Nucleotide Differences Research Articles

Into the Wild: A novel wild-derived inbred strain resource expands the genomic and phenotypic diversity of laboratory mouse models.

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for over a century. However, laboratory mice capture only a subset of the genetic variation found in wild mouse populations, ultimately limiting the potential of classical inbred strains to uncover phenotype-associated variants and pathways. Wild mouse populations are reservoirs of genetic diversity that could facilitate the discovery of new functional and disease-associated alleles, but the scarcity of commercially available, well-characterized wild mouse strains limits their broader adoption in biomedical research. To overcome this barrier, we have recently developed, sequenced, and phenotyped a set of 11 inbred strains derived from wild-caught Mus musculus domesticus. Each of these "Nachman strains" immortalizes a unique wild haplotype sampled from one of five environmentally distinct locations across North and South America. Whole genome sequence analysis reveals that each strain carries between 4.73-6.54 million single nucleotide differences relative to the GRCm39 mouse reference, with 42.5% of variants in the Nachman strain genomes absent from current classical inbred mouse strain panels. We phenotyped the Nachman strains on a customized pipeline to assess the scope of disease-relevant neurobehavioral, biochemical, physiological, metabolic, and morphological trait variation. The Nachman strains exhibit significant inter-strain variation in >90% of 1119 surveyed traits and expand the range of phenotypic diversity captured in classical inbred strain panels. These novel wild-derived inbred mouse strain resources are set to empower new discoveries in both basic and preclinical research.

Molecular identification and genetic diversity of ten Pierid butterflies based on mitochondrial COI gene

The IUCN Bangladesh has documented a total of 31 species belonging to the Pieridae family in Bangladesh. There is currently limited knowledge regarding the genetic diversity and molecular relationships among these butterflies. The present investigation involved the generation of mitochondrial COI (mtCOI) gene sequences for ten species of butterflies belonging to the Pierid family. After that, BLAST analysis showed 96% to 100% similarity, and then the sequences were submitted to NCBI’s GenBank. MEGA10 and BioEdit were used to identify a 584 bp COI gene fragment with 179 variable sites and 128 parsimony-informative sites. The COI sequences' average base compositions were 38.56% T, 17.12% C, 30.18% A, and 14.12% G. While a significant AT bias (68.74%) existed among the Pierid species. The genetic distance between ten Pierid species was assessed using the Kimura 2-Parameter (K2P) algorithm, and the nucleotide differences ranged from 0.00172% to 0.24261%. Afterwards, the Maximum Likelihood (ML) method was used to construct a phylogenetic tree using ten sequences of Pierid species. These species belongs to two subfamilies, Pierinae and Coliadinae in Bangladesh. On the tree, the Pierinae subfamily of Pieridae formed a paraphyletic arrangement whereas the Coliadinae subfamily was shown to be monophyletic. The data analyses support the following relationships between the two subfamilies as follows: [(Appias + Leptosia) + (Pieris + Appias) + (Delias) + (Eurema + Catopsilia) + (Pareronia)]. This supported the theory that Pierinae and Coliadinae are sister taxa. Nonetheless, Pareronia hippia remains outside the main Pierinae group, requiring further study to resolve this issue. Finally, these studies generated ten mtCOI gene sequences that have the potential to serve as valuable references for the accurate identification of Pierid species. In addition, this result could be used in the future to reveal the subfamily relationships within the Pieridae taxonomic classification. Bangladesh J. Zool. 51(3): 289-299, 2023

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

Uganda chicken genetic resources: II. genetic diversity and population demographic history inferred from mitochondrial DNA D-loop sequences.

The genetic diversity of indigenous chickens, which comprise over 80% of the chicken resources in Uganda, is largely not well-characterized for their genetic contribution. This study assessed the genetic diversity and population structure of the indigenous chicken population in Uganda to serve as an essential component for improvement and conservation strategies. A set of 344 mitochondrial DNA (mtDNA) D-loop sequences among 12 Ugandan chicken populations was evaluated. Twenty-eight polymorphic sites, accounting for 4.26% of the total analyzed loci of 658bp, defined 32 haplotypes. The haplotype diversity (Hd) was 0.437, with a nucleotide diversity (π) of 0.0169, while the average number of nucleotide differences (k) was 0.576, indicating a population that is moderately genetically diverse. Analysis of molecular variance found 98.39% (ρ < 0.01) of the total sequence variation among the chicken haplotypes within populations, 1.08% (ρ < 0.05) among populations, and 0.75% (ρ > 0.05) among populations within regions. This revealed subtle genetic differentiation among the populations, which appeared to be influenced by population fragmentation, probably due to neutral mutation, random genetic drift, and/or balancing selection. All the haplotypes showed affinity exclusively to the haplogroup-E mtDNA phylogeny, with haplotype UGA01 signaling an ancestral haplotype in Uganda. Neutrality tests Tajima's D (-2.320) and Fu's Fs (-51.369), augmented with mismatch distribution to measure signatures of recent historical demographic events, supported a population expansion across the chicken populations. The results show one matrilineal ancestry of Ugandan chickens from a lineage widespread throughout the world that began in the Indian subcontinent. The lack of phylogeographic signals is consistent with recent expansion events with extensive within-country genetic intermixing among haplotypes. Thus, the findings in this study hold the potential to guide conservation strategies and breeding programs in Uganda, given that higher genetic diversity comes from within the chicken population.

Sequence signatures within the genome of SARS-CoV-2 can be used to predict host source.

We conducted an in silico analysis to better understand the potential factors impacting host adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer, humans, and mink due to the strong evidence of sustained transmission within these hosts. Classification models trained on single nucleotide and amino acid differences between samples effectively identified white-tailed deer-, human-, and mink-derived SARS-CoV-2. For example, the balanced accuracy score of Extremely Randomized Trees classifiers was 0.984 ± 0.006. Eighty-eight commonly identified predictive mutations are found at sites under strong positive and negative selective pressure. A large fraction of sites under selection (86.9%) or identified by machine learning (87.1%) are found in genes other than the spike. Some locations encoded by these gene regions are predicted to be B- and T-cell epitopes or are implicated in modulating the immune response suggesting that host adaptation may involve the evasion of the host immune system, modulation of the class-I major-histocompatibility complex, and the diminished recognition of immune epitopes by CD8+ T cells. Our selection and machine learning analysis also identified that silent mutations, such as C7303T and C9430T, play an important role in discriminating deer-derived samples across multiple clades. Finally, our investigation into the origin of the B.1.641 lineage from white-tailed deer in Canada discovered an additional human sequence from Michigan related to the B.1.641 lineage sampled near the emergence of this lineage. These findings demonstrate that machine-learning approaches can be used in combination with evolutionary genomics to identify factors possibly involved in the cross-species transmission of viruses and the emergence of novel viral lineages.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus capable of infecting and establishing itself in human and wildlife populations, such as white-tailed deer. This fact highlights the importance of developing novel ways to identify genetic factors that contribute to its spread and adaptation to new host species. This is especially important since these populations can serve as reservoirs that potentially facilitate the re-introduction of new variants into human populations. In this study, we apply machine learning and phylogenetic methods to uncover biomarkers of SARS-CoV-2 adaptation in mink and white-tailed deer. We find evidence demonstrating that both non-synonymous and silent mutations can be used to differentiate animal-derived sequences from human-derived ones and each other. This evidence also suggests that host adaptation involves the evasion of the immune system and the suppression of antigen presentation. Finally, the methods developed here are general and can be used to investigate host adaptation in viruses other than SARS-CoV-2.

The surface of leaves and fruits of Peruvian cacao is home for several Hannaella yeast species, including the new species Hannaella theobromatis sp. nov.

As part of a long-term study aiming to isolate and identify yeast species that inhabit the surface of leaves and fruits of native fine-aroma cacao in the department of Amazonas, Peru, we obtained multiple isolates of Hannaella species. Yeasts of the genus Hannaella are common inhabitants of the phyllosphere of natural and crop plants. On the basis of morphological,and physiological characteristics, and sequence analysis of the D1/D2 domains of the large subunit rRNA gene (LSU) and the internal transcribed spacerregion (ITS), we identified five species of Hannaella from the phyllosphere of Peruvian cacao. Four have been previously described: H. phyllophila (isolates KLG-073, KLG-091), H. pagnoccae (KLG-076), H. sinensis (KLG-121), and H. taiwanensis (KLG-021). A fifth, represented by eight isolates (KLG-034, KLG-063, KLG-074, KLG-078, KLG-79, KLG-082, KLG-084, KLG-085), is not conspecific with any previously described Hannaella species,and forms the sister clade to H. surugaensis in thephylogenetic analysis. It has 2.6-3.9% (18-27 substitutions, 2-4 deletions, and 1-3 insertions in 610-938 bp-long alignments), and 9.8-10.0% nucleotide differences (37 substitutions and 14 insertions in 511-520 bp-long alignments) in the LSU andITS regions, respectively, to H. surugaensis type strain, CBS 9426. Herein, the new species Hannaella theobromatis sp. nov. is described and characterised. The species epithet refers to its epiphytic ecology on its host Theobroma cacao.

Genetic differentiation of Plasmodium vivax duffy binding protein in Ethiopia and comparison with other geographical isolates

BackgroundPlasmodium vivax Duffy binding protein (PvDBP) is a merozoite surface protein located in the micronemes of P. vivax. The invasion of human reticulocytes by P. vivax merozoites depends on the parasite DBP binding domain engaging Duffy Antigen Receptor for Chemokine (DARC) on these red blood cells (RBCs). PvDBPII shows high genetic diversity which is a major challenge to its use in the development of a vaccine against vivax malaria.MethodsA cross-sectional study was conducted from February 2021 to September 2022 in five study sites across Ethiopia. A total of 58 blood samples confirmed positive for P. vivax by polymerase chain reaction (PCR) were included in the study to determine PvDBPII genetic diversity. PvDBPII were amplified using primers designed from reference sequence of P. vivax Sal I strain. Assembling of sequences was done using Geneious Prime version 2023.2.1. Alignment and phylogenetic tree constructions using MEGA version 10.1.1. Nucleotide diversity and haplotype diversity were analysed using DnaSP version 6.12.03, and haplotype network was generated with PopART version 1.7.ResultsThe mean age of the participants was 25 years, 5 (8.6%) participants were Duffy negatives. From the 58 PvDBPII sequences, seven haplotypes based on nucleotide differences at 8 positions were identified. Nucleotide diversity and haplotype diversity were 0.00267 ± 0.00023 and 0.731 ± 0.036, respectively. Among the five study sites, the highest numbers of haplotypes were identified in Arbaminch with six different haplotypes while only two haplotypes were identified in Gambella. The phylogenetic tree based on PvDBPII revealed that parasites of different study sites shared similar genetic clusters with few exceptions. Globally, a total of 39 haplotypes were identified from 223 PvDBPII sequences representing different geographical isolates obtained from NCBI archive. The nucleotide and haplotype diversity were 0.00373 and 0.845 ± 0.015, respectively. The haplotype prevalence ranged from 0.45% to 27.3%. Two haplotypes were shared among isolates from all geographical areas of the globe.ConclusionsPvDBPII of the Ethiopian P. vivax isolates showed low nucleotide but high haplotype diversity, this pattern of genetic variability suggests that the population may have undergone a recent expansion. Among the Ethiopian P. vivax isolates, almost half of the sequences were identical to the Sal-I reference sequence. However, there were unique haplotypes observed in the Ethiopian isolates, which does not share with isolates from other geographical areas. There were two haplotypes that were common among populations across the globe. Categorizing population haplotype frequency can help to determine common haplotypes for designing an effective blood-stage vaccine which will have a significant role for the control and elimination of P. vivax.

Erysipelothrix amsterdamensis sp. nov., associated with mortalities among endangered seabirds.

Infectious diseases threaten endangered species, particularly in small isolated populations. Seabird populations on the remote Amsterdam Island in the Indian Ocean have been in decline for the past three decades, with avian cholera caused by Pasteurella multocida proposed as the primary driver. However, Erysipelothrix species have also been sporadically detected from albatrosses on Amsterdam Island and may be contributing to some of the observed mortality. In this study, we genomically characterized 16 Erysipelothrix species isolates obtained from three Indian yellow-nosed albatross (Thalassarche carteri) chick carcasses in 2019. Histological analyses suggest that they died of bacterial septicaemia. Two isolates were sequenced using both Illumina short-read and MinION long-read approaches, which - following hybrid assembly - resulted in closed circular genomes. Mapping of Illumina reads from the remaining isolates to one of these new reference genomes revealed that all 16 isolates were closely related, with a maximum of 13 nucleotide differences distinguishing any pair of isolates. The nucleotide diversity of isolates obtained from the same or different carcasses was similar, suggesting all three chicks were likely infected from a common source. These genomes were compared with a global collection of genomes from Erysipelothrix rhusiopathiae and other species from the same genus. The isolates from albatrosses were phylogenetically distinct, sharing a most recent common ancestor with E. rhusiopathiae. Based on phylogenomic analysis and standard thresholds for average nucleotide identity and digital DNA-DNA hybridization, these isolates represent a novel Erysipelothrix species, for which we propose the name Erysipelothrix amsterdamensis sp. nov. The type strain is A18Y020dT (=CIP 112216T=DSM 115297T). The implications of this bacterium for albatross conservation will require further study.

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs featureimproved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are providedby a double-stranded DNA fragment,a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and applicationin nucleic acid detection assays.

Nanoporous Dna Field Effect Transistor with Potential for Random‐Access Memory Applications: A Selectivity Performance Evaluation

AbstractMethods to encode digital data items as strands of synthetic DNA followed by selective data retrieval have been demonstrated. However, these initially bio‐oriented processes remain slow and not optimized. DNA field‐effect transistor (DNA‐FET) is studied here as a possible random‐access memory (RAM) device for simple, selective and rapid ssDNA fragment retrieval used as data pool identifier. The DNA‐FET is based on a co‐planar Au‐gated fully organic transistor appended with short single‐stranded DNA (ssDNA) probes bearing a blocking molecule to prevent partial hybridization and achieve near perfect selectivity for short length ssDNA (up to 45 nt). Examination of transconductance of the novel active layer incorporating a DNA nanopore architecture reveals enhanced binding site accessibility. This, in turn, facilitates discriminatory hybridization, particularly in the physical retrieval of short‐length ssDNA from a competitive, concentrated ssDNA background pool consisting of nine different sequences, with at least one nucleotide difference. The DNA‐FET exhibits rapid operation (9 min) in the millivolt range, low detection limit (sub‐femtomolar), high selectivity and reusability. Considering the straightforward concept, near error‐free identification capacity and hypothetically outstanding scalability, the DNA‐FET described here has potential as a foundation for further exploration of advanced RAM technology in the DNA data storage process.

Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoea isolates in Ghana.

Gonorrhoea is a disease associated with humans and caused by Neisseria gonorrhoea. N. gonorrhoea's ability to evolve and evade various treatment regimens can lead to untreatable gonorrhoea. In the absence of a viable vaccine and a national database on the antimicrobial resistance (AMR) and molecular characteristics of N. gonorrhoea, and with reliance on a syndromic management regime, continuous national antimicrobial resistance surveillance and molecular characterization of N. gonorrhoea remain imperative. Only two gonococcal studies have described N. gonorrhoea's molecular characteristics linked to AMR in Ghana. Secondary N. gonorrhoea isolates (n=4) were collected from two metropolises in Ghana: Tamale in the northern sector (n=1) and Accra in the southern sector (n=3). The isolates were confirmed and characterized using polymerase chain reaction (PCR) targeting the porB and tbpB genes, and the disc diffusion method was used to evaluate AMR. N. gonorrhoea multi-antigen sequence typing (NG-MAST) and porin B (porB) gene sequence analyses were employed to reveal the molecular epidemiology and evolutionary trajectory, respectively. All four isolates showed resistance to at least four of the tested antibiotics. One isolate showed resistance to all seven antibiotics, i.e. ceftriaxone, azithromycin, ciprofloxacin, tetracycline, erythromycin, togamycin and penicillin. NG-MAST typing revealed isolate S3 (MZ313864) as ST211. The locus of S2 (MZ313863) (transferrin-binding protein B; tbpB) was identified as tbpB1844, and its porB locus, as porB6412, with only 4 closely related variants but with 15 nucleotide differences. However, its sequence type does not exist. The porB analysis identified isolate S3 (MZ313864) to be found globally, while S2 (MZ313863) is unique to this study. Despite the small number of isolates tested, this study recorded multidrug resistance and previously unknown gonococcal variants based on porB gene. Additionally, the molecular typing schemes revealed a disparity between NG-MAST and the National Center for Biotechnology Information (NCBI) platforms. There is a need for continuous gonococcal AMR and molecular surveillance in Ghana to contribute to the global efforts to describe circulating strains and support proper application of the syndromic management regime to gonorrhoea.

First report on the molecular detection and genetic diversity of Anaplasma marginale in healthy dairy cattle in Khon Kaen province, Thailand

Background and Aim: Bovine anaplasmosis (BA) is one of the most important diseases of ruminants worldwide, causing significant economic losses in the livestock industry due to the high morbidity and mortality in susceptible cattle herds. Anaplasma marginale is the main causative agent of BA occurring worldwide in tropical and subtropical regions. This study aimed to investigate the first molecular detection and genetic diversity of A. marginale in dairy cattle in Khon Kaen Province, Thailand. Materials and Methods: Blood samples were collected from 385 lactating cows from 40 dairy farms in five districts of Khon Kaen, regardless of age and health status. To detect A. marginale, all DNA preparations were used for molecular diagnosis using a single polymerase chain reaction with the msp4 gene target. A phylogenetic tree was constructed from the msp4 gene sequences using molecular genetic characterization. Genetic diversity was calculated as haplotype diversity, haplotype number, number of nucleotide differences, nucleotide diversity, and average number of nucleotide differences. Results: The overall prevalence of A. marginale was 12.72% (49/385). The highest prevalence (17.19%) was found in Ubolratana district, followed by Muang, Kranuan, Khao Suan Kwang, and Nam Phong districts (14.94%, 14.74%, 13.79%, and 3.70%, respectively). Phylogenetic analysis showed that A. marginale was closely related to isolates from Australia (98.96%), China (99.68%), Spain (99.74%), and the USA (99.63%). Conclusion: The molecular prevalence of BA in dairy cattle is the first to be observed in this area, and the genetic variability with separated clusters shown in the msp4 gene of A. marginale revealed species variation in dairy cattle. This significant genetic diversity contributes to the understanding of the diversity of A. marginale and will be important for the control and prevention of A. marginale in dairy cattle. Keywords: Anaplasma marginale, bovine anaplasmosis, dairy cattle, molecular prevalence.

First report on the molecular phylogenetics and population genetics of Aedes aegypti in Iran

BackgroundAedes aegypti, the primary vector of various human arboviral diseases, is a significant public health threat. Aedes aegypti was detected in Iran in 2018, in Hormozgan province, but comprehensive information regarding its genetic diversity and origin within the country remains scarce. This study aimed to determine the origin and genetic diversity of Ae. aegypti in southern Iran.MethodsAedes aegypti mosquitoes were collected from Bandar Abbas City, Hormozgan Province, southern Iran, between May and July 2022. Specimens were morphologically identified. Origin and assess genetic diversity were assessed based on the mitochondrial DNA-encoded cytochrome c oxidase subunit I (mtDNA-COI) gene.ResultsBLAST (basic local alignment search tool) analysis confirmed the accuracy of the morphological identification of all specimens as Ae. aegypti, with 100% similarity to GenBank sequences. Calculated variance and haplotype diversity were 0.502 and 0.00157, respectively. Among the 604 examined nucleotide sequences, only a single site was non-synonymous. Total nucleotide diversity and average pairwise nucleotides were determined as 0.00083 and 0.502, respectively. Fu and Li's D test values were not statistically significant. Strobeck’s S statistic value was 0.487, and Tajima’s D value was 1.53395; both were not statistically significant (P > 0.10).ConclusionsPhylogenetic analysis revealed two distinct clades with minimal nucleotide differences and low haplotype diversity, suggesting the recent establishment of Ae. Aegypti in the southern region of Iran. The phylogenetic analysis also indicated an association between Ae. aegypti populations and mosquitoes from Saudi Arabia and Pakistan.Graphical

Ratiometric sandwich-type assays for RNAs with a point mutation using benzo[a]pyrene-modified probes.

Benzo[a]pyrene-modified oligonucleotides were developed for the detection of RNAs with a point mutation. The probes produced two distinct fluorescence signals in response to single nucleotide differences in the RNA sequences, allowing for discrimination between the matched and single base mismatched RNA sequences in colorimetric and ratiometric manners.

Phylogeny and molecular evolution of the first local monkeypox virus cluster in Guangdong Province, China

The first local mpox outbreak in Guangdong Province, China occurred in June 2023. However, epidemiological data have failed to quickly identify the source and transmission of the outbreak. Here, phylogeny and molecular evolution of 10 monkeypox virus (MPXV) genome sequences from the Guangdong outbreak were characterized, revealing local silent transmissions that may have occurred in Guangdong whose mpox outbreaks suggested a molecular epidemiological correlation with Portugal and several regions of China during the same period. The lineage IIb C.1, which includes all 10 MPXV from Guangdong, shows consistent temporal continuity in both phylogenetic characteristics and unique molecular evolutionary mutation spectrum, reflected in the continuous increase of single nucleotide polymorphisms (SNPs) and shared mutations over time. Compared with the Japan MPXV, the Guangdong MPXV showed higher genomic nucleotide differences and separated 14 shared mutations from the B.1 lineage, comprising 6 non-synonymous mutations in genes linked to host regulation, virus infection, and virus life cycle. The unique mutation spectrum with temporal continuity in IIb C.1, related to apolipoprotein B mRNA-editing catalytic polypeptide-like 3, promotes rapid viral evolution and diversification. The findings contribute to understanding the ongoing mpox outbreak in China and offer insights for developing joint prevention and control strategies.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Xanthium orientale in Korea.

Xanthium orientale L. (syn. Xanthium canadense Mill., Asteraceae), known as cocklebur, is an annual weed native to North America, which is now a neophyte distributed throughout the world. This plant was accidentally introduced to Korea in the late 1970s ( So et al. 2008) and is considered a problematic exotic weed in orchards, for which many herbicides are ineffective (Kim et al. 2020). In September 2018, powdery mildew was observed on X. orientale in Jeju, Korea. The disease incidence ranged from 40 to 60%. Voucher specimens were deposited in the Korea University Herbarium (Accession No. KUS-F30795) and Kunsan National University Herbarium (KSNUH1988). Symptoms appeared as round to irregular white patches with abundant hyphal growth on the leaf surface. Hyphal appressoria were nipple-shaped, and 3 to 6 μm diam. Conidiophores (n = 30) were 145 to 206 × 9 to 11.6 µm and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot-cells of the conidiophores were straight, cylindrical, and 43 to 100.9 μm long. Conidia (n = 30) were ellipsoid-ovoid, doliiform to somewhat limoniform, 25.2 to 31.8 × 13.6 to 16.8 μm (l/w 1.6 to 2.1), and devoid of distinct fibrosin bodies. The morphological characteristics corresponded to those of Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, under Golovinomyces spadiceus (Beck. & M.A. Curtis) U. Braun; Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), large subunit (LSU) (Bradshaw and Tobin 2020), the intergenic spacer (IGS) of rDNA, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Bradshaw et al. 2022) were amplified for a herbarium specimen (KUS-F30795). A BLASTn search of these sequences revealed 100% identity with reference sequences of G. ambrosiae on diverse Asteraceae plants (AB077644 for ITS, AB077643 for LSU, ON361171 for IGS, and ON075648 for GAPDH). However, there was a single nucleotide difference on both the IGS and GAPDH sequences when compared to the closely related species Golovinomyces latisporus. The sequences were deposited in GenBank (Accession No. OQ165157 (ITS), OQ165164 (LSU), OR050524 (IGS), and OR086076 (GAPDH)). Phylogenetic analyses of ITS, LSU, IGS, and GAPDH sequences revealed the Korean sample formed a well-supported group with other G. ambrosiae sequences, confirming its identity. A pathogenicity test was performed through inoculation by gently pressing diseased leaves onto the leaves of five healthy plants. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25±2°C. Powdery mildew colonies developed on the inoculated plants after ten days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the initially diseased leaves, fulfilling Koch's postulates. Powdery mildew on X. orientale has previously been reported as Golovinomyces cichoracearum (≡ Erysiphe cichoracearum) sensu lato in the USA, G. ambrosiae (= G. spadiceus) throughout all continents, and Podosphaera fusca sensu lato (now P. xanthii) in Korea (Braun and Cook 2012; Farr and Rossman 2023). To date, powdery mildew in Korea has been reported only on Xanthium strumarium as G. cichoracearum s. lat. and Podosphaera xanthii (KSPP 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on X. orientale in Korea.

Plasmodium falciparum population structure and genetic diversity of cell traversal protein for ookinetes and sporozoites (CelTOS) during malaria resurgences in Dielmo, Senegal

The ability to accurately measure the intensity of malaria transmission in areas with low transmission is extremely important to guide elimination efforts. Plasmodium falciparum Cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an important conserved sporozoite antigen reported as one of the promising malaria vaccine candidates, and could be used to estimate malaria transmission intensity. This study aimed at determining whether the diversity of PfCelTOS gene reflects the changes in malaria transmission that occurred between 2007 and 2014 in Dielmo, a Senegalese village, before and after the implementation of insecticide treated bed nets (ITNs). Of the 109 samples positive for PfCelTOS PCR, 96 (88%) were successfully sequenced and analysed for polymorphisms and population diversity. The number of segregating sites was higher during the pre-intervention period (13) and the malaria resurgences (11) than during the intervention period (5). Similarly, the number and diversity of haplotypes were higher during the pre-intervention period (16 and 0.914, respectively) and the malaria resurgences (6 and 0.821, respectively) than during the intervention period (4 and 0.758, respectively). Moreover, the average number of nucleotide differences was higher during the pre-intervention (3.792) and during malaria resurgences (3.467) than during the intervention period (2.189). The 3D7 KSSFNEP haplotype was only observed during the intervention period. Only two haplotypes were shared in both the pre-intervention and intervention periods while four haplotypes were shared between the pre-intervention and the malaria resurgences. The Fst values indicate moderate differentiation between pre-intervention and intervention periods (0.17433), and between intervention and malaria resurgences period (0.19198) as well as between pre-intervention and malaria resurgences periods (0.06607).PfCelTOS genetic diversity reflected changes of malaria transmission, with higher polymorphisms recorded before the large-scale implementation of ITNs and during the malaria resurgences. PfCelTOS is also a candidate vaccine; mapping its diversity across multiple endemic environments will facilitate the design and optimisation of a broad and efficacious vaccine.

Molecular Epidemiology and Evolution of Coxsackievirus A14.

As the proportion of non-enterovirus 71 and non-coxsackievirus A16 which proportion of composition in the hand, foot, and mouth pathogenic spectrum gradually increases worldwide, the attention paid to other enteroviruses has increased. As a member of the species enterovirus A, coxsackievirus A14 (CVA14) has been epidemic around the world until now since it has been isolated. However, studies on CVA14 are poor and the effective population size, evolutionary dynamics, and recombination patterns of CVA14 are not well understood. In this study, 15 CVA14 strains were isolated from HFMD patients in mainland China from 2009 to 2019, and the complete sequences of CVA14 in GenBank as research objects were analyzed. CVA14 was divided into seven genotypes A-G based on an average nucleotide difference of the full-length VP1 coding region of more than 15%. Compared with the CVA14 prototype strain, the 15 CVA14 strains showed 84.0-84.7% nucleotide identity in the complete genome and 96.9-97.6% amino acid identity in the encoding region. Phylodynamic analysis based on 15 CVA14 strains and 22 full-length VP1 sequences in GenBank showed a mean substitution rate of 5.35 × 10-3 substitutions/site/year (95% HPD: 4.03-6.89 × 10-3) and the most recent common ancestor (tMRCA) of CVA14 dates back to 1942 (95% HPD: 1930-1950). The Bayesian skyline showed that the effective population size had experienced a decrease-increase-decrease fluctuation since 2004. The phylogeographic analysis indicated two and three possible migration paths in the world and mainland China, respectively. Four recombination patterns with others of species enterovirus A were observed in 15 CVA14 strains, among which coxsackievirus A2 (CVA2), coxsackievirus A4 (CVA4), coxsackievirus A6 (CVA6), coxsackievirus A8 (CVA8), and coxsackievirus A12 (CVA12) may act as recombinant donors in multiple regions. This study has filled the gap in the molecular epidemiological characteristics of CVA14, enriched the global CVA14 sequence database, and laid the epidemiological foundation for the future study of CVA14 worldwide.

Assessment of genetic diversity and phylogenetic relationship of local coffee populations in southwestern Saudi Arabia using DNA barcoding.

The genetic diversity of local coffee populations is crucial to breed new varieties better adapted to the increasingly stressful environment due to climate change and evolving consumer preferences. Unfortunately, local coffee germplasm conservation and genetic assessment have not received much attention. Molecular tools offer substantial benefits in identifying and selecting new cultivars or clones suitable for sustainable commercial utilization. New annotation methods, such as chloroplast barcoding, are necessary to produce accurate and high-quality phylogenetic analyses. This study used DNA barcoding techniques to examine the genetic relationships among fifty-six accessions collected from the southwestern part of Saudi Arabia. PCR amplification and sequence characterization were used to investigate the effectiveness of four barcoding loci: atpB-rbcl, trnL-trnF, trnT-trnL, and trnL. The maximum nucleotide sites, nucleotide diversity, and an average number of nucleotide differences were recorded for atpB-rbcl, while trnT-trnL had the highest variable polymorphic sites, segregating sites, and haploid diversity. Among the four barcode loci, trnT-trnL recorded the highest singleton variable sites, while trnL recorded the highest parsimony information sites. Furthermore, the phylogenetic analysis clustered the Coffea arabica genotypes into four different groups, with three genotypes (KSA31, KSA38, and KSA46) found to be the most divergent genotypes standing alone in the cluster and remained apart during the analysis. The study demonstrates the presence of considerable diversity among coffee populations in Saudi Arabia. Furthermore, it also shows that DNA barcoding is an effective technique for identifying local coffee genotypes, with potential applications in coffee conservation and breeding efforts.

APOBEC3 deaminase editing in mpox virus as evidence for sustained human transmission since at least 2016.

Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.