitis Histologic Score), for more precise prediction of response to therapy and their future potential in predictive, preventive, and personalized patient management. Results: We enrolled 44 patients with SAH (NR + NE to CS) who were intended to be treated with FMT and analyzed 33 patients per protocol (after an additional 11 being excluded for receiving less than 5 doses of FMT). The mean age was 49.6y, 11 patients (33.3%) were females. The median MELD-Na score was 29, and ACLF of any degree had 27 patients (81.8%). FMT improved 30-day survival over the historical control group (p = 0.0204); ninety-day survival was improved but did not reach statistical significance (p = 0.4386). As predictors of FMT failure - evaluated by the surrogate of short-term mortality -, we identified baseline MELD ≥30, MDR ≥ 90, and ACLF grade > 1, respectively (p=0.016; p=0.024; p=0.01). Survival was not associated with baseline liver frailty index (LFI), hepatic venous-portal gradient (HVPG), or Alcoholic Hepatitis Histological Score (AHHS). Conclusion: Severe alcohol-associated hepatitis in patients beyond CS was confirmed to be associated with high short-term mortality. In this difficult-to-treat cohort, FMT improved 30-day survival over historical controls chosen by propensity score matching. Factors associated with better outcomes were MELD-Na ≤ 30, MDR ≤ 90, and ACLF < 2. Therefore, our results lend support to the notion that FMT can be considered a 3 PM approach: albeit more data is needed, FMT and the described factors are good candidates for the unmet need of preventing death, predicting therapeutic response, and personalized management of SAH.

Severe alcohol-associated hepatitis (SAH) is the most critical, acute, inflammatory phenotype within the alcohol-associated liver disease (ALD) spectrum, characterized by high 30- and 90-day mortality. Since several decades, corticosteroids (CS) are the only approved pharmacotherapy offering highly limited survival benefits. Contextually, there is an evident demand for 3PM innovation in the area meeting patients’ needs and improving individual outcomes. Fecal microbiota transplantation (FMT) has emerged as one of the new potential therapeutic options. In this study, we aimed to address the crucial 3PM domains in order to assess (i) the impact of FMT on mortality in SAH patients beyond CS, (ii) to identify factors associated with the outcome to be improved (iii) the prediction of futility, (iv) prevention of suboptimal individual outcomes linked to increased mortality, and (v) personalized allocation of therapy. We conducted a prospective study (NCT04758806) in adult patients with SAH who were non-responders (NR) to or non-eligible (NE) for CS between January 2018 and August 2022. The intervention consisted of five 100 ml of FMT, prepared from 30 g stool from an unrelated healthy donor and frozen at − 80 °C, administered daily to the upper gastrointestinal (GI) tract. We evaluated the impact of FMT on 30- and 90-day mortality which we compared to the control group selected by the propensity score matching and treated by the standard of care; the control group was derived from the RH7 registry of patients hospitalized at the liver unit (NCT04767945). We have also scrutinized the FMT outcome against established and potential prognostic factors for SAH — such as the model for end-stage liver disease (MELD), Maddrey Discriminant Function (MDF), acute-on-chronic liver failure (ACLF), Liver Frailty Index (LFI), hepatic venous-portal pressure gradient (HVPG) and Alcoholic Hepatitis Histologic Score (AHHS) — to see if the 3PM method assigns them a new dimension in predicting response to therapy, prevention of suboptimal individual outcomes, and personalized patient management. We enrolled 44 patients with SAH (NR or NE) on an intention-to-treat basis; we analyzed 33 patients per protocol for associated factors (after an additional 11 being excluded for receiving less than 5 doses of FMT), and 31 patients by propensity score matching for corresponding individual outcomes, respectively. The mean age was 49.6 years, 11 patients (33.3

Carbon quantum dots (CQDs) are promising therapeutic agent due to their pro-oxidant, antioxidant, antiviral, antibacterial, and anticancer properties when exposed to visible light irradiation. Oxidative stress in bacteria is the main reason for bacteria death after exposure to blue light photoexcited quantum dots. Herein, we present the antibacterial activities of hydrophobic carbon quantum dots/polydimethylsiloxane nanocomposites, hydrophilic citric acid CQDs, and combinations of CQDs with methylene blue. We investigated the antirickettsial effect of hydrophilic and hydrophobic CQDs against Rickettsia slovaca, a tick-borne bacterial pathogen. Photodynamic activity against on rickettsiae reached 99.66% using CQDs with 470 nm blue light irradiation. Combining methylene blue with CQDs further enhanced the effect on rickettsial infection, achieving 99,98% efficacy. The obtained results reveal the in vitro antirickettsial properties of CQDs. Sequencing analysis on the genomic level of control and treated samples showed single nucleotide variants (SNVs). Based on snippy analysis SNVs were assigned to the rRNA genes, 16S rRNA and 30S rRNA genes. By freebayes analysis in treated samples, a stop-lost mutation was detected in pseudogene (RSL_RS06070), while the possible effect on down-stream genes including tsaD, acyl-CoA-desaturase, 30S ribosomal protein S6 and DUF424 family protein. The frameshift mutation was localized within clpB pseudogene belonging to stress-response heat-shock proteins.

Analysis of the viromes of three symptomatic Fabaceae plants, i.e., red clover (Trifolium pratense L.), pea (Pisum sativum L.), and common bean (Phaseolus vulgaris L.), using high-throughput sequencing revealed complex infections and enabled the acquisition of complete genomes of a potyvirus, bean yellow mosaic virus (BYMV). Based on phylogenetic analysis, the Slovak BYMV isolates belong to two distinct molecular groups, i.e., VI (isolate FA40) and XI (isolates DAT, PS2). Five commercial pea genotypes (Alderman, Ambrosia, Gloriosa, Herkules, Senator) were successfully infected with the BYMV-PS2 inoculum and displayed similar systemic chlorotic mottling symptoms. Relative comparison of optical density values using semi-quantitative DAS-ELISA revealed significant differences among virus titers in one of the infected pea genotypes (Ambrosia) when upper fully developed leaves were tested. Immunoblot analysis of systemically infected Alderman plants showed rather uneven virus accumulation in different plant parts. The lowest virus accumulation was repeatedly detected in the roots, while the highest was in the upper part of the plant stem.

During the screening of lettuce (Lactuca sativa L.) samples from the United Kingdom in 2015 using high throughput sequencing (HTS) technology, the genome of a novel virus was identified in a plant showing symptoms of stunting and yellowing. The virus genome consisted of two RNA molecules; an RNA1 of 6001 nt encoding a polyprotein with protein cofactor, helicase, protease and RNA-dependent RNA polymerase motifs and an RNA2 of 6553 nt encoding a polyprotein with viral movement and coat protein motifs. Independently, in 2018, an analysis of ribodepleted total RNA from a dandelion (Taraxacum officinale L.) displaying leaf malformation and puckering revealed a complex infection involving a secovirus related to the UK lettuce virus. Moreover, a small survey confirmed the presence of the secovirus in environmental dandelion samples in Slovakia. Detailed analysis of these sequences suggests they all belonged to the same putative species, a member of the subgenus Stramovirus, genus Sadwavirus, family Secoviridae named lettuce secovirus 1 with the tentative Latin binomial Sadwavirus lactucae.

Biological aging is linked to altered body composition and reduced neuroactive steroid hormones like dehydroepiandrosterone sulfate (DHEAS), which can stimulate the GABA signaling pathway via gut microbiota. Our study examined the association of gut microbiota with lifespan in mice through comprehensive analysis of its composition and functional involvement in cholesterol sulfate, a precursor of DHEAS, metabolism. We used 16S rRNA and metagenomic sequencing, followed by metabolic pathway prediction and thin layer chromatography and MALDI-TOF cholesterol sulfate identification. Significant increases in bacteria such as Bacteroides, typical for long-lived and Odoribacter and Colidextribacter, specific for short-lived mice were detected. Furthermore, for males (Rikenella and Alloprevotella) and females (Lactobacillus and Bacteroides), specific bacterial groups emerged as predictors (AUC = 1), highlighting sex-specific patterns. Long-lived mice showed a strong correlation of Bacteroides (0.918) with lipid and steroid hormone metabolism, while a negative correlation of GABAergic synapse with body weight (-0.589). We found that several Bacteroides species harboring the sulfotransferase gene and gene cluster for sulfonate donor synthesis are involved in converting cholesterol to cholesterol sulfate, significantly higher in the feces of long-lived individuals. Overall, we suggest that increased involvement of gut bacteria, mainly Bacteroides spp., in cholesterol sulfate synthesis could ameliorate aging through lipid metabolism.

The gut microbiota of paediatric oncology patients undergoing a conditioning regimen before hematopoietic stem cell transplantation is recently considered to play role in febrile neutropenia. Disruption of commensal microbiota and evolution of opportune pathogens community carrying a plethora of antibiotic-resistance genes play crucial role. However, the impact, predictive role and association of patient´s gut resistome in the course of the therapy is still to be elucidated. We analysed gut microbiota composition and resistome of 18 paediatric oncology patients undergoing hematopoietic stem cell transplantation, including 12 patients developing febrile neutropenia, hospitalized at The Bone Marrow Transplantation Unit of the National Institute of Children´s disease in Slovak Republic and healthy individuals (n = 14). Gut microbiome of stool samples obtained in 3 time points, before hematopoietic stem cell transplantation (n = 16), one week after hematopoietic stem cell transplantation (n = 16) and four weeks after hematopoietic stem cell transplantation (n = 14) was investigated using shotgun metagenome sequencing and bioinformatical analysis. We identified significant decrease in alpha-diversity and nine antibiotic-resistance genes msr(C), dfrG, erm(T), VanHAX, erm(B), aac(6)-aph(2), aph(3)-III, ant(6)-Ia and aac(6)-Ii, one week after hematopoietic stem cell transplantation associated with febrile neutropenia. Multidrug-resistant opportune pathogens of ESKAPE, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli found in the gut carried the significant subset of patient’s resistome. Over 50% of patients treated with trimethoprim/sulfamethoxazole, piperacillin/tazobactam and amikacin carried antibiotic-resistance genes to applied treatment. The alpha diversity and the resistome of gut microbiota one week after hematopoietic stem cell transplantation is relevant predictor of febrile neutropenia outcome after hematopoietic stem cell transplantation. Furthermore, the interindividual diversity of multi-drug resistant opportunistic pathogens with variable portfolios of antibiotic-resistance genes indicates necessity of preventive, personalized approach.

Tellurite resistance gene clusters have been identified in numerous pathogenic bacteria, including clinical isolates of Escherichia coli. The rareness of tellurium in host organisms and the noncontaminated environment raises a question about the true functionality of tellurite resistance gene clusters in pathogenesis and their possible contribution to bacterial fitness. The study aims to point out the beneficial effects of the tellurite resistance gene cluster of pathogenic bacteria to survive in ROS-rich environments. Here, we analysed the bacterial response to oxidative stress conditions with and without tellurite resistance gene clusters, which are composed of terWY1XY2Y3 and terZABCDEF genes. By measuring the levels of protein carbonylation, lipid peroxidation, and expression changes of oxidative stress genes upon oxidative stress, we propose a tellurite resistance gene cluster contribution to the elimination of oxidative damage, potentially increasing fitness and resistance to reactive oxygen species during macrophage attack. We have shown a different beneficial effect of various truncated versions of the tellurite resistance gene cluster on cell survival. The terBCDEF genes increased the survival of E. coli strain MC4100 by 13.21%, terW and terZABCDEF by 10.09%, and terWY1XY2Y3 and terZABCDEF by 25.57%, respectively. The ability to survive tellurite treatment is the most significant at 44.8% in wild clinical strain KL53 compared to laboratory strain E. coli MC4100 due to a complete wild-type plasmid presence.

Is the time ripe for microbiome to be levered in clinical hepatology? Is the preventive, predictive, and personalized medicine (3PM/PPPM) realm offering a framework for just an intellectual exercise in the world of natura cogitans, or can we already predict, personalize, and prevent liver diseases by targeting microbiome? Can the knowledge be extrapolated to other branches of clinical medicine? In the following, these and other questions are approached from the perspective of a clinical hepatology enrouted in a resource-aware healthcare setting—an environment not usually perceived as being conducive to the translation of ideas from the bench to bedside. And that is just it: to include microbiome into mindsets possessed by 3PM irrespective of the setting. This chapter is offered to the readers who hesitate at the front of the door of perception thinking that microbiome is not for them and that they can do as well without it. Microbiome science seems to be entering medicine with the new millennium but Hippocrateses “All disease origins in the gut” and ancient’s China “Yellow soup for the soul” could well be its predecessors. However, there is no doubt that the advances in molecular biology and computer science enabled us to look beyond the Petri dishes and discover the microbiome’s unknown universe. Trillions of microbes outnumber body cells and genes by the factors of 1.3 and one-two orders, respectively. Ninety-five percent of diseases have their pathophysiology linked to a deranged microbiome which—weighing more than the brain, became the heaviest organ of the body. Fascination with these numbers notwithstanding, it is now clear that what matters more is the microbiome composition, function, nutrients, predators, metabolites, and interactions. We know that the “healthy microbiome” is a relatively stable ecosystem entrenched in a baby gut as a relatively distinct enterotype by the means of mode of birth, mother’s touch, and early-life events. With its abundance and diversity, it resembles a rainforest. Its metabolism helps the whole body thrive from the very first breastfeeding. Healthy microbiome keeps the integrity of host–environment barriers steadfast, systemic inflammation low, and liver resilience high. When dynamics underlying the microbiome composition and function change, dysbiosis ensues. This state is epitomized by a desert losing the abundance of species and their diversity. Dysbiosis can be induced by factors such as hyperhygienic lifestyle, low physical activity, highly processed food, alcohol, antibiotics, stress, etc. Dysbiosis is known or a proposed causative factor of a host of liver diseases, from hepatitis to cirrhosis, acute -on-chronic liver failure, and cancer. Many liver syndromes are preceded by a relatively specific patterns of dysbiosis which are possible targets for prevention, prediction, and personalization. Obesity, non-alcoholic fatty liver disease, alcohol use disorder, alcohol-associated liver disease, ACLF, hepatic encephalopathy, and autoimmune liver disease - to name but a few. As for the domain of 3PM in liver diseases, huge endeavors are in place on the platforms of consortia such as MicrobPredict and alike.

Background: Gut microbial composition seems to change in association with prediabetes. The purpose of this prospective cross-sectional study was to compare the composition of gut microbiota and energy metabolites between individuals with class III obesity but without type 2 diabetes mellitus (OB) and healthy normal weight controls. Methods: The subjects of this prospective cross-sectional study were participants recruited from a previous clinical trial (No: NCT02325804), with intervention focused on weight loss. We recruited 19 OB [mean age ± standard deviation (SD) was 35.4 ± 7.0 years, mean body mass index (BMI) ± SD was 48.8 ± 6.7 kg/m2] and 23 controls (mean age ± SD was 31.7 ± 14.8 years, mean BMI ± SD was 22.2 ± 1.7 kg/m2). Their fecal microbiota was categorized using specific primers targeting the V1-V3 region of 16S rDNA, whereas serum metabolites were characterized by nuclear magnetic resonance spectroscopy. Multivariate statistical analysis and Random Forest models were applied to discriminate predictors with the highest variable importance. Results: We observed a significantly lower microbial α-diversity (P = 0.001) and relative abundance of beneficial bacterium Akkermansia (P = 0.001) and the short-chain fatty acid-producing bacteria Eubacterium hallii (P = 0.019), Butyrivibrio (P = 0.024), Marvinbryantia (P = 0.010), and Coprococcus (P = 0.050) and a higher abundance of the pathogenic bacteria Bilophila (P = 0.018) and Fusobacterium (P = 0.022) in OB compared with controls. Notably, the Random Forest machine learning analysis identified energy metabolites (citrate and acetate), HOMA-IR, and insulin as important predictors capable of discriminating between OB and controls. Conclusions: Our results suggest that changes in gut microbiota and in serum acetate and citrate are additional promising biomarkers before progression to Type 2 diabetes. The non-invasive manipulation of gut microbiota composition in OB through a healthy lifestyle, thus, offers a new approach for managing class III obesity and associated disorders. ClinicalTrials.gov identifier: NCT02325804.

With the rapid growth of massively parallel sequencing technologies, still more laboratories are utilising sequenced DNA fragments for genomic analyses. Interpretation of sequencing data is, however, strongly dependent on bioinformatics processing, which is often too demanding for clinicians and researchers without a computational background. Another problem represents the reproducibility of computational analyses across separated computational centres with inconsistent versions of installed libraries and bioinformatics tools. We propose an easily extensible set of computational pipelines, called SnakeLines, for processing sequencing reads; including mapping, assembly, variant calling, viral identification, transcriptomics, and metagenomics analysis. Individual steps of an analysis, along with methods and their parameters can be readily modified in a single configuration file. Provided pipelines are embedded in virtual environments that ensure isolation of required resources from the host operating system, rapid deployment, and reproducibility of analysis across different Unix-based platforms. SnakeLines is a powerful framework for the automation of bioinformatics analyses, with emphasis on a simple set-up, modifications, extensibility, and reproducibility. The framework is already routinely used in various research projects and their applications, especially in the Slovak national surveillance of SARS-CoV-2.

The genus Elaphe Fitzinger, 1833 includes 17 species of charismatic, large-sized, non-venomous, Eurasian snakes. In the Western Palearctic, the genus is represented by three species from the Elaphe quatuorlineata group ranging from the Apennine peninsula to Central Asia. The southernmost population of this group is distributed in the mountains of the Southern Levant, with more than 400 km gap to other Elaphe populations. This population has been known to science for only 50 years and is virtually unstudied due to its extreme rarity. We studied these snakes' morphological and genetic variation from the three countries where they are known to occur, i.e., Israel (Hermon, the Israeli-controlled Golan Heights), Lebanon, and Syria. We used nine mitochondrial and nuclear genes, complete mitogenome sequences, and a comprehensive morphological examination including published data, our own field observations, and museum specimens, to study its relationship to other species in the group. The three currently recognized species of the group (E. quatuorlineata, E. sauromates, E. urartica), and the Levant population, form four deeply divergent, strongly supported clades. Three of these clades correspond to the abovementioned species while the Southern Levant clade, which is genetically and morphologically distinct from all named congeners, is described here as a new species, Elaphe druzei sp. nov. The basal divergence of this group is estimated to be the Late Miocene with subsequent radiation from 5.1 to 3.9 Mya. The revealed biogeography of the E. quatuorlineata group supports the importance of the Levant as a major center of endemism and diversity of biota in Eurasia. The new species is large-sized and is one of the rarest snakes in the Western Palearctic. Because of its small mountain distribution range, in an area affected by land use and climate change, the new Elaphe urgently needs strict protection. Despite political issues, we hope this will be based on the cooperation of all countries where the new species occurs.