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PubMed2025

Demixer: a probabilistic generative model to delineate different strains of a microbial species in a mixed infection sample.

MOTIVATION: Multi-drug resistant or hetero-resistant tuberculosis (TB) hinders the successful treatment of TB. Hetero-resistant TB occurs when multiple strains of the TB-causing bacterium with varying degrees of drug susceptibility are present in an individual. Existing studies predicting the proportion and identity of strains in a mixed infection sample rely on a reference database of known strains. A main challenge then is to identify de novo strains not present in the reference database, while quantifying the proportion of known strains. RESULTS: We present Demixer, a probabilistic generative model that uses a combination of reference-based and reference-free techniques to delineate mixed infection strains in whole genome sequencing (WGS) data. Demixer extends a topic model widely used in text mining to represent known mutations and discover novel ones. Parallelization and other heuristics enabled Demixer to process large datasets like CRyPTIC (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium). In both synthetic and experimental benchmark datasets, our proposed method precisely detected the identity (e.g. 91.67% accuracy on the experimental in vitro dataset) as well as the proportions of the mixed strains. In real-world applications, Demixer revealed novel high confidence mixed infections (101 out of 1963 Malawi samples analysed), and new insights into the global frequency of mixed infection (2% at the most stringent threshold in the CRyPTIC dataset) and its significant association to drug resistance. Our approach is generalizable and hence applicable to any bacterial and viral WGS data. AVAILABILITY AND IMPLEMENTATION: All code relevant to Demixer is available at https://github.com/BIRDSgroup/Demixer.

Mycobacterium tuberculosis↗

PubMed2025

Deciphering mixed infections by plant RNA virus and reconstructing complete genomes simultaneously present within-host.

Local co-circulation of multiple phylogenetic lineages is particularly likely for rapidly evolving pathogens in the current context of globalisation. When different phylogenetic lineages co-occur in the same fields, they may be simultaneously present in the same host plant (i.e. mixed infection), with potentially important consequences for disease outcome. This is the case in Burkina Faso for the rice yellow mottle virus (RYMV), which is endemic to Africa and a major constraint on rice production. We aimed to decipher the distinct RYMV isolates that simultaneously infect a single rice plant and to sequence their genomes. To this end, we tested different sequencing strategies, and we finally combined direct cDNA ONT (Oxford Nanopore Technology) sequencing with the bioinformatics tool RVhaplo. This method was validated by the successful reconstruction of two viral genomes that were less than a hundred nucleotides apart (out of a genome of 4450nt length, i.e. 2-3%), and present in artificial mixes at a ratio of up to a 99/1. We then used this method to subsequently analyze mixed infections from field samples, revealing up to three RYMV isolates within one single rice plant sample from Burkina Faso. In most cases, the complete genome sequences were obtained, which is particularly important for a better estimation of viral diversity and the detection of recombination events. The method described thus allows to identify various haplotypes of RYMV simultaneously infecting a single rice plant, obtaining their full-length sequences, as well as a rough estimate of relative frequencies within the sample. It is efficient, cost-effective, as well as portable, so that it could further be implemented where RYMV is endemic. Prospects include unravelling mixed infections with other RNA viruses that threaten crop production worldwide.

Genome, Viral↗

PubMed2026

Occurrence and Characterization of Graft-Transmissible Pathogens of Citrus in Orchards and Urban Citrus in Chile with a Focus on the Apscaviroid nanocitri.

Apscaviroid nanocitri (citrus dwarfing viroid, CDVd) is a graft-transmissible pathogen that frequently coexists with other citrus viroids and affects tree vigor. This study evaluated the occurrence of citrus viroids in small growers and commercial orchards in Chile and characterized CDVd strains obtained from urban Citrus spp. trees, with unknown propagation history following the discontinuation of the national citrus certification program. A total of 301 samples from five citrus-producing regions were analyzed, revealing that CDVd is widely distributed and commonly associated with Hostuviroid impedihumuli (hop stunt viroid, HSVd). Single infections were the most frequent (41%), followed by double (7.3%) and triple infections (3.0%), indicating that mixed infections are common. CDVd strains from urban trees were characterized using biological indexing on 'Etrog' citron Arizona 861-S-1 indicator plants combined with RT-PCR detection and phylogenetic analysis. Symptom development and pathogen detection were evaluated at 6 and 18 months post-inoculation. All graft-inoculated plants developed symptoms consistent with viroid infection, whereas control plants remained asymptomatic. CDVd was consistently detected at 18 months but not at earlier stages, suggesting delayed systemic accumulation. Although Closterovirus tristezae (citrus tristeza virus, CTV) was present in the source plants, it was not detected in indicator plants at either time point, possibly associated with the higher temperatures required for viroid symptom development during bioindexing. The frequent occurrence of mixed infections highlights the epidemiological relevance of viroid interactions and supports the need for reinstatement and strengthening of the national citrus certification program.

Apscaviroid nanocitri↗

PubMed2026

Development and epidemiological investigation of a TaqMan-based multiplex real-time quantitative PCR assay for simultaneous detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV).

INRODUCTION: Infectious diseases caused by bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine norovirus (BNoV), bovine enterovirus (BEV), and bovine coronavirus (BCoV) significantly threaten the cattle industry, resulting in substantial economic losses. These pathogens often present similar clinical signs, such as diarrhea, vomiting, and reproductive disorders in pregnant cattle, and frequent covert or mixed infections further complicate accurate diagnosis. Therefore, rapid, sensitive, and field‑deployable diagnostic methods are essential for effective disease surveillance and control in the cattle industry. METHODS: In this study, we report for the first time the establishment of a TaqMan‑based real‑time quantitative PCR (qPCR) assay that enables simultaneous detection of these five bovine viruses. Multiple sequence alignment of conserved genomic regions was performed, and virus‑specific primers and probes were designed and optimized using Beacon Designer 7 software. Subsequently, a TaqMan‑based multiplex real‑time qPCR assay was established for simultaneous detection of BVDV, AKAV, BNoV, BEV, and BCoV. The established detection method was applied to 200 clinical samples collected from 10 farms in multiple regions of Jilin Province. RESULTS: The results showed that the detection rates for BVDV, AKAV, BNoV, BEV, and BCoV were 33.50%, 0.50%, 4.50%, 7.50%, and 12.00%, respectively. Mixed infections were detected in 9 samples co‑infected with two of the five pathogens, with an overall mixed infection rate of 4.50%. Compared with conventional PCR, coincidence rates were 100% for BVDV, AKAV, BNoV, BEV, and BCoV. DISCUSSION: These findings indicate that the TaqMan multiplex real‑time qPCR assay developed here demonstrates favorable specificity, sensitivity, and reproducibility. This assay enables efficient detection and surveillance of bovine viruses, offering a reliable technical tool for the diagnosis and control of corresponding viral diseases in cattle.

Akabane virus (AKAV)↗

PubMed2026

Hybridization capture increases on-target nanopore sequencing of plant RNA tobamovirus- derived cDNA libraries.

High-throughput sequencing (HTS) can support plant virus surveillance, but host nucleic acids often reduce on-target read recovery. We evaluated a targeted hybridization-capture workflow in which barcoded double-stranded cDNA (ds-cDNA) libraries generated from plant RNA extracts spiked with lyophilized tobamovirus-positive controls were enriched before Oxford Nanopore sequencing. Biotinylated probes targeted conserved regions of cucumber green mottle mosaic virus (CGMMV), species Tobamovirus viridimaculae; pepper mild mottle virus (PMMoV), species Tobamovirus capsici; and tobacco mosaic virus (TMV), species Tobamovirus tabaci. Across four pairs per virus, relative target-read abundance increased after capture from 0.76 ± 0.33% to 37.62 ± 15.72% for CGMMV, 8.16 ± 3.86% to 24.68 ± 12.34% for PMMoV, and 15.62 ± 10.40% to 36.83 ± 30.33% for TMV. Exact two-sided Wilcoxon signed-rank tests yielded P = 0.125 for each virus; with four nonzero differences in a common direction, this was the minimum attainable two-sided P value. Genome-coverage breadth was maintained. Retrospective duplex qPCR supported an increased virus-to-18S ratio for CGMMV, showed a variable PMMoV response, and showed a decreased virus-to-18S ratio for TMV because the 18S signal shifted earlier by as much as or more than the TMV signal. The findings provide proof-of-concept evidence for target-dependent library enrichment but do not establish analytical sensitivity, diagnostic performance, or field validity. Validation with naturally infected, low-titer, and mixed-infection samples and comparison with simpler targeted workflows are required.

biosecurity↗

PubMed2024

Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity.

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover ~92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

Cryptosporidium↗

PubMed2026

Comparison of phylogenetic metrics of transmission between symptomatic and asymptomatic tuberculosis in individuals who were incarcerated in Brazil in 2008-24: a retrospective genomic epidemiology study.

BACKGROUND: Tuberculosis control efforts have traditionally targeted symptomatic individuals; however, the role of asymptomatic cases in sustaining transmission is increasingly recognised. We aimed to quantify the contribution of asymptomatic tuberculosis to recent transmission using genomic and epidemiological data from a high-transmission setting. METHODS: We conducted a retrospective genomic epidemiology study of Mycobacterium tuberculosis isolates collected in Mato Grosso do Sul, Brazil, between Aug 25, 2008, and March 19, 2024. Available isolates underwent whole-genome sequencing. Demographic, clinical, incarceration history, and laboratory metadata were obtained from surveillance records. From Jan 1, 2017, to March 19, 2024, active case finding was conducted in the state's three largest prisons (all male-only facilities), during which sputum samples were collected from individuals irrespective of symptoms and tested using GeneXpert and culture. Comparisons of transmission between individuals with and without symptoms were restricted to individuals who were incarcerated and were identified through active case finding and for whom high-quality, M tuberculosis lineage 4 genomes were available. Metrics of recent transmission included phylogenetic clustering, time-scaled haplotype density (THD), local branching index (LBI), and transmission probabilities inferred using Bayesian Reconstruction and Evolutionary Analysis of Transmission Histories. FINDINGS: 4448 tuberculosis cases were notified in Mato Grosso do Sul in 2008-24. After excluding cases for which M tuberculosis isolates were not available or had low sequencing quality, who had contaminated cultures or mixed infection, or who were infected with non-lineage 4 M tuberculosis, we included 2362 lineage 4 M tuberculosis isolates with high-quality genome sequences. 1849 (78·3%) of 2362 isolates were part of a genomic cluster. Among 2362 individuals with tuberculosis, 1137 (48·1%) were incarcerated at diagnosis. Of these individuals, 505 were identified through active case finding in three male-only prisons. The median age was 30 years (IQR 25-37); 304 (60·2%) had mixed ethnicity, 90 (17·8%) were White, 56 (11·1%) were Black, 13 (2·6%) were Indigenous, and six (1·2%) were Asian. 277 (54·9%) had symptomatic disease and 228 (45·1%) had asymptomatic tuberculosis. There were no significant differences between symptomatic and asymptomatic individuals in phylogenetic clustering (213 [76·9%] of 277 vs 195 [85·5%] of 228; p=0·37), THD (median 0·39 [IQR 0·06-0·62] vs 0·50 [0·09-0·65]; p=0·12), or LBI (0·00863 [0·00810-0·00988] vs 0·00871 [0·00829-0·01020]; p=0·088). Bayesian transmission trees showed no significant difference in the number of secondary infections inferred from symptomatic compared with asymptomatic individuals (p=0·56). These findings were consistent across genomic clusters and robust to model assumptions. INTERPRETATION: We identified no differences in transmission between individuals who were symptomatic and those who were asymptomatic using multiple genomic measures. In this high-transmission setting, where systematic screening is implemented, our findings indicate that asymptomatic tuberculosis substantially contributes to tuberculosis transmission at the population level. These results suggest that symptom-based case detection alone is likely to be insufficient to interrupt transmission and highlight the importance of expanded screening strategies in high-risk populations. FUNDING: US National Institutes of Health and the Brazilian National Research Council (CNPq).

Humans↗

PubMed2025

GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (<40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of human-infecting Cryptosporidium species ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium spp. DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage and the reliability of detecting species and mixed infections within a sample, and allows assessment of genetic variation via SNP calling, while decreasing costs.

Journal Article↗

PubMed2026

Arginine-substituted Mastoparan-C derivatives combat dual bacterial pathogens: in vitro mechanistic insights and in vivo efficacy in polymicrobial wounds.

UNLABELLED: The synergistic interactions in multi-pathogen infections compromise wound healing and limit therapeutic efficacy. In this study, we designed and synthesized arginine-substituted derivatives of the antimicrobial peptide Mastoparan-C (MP-C). Among them, Arg²MP-C and Arg4.11.12MP-C exhibited potent, broad-spectrum activity against both Escherichia coli and Staphylococcus aureus. Their enhanced antibacterial activity is associated with increased positive charge and optimized hydrophobicity. Mechanistically, both peptides employ a dual-target strategy, disrupting bacterial membranes and binding genomic DNA; Arg²MP-C acted most rapidly against the E. coli envelope, while Arg4.11.12MP-C caused the strongest membrane damage to S. aureus. In a murine polymicrobial wound model, Arg²MP-C treatment nearly achieved complete wound closure by day 10, significantly reduced bacterial loads, and promoted tissue regeneration. This study demonstrates that arginine engineering can yield peptides with potent, multi-mechanistic action, identifying Arg²MP-C as a promising candidate for combating polymicrobial wound infections. IMPORTANCE: Wounds infected with multiple bacterial species are notoriously difficult to treat, often leading to poor healing and limited effectiveness of existing therapies. In this study, we developed new antimicrobial peptides by introducing arginine substitutions into a natural peptide called Mastoparan-C. Two of our engineered peptides, Arg²MP-C and Arg4.11.12MP-C, showed potent activity against two common wound pathogens, Escherichia coli and Staphylococcus aureus. These peptides work through a dual mechanism: disrupting bacterial membranes and binding to bacterial DNA. In a mouse model of mixed-infection wounds, treatment with Arg²MP-C led to nearly complete wound closure by day 10, drastically reduced bacterial counts, and promoted tissue repair. Our findings highlight arginine engineering as a promising strategy to create next‑generation antimicrobial agents that can effectively combat complex polymicrobial wound infections, addressing a critical unmet need in clinical wound care.

Animals↗

PubMed2025

Hierarchical integration of mNGS, PCR, and other conventional methods for precision TB diagnostics.

UNLABELLED: This study systematically compared the diagnostic accuracy of seven assays for detecting the Mycobacterium tuberculosis complex, including metagenomic next-generation sequencing (mNGS), droplet digital polymerase chain reaction, real-time quantitative polymerase chain reaction, EasyNAT MTC, GeneXpert MTB/RIF, interferon-gamma release assay (IGRA), and acid‒fast staining (AFS). We try to select appropriate combinations of tuberculosis (TB) detection methods for regions with varying levels of medical resources, based on sensitivity, cost-effectiveness, and operational feasibility. A retrospective analysis was conducted on 141 samples collected from patients with suspected active TB at The First Affiliated Hospital of Sun Yat-sen University between April 2022 and April 2024. Among these samples, there were 100 cases assigned to the case group and 41 cases to the control group, based on the tuberculosis diagnostic criteria. Historical data for Xpert, IGRA, and AFS were collected, and parallel experiments using mNGS, droplet digital PCR (ddPCR), real-time quantitative polymerase chain reaction (RT-qPCR), and EasyNAT were conducted on all samples. Diagnostic performance was evaluated by comparing it with the final clinical diagnoses. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operating characteristic (ROC) curve analysis were conducted, along with DeLong tests for statistical comparison. Compared with the final clinical diagnosis, mNGS demonstrated the highest sensitivity (100%), followed by IGRA (79.2%), EasyNAT (79.1%), RT-qPCR (78.0%), ddPCR (75.8%), Xpert (75.3%), and AFS (16.7%). The specificity was 100% for both Xpert and AFS, followed by ddPCR (97.6%), RT-qPCR (95.1%), EasyNAT (92.7%), IGRA (72.7%), and mNGS (75.6%). ROC analysis revealed a significantly greater area under the ROC curve for mNGS (0.878) than for ddPCR (0.817, P = 0.031). DeLong tests revealed statistically significant differences in diagnostic performance between mNGS and ddPCR (P < 0.05) and between IGRA and AFS (P < 0.01). mNGS uniquely identified the pathogens involved in co-infection and quantified pathogen-specific sequencing reads. Through a comprehensive evaluation of the diagnostic efficacy, cost-effectiveness, and timeliness of tuberculosis detection methods, we propose corresponding combinations of TB testing approaches for regions with different healthcare resources. For undeveloped regions with limited resources, a combination of AFS +EasyNAT + chest X-ray is recommended. Primary care facilities may additionally employ IGRA + RT-qPCR. Intermediate-level hospitals can incorporate Xpert MTB/RIF for drug resistance testing, while tertiary hospitals or specialized centers should, on the basis of these fundamental tests, utilize mNGS for diagnosis and ddPCR for therapeutic monitoring in patients with complex mixed infections. IMPORTANCE: This study is the first to comprehensively evaluate the diagnostic efficacy, cost-effectiveness, and timeliness of seven TB detection methods in a single-center cohort. Our findings provide actionable solutions for optimizing TB diagnostics in diverse healthcare ecosystems, aligning with the WHO's End TB Strategy to ensure equitable access to rapid diagnostics.

Humans↗

PubMed2026

Phylogenetic inconsistency of pairwise SNP clustering for inferring tuberculosis transmission in a high-burden, endemic setting: a case study from Thailand.

Whole-genome sequence analysis is now widely used to delineate tuberculosis transmission clusters. A standard practice is to cluster bacterial isolates based on a fixed maximum genome-wide pairwise single nucleotide polymorphism (pwSNP) distance threshold. In this study, we evaluated the phylogenetic consistency of pwSNP-distance clustering with thresholds ranging between 1 and 25 single nucleotide polymorphisms (SNPs) using two contrasting data sets: (i) a data set from the UK (N = 390) published by T. M. Walker, C. L. C. Ip, R. H. Harrell, J. T. Evans, et al. (Lancet Infect Dis 13:137-146, 2013, https://doi.org/10.1016/S1473-3099(12)70277-3), which was foundational to the establishment of this method, and (ii) a data set from Thailand (N = 3,341), characterized by persistent transmission and sparse, non-systematic sampling. For the UK data set, the standard pwSNP-distance clustering using thresholds of ≥12 SNPs yielded entirely monophyletic clusters and showed high concordance with a comparative monophyly constrained, tree-based method. In contrast, for the Thai data set, pwSNP-distance clustering often generated non-monophyletic clusters, even by the 25-SNP threshold. The pwSNP-distance and comparative tree-based clustering methods only showed large consistency at thresholds of ≥22 SNPs. This suggests that SNP clusters defined by low distance thresholds (i.e., <12 SNPs for the UK data set, and <22 SNPs for the Thai data set) may lack robustness, and the problem is particularly severe for data sets characterized by persistent transmission, likely due to poorer cluster separation. Moreover, our findings indicate that large cluster sizes, high maximum intra-cluster genetic distances, and broad sample collection time spans may serve as useful indicators of potentially non-monophyletic clusters. We also demonstrate that mixed infections can produce spurious, phylogenetically long-range SNP linkages, underscoring the necessity of strict sequence quality control.IMPORTANCEFixed-threshold pairwise single nucleotide polymorphism (pwSNP)-distance clustering is commonly used to delineate tuberculosis transmission clusters. From an epidemiological perspective, a genuine transmission cluster must be monophyletic, originating from a single source. However, pwSNP-distance clustering is inherently simplistic and can therefore violate this principle, making the assessment of its phylogenetic consistency critical. Our results demonstrate that while this method effectively delineated complete transmission clusters for the data set from the UK, a low-burden and non-persistent transmission setting, it frequently generated non-monophyletic clusters when applied to the Thai data set, characterized by persistent transmission alongside sparse and non-systematic sampling. Furthermore, we found that clusters derived using low distance thresholds could notably vary between the pwSNP-distance and comparative tree-based clustering methods, suggesting limited reliability and robustness. To accurately delineate tuberculosis transmission clusters, especially for complex data from high-burden, endemic settings, we recommend transitioning from pwSNP-distance clustering toward more robust, phylogenetic clustering that respects evolutionary descent.

Mycobacterium tuberculosis↗

PubMed2025

Advances in diagnosis of diseases causing diarrhea in newborn calves.

Diarrhea in newborn calves is a serious global health problem. It poses challenges for animal industry, veterinarians and researchers due to the rapid onset of dehydration. Mixed infections make treatment complicated, and many young calves suffer high rates of illness and death from this condition. Numerous enteropathogens are associated with diarrhea in newborn calves, encompassing viruses, bacteria, parasites, and protozoa. Their occurrence differs by region, yet the most prevalent infections include E. coli, Salmonella species, Clostridium perfringens, Clostridium difficile, Rotavirus, Coronavirus, Cryptosporidium, Toxocara, Giardia and Eimeria. This review outlines the diagnostic techniques for diseases that lead to diarrhea in newborn calves. Diagnosis is based on clinical manifestations; however, the laboratory identification of etiological items is the only valid way for detecting the illness's aetiology and initiating treatment protocols. Classic methods such as bacterial culturing, fecal flotation, direct microscopy, and virus isolation help us understand pathogens better. Immunological assays like ELISA and immunochromatography are fast, accurate, affordable, and useful for on-farm detection. They help identify specific antigens or antibodies efficiently. Molecular methods including PCR (standard, multiplex, real time and digital), LAMP assays, DNA microarrays and whole-genome sequencing allow highly accurate and sensitive detection. They can identify pathogens effectively, even at very low levels. Nanotechnology-based assays introduce a novel level of sensitivity and specificity, often yielding quick results with minimal sample volumes. In conclusion, accurate and rapid diagnosis using advanced techniques is critical for managing and preventing diseases that lead to diarrhea in newborn calves.

Animals↗

PubMed2026

Strain Diversity and Resistance to Cucumber Green Mottle Mosaic Virus (CGMMV) in Cucumber.

Cucumber green mottle mosaic virus (CGMMV) is a tobamovirus that causes disease in cucumber crops worldwide, leading to significant economic losses. To study the variability of CGMMV in southeastern Spain, partial genome sequences were obtained from isolates collected in 2017 and 2020 from cucumber crops. Phylogenetic analyses revealed that isolates clustered into two major groups, Asian (AS)-like and European (EU)-like CGMMV isolates. These two groups coexisted in the same area, crops, and even individual plants, although the AS type predominated. The accumulation and symptom expression of molecularly cloned isolates from these two groups were assessed in two cucumber cultivars (resistant and susceptible) under both summer and winter conditions. An in planta antagonistic interaction was detected between the AS and EU isolates, in which the accumulation of CGMMV-EU was suppressed during mixed infections. A multivariate analysis did not identify statistically significant differences because of variations in environmental conditions. Unlike CGMMV-EU, CGMMV-AS did not show significant differences in accumulation based on the plant genotype. To further investigate this, the response to CGMMV-AS infection was analyzed in additional susceptible and resistant cultivars. All cultivars appeared to be similarly susceptible to CGMMV-AS, in contrast to CGMMV-EU, which accumulated to a much lower extent in resistant compared with susceptible plants. These results reinforce the need to continue epidemiological surveillance, identify new sources of resistance, and implement strict control of infected seed trade, given the growing threat that CGMMV-AS isolates pose to cucumber cultivation.

agroinfectious clone↗

PubMed2025

Human papillomavirus (HPV) genotypes extended prevalence in the female population from a city in Northern Chile.

BACKGROUND: Cervical cancer is primarily associated with the presence of human papillomavirus (HPV), with high-risk genotypes HPV-16 and HPV-18 being the focus of vaccination programs in developing countries such as Chile. Preventive screening for cervical cancer in women aged 25 to 64 years remains centered on cytological techniques and is primarily performed based on clinical suspicion of cervical lesions. However, extended screening for HPV genotypes using DNA amplification methods is not routinely applied to the Chilean female population yet. This study aimed to determine the prevalence of high- and low-risk HPV genotypes in women without known risk factors in a city in northern Chile. METHODS: Cervicovaginal brushing samples were obtained from 390 women from Antofagasta city, Northern Chile, aged between 25 and 64 years; genomic DNA was extracted, and multiplex real-time PCR analysis was used to identify a larger group of high- and low-risk HPV genotypes. RESULTS: Among 390 samples, HPV prevalence was 36.9%, of which 54.9% were high-risk genotypes, 18.7% were low-risk genotypes, and 26.4% showed mixed infection with both high- and low-risk genotypes. High-risk genotypes 16, 58, 39, and 31 were the most frequently identified among HPV-positive samples. Furthermore, a significant association was observed between HPV presence and both age and suspicion of cervical alteration, and women testing positive for other sexually transmitted infections (STIs) were more likely to acquire HPV. CONCLUSIONS: Implementing a screening program that incorporates extended HPV genotyping in Chile, including testing for high-risk genotypes 16, 18, 31, 39, and 58, is crucial to optimize control, early detection, and vaccination efforts for Chilean circulating HPV genotypes that are not covered by the actual vaccine, thus contributing to a more effective reduction in the burden of disease associated with the virus.

Humans↗

PubMed2026

Virome of the Russian Grapevine Germplasm: A Final Study and Summary.

Ampelographic collections play an important role in the conservation of grapevine genetic resources and therefore require continuous phytosanitary monitoring. In this study, the virome of grapevines from the Magarach ampelographic collection in Russia was analyzed using total RNA high-throughput sequencing. A total of twenty-seven grapevine viruses and four viroids were identified. Two viruses were characterized as putative novel species: (+) ssRNA grapevine umbra-like virus 5 (GULV-5) and the bipartite (+) ssRNA grapevine Magarach secovirus (GMSV), which, together with related viruses, may represent a novel genus within the family Secoviridae. Among the economically important viruses, the most prevalent were grapevine fanleaf virus (76%), grapevine leafroll-associated virus 1 (39%), and grapevine virus A (33%). Mixed infections involving two or three of these viruses were detected in 50% of the analyzed grapevines. Grapevine virus D was detected in Russia for the first time. Phylogenetic analysis of 222 assembled virus and viroid genome sequences revealed high genetic diversity. The obtained results were summarized and compared with previous virome studies conducted on four Russian ampelographic collections.

RNA-Seq↗

PubMed2026

Longitudinal characterization of mixed-genotype SARS-CoV-2 infections in a military cohort reveals compartmentalized viral populations.

UNLABELLED: Mixed-genotype severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are a concern due to the potential generation of novel recombinants that give rise to new variants. To better understand intra-host viral dynamics, we analyzed specimens from 24 participants from the U.S. Military Health System's Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential COVID-19 cohort with suspected mixed-genotype SARS-CoV-2 infections. From an initial 24 suspected cases, we confirmed 17 as genuine coinfections and graded them by evidence: 7 were "strong"; 4 were "moderate"; 6 were "weak"; and 7 were deemed unlikely to be true mixed-genotype infections. Access to swabs from multiple body sites across the course of infection allowed us to observe compartmentalization and shifts in variant dominance that would have been missed by a single-timepoint analysis, as well as one recombinant Omicron BA.1/BA.2 genome. By using an evidence-based bioinformatic framework to assess sequencing data from well-characterized clinical cases, we distinguished genuine coinfections from bioinformatic artifacts. Our findings emphasize the importance of both extensive specimen collection and careful bioinformatic approaches in ascertaining dual genotype infections. IMPORTANCE: Novel recombinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) arise from coinfections with different lineages, but mixed infections are not screened for despite risk to public health, and most surveillance relies on single swabs. We analyzed a longitudinal data set with specimens from multiple body sites, providing an opportunity to assess intra-host dynamics. To distinguish true coinfection from bioinformatic artifacts with confidence, we applied a framework that grades evidence for mixed genotypes by incorporating lineage and clade with manually validated variant calls. This allowed investigation beyond abundance levels of mixed genotypes within a single specimen, including observations of compartmentalization and a recombinant virus. This work enables further study of evolutionary, immunological, and clinical implications of mixed SARS-CoV-2 genotypes. Detecting dual-genotype infections and discriminating between true dual-genotype infection vs potential bioinformatics-based artifacts support public health and military readiness. These efforts provide evidence to bolster decision-making in molecular epidemiological studies to track transmission and for the choice of effective countermeasures.

SARS-CoV-2↗

PubMed2026

Whole-Genome Sequencing Reveals Co-Infection with Bovine Viral Diarrhea Virus, Bovine Enterovirus, and Caprine Parainfluenza Virus Type 3 in a Calf from a Cattle Herd in Xizang, China.

Although mixed viral infections are increasingly recognized as contributors to bovine diarrhea syndrome, diagnosing such co-infections remains challenging, particularly in high-altitude regions where surveillance is limited. In July 2024, a calf presenting with severe diarrhea and respiratory distress was identified on a cattle farm in Linzhi, Xizang, China. Using unbiased whole-genome sequencing (WGS) of the fecal sample, we assembled near-complete genomes of three distinct RNA viruses: two bovine viral diarrhea virus type 1 (BVDV-1) strains (subtypes 1v and 1q, designated BVDV-1/XZ87 and XZ87), one bovine enterovirus (genotype EV-E, designated BEV/XZ87), and one caprine parainfluenza virus type 3 (CPIV3/XZ87). The CPIV3/XZ87 genome exhibited 99.9% nucleotide identity to the goat-derived GS2017-2 strain from Jiangsu, China, raising the possibility of viral spread through livestock trade. Quantitative real-time PCR (RT-qPCR) confirmed the presence of all three pathogens (Ct values: 24.78 for BEV, 25.98 for CPIV3, and 31.28 for BVDV). This study provides the genomic evidence of a triple co-infection involving BVDV-1, BEV, and CPIV3 in Xizang. It illustrates the potential of WGS for unbiased pathogen detection in complex clinical specimens. The near-complete genomes generated here fill critical gaps in the virological surveillance of this epidemiologically under-sampled high-altitude region.

bovine enterovirus↗

PubMed2025

Genotypic Analysis and Clinical Findings of Sapovirus-Associated Acute Gastroenteritis in Mie Prefecture, Japan, 2010-2022.

Sapovirus (SaV) is one of the major viruses causing acute gastroenteritis. Of the 1981 fecal specimens collected through sentinel pediatric acute gastroenteritis pathogen surveillance in Mie Prefecture, Japan (2010-2022), 236 were positive for SaV, according to PCR screening. Whole or near-whole genome sequences were determined for 158 strains by next-generation sequencing. Genotype GI.1 was the most common of the nine SaV genotypes detected, followed by GII.3 and GII.1. Phylogenetic analysis showed that SaVs of these three genotypes separated into three different clusters depending on the year of detection, suggesting continuous genetic changes in the same genotype. Coinfections involving different SaV genotypes, as well as reinfections with SaV in the same individual, were observed in this study. The main clinical manifestations were diarrhea (68.4%) and vomiting (61.6%), with an increased rate of emesis, particularly in patients over 3 years of age. In addition, 18.1% of the children had fever. This study clarified the prevalence of viral genotypes as well as clinical findings of SaV-positive gastroenteritis in children, and revealed trends by age.

Humans↗