Phylogenetic Link Research Articles

Resistome phylodynamics of multidrug-resistant Shigella isolated from diarrheal patients.

Multi-drug resistance (MDR) in Shigella continues to pose a significant public health challenge, particularly in developing countries. Recent advances in genomics strengthen the surveillance of MDR-pathogens and antimicrobial resistance (AMR) mediators. However, genome-based investigations into resistome dynamics in Shigella are limited, specifically in Bangladesh. Therefore, we investigated MDR-Shigella resistomes to evaluate their AMR transmission and phylodynamics. Clinical Shigella strains were screened for MDR phenotypes through susceptibility tests against 28 antibiotics from 10 different classes. Whole-genome sequencing (WGS) and bioinformatics approaches were performed to unveil the resistome dynamics: >500 global plasmid entities and >1,000 plasmid-mediated resistance gene clusters from global databases were included in this study. We identified 28 distinct antimicrobial resistance genes (ARGs) from nine antibiotic classes, with 75% originating from plasmids. Notably, two conjugative MDR plasmids included nearly all potential ARGs, conferring resistance to first-line drugs for shigellosis. Two third-generation cephalosporin-resistant [wubC-blaCTX-M-15-ISEcp1 and blaTEM-1] and two macrolide-resistant mobile genomic islands (GIs) [mphA-mrx-mph(R)A-IS6100 and mphE-msrE-IS482-IS6] had emerged in Shigella in Bangladesh. In addition, trimethoprim-aminoglycoside-streptothricin-sulfonamide-resistant dfrA1-sat1-aadA1 and aph3-dfrA14-aph6-sul2 were in conjugative plasmids in Bangladesh. The MDR plasmids and resistant GIs were phylogenetically relevant to Europe, USA, or China-derived isolates, indicating carry-over of the emerging ARGs from heavily industrialized countries and MSM-burdened (men who have sex with men) populations. The global burden of resistance GIs has increased sharply, especially after 2014. Emerging resistance mediators were most frequent (>80%) in human-associated Escherichia coli and Klebsiella pneumoniae. We infer ARGs horizontally propagate among Enteropathogens: informing treatment strategies and supporting policymakers in strengthening AMR-containment efforts utilizing the phylodynamics network.IMPORTANCEThe world is suffering from a high burden of MDR enteropathogens. Healthcare providers in low- and middle-income countries (LMICs) often face trouble finding effective drugs among the many antibiotics introduced in diarrheal treatment. Resistance-mediated drug inactivation is more rapid than the advent of new antimicrobials, leaving enteritis treatment on the edge. In Bangladesh, where one-third of users are self-prescribing antibiotics and thousands are dying due to resistance-related treatment failure, phylogenomic evidence of AMR transmission root is scarce. Therefore, investigating the resistomes of MDR-Shigella, the leading cause of diarrheal deaths in Bangladesh, is crucial. We identified several emerging resistance mediators and their phylogenetic links to global entities, which is significant for improving shigellosis treatment and enhancing AMR containment strategies. Understanding the MDR mechanism in Shigella will help physicians choose effective drugs and anticipate resistance-mediated changes in treatment approaches; the spatiotemporal phylodynamics of AMR mediators aid policymakers in setting effective checkpoints in the AMR transmission network.

Mitochondrial Genome Assembly and Comparative Analysis of Chionanthus Retusus (Oleaceae)

Background/Objectives: Chionanthus retusus Lindl. & Paxton is an ornamental tree species native to North China. Research on the mitochondrial genome can elucidate the evolution and biological characteristics of C. retusus and better protect this important species. Methods and Results: This work aimed to clarify the evolutionary and phylogenetic links by sequencing, assembling, annotating, and analyzing the entire mitochondrial genome of C. retusus. The single-loop structure that made up the mitochondrial genome had a total length of 657,640 bp and a GC content of 44.52%. In total, 37 unique protein-coding genes, 20 tRNA genes, and 3 rRNA genes were identified. Numerous repeat sequences and migrating fragments of chloroplast sequences were found. Using the mitochondrial protein-coding genes to construct evolutionary trees, it was found that the closest relative of C. retusus is C. rupicola (Lingelsh.) Kiew. Conclusions: This research represents the first comprehensive set of data on the mitochondrial genome of an ancient (>500 yr) C. retusus specimen. In addition to elucidating the biological characteristics of C. retusus. The findings contribute to the Oleaceae mitochondrial genome database and offer valuable insights for future studies in molecular breeding, evolutionary biology, and genetic diversity conservation.

Prevalence and Antibiotic Susceptibility of Nontyphoidal Salmonella from Food-Animal Products in Bejaia, Algeria.

Nontyphoidal Salmonella presents a significant threat to animal and human health as a food-borne infectious agent. This study focused on the characterization of Salmonella isolates obtained from fresh animal products consumed in Bejaia, Algeria. In total, 495 beef products, 600 chicken products, and 355 dairy products were collected from retail outlets and slaughterhouses in the region. Out of the 1450 samples collected, a Salmonella positivity rate of 3.5% (51/1450) was observed. Traditional Algerian sausages exhibited the highest prevalence of Salmonella contamination (14.1%, 36/256). Fifteen different serotypes were identified, with S. Kentucky (n = 12), S. Anatum (n = 11), and S. Bredeney (n = 9) being the most prevalent. Antimicrobial susceptibility testing showed resistance to diverse antibiotics, particularly against tetracycline, ampicillin, nalidixic acid, and sulphonamides. Whole-genome sequencing conducted on 21 isolates enabled the comparison of phylogenetic links between isolates. We notably identified clones circulating across the region in different locations and food types, suggesting contamination at the early stages of the food chain (in the herd or slaughterhouses) that disseminated to numerous butcher shops in various cities. We also identified acquired antibiotic resistance genes and point mutations in the quinolone resistance-determining region genes, contributing to the observed resistance patterns. This study describes the genomic and phylogenetic characteristics of several Salmonella clones found in diverse food samples in Algeria. It suggests potential transmission dynamics that could better understand Salmonella's contamination routes.

Characterization of Tomato Leaf Curl New Delhi Virus Associated with Leaf Curl and Yellowing Disease of Bitter Gourd from Karnataka, India

Aims: The objective of this study was to identify the ToLCNDV isolate responsible for leaf curl and yellowing disease in bitter gourd crops in Karnataka, India. Study Design: Molecular characterisation and transmission study. Place and Duration of Study: The laboratory studies were conducted in the Department of Plant Pathology, College of Agriculture, University of Agricultural Sciences, GKVK, Bangalore-560065. Methodology: The sample was collected and subjected to molecular characterization using specific primer for ToLCNDV and sent for sequencing. Vector and sap transmission studies were also carried out. Results: PCR-mediated detection revealed the presence of ToLCNDV, although it was not coupled with any satellite molecules. The partial genome sequencing of the AV1 gene revealed more than 97% similarity with ToLCNDV isolates. The isolate under investigation was identified as a ToLCNDV isolate and given the name ToLCNDV_GKVK_BT due to high sequence identities and phylogenetic links between the incomplete DNA-A genome and ToLCNDV isolates. The transmission experiments demonstrated that it was effectively transmitted via vector and sap transfer. Conclusion: This study identified the ToLCNDV isolate responsible for leaf curl and yellowing disease in bitter gourd crops in Karnataka, India. Identifying, characterizing at the molecular level, and understanding the transmission of begomoviruses are essential steps in developing effective management approaches to minimize agricultural losses caused by these viruses. Therefore, these findings are important for identifying the leaf curl disease of bitter gourd.

Environmental contamination with carbapenem resistant Acinetobacter baumannii in healthcare settings in Fiji: a potential source of infection.

There are multiple ongoing outbreaks of carbapenem resistant Acinetobacter baumannii (CRAb) infection in Fiji's hospitals. CRAb is able to colonize and persist on various hospital surfaces for extended periods. We conducted a study to understand the extent of hospital environmental contamination and phylogenetic links with clinical isolates. Swabs were collected from high-touch surfaces at Colonial War Memorial Hospital (CWMH) September 2021 and December 2022; Lautoka Hospital (LTKH) August 2022; and Labasa Hospital (LBSH) November 2022. All bacterial isolates were identified, and antimicrobial susceptibility testing (AST) performed; isolates resistant to carbapenems and producing a carbapenemase underwent whole genome sequencing. Comparison was made to clinical isolates obtained from CWMH in 2016-2017 and 2019-2021 and from LTKH and LBSH from 2020-2021. From the 180 environmental samples collected, ten (5.6%) CRAb were isolated; no other carbapenem-resistant gram-negative organisms were isolated. Seven (70%) of the CRAb were isolated from CWMH and three (30%) from LTKH; no CRAb were isolated from LBSH. Of the seven CWMH CRAb, two were sequence type 2 (ST2), three ST25, and two ST499. All LTKH isolates were ST499. The two environmental CRAb ST2 isolates were closely genetically linked to isolates obtained from patients in CWMH, LTKH, and LBSH 2020-2021. Similarly, the three environmental CRAb ST25 isolates were closely genetically linked to isolates obtained from patients admitted to CWMH in 2019-2021 and LBSH in 2020. The environmental CRAb ST499 isolates represented two distinct clones, with clone 1 comprising two genetically identical isolates from CWMH and clone 2 the three isolates from LTKH. Although no genetic linkages were observed when comparing environmental ST499 isolates to those from CWMH patients in 2020-2021, both clone 1 isolates were genetically identical to an isolate obtained from a patient admitted during the sampling period. Our study highlights the contamination of high-touch surfaces within Fiji hospitals with CRAb, suggesting that these may serve as important sources for CRAb. Phylogenetic linkages to CRAb isolated from patients since 2019 underscores the persistence of this resistant pathogen in hospital settings and the ongoing risk for hospital-acquired infections.

In silico analyses identify sequence contamination thresholds for Nanopore-generated SARS-CoV-2 sequences.

The SARS-CoV-2 pandemic has brought molecular biology and genomic sequencing into the public consciousness and lexicon. With an emphasis on rapid turnaround, genomic data informed both diagnostic and surveillance decisions for the current pandemic at a previously unheard-of scale. The surge in the submission of genomic data to publicly available databases proved essential as comparing different genome sequences offers a wealth of knowledge, including phylogenetic links, modes of transmission, rates of evolution, and the impact of mutations on infection and disease severity. However, the scale of the pandemic has meant that sequencing runs are rarely repeated due to limited sample material and/or the availability of sequencing resources, resulting in the upload of some imperfect runs to public repositories. As a result, it is crucial to investigate the data obtained from these imperfect runs to determine whether the results are reliable prior to depositing them in a public database. Numerous studies have identified a variety of sources of contamination in public next-generation sequencing (NGS) data as the number of NGS studies increases along with the diversity of sequencing technologies and procedures. For this study, we conducted an in silico experiment with known SARS-CoV-2 sequences produced from Oxford Nanopore Technologies sequencing to investigate the effect of contamination on lineage calls and single nucleotide variants (SNVs). A contamination threshold below which runs are expected to generate accurate lineage calls and maintain genome-relatedness and integrity was identified. Together, these findings provide a benchmark below which imperfect runs may be considered robust for reporting results to both stakeholders and public repositories and reduce the need for repeat or wasted runs.

The Miocene primate Pliobates is a pliopithecoid

The systematic status of the small-bodied catarrhine primate Pliobates cataloniae, from the Miocene (11.6 Ma) of Spain, is controversial because it displays a mosaic of primitive and derived features compared with extant hominoids (apes and humans). Cladistic analyses have recovered Pliobates as either a stem hominoid or as a pliopithecoid stem catarrhine (i.e., preceding the cercopithecoid–hominoid divergence). Here, we describe additional dental remains of P. cataloniae from another locality that display unambiguous synapomorphies of crouzeliid pliopithecoids. Our cladistic analyses support a close phylogenetic link with poorly-known small crouzeliids from Europe based on (cranio)dental characters but recover pliopithecoids as stem hominoids when postcranial characters are included. We conclude that Pliobates is a derived stem catarrhine that shows postcranial convergences with modern apes in the elbow and wrist joints—thus clarifying pliopithecoid evolution and illustrating the plausibility of independent acquisition of postcranial similarities between hylobatids and hominids.

Abstract 3778: Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact

Abstract Introduction: Recent studies on disseminated cancer cells (DCCs) in Non-Small-Cell Lung Cancer (NSCLC) patients demonstrated their clinical relevance even in early tumor stages. However, their molecular and phylogenetic link to the primary tumor is currently unclear. Certain genetic alterations, such as TP53 or KRAS mutations, have been proposed to be clonal events and drivers of dissemination and metastasis. In this study, we analyzed the genome and transcriptome of single DCCs to identify the characteristics of early DCCs and their translational clinical value. Materials and methods: From 2011 to 2019 we prospectively collected and analyzed bone marrow aspirates and lymph node samples from 296 newly diagnosed NSCLC patients undergoing tumor resection with curative intention. Presence of DCC was examined by i) immunocytology (IC) for Cytokeratin (clone A45-B/B3, bone marrow, BM-DCCs) or EpCAM (clone BerEp4, lymph node, LN-DCCs) and ii) immunofluorescent (IF) staining for EpCAM in a live cell suspension (clone HEA-125) from BM or LN. Single cells were isolated using micromanipulation and subjected to Whole Genome Amplification or combined Whole Genome/Transcriptome Amplification for IC and IF, respectively. Sanger sequencing for known hotspot mutations in TP53 and KRAS was performed on single DCCs. Gene expression profiles of DCCs were analyzed by single cell RNA sequencing and the results were compared with clinical data with a mean follow-up of 2.93 years (range 0.03-7.22). Results: LN-DCCs were found in 44.8% and 59.6% by IC and IF, respectively. CKIC pos BM-DCCs were found in 45.2%, while EpCAMIF pos BM-DCCs were present in 52.8%. LN-DCCs correlated with reduced tumor-specific survival (TSS, p=0.009) and progression-free survival (PFS, p=0.025). EpCAMIF pos, but not CKIC pos BM-DCCs correlated with reduced TSS (p=0.003) and PFS (p=0.083). Genetic analysis of single DCCs revealed that TP53 or KRAS mutations could be found in 3.6% and 2.8% of analyzed BM-DCCs and 5.0% and 29.3% of LN-DCCs for IF and IC, respectively. Single cell RNA-seq showed distinct transcriptomic profiles for LN- and BM-DCCs, and identified a DCC phenotype with high stemness score in the bone marrow. Further analysis of potential transcriptome-based biomarkers identified a novel candidate marker for activated EpCAMpos BM-DCCs that strongly correlates with PFS and TSS (p<0.001). Conclusions: DCCs in early-stage NSCLC have high prognostic value and predict reduced PFS and TSS. While genetic alterations in the TP53 and KRAS gene were not detected in the majority of analyzed DCCs, transcriptomic phenotypes provided relevant information and identified a subset of activated DCCs that correlate strongly with relapse and poor outcome. Citation Format: Tobias Mederer, Daniel Spitzl, Felix Elsner, Bernhard Polzer, Michael Ried, Reiner Neu, Tobias Robold, Hans-Stefan Hofmann, Christoph A. Klein. Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3778.

Unveiling the impacts of land use on the phylogeography of zoonotic New World Hantaviruses

Billions of genomic sequences and records of species occurrence are available in public repositories (e.g. National Center for Biotechnology Information, NCBI and the Global Biodiversity Information Facility, GBIF). By implementing analytical tools from different scientific disciplines, data mining these databases can aid in the global surveillance of zoonotic pathogens that circulate among wildlife. We illustrate this by investigating the Hantavirus–rodent system in the Americas, i.e. New World Hantaviruses (NWH). First, we considered the circulation of pathogenic NWH among Cricetidae rodents, by inferring the phylogenetic links among 277 genomic samples of the S segment (N protein) of NWH found in 55 species. Second, we used machine learning to assess the impact of land use on the probability of presence of the rodent species linked with reservoirs of pathogenic Hantaviruses. Our results show that hosts are widely present across the Americas. Some hosts are present in the primary forest and agricultural land, but not in the secondary forest, whereas other hosts are present in secondary forest and agricultural land. The diversity of host species allows Hantavirus to circulate in a wide spectrum of habitats, in particular rural rather than urban. We highlight that public repositories of genomic data and species occurrence are very useful resources for monitoring potential enzootic transmission and spillover of zoonotic viruses in relation with the changes that humans produce in the biosphere.

Genetic variation of Plasmodium and Haemoproteus parasites in birds of Iran

Background: Plasmodium and Haemoproteus are common parasites transferred to birds by Culicidae, Ceratopogonidae, and Hippoboscidae vectors. These parasites are of particular concern to healthcare researchers and veterinarians due to their impact on avian malaria, association with other animals and humans, and phylogenetic link with human Plasmodium species. Methods: This bibliographic review article was designed to deliver a report on the avian malaria infections of Plasmodium spp. and Haemoproteus spp. in birds in Iran. Published reports in Google Scholar, PubMed, and Science Direct were used for this revision until April 2023. Results: The highest number of recorded infections with Haemoproteus spp. and Plasmodium spp. was found in Passeridae, as revealed by genetic analysis of Cytb DNA sequences. In this revision, following preceding investigations, the overall prevalence of Haemoproteus spp. was greater than that of Plasmodium spp. discovered by molecular techniques in Iran. Besides, the frequency of published genetic variants (lineages) of Haemoproteus is higher than that of Plasmodium. Our result indicated that the frequency of novel genetic variants Haemoproteus and Plasmodium in published reports was 38% and 12.5% of diagnosed lineages, respectively. Conclusion: The region needs more studies to detect parasites in unexplored hosts and vectors, as there is a lack of research. Understanding the prevalence, epidemiology, transmission approach, and vectors of avian blood parasites is crucial for pet health care, zoos, parks, aviaries, and rehabilitation facilities.

Ocorrência de reovírus aviário e picobirnavirus em aves silvestres de uma área de proteção ambiental na Amazônia brasileira

ABSTRACT Wild birds have great prominence in the transmission of diseases to humans, mainly due to their ease of access to human populations, raising concerns about the potential impact of that proximity on public health. The present study reports ARV and PBV occurrence in wild birds from an environmental protection area in the Amazon biome, in Pará state, Brazil. We obtained 155 fecal specimens from 47 species of wild birds for RNA extraction, ARV and PBV detection utilizing molecular methods, nucleotide sequencing, and phylogenetic analysis. ARV prevalence was 0.6% (1/155), being positive in an individual of Myrmotherula longipennis, and PBV prevalence was 1.29% (2/155), affecting two individuals of Guira guira. The PBV strains were assigned to genogroup I based on phylogenetic analysis, and they shared a strong phylogenetic link with strains isolated from different geographic locations. The ARV strain was more closely related to strains that had previously circulated in the same region. The presence of ARV and PBV in this habitat suggests that infection cycles of these agents occur naturally in the wild ecosystem, potentially triggering transmission events between bird species and humans. This is the first study on ARV detection in wild birds in Brazil and the first report on the occurrence of PBV in wild Guira guira. Additional studies are required to determine the epidemiology, origin, evolution, and emergence of new potentially pathogenic viruses in the Amazon.

Disjunct habitat of cryptic Terebellides (Annelida, Trichobranchidae) species shows a phylogenetic link between polychaetes from the White and the North Seas

Understanding the distribution and biodiversity of marine species is crucial for developing effective conservation strategies and maintaining the health of global ecosystems. Advancements in molecular data utilization have significantly improved our understanding of biodiversity within the genus Terebellides. In this study, we conducted a phylogenetic analysis on polychaete samples from the Kandalaksha Bay, White Sea, revealing their affiliation with a putative undescribed species of the genus Terebellides found in two locations of the North Sea. Interestingly, this species was not detected in the Norwegian and Barents Seas, leading us to propose a disjunct distribution scenario for this Terebellides species. This unique distribution pattern might be attributed to the succession of polychaetes by new species, facilitated by the Gulf Stream and a climate change role in driving shifts in species' ranges and altering marine ecosystem dynamics.

Molecular characterization of Newcastle disease virus obtained from Mawenzi live bird market in Morogoro, Tanzania in 2020–2021

Newcastle disease (ND) is among the most important poultry diseases worldwide. It is the major threat to poultry production in Africa and causes major economic losses for both local and commercial chickens. To date, half of ND class II genotypes have been reported in Africa (I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII, and XXI). The information on the circulating NDV genotypes is still scarce despite the endemic nature of ND in most countries on the African continent.A total of 659 oro-cloacal swabs were collected from local chickens in Mawenzi live bird market located in Morogoro, Tanzania, between June 2020 and May 2021. Newcastle disease virus was detected by using reverse transcription real-time polymerase chain reaction (RT-qPCR) and conventional PCR followed by sequencing of PCR products. The prevalence of NDV in the surveilled live bird markets was 23.5%. Sequencing and phylogenetic analysis revealed the presence of sub-genotype VII.2. The detected sub-genotype VII.2 has phylogenetic links to Zambian NDV strains implying a Southeast dissemination of the virus, considering that it was first detected in Mozambique. This study underscores the need of active NDV surveillance to determine the distribution of this NDV genotype in the country and monitor its spread and contribution to the emergence of new ND viruses.

CLONAL ARCHITECTURE OF RELAPSED OR REFRACTORY FOLLICULAR HELPER T‐CELL LYMPHOMA: AN ANCILLARY STUDY OF THE ORACLE TRIAL, A LYSA STUDY

Follicular helper T-cell lymphoma (TFHL) results from the oncogenic transformation of a TFH cell, driven by mutations in genes involved in epigenetic regulation (TET2, DNMT3A, IDH2) and T-cell signaling (RHOA). Demonstration of TET2 and DNMT3A mutations in B cells, myeloid cells, or hematopoietic progenitor cells have suggested that TFHL can emerge from clonal hematopoiesis (CH) in a multi-step process. However, the frequency, the extension, and the relevance of such CH in TFHL oncogenesis are unclear. To describe and characterize the clonal architecture of refractory/relapsed (R/R) TFHL, we collected bone marrow samples of patients included in the ORACLE trial (NCT03593018), a phase 3 trial evaluating the oral 5-azacitidine (CC-486) compared to single-agent chemotherapy in patients with R/R TFHL. Bone marrow (BM) cells from 32 patients at inclusion were cultured in methylcellulose supplemented with standard growth factors. BFU-E, CFU-GM, and CFU-GEMM were counted and individually harvested. A total of 517 colonies derived from the 32 patients were then individually genotyped by next-generation sequencing (NGS) using a capture-based panel (limit of detection: 1%) covering 43 genes recurrently mutated in CH and/or TFHL, including TET2, DNMT3A, IDH2, and RHOA. Meanwhile, bulk BM cells from 29 patients were sequenced with the same NGS panel and then compared to the mutations found in cfDNA (31 patients) and tumor biopsies (28 patients). Overall, the frequency of CH and the phylogenetic link between the myeloid progenitors and the TFHL was evaluable in 30 TFHL patients. We successfully genotyped 471/517 (91%) colonies. CH defined by at least one somatic mutation detected in at least one myeloid colony or total BM cells was found in 29/30 (97%) patients. Among the 26/29 patients with mutated myeloid colonies, the median percentage of mutated colonies was 21% (IQR 7%–53%) and 7 had subclonal architecture with a median of 3 different clones. The 2 most frequent mutations were DNMT3A and TET2, detected in 24/30 (80%) and 18/30 (60%) patients' BM, respectively. We confirmed the absence of RHOAG17V and IDH2R172 mutations in BM cells of TFHL patients. While 17/18 patients with a detectable TET2 mutation in CH had a TET2 mutated TFHL, only 13/23 patients with a DNMT3A mutated CH had a DNMT3A mutated TFHL. We thus identified 3 groups of patients according to the distribution of mutations found in the BM and the LN lymph nodes: 16/30 (53.3%) patients had a TFHL clonally derived from CH (CH-related TFHL), 13 (43.3%) had TFHL, and clonally distinct CH (CH-unrelated TFHL), and one patient (3.3%) had undetectable CH. No significant difference in patient outcome was observed regarding the presence of CH-related or unrelated TFHL, but our results are limited by the small number of patients and treatment heterogeneity. Encore Abstract—previously submitted to EHA 2023 Keywords: aggressive T-cell non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Kitaviruses: A Window to Atypical Plant Viruses Causing Nonsystemic Diseases.

Kitaviridae is a family of plant-infecting viruses that have multiple positive-sense, single-stranded RNA genomic segments. Kitaviruses are assigned into the genera Cilevirus, Higrevirus, and Blunervirus, mainly on the basis of the diversity of their genomic organization. Cell-to-cell movement of most kitaviruses is provided by the 30K family of proteins or the binary movement block, considered an alternative movement module among plant viruses. Kitaviruses stand out for producing conspicuously unusual locally restricted infections and showing deficient or nonsystemic movement likely resulting from incompatible or suboptimal interactions with their hosts. Transmission of kitaviruses is mediated by mites of many species of the genus Brevipalpus and at least one species of eriophyids. Kitavirus genomes encode numerous orphan open reading frames but RNA-dependent RNA polymerase and the transmembrane helix-containing protein, generically called SP24, typify a close phylogenetic link with arthropod viruses. Kitaviruses infect a large range of host plants and cause diseases of economic concern in crops such as citrus, tomato, passion fruit, tea, and blueberry.

Spanish Outbreak Isolates Bridge Phylogenies of European and American Bacillus anthracis.

The geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.

GENETIC FEATURES OF TARPAN-LIKE HORSES OF THE POLISH PRIMITIVE (THE KONIK) BREED AND ESTABLISHMENT OF PHYLOGENETIC OF ANCIENT EGUIDS USING THE ISSR-PCR MARK

In the development of human society, the horse, compared to other domestic animals, played a central role. It became an integral part of the economic and intellectual development of ancient human settlements, a symbol of the transformation of subsequent great epochs. The last wild horse that survived in Ukraine until the end of the XIX century, according to many paleontologists, was the tarpan. However, this statement is still questioned. In the books and documents of old chroniclers and researchers we find descriptions of wild horses called tarpans, which could be found in the wooded areas of Poland, Lithuania and Prussia in the XVIII century. Emphasizing the forest existence of these horses, they received the Latin name Equii silvestris (forest horses). According to historical records, tarpans survived the longest in the wild in the vicinity of Bialowieza Forest, more or less until 1780, when they were finally caught and transferred to the Zoo of the Counts of Zamoyski (an influential Polish count) in the town of Zwierzyniec near the town of Bilhora in Lublin Voivodeship in eastern Poland. Around 1806, the zoo ceased to exist, and the horses were distributed to local peasants. Thus, we can assume that this is the last case of domestication of wild horses in Poland. The World Wide Fund for Nature (WWF) has launched a program to return the tarpan-like Polish horse to nature. Therefore, tarpan-like horses were brought to the Belarusian part of Belovezhskaya Pushcha and to the south-west of Latvia (1999) [11]. In 2009, with the consent of the Ministry of Environment of Ukraine and the Ministry of Nature Protection of Poland, horses of the descendants of the wild tarpan horse were brought to the territory of Yavoriv National Nature Park from Roztochansky National Park to preserve and reproduce the gene pool of these horses. However, the origin of the domestic horse and the history of most modern breeds remains unclear to this day. This is due to the fact that the bones of wild and domesticated horses are almost identical. The lack of diagnostic, anatomical and biometric criteria does not allow to determine the archaeozoological remains of the horse. To solve these problems molecular genetic analysis of horse DNA is used. With the use of molecular markers in research, there are new opportunities to study genetic diversity, as well as the definition of phylogenetic relationships at both intra- and interspecific levels. One of the types of genetic polymorphism research is ISSR-PCR - amplification of intermicrosatellite DNA fragments, the purpose of which is to conduct genetic monitoring in rocks in order to preserve the allelofund of a few rocks. (3) The aim of the work was to identify intraspecific genetic variability of tarpan-like horses of the Polish konik breed and to establish phylogenetic links between ancient equids (Pleistocene horse, real tarpan) using ISSR - fingerprinting. The material for the assessment of intraspecific and interspecific genetic variability and the establishment of phylogenetic relationships between modern horses and fossil remains of ancient horses were horses of the Polish konik breed (10 heads) of the Yavoriv National Nature Park of the Lviv Region. and fossil bones of horses of the Pleistocene period (about 10 thousand years BC), metacarpal bone (os. tarsicentral). The bone was found in Novgorod-Siversky, Chernihiv region during quarry construction work. The excavations were carried out by PI Borisovsky in 1935. A tooth found in the village of Tarpan was used to study a wild tarpan horse (4.5 thousand years BC). Skibnytsia, Trostyanets district, Vinnytsia region. Excavations were conducted in 1959 by VM Danylenko. The paleontological material that was studied was provided by the Kyiv National Museum of Natural History of the National Academy of Sciences of Ukraine, Department of Paleontology. It was found that the species-specific spectra of PCR products obtained by ISSR-PCR for species E.caballus are fragments of 380-400 np and 500-520 np. The breed-specific fragments of the DNA molecule inherent in horses of the Polish konik breed include spectra of amplification products with a size of 680-710 np. It was found that the genetic distances between the amplicons of ISSR-PCR markers of Polish horses and fossil remains of a Pleistocene horse were 0.0881 and between the remains of a real tarpan - 0.0845. The low value of the share of polymorphic loci (P) and the index of polymorphic information content (PIC) was revealed, which indicates a high degree of genetic consolidation of horses of the Polish konik breed and possible reproductive isolation of the population. It was established that the spectra of amplification products of horses of the Polish horse and fossil remains of Pleistocene horse and real tarpan by 97% on the universal scale of dimension of ISSR fragments in np belonged to "light" (A-35 - A-25) and "medium" -25 - A-13), which is a specific characteristic of E. caballus species.

Re-Emergence of Dengue Serotype 3 in the Context of a Large Religious Gathering Event in Touba, Senegal.

Dengue virus (DENV) was detected in Senegal in 1979 for the first time. Since 2017, unprecedented frequent outbreaks of DENV were noticed yearly. In this context, epidemiological and molecular evolution data are paramount to decipher the virus diffusion route. In the current study, we focused on a dengue outbreak which occurred in Senegal in 2018 in the context of a large religious gathering with 263 confirmed DENV cases out of 832 collected samples, including 25 life-threatening cases and 2 deaths. It was characterized by a co-circulation of dengue serotypes 1 and 3. Phylogenetic analysis based on the E gene revealed that the main detected serotype in Touba was DENV-3 and belonged to Genotype III. Bayesian phylogeographic analysis was performed and suggested one viral introduction around 2017.07 (95% HPD = 2016.61-2017.57) followed by cryptic circulation before the identification of the first case on 1 October 2018. DENV-3 strains are phylogenetically related, with strong phylogenetic links between strains retrieved from Burkina Faso and other West African countries. These phylogenetic data substantiate epidemiological data of the origin of DENV-3 and its spread between African countries and subsequent diffusion after religious mass events. The study also highlighted the usefulness of a mobile laboratory during the outbreak response, allowing rapid diagnosis and resulting in improved patient management.

Nectar Secretion, Morphology, Anatomy and Ultrastructure of Floral Nectary in Selected Rubus idaeus L. Varieties

The distinctive features of floral nectaries facilitate identification of ecological and phylogenetic links between related taxa. The structure and functioning of nectaries determine the relationships between plants, pollinators, and the environment. The aim of the study was to determine and compare the micromorphology of the epidermis in the floral nectaries of six Rubus idaeus cultivars belonging to biennial (‘Glen Ample’, ‘Laszka’, ‘Radziejowa’) and repeated fruiting (‘Pokusa’, ‘Polana’, ‘Polka’) groups. Another objective was to characterize the cuticle ornamentation and stomatal morphology, the anatomy of the nectary epidermis, parenchyma, and sub-nectary parenchyma in the initial nectar secretion phase, as well as the ultrastructure of the nectary epidermis and parenchyma cells in the initial and full nectar secretion phases. The study was carried out using light, fluorescence, scanning and transmission-electron microscopy techniques. Semi-thin and ultrathin sections were used for the microscopic analyses. The cuticular ornamentation and stomatal morphology may be helpful elements in the identification of relatedness between Rubus species. The interaction of the extensive system of endoplasmic reticulum membranes, mitochondria, and Golgi apparatus indicates high metabolic activity, and the fusion of transport vesicles with the membrane suggests granulocrine nectar secretion. The results bring new data to the biology of plants.

Dissemination of carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan.

Carbapenems are considered last-line beta-lactams for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, their activity is compromised by the rising prevalence of carbapenemase-producing Enterobacterales (CPE), which are especially marked in the Indian subcontinent. In Pakistan, previous reports have warned about the possible spread of CPE in the community, but data are still partial. This study was carried out to analyse the prevalence of CPE, the genetic characterisation, and phylogenetic links among the spreading CPE in the community. In this cohort study, we collected 306 rectal swabs from patients visiting Benazir Bhutto hospital, Rawalpindi. CPEs were screened by using ertapenem-supplemented MacConkey agar. Identification was performed by using conventional biochemical tests, and genomes were sequenced using Illumina chemistry. Antibiotic resistance genes, plasmid incompatibility groups, and Escherichia coli phylogroups were determined in silico. Sequence types were determined by using MLST tool. The prevalence of CPE carriage observed was 14.4% (44/306 samples). The most common carbapenemase-encoding gene was bla-NDM-5 (n = 58) followed by blaNDM-1 (n = 7), blaNDM (non-assigned variant, n = 4), blaOXA-181 (n = 3), blaOXA-232 (n = 3) and blaNDM-7 (n = 1). Most of the CPE were E. coli (55/64, 86%), and the genomic analysis revealed a pauciclonal diffusion of E. coli with ST167 (n = 14), 405 (n = 10), 940 (n = 8), 648 (n = 6) and 617 (n = 5). We obtained a second sample from 94 patients during their hospital stay in whom carriage was negative at admission and found that 7 (7.4%) acquired a CPE. Our results indicate that the prevalence of CPE carriage in the Pakistani urban community was high and driven by the dissemination of some E. coli clones, with ST167 being the most frequent. The high CPE carriage in the community poses a serious public health threat and calls for implementation of adequate preventive measures.