Infection, Genetics and Evolution

BackgroundThe invasive mosquito Aedes koreicus has established populations in several European countries during the past decade, raising increasing public health concerns due to its potential role as a vector of pathogens. While species identification is primarily based on morphological characters, Ae. koreicus exhibits distinct morphological variants originating from mainland Korea and Jeju Island, which complicates surveillance and may lead to misidentification, particularly in regions where closely related species co-occur. To date, the genetic basis and population-level relevance of these morphological forms in Europe remain poorly understood. MethodsWe investigated the co-occurrence of two morphological forms of Ae. koreicus in Hungary, representing the first confirmed European location where both forms were detected sympatrically and even within the same breeding sites. Adult mosquitoes were morphologically characterised using diagnostic traits, and individuals representing both morphotypes were subjected to comprehensive genetic analyses. We sequenced multiple mitochondrial markers (COX1, COX2, COX3, ATP6, ND1, ND3) and the nuclear ITS2 region using Oxford Nanopore long-read sequencing. Phylogenetic reconstructions and haplotype network analyses were applied to assess genetic differentiation between morphotypes and to compare them with Aedes japonicus as a closely related reference species. ResultsAcross all analysed mitochondrial and nuclear markers, no genetic differentiation was detected between specimens identified as the "mainland" or "Jeju-do" morphological forms of Ae. koreicus. Phylogenetic and haplotype network analyses consistently grouped individuals independently of their morphotype, indicating a shared genetic background at the population level. In contrast, Ae. japonicus formed a clearly distinct genetic lineage, confirming the robustness of the applied markers for interspecific discrimination. ConclusionsOur results demonstrate that the observed morphological variability in Ae. koreicus populations are not underpinned by detectable genetic differentiation using commonly applied mitochondrial and genomic markers. These findings highlight the limitations of using morphology alone to infer population origin or structure and emphasise the need for heightened awareness of intraspecific variability in routine surveillance. Accurate morphological identification remains critical, particularly in citizen science-based monitoring programmes and AI-based automatization platforms, such as Mosquito Alert, to avoid confusion with morphologically similar invasive species. Further studies integrating genomic and ecological approaches are required to elucidate the mechanisms underlying morphological variability in this emerging vector species.

Pathogenesis inflicted by Entamoeba histolytica causes amoebic diarrhea and liver abscesses and is one of the leading causes of mortality from parasitic disease worldwide. Preventive therapeutics in the form of a vaccine could be highly effective at providing umbrella protection for a vulnerable community. In this study, a group of ten putative hypothetical surface N-linked glycoproteins was examined to assess their potential as vaccine candidates and/or diagnostic markers against amoebic intestinal colitis and liver abscesses. To evaluate the immunogenicity of putative surface glycoproteins, we used Entamoeba histolytica-infected patients sera from Bangladesh and then assessed the titer of antibody these glycoproteins elicited in patients by enzyme-linked immunosorbent assay (ELISA) and immunoblot. Based on this study, eight of ten surface glycoproteins were found immunogenic, as specific antibodies against these glycoproteins were detected in patients sera. Three of the immunogenic glycoproteins showed strong IgG antibody responses in both patients with intestinal amoebiasis and those with liver abscess. On the other hand, the other two glycoproteins showed the presence of specific serum antibodies exclusively in patients with amoebic liver abscesses, not in individuals with amoebic colitis. The remaining two glycoproteins showed more specific sera antibodies against ALA, but the preference was not very distinct. Immunolocalization with a specific antibody against the most immunogenic glycoproteins further confirmed their presence in the cell membrane. This differential immunogenicity of these glycoproteins in the two groups of patients qualifies them to become prospective sera-based diagnostic markers and also has the potential to become vaccine candidates for amoebiasis protection.

BackgroundAedes albopictus is among the worlds most invasive mosquito species and an important vector of dengue, Zika, and chikungunya viruses. Its global spread has been strongly associated with human-mediated transport and international trade, particularly through commodities such as used tires and ornamental plants. However, integrative studies combining population genetics, microbial symbiosis, and trade connectivity remain limited in Latin America, constraining understanding of invasion dynamics and dispersal processes. MethodsAedes albopictus populations from five Colombian departments sampled between 2019 and 2023 were analyzed using eight microsatellite loci and a [~]1.3-kb mitochondrial COI fragment. Wolbachia infection and lineage composition (wAlbA/wAlbB) were evaluated by PCR, and arbovirus detection (DENV, CHIKV, ZIKV) was performed using multiplex RT-PCR. Nuclear and mitochondrial differentiation (FST, {Phi}ST), mito-nuclear discordance, and trade-related connectivity were evaluated in relation to geographic, national transport, and international trade variables derived from customs databases. ResultsMicrosatellite analyses revealed admixed but structured populations consistent with multiple introductions and contemporary gene flow. Colombian populations showed nuclear genetic affinities with Asian, European, and North American populations, supporting a complex invasion history involving multiple geographically distributed lineages. In contrast, mitochondrial COI data showed evidence of regional genetic structure and heterogeneous mito-nuclear discordance among several population pairs. Notably, nuclear and mitochondrial markers captured contrasting dimensions of the invasion process: nuclear differentiation was positively associated with international trade intensity, particularly shipment frequency and used tire importation, whereas mitochondrial differentiation retained stronger regional structure and showed no detectable association with trade-related variables. Wolbachia prevalence ranged from 34% to 100% across departments and showed exploratory patterns consistent with localized mitochondrial differentiation. Natural detection of DENV and CHIKV RNA in larvae provided evidence of local arbovirus circulation. ConclusionsIntegrating population genetics, trade connectivity, and Wolbachia screening supports a scenario in which the Colombian invasion of Ae. albopictus has been shaped by multiple introductions, contemporary human-mediated connectivity, and partially discordant mito-nuclear histories. These findings highlight how different genomic compartments retain complementary signatures of invasion dynamics, with contemporary trade-associated connectivity primarily reflected in nuclear structure and regional lineage persistence retained in mitochondrial variation. Author SummaryThe Asian tiger mosquito, Aedes albopictus, is one of the worlds most invasive mosquito vectors and continues to expand across Latin America through human transportation and trade networks. However, the processes shaping its spread in the region remain poorly understood. We combined population genetics, international trade data, Wolbachia screening, and arbovirus surveillance to investigate the invasion dynamics of Ae. albopictus in Colombia. Our results revealed evidence of multiple introductions and ongoing genetic admixture, with international trade connectivity emerging as an important predictor of contemporary nuclear genetic structure. In contrast, mitochondrial DNA retained stronger regional patterns, generating heterogeneous mito-nuclear discordance among populations. These findings suggest that different genomic compartments retain distinct signatures of the invasion process, with trade-associated connectivity reflected primarily in nuclear variation and stronger regional structure preserved in mitochondrial lineages. More broadly, our study highlights the complex invasion dynamics of Ae. albopictus in Latin America illustrates how integrating genetics and human connectivity data can improve understanding of invasive vector spread.

Influenza C (ICV) and influenza D (IDV) viruses belong to the Orthomyxoviridae family and are classified in the genera Gammainfluenzavirus and Deltainfluenzavirus, respectively. Although the main reservoir of ICV is humans, IDV is mainly found in cattle. To date, the zoonotic potential of IDV has not been fully elucidated. ICV and IDV share about 50% homology at the genetic level, and both express hemagglutinin esterase fusion (HEF) glycoproteins on the surface for the dual purpose of binding the receptor and releasing new virions. Using pseudotyped viruses (PVs) in a pseudotyped virus-based microneutralisation assay (pMN), some bovine serum samples showed strong neutralisation of both ICV and IDV. In silico analyses were performed to explore the molecular basis of this phenomenon. HEF structures were recovered from the Protein Data Bank, epitopes were predicted using BepiPred, and sialic acid receptor docking was evaluated with HDOCK. Five potential epitopes were selected, and mutual substitutions of amino acid residues were introduced to generate mutant ICV and IDV HEFs and corresponding PVs. Although only mutant IDV PVs were successfully produced, a reference ICV antiserum showed high neutralising activity against one construct, indicating the exposure of an ICV-like antigenic site within the IDV framework. Herein we provide evidence consistent with the existence of antigenic sites shared between ICV and IDV, which could be exploited for cross-protective vaccine design, through integrated computational and experimental investigations.

BackgroundHelicobacter pylori coloniz the gastric mucosa of nearly half of the global population and is classified as a Group I carcinogen by the World Health Organization due to its strong association with gastric cancer. The growing prevalence of antibiotic-resistant H. pylori strains significantly compromises current therapeutic strategies, emphasizing the urgent need for effective prophylactic approaches. Research design and methodsIn this study, a novel multi-epitope vaccine was designed targeting H. pylori, incorporating epitopes from four key virulence proteins: BabB, SabB, SabA, and VacA. Using an immunoinformatics-guided structural vaccinology approach, B- and T-cell epitopes were predicted, prioritized based on immunogenicity, conservation, population coverage, and non-homology to human proteins, and assembled into the final vaccine construct. To enhance immunogenicity and specifically stimulate mucosal immune responses, the cholera toxin B subunit (CTB) was fused at the N-terminal via an EAAAK linker, a novel application in H. pylori multi-epitope vaccines. The PADRE universal epitope and additional linkers were incorporated to optimize epitope presentation and helper T-cell activation. ResultsComprehensive evaluations of physicochemical, antigenic, allergenic, and toxic properties were conducted, followed by secondary and tertiary structure modeling, refinement, and validation. Conformational B-cell epitopes were mapped, and molecular docking, binding affinity analysis, energy minimization, and molecular dynamics simulations confirmed structural stability and re-ceptor interactions. Codon optimization and in silico cloning predicted efficient expression in Escherichia coli, while immune simulations suggested robust humoral and cellular responses. ConclusionsThis study presents a promising multi-epitope vaccine candidate against H. pylori, offering a rational framework for future experimental validation and potential clinical application.

Lumpy skin disease (LSD) is an emerging cattle disease caused by lumpy skin disease virus (LSDV), with major impacts on the industry, being classified as a Category A disease. Although it was historically confined to Africa, LSD has expanded into the Middle East, Asia and Europe. Here, we report two LSDV genomes from the first outbreak detected in Catalonia, Spain, in October 2025. The genomes were assembled from high-throughput sequencing data generated from two homogenized skin nodules. Comparative phylogenetic analyses were performed using all available complete LSDV genomes and rpo30 gene sequences. These analyses placed the LSDV isolates detected in Catalonia within clade 1.2, closely related to the isolates recently reported in Sardinia, Italy. Our findings also support a connection between recent south-western Europe and central African strains, possibly through northern Africa, and highlight the need for more complete genomes to clarify the origin and connections among recent LSDV outbreaks.

Rabies remains a significantly underreported fatal zoonosis in India, where the Arctic-like 1a (AL1a) lineage predominates in dog populations. While atypical clinical presentations in dogs can delay diagnosis and increase human exposure risk, genomic and clinical data on neonatal canine rabies remain limited. This study reports an exceptional case of rabies in a 54-day old unvaccinated German shepherd puppy which presented with severe pruritus and self-biting behaviour. The puppy was euthanized due to poor clinical response. Post-mortem testing revealed viral antigen (by Direct Fluorescent Antibody Test) and viral RNA (by real-time RTPCR) in the brain tissue. Whole-genome sequencing recovered a near-complete rabies virus genome (11,947 nucleotides; 99.5% genome coverage), classified within the AL1a_A1.1 sublineage. Phylogenetic analysis revealed close genetic relatedness to contemporary Indian rabies virus strains. Comparative genomic analysis identified 4, 3, 6, and 8 non-synonymous substitutions in the phosphoprotein, matrix, glycoprotein, and polymerase genes, respectively. This case is one of the youngest documented cases of canine rabies with atypical manifestations, caused by the AL1a viral clade. Our findings highlight the risks associated with neonatal canine rabies, the need for heightened clinical suspicion in atypical cases, and the importance of genomic surveillance to monitor evolving rabies virus lineages in endemic regions.

BackgroundLyme borreliosis is a common tick-borne disease in Europe caused by spirochetes of the Borrelia burgdorferi sensu lato complex, including Borrelia afzelii, which is maintained in nature through interactions with rodent reservoir hosts. These spirochetes have evolved several surface proteins to manipulate rodent host immunity, some of which remain polymorphic in Borrelia populations. Among these proteins, OspE, which binds the host complement-regulating factor CFH to evade destruction by complement, is one of the most variable. Yet, what evolutionary forces maintain this polymorphism is not well understood. Motivated by a recent discovery of CFH polymorphism in the bank vole (Clethrionomys glareolus), the main reservoir host of B. afzelii, we hypothesized that the polymorphism is maintained by host-parasite coevolution involving specific associations between host and parasite genetic variants. MethodsWe analyzed associations between bank vole CFH alleles and B. afzelii OspE variants across three datasets sampled in Poland. Selection acting on OspE was evaluated using omegaMap. Host-pathogen genotype associations were tested using partial redundancy analysis (RDA), and co-structure was assessed using co-correspondence analysis (CoCA). ResultsWe found that OspE evolves under positive selection, however, we found no evidence for an association between OspE and host CFH variants at the individual level based on RDA or at the population level based on CoCA. ConclusionsDespite evidence of positive selection acting on OspE, we found no support for specific genetic matching between B. afzelii and its bank vole host at the CFH-OspE interface. These results suggest that the evolution of CFH and OspE may be shaped by broader selective pressures, potentially including interactions with multiple host species.

Rodents are an important reservoir of zoonotic viruses and are ubiquitously present in densely populated urban areas. Betacoronaviruses in the Embecovirus lineage are well known to infect both humans and animals and have established rodent reservoirs. Here three Betacoronavirus gravedinis genomes were sequenced and characterized in white footed mice (Peromyscus leucopus, commonly white footed mice) collected in New York City, the second most populous city in North America. The genomes were distinct from mouse hepatitis virus (MHV), the prototype mouse betacoronavirus, and highly similar and identical in one case to previously characterized B. gravedinis sequences from white footed mice in Connecticut. Codon aware evolutionary models were used to identify specific sites under positive selection within the spike protein of B. gravedinis. A novel method was developed to predict the probable geographic distribution of the virus using publicly available data from the Global Biodiversity Information Facility to generate a weighted distribution map highlighting overlapping potential host ranges based on the evolutionary distance using a high resolution cytocrome B (CYTB) phylogeny of rodent species with potentially overlapping ranges. Our models predict three current hotspots of circulation in North America under different possible transmission regimes, and an additional fourth hotspot was predicted to arise in a warming future. This study highlights the continued need for biodiversity-informed surveillance of potential zoonotic pathogens in rodents.

Avian malaria parasites form a highly prevalent and genetically diverse group within the haemosporidians, yet they have long been overlooked relative to their human- and rodent-infecting counterparts. Among these, parasites of the genus Haemoproteus (Haemosporida, Haemoproteidae) are widespread and prevalent blood parasites of birds, transmitted by louse flies (Hippoboscidae) and biting midges (Ceratopogonidae). Recent phylogenomic analyses place Haemoproteus parasites at the root of the haemosporidian tree, making genomic data from these taxa essential for understanding the evolutionary origins of malaria parasites. To date, only two avian Plasmodium and one avian Haemoproteus genomes have been sequenced. We present the first assembled genome of Haemoproteus majoris (lineage WW2), a common blood parasite of passerine birds. As avian erythrocytes are nucleated, parasite DNA was enriched by FACS-based sorting to discriminate and isolate the parasite from host cell nuclei prior to whole-genome amplification. The genome was assembled using Nanopore long-read sequencing and polished with Illumina short-reads, yielding 145 contigs with a total assembly size of 23.9Mb and a G+C content of 27.85%. Genome annotation identified 5501 protein-coding genes, 69 non-coding RNA genes, and 57 long terminal repeat retrotransposons (LRT-RTs), including one full-length element. This genomic resource represents a critical step towards elucidating the evolutionary history and genomic architecture of avian malaria parasites. SIGNIFICANCE STATEMENTWe present the first assembled genome of Haemoproteus majoris (lineage WW2), a prevalent and generalist avian malaria parasite. Taxonomic resolution of this genus is difficult as there are few distinct morphological differences among closely-related species. This genome provides a valuable resource for studying the evolution within the Haemoproteus genus and to elucidate the evolutionary history of malaria parasites.

Group A Streptococcus (GAS), or Streptococcus pyogenes is almost exclusive and greatly adapted human pathogen. It causes a wide array of clinical symptoms, ranging from minor infections of the skin and soft tissues to pharyngitis, meningitis, pneumonia, bacteraemia, cellulitis, puerperal sepsis, and necrotising fasciitis. The risk of S. pyogenes infection is known to be influenced by several host characteristics, including age, underlying diseases like diabetes, varicella, or skin lesions, both chronic and acute, and certain risk behaviours such as use of drugs. Household size and overcrowding are two environmental factors that significantly affect the transmission of S. pyogenes. The majority of cases occur spontaneously in the community, and preventative opportunities are still limited. A large portion of GAS-related mortality is found in low-income areas and communities. Based on aforementioned public health risk, the creation of effective therapeutic vaccines would be an excellent addition to current control measures. The purpose of this work is to address the need for new instruments to aid in the elimination of S. pyogenes infections. The discovery of high antigenic regions in several highly conserved proteins brings us one step closer to developing peptide vaccines capable of influencing the different phases of S. pyogenes infection, providing more effective defence and greater serotype coverage. This study used various techniques of immunoinformatics to design an effective multi-epitope vaccine that produced neutralising antibodies against multiple strains of S. pyogenes.

Andes virus (ANDV) caused a multi-country outbreak of hantavirus pulmonary syndrome among passengers and crew of a cruise ship in 2026. To investigate the origin and evolutionary history of the virus responsible for the outbreak, we analyzed complete ANDV small (S), medium (M), and large (L) genome segment sequences from Chile alongside outbreak-associated and publicly available genome sequences. Across all three segment-specific phylogenies, the outbreak virus clustered within an ANDV Clade III cluster spanning southern Chile and northern Patagonia in Argentina and were most closely related to a human-derived ANDV (p1236) collected in Los Rios Region of Chile in 2012, representing the closest known historical relative of the outbreak-associated virus. Phylogeographic analysis showed that the genetically distinct ANDV Clade V lineage circulating in central Chile was not closely related to the cruise ship outbreak-associated genomes, thereby reducing the likelihood that the index cases acquired infection through zoonotic spillover while traveling through the Maule Region. These findings trace the geographic origin of the outbreak-associated virus to a defined corridor, the Hua Hum Pass, a cross-border zone connecting Neuquen Province in Argentina with the Los Rios and La Araucania Regions of Chile.

Influenza A viruses H3N2 and H10N5 represent, respectively, a persistently dominant seasonal pathogen and a newly documented zoonotic threat with the latter strain variants responsible for the first confirmed human fatality in January 2024, yet no vaccine platform currently addresses co-protection against both subtypes within a unified immunogen. We report here the immunoinformatics based vaccine design and multi-layered computational validation of a 419-amino-acid multi-epitope subunit vaccine construct targeting conserved hemagglutinin (HA) and neuraminidase (NA) antigens identified through multiple sequence alignment of the avian H10N5 (A/swine/Hubei/10/2008) and H3N2 human reference strain sequences to identify viral agents undergoing mammalian adaptations. Linear B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were predicted using ABCpred, BCEpred, BepiPred 2.0, NetMHCpan 2.1, and NetMHCpan 4.0, then filtered through VaxiJen 3.0, AllerTOP v2.1, and ToxinPred to retain only antigenic, non-allergenic, non-toxic candidates. The final construct, incorporating an avian {beta}-defensin N-terminal adjuvant with GPGPG, AAY, and EAAAK linkers, exhibited a molecular weight of 43.9 kDa, instability index of 31.15, and SOLPro solubility probability of 0.763. Tertiary structure modeling via I-TASSER and GalaxyRefine achieved 84.4% Ramachandran-favored residues. Molecular docking against TLR3 and TLR7 yielded binding free energies of -16.1 and -16.8 kcal/mol with picomolar dissociation constants. Molecular dynamics simulations confirmed complex stability over extended trajectories. Furthermore, codon optimization produced a Codon Adaptation Index of 1.0 for E. coli K12 expression. In silico immune simulation demonstrated robust activation of humoral and cellular immunity including elevated IgG1, IgM, IFN-{gamma}, IL-2, rapid NK cell expansion, and broad B-cell clonal diversity. These findings establish a computationally validated candidate capable of providing protection against influenza in multiple host organisms, warranting experimental advancement.

Bangladesh reported a nationwide measles outbreak in April 2026 involving over 19,000 suspected cases, despite high reported first-dose vaccine coverage ([≥]95%). We assessed whether publicly available molecular data could support epidemiologic interpretation of this resurgence and evaluated broader sequence sharing practices across South Asia. We analyzed public outbreak reports, WHO/UNICEF Estimates of National Immunization Coverage (WUENIC), PubMed indexed literature, and NCBI GenBank records from nine regional countries. Public sequence visibility across the region was highly uneven. While India and Pakistan associated records dominated the public dataset, only 32 Bangladesh origin records were retrieved, and notably, none were collected after 2019. The sole 2026 Bangladesh linked molecular record was a travel associated genotype B3 genome isolated in Australia (PZ189094.1). Its closest public N450 relative was a contemporaneous Pakistan sequence (2-nucleotide difference). The historical Bangladesh sequences were more distant, precluding robust phylogenetic inference regarding local viral persistence, cross-border importation, or transmission direction. Immunization data revealed a high regional baseline but highlighted subnational vulnerability and a significant pandemic era coverage collapse in neighboring Myanmar. The absence of timely, publicly available genomic data during the critical early months of the outbreak highlights a severe genomic surveillance gap. Public molecular records were historically sparse and insufficient to reconstruct outbreak transmission dynamics. To support elimination goals, establishing targeted sequencing pipelines, enforcing minimum metadata standards, and ensuring rapid public data deposition are urgently needed. Keywords: measles; Bangladesh; South Asia; genomic surveillance; molecular epidemiology; immunization coverage

Antimalarial resistance is one of the greatest threats to malaria elimination. Following an outbreak of Plasmodium falciparum infection in Sabah Malaysia in 2019, 98 samples were sequenced to search for adaptations driving the outbreak. The genomic data revealed evidence of clonal expansion of a strain carrying multiple copies of the chloroquine resistance transporter gene (pfcrt) in 86 cases. A TaqMan qPCR assay was developed to quantify pfcrt copy number, confirming the in silico evidence of duplication. Whilst point mutations in pfcrt have been associated with resistance to chloroquine and other drugs, copy number amplification has not been widely explored as a resistance mechanism. We investigated the genetic architecture of the duplication, revealing a wildtype haplotype (3D7 reference-type with 76K variant) and a novel mutant (with 76T mutation). Application of the TaqMan assay in 43 P. falciparum cases from neighbouring Palawan Island, the Philippines, identified a further two cases with pfcrt duplication. Assessment of the global genomic data in the MalariaGEN Pf8 repository identified a further 86 cases, including 73 (85%) from West Africa with evidence of pfcrt duplication. Amongst 47 monoclonal MalariaGEN cases, majority (95%) comprised wild type and mutant variants at codon 76. Our study reveals a potential previously unconsidered antimalarial resistance mechanism for P. falciparum and provides an assay for surveillance in other populations.

Species-level delimitation within the genus Trichomycterus remains one of the main systematic challenges within the Trichomycterinae, particularly in lineages characterised by conservative external morphology, high apparent intraspecific variation, and historical diagnostic criteria based primarily on body proportions, colouration and a few meristic characters. In central Chile, Trichomycterus areolatus has traditionally been interpreted as a widely distributed and morphologically variable species, whilst T. maculatus, originally described from "Santiago du Chili", has remained subordinate to this broad conception without a modern phylogenetic reassessment. Here we reassess the specific boundaries of T. areolatus sensu lato using an integrative approach that combines complete mitogenomes, estimates of genetic divergence and comparative morphology of the cephalic laterosensory system associated with the neurocranium. Phylogenetic analyses reveal T. areolatus sensu lato to be non-monophyletic and identify a deeply divergent lineage, geographically coherent and attributable to T. maculatus. This lineage differs from restricted T. areolatus by extensive mitochondrial divergence, comparable to that observed between recognised species of Trichomycterinae, and by discrete characters of the cephalic lateral line system, primarily related to the continuity of the supraorbital canal and the arrangement of the associated pores. The congruence between mitogenomic, nuclear and morphological evidence supports the revalidation of Trichomycterus maculatus Valenciennes, 1846, and calls for a more restricted geographic circumscription of T. areolatus. These results demonstrate that the diversity of Trichomycterinae in central Chile has been underestimated, modify previous interpretation of the distribution of the species involved, and highlight the value of integrating mitogenomics and neurocranial/laterosensory characters into the taxonomy of morphologically conservative siluriform lineages.

Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) remains one of the most important transboundary pathogens affecting swine production in Vietnam; however, it remains poorly understood how long-term evolutionary dynamics were impacted by the African swine fever (ASF) epidemic, a period of time where swine population demographics and movement were heavily perturbed. We investigated the molecular epidemiology, evolutionary history, and phylogeographic dynamics of PRRSV-2 circulating in Vietnam between 2007 and 2024 by integrating 366 Vietnamese ORF5 sequences with a globally curated lineage reference. Maximum-likelihood phylogenetic, Bayesian phylodynamic, and discrete phylogeographic analyses revealed that the Vietnamese PRRSV-2 population underwent substantial reshaping after the ASF epidemic, shifting from a predominantly endemic sub-lineage L8E population to a genetically diverse viral community comprising multiple established and newly emerging sub-lineages. Despite these epidemiological changes, the endemic sub-lineage L8E population maintained a relatively stable evolutionary rate across the pre- and post-ASF periods, suggesting that ASF reshaped viral population structure rather than intrinsic evolutionary dynamics. Two previously unclassified viral clusters circulating in Vietnam and Thailand fulfilled all criteria for formal designation and were recognized as the novel sub-lineages L1M and L10B by the International PRRSV-2 Nomenclature Consortium. Phylogeographic reconstruction further demonstrated contrasting transmission patterns among major sub-lineages, including long-term endemic persistence of L8E, repeated unidirectional introductions of sub-lineages L1M and L10B from Thailand, and bidirectional transpacific dissemination of sub-lineage L1A linking Southeast Asia and North America. Collectively, these findings demonstrate that the ASF epidemic coincided with a fundamental reshaping of the PRRSV-2 epidemiological landscape in Vietnam while revealing Southeast Asia as an active center of ongoing viral diversification. This study provides an updated evolutionary framework for PRRSV-2 surveillance and highlights the importance of continuous genomic monitoring and regional collaboration for the early detection and control of emerging transboundary variants.

Xenorhabdus bovienii is a symbiotic bacterium associated with entomopathogenic nematodes of the genus Steinernema. Comparative genomic analyses of closely related isolates provide an opportunity to investigate fine-scale diversification, genome plasticity, and the evolutionary processes shaping symbiotic bacterial populations. Here, we analyzed four X. bovienii isolates (XenUTI4.1-XenUTI4.4) recovered from a single Steinernema feltiae isolation event using comparative genomics approaches integrating average nucleotide identity (ANI), single-nucleotide polymorphism (SNP) analyses, pangenome reconstruction, biosynthetic gene cluster (BGC) prediction, and mobile element-associated annotation screening. Whole-genome comparisons revealed extremely high genomic similarity among isolates, with ANI values exceeding 99.84%. Read-based SNP analyses identified only 23-36 annotated variants relative to the XenUTI4.1 reference genome, indicating limited sequence divergence despite detectable microvariation. Functional annotation of these variants showed that most corresponded to missense or synonymous substitutions affecting a small number of coding sequences. Pangenome analysis identified 4,712 orthologous gene clusters, including a highly conserved core genome of 4,256 clusters (90.3%) shared by all isolates and a relatively small accessory genome comprising 456 clusters. antiSMASH analyses revealed broadly conserved secondary metabolite biosynthetic potential across the four genomes, whereas screening of genome annotations identified abundant phage-related, transposase-associated, and recombination-associated genes consistent with ongoing genome plasticity. Collectively, these results demonstrate that the analyzed X. bovienii isolates represent a highly conserved population exhibiting limited but detectable genomic microdiversification. The coexistence of a large core genome, a modest accessory gene complement, and numerous mobile element-associated functions suggests that localized sequence variation and mobile genetic elements contribute to genomic diversification within S. feltiae-associated X. bovienii populations.

Antimalarial drug resistance remains one of the most significant challenges to global malaria control. The emergence of resistance to chloroquine, the first truly globally distributed antimalarial, has been extensively studied but is still not fully understood. While mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) primarily drive resistance, the phenotype is complex and multigenic, with mutations in the putative amino acid transporter PfAAT1 recently confirmed to play a modulatory role. To date studies have focused on PfAAT1 mutations found in African and Southeast Asian P. falciparum lineages, but chloroquine resistance emerged independently in South America, where there may be novel PfAAT1 polymorphisms that are functionally important. We used AlphaFold modelling to reveal high homology between PfAAT1 and the human lysosomal arginine transporter SLC38A9, which allows prediction of membrane orientation and identifies a partially open channel accessible from the cytoplasm. Several PfAAT1 mutations found only in South American isolates sit near the entrance of this pore, most notably V231 where mutation to aspartate is predicted to alter channel conformation and influence transport, while nearby P446A (which is always found in combination with V231D) and I248T are predicted to impact pore flexibility and local structural stability. To functionally validate these insights, we employed CRISPR/Cas9 gene editing across parasite strains with diverse geographic origins. Reverting the regional V231D mutation in the South American 7G8 strain significantly reduced CQ resistance, providing the first functional evidence that this residue modulates drug susceptibility. Furthermore, introducing the apparently Colombia-specific I248T mutation significantly enhanced parasite multiplication rates in 7G8, demonstrating complex fitness and sensitivity trade-offs. Our findings reinforce the distinct evolutionary trajectory for South American CQ resistance and highlight the necessity of including additional pfaat1 mutations in global molecular surveillance strategies. Author SummaryAntimalarial drug resistance is a major threat to global public health. Chloroquine was used widely in the 1950s-60s as part of a global malaria eradication campaign, but resistance emerged in multiple places independently and chloroquine resistant parasites directly led to the death of millions of children. Chloroquine resistance is primarily driven by mutations in the transporter PfCRT which are thought to increase export of chloroquine from the digestive vacuole, where chloroquine acts to prevent the ability of the parasite to digest haemoglobin as a source of energy. However, it is becoming increasingly clear that additional vacuolar transporters can also modulate chloroquine resistance. In this study we focused on mutations in the putative amino acid transporter 1 (PfAAT1) which are specific to South America, where chloroquine resistance emerged independently from Southeast Asia. By integrating AlphaFold structural predictions with CRISPR/Cas9 gene editing across geographically diverse parasite backgrounds we provide the first functional evidence that the region-specific V231D mutation significantly diminishes chloroquine resistance. These findings emphasise that chloroquine resistance in South America followed a unique trajectory and that PfAAT1 is involved in complex fitness and sensitivity trade-offs. This work emphasises the benefits of integrating diverse experimental and modelling approaches with global molecular surveillance.

Carbapenem-resistant Escherichia coli (CREc) recovered sequentially from one patient typically retain the same carbapenemase, with escalating resistance usually attributed to porin loss combined with pre-existing {beta}-lactamase expression. We used whole-genome sequencing to characterize a clonal pair of CREc isolates, CAEC145 and CAEC155, recovered 25 days apart from a hospitalized patient with sequential urinary and bloodstream infection. Both belonged to sequence type 361 (ST361), phylogroup A, serotype O-nontypeable:H30, and were separated by only 28 core-genome SNPs, confirming clonal relatedness. Despite this, the isolates differed sharply in carbapenemase content. CAEC145 carried blaOXA-1207, a recently described OXA-48-family variant, on a conjugative IncFII(pCoo)/ColKP3 plasmid, whereas CAEC155 lacked this determinant and instead harbored blaNDM-4 on a conserved IncX3 plasmid nearly identical to pJEG027, a member of a globally disseminated IncX3 lineage. This genotypic shift tracked a clear phenotypic transition. CAEC145 remained susceptible to imipenem and meropenem while resistant to ertapenem, whereas CAEC155 showed uniform high-level resistance to all three carbapenems and to ceftazidime-avibactam. Both isolates, however, remained susceptible to imipenem-relebactam, meropenem-vaborbactam, and cefiderocol. Comparative genomics linked the blaOXA-1207 element to a {Delta}Tn6361 transposon structure also found in the original German isolates where blaOXA-1207 was first described, and a 137-genome core-genome phylogeny placed both isolates within a globally disseminated ST361 lineage carrying multiple carbapenemase classes. These findings document, to our knowledge, the first within-host succession from an OXA-48-like to an NDM-type carbapenemase during a single sequential E. coli infection, driven by plasmid-level displacement rather than in-place gene evolution, with implications for genomic surveillance and antibiotic selection.