scRNA-seq data from the larval Drosophila ventral cord provides a resource for studying motor systems function and development.

The Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. Here, we use high-quality scRNA-seq, validated by anatomical identification, to create a comprehensive census of larval VNC cell types. We show that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separate all motor neuron bundles and link individual neuron clusters to morphologically characterized known subtypes. We discovered a glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. We describe larval glia and endorse the general view that glia perform consistent activities throughout development. This census represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination.

Genetic diversity of Mycobacterium tuberculosis strains isolated from spiritual holy water site attendees in Northwest Ethiopia. A cross-sectional study.

Background: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains was characterized among isolates from individuals with pulmonary tuberculosis (PTB) symptoms attended holy water sites (HWSs) in the Amhara region, Ethiopia. Methods: A cross-sectional study was done from June 2019 to March 2020 to describe the genetic diversity and drug-resistance profiles of MTBC isolates. Sputum specimens were collected and cultured in the Löwenstein-Jensen culture medium. Line Probe Assay, MTBDRplus VER 2.0, and MTBDRsl VER 2.0 were used to detect first-and second-line anti-TB drug-resistance patterns. A spoligotyping technique was utilized to characterize the genetic diversity. Statistical analysis was performed using STATA 15. Results: Of 560 PTB-symptomatic participants, 122 (21.8%) were culture-positive cases. Spoligotyping of 116 isolates revealed diverse MTBC sublineages, with four major lineages: Euro-American (EA) (Lineage 4), East-African-Indian (EAI) (Lineage 3), Ethiopian (ETH) (Lineage 7), East Asian (EA) (Lineage 2). The majority (96.6%) of the isolates were EA (lineage 4) and EAI, with proportions of 54.3% and 42.2%, respectively. A total of 31 spoligotype patterns were identified, 26 of which were documented in the SITVIT2 database. Of these, there were 15 unique spoligotypes, while eleven were grouped with 2-17 isolates. SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), SIT25/CAS1-DELHI (n = 12), and SIT52/T2 (n = 11) spoligotypes were predominant. A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. The overall clustering rate of sub-lineages with known SIT was 76.4%.Of the 122 culture-positive isolates tested, 16.4% were resistant to rifampicin (RIF) and/or isoniazid (INH). Multidrug-resistant TB (MDR-TB) was detected in 12.3% of isolates, five of which were fluoroquinolones (FLQs) resistant. SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. Conclusions: Diverse MTBC spoligotypes were identified, with the T and CAS families and EA (lineage 4) predominating. A high prevalence of drug-resistant TB, with SIT149/T3-ETH and CAS1-KILI sublineages comprising a greater share, was observed. A study with large sample size and a sequencing method with stronger discriminatory power is warranted to understand better the genetic diversity of circulating MTBC in this cohort of study, which would help to adopt targeted interventions.

Classification and counting of cells in brightfield microscopy images: an application of convolutional neural networks.

Microscopy is integral to medical research, facilitating the exploration of various biological questions, notably cell quantification. However, this process's time-consuming and error-prone nature, attributed to human intervention or automated methods usually applied to fluorescent images, presents challenges. In response, machine learning algorithms have been integrated into microscopy, automating tasks and constructing predictive models from vast datasets. These models adeptly learn representations for object detection, image segmentation, and target classification. An advantageous strategy involves utilizing unstained images, preserving cell integrity and enabling morphology-based classification-something hindered when fluorescent markers are used. The aim is to introduce a model proficient in classifying distinct cell lineages in digital contrast microscopy images. Additionally, the goal is to create a predictive model identifying lineage and determining optimal quantification of cell numbers. Employing a CNN machine learning algorithm, a classification model predicting cellular lineage achieved a remarkable accuracy of 93%, with ROC curve results nearing 1.0, showcasing robust performance. However, some lineages, namely SH-SY5Y (78%), HUH7_mayv (85%), and A549 (88%), exhibited slightly lower accuracies. These outcomes not only underscore the model's quality but also emphasize CNNs' potential in addressing the inherent complexities of microscopic images.

Epidemiological and clinical insights into the enterovirus D68 upsurge in Europe 2021/22 and the emergence of novel B3-derived lineages, ENPEN multicentre study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 and its clinical impact during the fall-winter season of 2021/22. From 19 European countries, 58 institutes reported 10,481 (6.8%) EV-positive samples of which 1,004 (9.6%) were identified as EV-D68 (852 respiratory samples). Clinical data was reported for 969 cases. 78.9% of infections were reported in children (0-5 years); 37.9% of cases were hospitalised. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases with six reported with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of two novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale EV-D68 European upsurge with severe clinical impact and the emergence of B3-derived lineages.

Wiskott-Aldrich syndrome protein expression in female WAS carriers: A flow cytometry study from North India.

INTRODUCTION: Wiskott-Aldrich syndrome (WAS) is a rare X-linked inborn error of immunity characterized by microthrombocytopenia, infections, eczema, and increased predisposition to develop autoimmunity and malignancy. Flow cytometric assay for determining WAS protein (WASp) is a rapid and cost-effective tool for detecting patients. However, very few studies described WASp expression in female carriers. Most WAS carriers are clinically asymptomatic. Active screening of female family members helps identify female carriers, distinguish de novo mutations, and to select appropriate donor prior to curative stem cell transplantation. This study was undertaken to evaluate the diagnostic capability of flow cytometry-based WASp expression in peripheral blood cells to identify carriers and compare WASp expression in different blood cell lineages. PATIENTS AND METHODS: Female patients, heterozygous for WAS gene, were enrolled in this study conducted at Pediatric Allergy Immunology Unit, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India. Flow cytometric assessment of WASp expression in lymphocytes, monocytes, and neutrophils was carried out and compared with healthy control and affected patients. The results were expressed in delta (Δ) median fluorescence intensity (MFI) as well as stain index (SI), which is the ratio of ΔMFI of patient and ΔMFI of control. RESULTS: Thirteen mothers and two sisters of genetically confirmed WAS patients were enrolled in the study. All enrolled females were clinically asymptomatic and did not have microthrombocytopenia. Low WASp expression (SI < 1) was seen in lymphocytes and monocytes in 10 (66.6%) carriers. Females with variants in proximal exons (exons 1 and 2) were found to have lesser expression than those with distal (exons 3-12) variants. CONCLUSION: Flow cytometry is a rapid, easily available, cost-effective tool for WASp estimation. Lymphocytes followed by monocytes are the best cell lineages for WASp estimation in carrier females. However, genetic testing remains the gold standard, as carrier females with variants in distal exons may have normal WASp expression.

Genetic variation associated with adult migration timing in lineages of Steelhead and Chinook Salmon in the Columbia River.

With the discovery of a major effect region (GREB1L, ROCK1) for adult migration timing in genomes of both Chinook Salmon and Steelhead, several subsequent studies have investigated the effect size and distribution of early and late migration alleles among populations in the Columbia River. Here, we synthesize the results of these studies for the major lineages of Chinook Salmon and Steelhead that include highly distinct groups in the interior Columbia River that exhibit atypical life histories from most coastal lineage populations of these two species. Whole-genome studies with high marker density have provided extensive insight into SNPs most associated with adult migration timing, and suites of markers for each species have been genotyped in large numbers of individuals to further validate phenotypic effects. For Steelhead, the largest phenotypic effect sizes have been observed in the coastal lineage (36% of variation for passage timing at Bonneville Dam; 43% of variation for tributary arrival timing) compared to the inland lineage (7.5% of variation for passage timing at Bonneville Dam; 8.4% of variation for tributary arrival timing) that overwinter in freshwater prior to spawning. For Chinook Salmon, large effect sizes have been observed in all three lineages for multiple adult migration phenotypes (Coastal lineage: percentage of variation of 27.9% for passage timing at Bonneville Dam, 28.7% for arrival timing for spawning; Interior ocean type: percentage of variation of 47.6% for passage timing at Bonneville Dam, 39.6% for tributary arrival timing, 77.9% for arrival timing for spawning; Interior stream type: percentage of variation of 35.3% for passage at Bonneville Dam, 9.8% for tributary arrival timing, 4.7% for arrival timing for spawning). Together, these results have extended our understanding of genetic variation associated with life history diversity in distinct populations of the Columbia River, however, much research remains necessary to determine the causal mechanism for this major effect region on migration timing in these species.

Temporal Dynamics and Genomic Landscape of SARS-CoV-2 After Four Years of Evolution.

Introduction Since its emergence, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone extensive genomic evolution, impacting public health policies, diagnosis, medication, and vaccine development. This study leverages advanced bioinformatics to assess the virus's temporal and regional genomic evolution from December 2019 to October 2023. Methods Our analysis incorporates 16,575 complete SARS-CoV-2 sequences collected from 214 countries. These samples were comparatively analyzed, with a detailed characterization of nucleic mutations, lineages, distribution, and evolutionary patterns during each year, using the Wuhan-Hu-1 strain as the reference. Results Our analysis has identified a total of 21,580 mutations that we classified into transient mutations, which diminished over time, and persistent mutations with steadily increasing frequencies. This mutation landscape led to a notable surge in the evolutionary rate, rising from 13 mutations per sample in 2020 to 96 by 2023, with minor geographic variations. The phylogenetic analysis unveiled three distinct evolutionary branches, each representing unique viral evolution pathways. These lineages exhibited a tendency for a reduced duration of dominance with a shortening prevalence period over time, as dominant strains were consistently replaced by more fit variants. Notably, the emergence of the Alpha and Delta variants in 2021 was followed by the subsequent dominance of Omicron clade variants that have branched into several recombinant variants in 2022, marking a significant shift in the viral landscape. Conclusion This study sheds light on the dynamic nature of SARS-CoV-2 evolution, emphasizing the importance of continuous and vigilant genomic surveillance. The dominance of recombinant lineages, coupled with the disappearance of local variants, underscores the virus's adaptability.

Hybrid lineages of CD4+ T cells: a handbook update.

CD4+ T lymphocytes have been classified into several lineages, according to their gene expression profiles and their effector responses. Interestingly, recent evidence is showing that many lineages could yield hybrid phenotypes with unique properties and functions. It has been reported that such hybrid lineages might underlie pathologies or may function as effector cells with protection capacities against molecular threats. In this work, we reviewed the characteristics of the hybrid lineages reported in the literature, in order to identify the expression profiles that characterize them and the markers that could be used to identify them. We also review the differentiation cues that elicit their hybrid origin and what is known about their physiological roles.

The opposed forces of differentiation and admixture across glacial cycles in the butterfly Aglais urticae.

Glacial cycles lead to periodic population interbreeding and isolation in warm-adapted species, which impact genetic structure and evolution. However, the effects of these processes on highly mobile and more cold-tolerant species are not well understood. This study aims to shed light on the phylogeographic history of Aglais urticae, a butterfly species with considerable dispersal ability, and a wide Palearctic distribution reaching the Arctic. Through the analysis of genomic data, four main genetic lineages are identified: European, Sierra Nevada, Sicily/Calabria/Peloponnese, and Eastern. The results indicate that the Sardo-Corsican endemic taxon ichnusa is a distinct species. The split between the relict lineages in southern Europe and the main European lineage is estimated to have happened 400-450 thousand years ago, with admixture observed during the Quaternary glacial cycles, and still ongoing, albeit to a much smaller extent. These results suggest that these lineages may be better treated as subspecific parapatric taxa. Ecological niche modelling supported the existence of both Mediterranean and extra-Mediterranean refugia during the glacial periods, with the main one located on the Atlantic coast. Nevertheless, gene flow between populations was possible, indicating that both differentiation and admixture have acted continuously across glacial cycles in this cold-tolerant butterfly, generally balancing each other but producing differentiated lineages in the southern peninsulas. We conclude that the population dynamics and the processes shaping the population genetic structure of cold-adapted species during the Quaternary ice ages may be different than those classically accepted for warm-adapted species.

Refined understanding of the impact of the Mycobacterium tuberculosis complex diversity on the intrinsic susceptibility to pretomanid.

Previous work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1-9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2-4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2-4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT. IMPORTANCE: This study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

Quantifying the HIV reservoir with dilution assays and deep viral sequencing.

People living with HIV on antiretroviral therapy often have undetectable virus levels by standard assays, but "latent" HIV still persists in viral reservoirs. Eliminating these reservoirs is the goal of HIV cure research. The quantitative viral outgrowth assay (QVOA) is commonly used to estimate the reservoir size, that is, the infectious units per million (IUPM) of HIV-persistent resting CD4+ T cells. A new variation of the QVOA, the ultra deep sequencing assay of the outgrowth virus (UDSA), was recently developed that further quantifies the number of viral lineages within a subset of infected wells. Performing the UDSA on a subset of wells provides additional information that can improve IUPM estimation. This paper considers statistical inference about the IUPM from combined dilution assay (QVOA) and deep viral sequencing (UDSA) data, even when some deep sequencing data are missing. Methods are proposed to accommodate assays with wells sequenced at multiple dilution levels and with imperfect sensitivity and specificity, and a novel bias-corrected estimator is included for small samples. The proposed methods are evaluated in a simulation study, applied to data from the University of North Carolina HIV Cure Center, and implemented in the open-source R package SLDeepAssay.

RNA barcode segments for SARS-CoV-2 identification from HCoVs and SARSr-CoV-2 lineages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for coronavirus disease 2019 (COVID-19), continues to evolve, giving rise to more variants and global reinfections. Previous research has demonstrated that barcode segments can effectively and cost-efficiently identify specific species within closely related populations. In this study, we designed and tested RNA barcode segments based on genetic evolutionary relationships to facilitate the efficient and accurate identification of SARS-CoV-2 from extensive virus samples, including human coronaviruses (HCoVs) and SARSr-CoV-2 lineages. Nucleotide sequences sourced from NCBI and GISAID were meticulously selected and curated to construct training sets, encompassing 1733 complete genome sequences of HCoVs and SARSr-CoV-2 lineages. Through genetic-level species testing, we validated the accuracy and reliability of the barcode segments for identifying SARS-CoV-2. Subsequently, 75 main and subordinate species-specific barcode segments for SARS-CoV-2, located in ORF1ab, S, E, ORF7a, and N coding sequences, were intercepted and screened based on single-nucleotide polymorphism sites and weighted scores. Post-testing, these segments exhibited high recall rates (nearly 100%), specificity (almost 30% at the nucleotide level), and precision (100%) performance on identification. They were eventually visualized using one and two-dimensional combined barcodes and deposited in an online database (http://virusbarcodedatabase.top/). The successful integration of barcoding technology in SARS-CoV-2 identification provides valuable insights for future studies involving complete genome sequence polymorphism analysis. Moreover, this cost-effective and efficient identification approach also provides valuable reference for future research endeavors related to virus surveillance.

Age-dependent influenza infection patterns and subtype circulation in Denmark, in seasons 2015/16 to 2021/22.

BackgroundInfluenza was almost absent for 2 years following the implementation of strict public health measures to prevent the spread of SARS-CoV-2. The consequence of this on infections in different age groups is not yet known.AimTo describe the age groups infected with the influenza virus in 2021/22, the first post-pandemic influenza season in Denmark, compared with the previous six seasons, and subtypes circulating therein.MethodsInfection and hospitalisation incidences per season and age group were estimated from data in Danish registries. Influenza virus subtypes and lineages were available from samples sent to the National Influenza Centre at Statens Serum Institut.ResultsTest incidence followed a similar pattern in all seasons, being highest in 0-1-year-olds and individuals over 75 years, and lowest in 7-14-year-olds and young people 15 years to late twenties. When the influenza A virus subtypes A(H3N2) and A(H1N1)pdm09 co-circulated in seasons 2015/16 and 2017/18 to 2019/20, the proportion of A(H1N1)pdm09 was higher in 0-1-year-olds and lower in the over 85-year-olds compared with the overall proportion of A(H1N1)pdm09 in these seasons. The proportion of A(H3N2) was higher in the over 85 years age group compared with the overall proportion of A(H3N2). The 2016/17 and 2021/22 seasons were dominated by A(H3N2) but differed in age-specific trends, with the over 85 years age group initiating the 2016/17 season, while the 2021/22 season was initiated by the 15-25-year-olds, followed by 7-14-year-olds.ConclusionThe 2021/22 influenza season had a different age distribution compared with pre-COVID-19 pandemic seasons.

Emergence and spread of SARS-CoV-2 variants of concern in Canada: a retrospective analysis from clinical and wastewater data.

BACKGROUND: The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2lineages have been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Variants of concern (VOCs) and SARS-CoV-2 lineages associated with increased transmissibility and/or severity are of particular interest. METHOD: Here, we consider clinical and wastewater data sources to assess the emergence and spread of VOCs in Canada retrospectively. RESULTS: We show that, overall, wastewater-based VOC identification provides similar insights to the surveillance based on clinical samples. Based on clinical data, we observed synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. CONCLUSION: In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence. In contrast, VOC Omicron achieved the same contribution in less than one month. This study provides significant benchmarks to enhance planning for future VOCs, and to some extent for future pandemics caused by other pathogens, by quantifying the rate of SARS-CoV-2 VOCs invasion in Canada.

The conserved RNA-binding protein Imp is required for the specification and function of olfactory navigation circuitry in Drosophila.

Complex behaviors depend on the precise developmental specification of neuronal circuits, but the relationship between genetic programs for neural development, circuit structure, and behavioral output is often unclear. The central complex (CX) is a conserved sensory-motor integration center in insects, which governs many higher-order behaviors and largely derives from a small number of type II neural stem cells (NSCs). Here, we show that Imp, a conserved IGF-II mRNA-binding protein expressed in type II NSCs, plays a role in specifying essential components of CX olfactory navigation circuitry. We show the following: (1) that multiple components of olfactory navigation circuitry arise from type II NSCs. (2) Manipulating Imp expression in type II NSCs alters the number and morphology of many of these circuit elements, with the most potent effects on neurons targeting the ventral layers of the fan-shaped body (FB). (3) Imp regulates the specification of Tachykinin-expressing ventral FB input neurons. (4) Imp is required in type II NSCs for establishing proper morphology of the CX neuropil structures. (5) Loss of Imp in type II NSCs abolishes upwind orientation to attractive odor while leaving locomotion and odor-evoked regulation of movement intact. Taken together, our findings establish that a temporally expressed gene can regulate the expression of a complex behavior by developmentally regulating the specification of multiple circuit components and provides a first step toward a developmental dissection of the CX and its roles in behavior.

Occurrence of Anaplasma spp. in wild lagomorphs from Southern Spain: Molecular detection of new Anaplasma bovis lineages.

Wild lagomorphs can act as reservoirs of several pathogens of public and animal health concern. However, the number of studies assessing the presence of Anaplasma spp. in these species is scarce. The aim of the present study was to molecularly identify Anaplasma spp. in wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) from Southern Spain and assess their epidemiological role in the maintenance of the bacterium. During 2017-2021, spleen samples of 394 wild rabbits and 145 Iberian hares were collected. Anaplasma DNA was detected using different PCR assays (16S rRNA and groEL) and phylogenetic analyses were carried out by Bayesian approach. The possible influence of lagomorph species, age and sex on the prevalence of Anaplasma spp. was evaluated by a multiple logistic regression model. The 9.4% of the rabbits were positive to Anaplasma bovis, but all the hares were negative. No significant differences were found in Anaplasma spp. prevalence regarding to age or sex. This is the first report of A. bovis in lagomorphs from Europe. The phylogenetic analysis of A. bovis confirms the existence of different clusters suggesting the existence of several lineages. In addition, a high divergence of nucleotide identity was observed within the lineage 4, which could result in the under-detection of some strains when using A. bovis-specific PCR, hindering its detection and characterization. Since this analysis is based on a limited number of nucleotide bases and sequences, more studies are needed for further characterize A. bovis, as well as its relationship with other Anaplasma spp.

Lassa virus in novel hosts: insights into the epidemiology of lassa virus infections in southern Nigeria.

Identification of the diverse animal hosts responsible for spill-over events from animals to humans is crucial for comprehending the transmission patterns of emerging infectious diseases, which pose significant public health risks. To better characterize potential animal hosts of Lassa virus (LASV), we assessed domestic and non-domestic animals from 2021-2022 in four locations in southern Nigeria with reported cases of Lassa fever (LF). Birds, lizards, and domestic mammals (dogs, pigs, cattle and goats) were screened using RT-qPCR, and whole genome sequencing was performed for lineage identification on selected LASV positive samples. Animals were also screened for exposure to LASV by enzyme-linked immunosorbent assay (ELISA). Among these animals, lizards had the highest positivity rate by PCR. Genomic sequencing of samples in most infected animals showed sub-lineage 2 g of LASV. Seropositivity was highest among cattle and lowest in pigs. Though the specific impact these additional hosts may have in the broader virus-host context are still unknown - specifically relating to pathogen diversity, evolution, and transmission - the detection of LASV in non-rodent hosts living in proximity to confirmed human LF cases suggests their involvement during transmission as potential reservoirs. Additional epidemiological data comparing viral genomes from humans and animals, as well as those circulating within the environment will be critical in understanding LASV transmission dynamics and will ultimately guide the development of countermeasures for this zoonotic health threat.

Genetic variability analysis of human papillomavirus 58: Novel sublineage identification and persistent infection association.

This study aims to characterize the genetic variability of HPV58, identify novel lineages and sublineages, and explore the association between persistent/multiple HPV58 infections and genetic variation. In this study, samples from 124 women with HPV58 infection in Eastern China were collected and 81 isolates of E6 and L1 full-length genes were successfully amplified from 55 samples. We evaluated the diversity of genetic variants and performed correlation analyses between genetic variability and pathology, vaccination, multiple infections, and persistent infections. Among the E6 and L1 gene sequences collected, the dominant prevailing sublineages were A1 (46.2%) and A2 (23.1%). In addition, we found two potential novel sublineages denoted as the A4 and A5 sublineage. A total of 50 nucleotide substitutions, including 28 synonymous substitutions and 22 nonsynonymous substitutions, were observed in the E6 and L1 genes. Among them, variants with A388C/K93N substitutions in the E6 gene correlated with persistent infection (≥1 and ≥2 years) (p < 0.005), and C307T/C66C was associated with persistent infection (≥2 years) (p < 0.005). Notably, two mutations above were detected in the isolate from the patient with breakthrough vaccine infection. Our study found two novel sublineages and sites of genetic variability in multiple and persistent infection variants. In addition, we identified two mutational sites associated with persistent infection. This study provides new insight into the clinical characteristics of HPV 58 genetic variations and offers new ideas for research on next-generation vaccines in Eastern China.