Genome Types Research Articles

Whole genome sequencing of M. tuberculosis for disease control in high-burden settings: study protocol for a cluster randomized controlled trial evaluating different community-wide intervention strategies in rural Madagascar

BackgroundRetrospective and descriptive molecular epidemiology studies have shown that Mycobacterium tuberculosis whole genome sequencing can identify outbreaks and disease transmission events with higher resolution than conventional epidemiological investigations. Those studies have strengthened our understanding of genomic polymorphisms correlating with person-to-person transmission and helped resolve putative transmission clusters. To date, systematic genomic surveillance programs implemented for M. tuberculosis were only implemented in low-incidence settings. The purpose of this study is to determine whether there is an impact of routine M. tuberculosis whole genome sequencing on tuberculosis case detection in a high-incidence setting.MethodsA cluster randomized controlled trial will be performed. Forty-eight rural village groups (or Fokontany) in the Vohibato district of Madagascar will be randomized to one of three interventions arms. Arm 1 (standard of care) involves healthcare facility-based passive case detection with smear microscopy testing. Arm 2 (best practice) consists of active case finding and Xpert MTB/RIF Ultra PCR testing followed by household contact investigations. Arm 3 (novel intervention) includes the same interventions as arm 2, with addition of sputum culture and M. tuberculosis whole genome sequencing for all newly diagnosed cases. In arm 3, molecular suggested putative outbreaks are investigated, and additional TB suspects are appropriately tested. The intervention observational period will be 2 years. The primary outcome will be the number of detected cases/100,000/year in each arm after 1 year of intervention.DiscussionThis study is designed to determine whether there is an impact of prospective whole genome sequencing-based molecular typing on tuberculosis case detection in high-incidence settings. Investigating potential outbreaks and focusing active case finding in spatiotemporal settings where disease transmission is suggested by genomic typing is hypothesized to improve case detection in rural communities.Trial registrationClinicalTrials.gov NCT05406453. Retrospectively registered on June 6, 2022.

Expanding the Triangle of U: Comparative analysis of the Hirschfeldia incana genome provides insights into chromosomal evolution, phylogenomics and high photosynthesis-related traits.

The Brassiceae tribe encompasses many economically important crops and exhibits high intraspecific and interspecific phenotypic variation. After a shared whole-genome triplication (WGT) event (Br-α, ~15.9 million years ago), differential lineage diversification and genomic changes contributed to an array of divergence in morphology, biochemistry, and physiology underlying photosynthesis-related traits. Here, the C3 species Hirschfeldia incana is studied as it displays high photosynthetic rates under high-light conditions. Our aim was to elucidate the evolution that gave rise to the genome of H. incana and its high-photosynthesis traits. We reconstructed a chromosome-level genome assembly for H. incana (Nijmegen, v2.0) using nanopore and chromosome conformation capture (Hi-C) technologies, with 409Mb in size and an N50 of 52Mb (a 10× improvement over the previously published scaffold-level v1.0 assembly). The updated assembly and annotation was subsequently employed to investigate the WGT history of H. incana in a comparative phylogenomic framework from the Brassiceae ancestral genomic blocks and related diploidized crops. Hirschfeldia incana (x=7) shares extensive genome collinearity with Raphanus sativus (x=9). These two species share some commonalities with Brassica rapa and B. oleracea (A genome, x=10 and C genome, x=9, respectively) and other similarities with B. nigra (B genome, x=8). Phylogenetic analysis revealed that H. incana and R. sativus form a monophyletic clade in between the Brassica A/C and B genomes. We postulate that H. incana and R. sativus genomes are results of hybridization or introgression of the Brassica A/C and B genome types. Our results might explain the discrepancy observed in published studies regarding phylogenetic placement of H. incana and R. sativus in relation to the "Triangle of U" species. Expression analysis of WGT retained gene copies revealed sub-genome expression divergence, likely due to neo- or sub-functionalization. Finally, we highlighted genes associated with physio-biochemical-anatomical adaptive changes observed in H. incana which likely facilitate its high-photosynthesis traits under high light. The improved H. incana genome assembly, annotation and results presented in this work will be a valuable resource for future research to unravel the genetic basis of its ability to maintain a high photosynthetic efficiency in high-light conditions and thereby improve photosynthesis for enhanced agricultural production.

A single-stranded based library preparation method for virome characterization

BackgroundThe gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent diversity of genome types (dsDNA, ssDNA, dsRNA, and ssRNA) and topologies (linear, circular, or fragments), with subsequently biases associated with current sequencing library preparation methods. To overcome these problems and improve reproducibility and comparability across studies, universal or standardized virome sequencing library construction methods are highly needed in the gut virome study.ResultsWe repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA + Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches.ConclusionWe introduce and improve a fast, simple, and efficient ligation-based single-stranded DNA library preparation for gut virome study. This method is compatible with Illumina sequencing platforms and only requires ligation reagents within 3-h library preparation, which is similar or even better than the advanced library preparation method (xGen). We hope this method can be further optimized, validated, and widely used to make gut virome study more comparable and reproducible.EMStnH64sPQJC5VtA73CKfVideo

Reexamining the Mycovirome of Botrytis spp.

Botrytis species cause gray mold disease in more than 200 crops worldwide. To control this disease, chemical fungicides are usually applied. However, more sustainable control alternatives should be explored, such as the use of hypovirulent mycovirus-infected fungal strains. To determine the mycovirome of two Botrytis species, B. cinerea and B. prunorum, we reanalyzed RNA-Seq and small RNA-Seq data using different assembly programs and an updated viral database, aiming to identify new mycoviruses that were previously not described in the same dataset. New mycoviruses were identified, including those previously reported to infect or be associated with B. cinerea and Plasmopara viticola, such as Botrytis cinerea alpha-like virus 1 and Plasmopara viticola lesion-associated ourmia-like virus 80. Additionally, two novel narnaviruses, not previously identified infecting Botrytis species, have been characterized, tentatively named Botrytis cinerea narnavirus 1 and Botrytis narnavirus 1. The analysis of small RNAs suggested that all identified mycoviruses were targeted by the antiviral fungal mechanism, regardless of the viral genome type. In conclusion, the enlarged list of newly found viruses and the application of different bioinformatics approaches have enabled the identification of novel mycoviruses not previously described in Botrytis species, expanding the already extensive list.

Identification of an immune cell infiltration-related gene signature for prognosis prediction in triple-negative breast cancer.

Triple-negative breast cancer TNBC with higher immunogenicity and tumor-infiltrating lymphocyte (TIL) enrichment can benefit from immunotherapy relative to other breast cancer subtypes. Our work was designed to identify the TIL-related hub genes in TNBC and construct a prognostic signature for TNBC. TNBC gene expression files were obtained from the TCGA database. CIBERSORT algorithm and random forest risk model were used for immune infiltration group division. The TIL-related differentially expressed genes (DEGs) were then selected and subject to GO, KEGG analyses and GSEA. Next, Lasso cox regression analyses were adopted for constructing a prognostic risk model, followed by evaluation using time-dependent ROC curves. The copy number variation between the two risk groups was also analyzed, and major genomic mutation types were identified. Additionally, the nomogram was constructed with calibration curve for clinical prognosis analysis. Our results showed that totally 113 TNBC samples were allocated into the high or low-immune risk groups. We identified 243 DEGs between groups, namely TIL-related DEGs, with 128 upregulated and 115 downregulated genes. Among the TIL-related DEGs, 6 hub genes (SLITRK3, PCDHGB3, NELL2, SRRM4, ASIC2 and B4GALNT2) were screened out and constructed a prognostic risk signature, which had good performance for long-term prognosis prediction. Analysis of genomic mutation showed that the TP53, PIK3CA, TTH, etc. showed high mutation frequency in the two prognostic risk groups. Moreover, the higher risk score of the prognostic risk model predicted poor overall survival in TNBC patients, and nomogram and calibration curve confirmed the potent prediction ability of this model. To sum up, six TIL-related biomarkers (SLITRK3, PCDHGB3, NELL2, SRRM4, ASIC2 and B4GALNT2) were identified and used for the construction of the prognostic risk model, which might provide novel insight for the clinical decisions.

Dothistroma septosporum and Dothistroma pini, the causal agents of Dothistroma needle blight, are infected by multiple viruses

Dothistroma septosporum and Dothistroma pini are severe foliar pathogens of conifers. They infect a broad spectrum of hosts (mainly Pinus spp.), causing chlorosis, defoliation of needles, and eventually the death of pine trees in extreme cases. Mycoviruses represent a novel and innovative avenue for controlling pathogens. To search for possible viruses hosted by Dothistroma spp. we screened a subset of isolates (20 strains of D. septosporum and one D. pini) originating from the Czech Republic, Slovenia, Italy, Austria and Ireland for viral dsRNA segments. Only five of them showed the presence of dsRNA segments. A total of 21 fungal isolates were prepared for total RNA extractions. RNA samples were pooled, and two separate RNA libraries were constructed for stranded total RNA sequencing. RNA-Seq data processing, pairwise sequence comparisons (PASC) and phylogenetic analyses revealed the presence of thirteen novel putative viruses with varying genome types: seven negative-sense single-stranded RNA viruses, including six bunya-like viruses and one new member of the order Mononegavirales; three positive-sense single-stranded RNA viruses, two of which are similar to those of the family Narnaviridae, while the genome of the third correspond to those of the family Gammaflexiviridae; and three double-stranded RNA viruses, comprising two novel members of the family Chrysoviridae and a potentially new species of gammapartitivirus. The results were confirmed with RT-PCR screening that the fungal pathogens hosted all the viruses and showed that particular fungal strains harbour multiple virus infections and that they are transmitted vertically. In this study, we described the narnavirus infecting D. pini. To our knowledge, this is the first virus discovered in D. pini.

The TELCoMB Protocol for High-Sensitivity Detection of ARG-MGE Colocalizations in Complex Microbial Communities.

Understanding the genetic basis of antimicrobial resistance is crucial for developing effective mitigation strategies. One necessary step is to identify the antimicrobial resistance genes (ARGs) within a microbial population, referred to as the resistome, as well as the mobile genetic elements (MGEs) harboring ARGs. Although shotgun metagenomics has been successful in detecting ARGs and MGEs within a microbiome, it is limited by low sensitivity. Enrichment using cRNA biotinylated probes has been applied to address this limitation, enhancing the detection of rare ARGs and MGEs, especially when combined with long-read sequencing. Here, we present the TELCoMB protocol, a Snakemake workflow that elucidates resistome and mobilome composition and diversity and uncovers ARG-MGE colocalizations. The protocol supports both short- and long-read sequencing and does not require enrichment, making it versatile for various genomic data types. TELCoMB generates publication-ready figures and CSV files for comprehensive analysis, improving our understanding of antimicrobial resistance mechanisms and spread. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Installing TELCOMB Locally Alternate Protocol: Installing TELCOMB on a SLURM Cluster Basic Protocol 2: Data Preprocessing Basic Protocol 3: Calculation of Resistome Distribution and Composition Basic Protocol 4: Identification of ARG-MGE Colocalizations.

Genomic evidence of two-staged transmission of the early seventh cholera pandemic

The seventh cholera pandemic started in 1961 in Indonesia and spread across the world in three waves in the decades that followed. Here, we utilised genomic evidence to detail the first wave of the seventh pandemic. Genomes of 22 seventh pandemic Vibrio cholerae isolates from 1961 to 1979 were completely sequenced. Together with 152 publicly available genomes from the same period, they fell into seven phylogenetic clusters (CL1–CL7). By multilevel genome typing (MGT), all were assigned to MGT2 ST1 (Wave 1) except three isolates in CL7 which were typed as MGT2 ST2 (Wave 2). The Wave 1 seventh pandemic expanded in two stages, with Stage 1 (CL1–CL5) spread across Asia and Stage 2 (CL6 and CL7) spread to the Middle East and Africa. Three non-synonymous mutations, one each, in three regulatory genes, csrD (global regulator), acfB (chemotaxis), and luxO (quorum sensing) may have critically contributed to its pandemicity. The three MGT2 ST2 isolates in CL7 were the progenitors of Wave 2 and evolved from within Wave 1 with acquisition of a novel IncA/C plasmid. Our findings provide new insight into the evolution and transmission of the early seventh pandemic, which may aid future cholera prevention and control.

Does combining numerous data types in multi-omics data improve or hinder performance in survival prediction? Insights from a large-scale benchmark study

BackgroundPredictive modeling based on multi-omics data, which incorporates several types of omics data for the same patients, has shown potential to outperform single-omics predictive modeling. Most research in this domain focuses on incorporating numerous data types, despite the complexity and cost of acquiring them. The prevailing assumption is that increasing the number of data types necessarily improves predictive performance. However, the integration of less informative or redundant data types could potentially hinder this performance. Therefore, identifying the most effective combinations of omics data types that enhance predictive performance is critical for cost-effective and accurate predictions.MethodsIn this study, we systematically evaluated the predictive performance of all 31 possible combinations including at least one of five genomic data types (mRNA, miRNA, methylation, DNAseq, and copy number variation) using 14 cancer datasets with right-censored survival outcomes, publicly available from the TCGA database. We employed various prediction methods and up-weighted clinical data in every model to leverage their predictive importance. Harrell’s C-index and the integrated Brier Score were used as performance measures. To assess the robustness of our findings, we performed a bootstrap analysis at the level of the included datasets. Statistical testing was conducted for key results, limiting the number of tests to ensure a low risk of false positives.ResultsContrary to expectations, we found that using only mRNA data or a combination of mRNA and miRNA data was sufficient for most cancer types. For some cancer types, the additional inclusion of methylation data led to improved prediction results. Far from enhancing performance, the introduction of more data types most often resulted in a decline in performance, which varied between the two performance measures.ConclusionsOur findings challenge the prevailing notion that combining multiple omics data types in multi-omics survival prediction improves predictive performance. Thus, the widespread approach in multi-omics prediction of incorporating as many data types as possible should be reconsidered to avoid suboptimal prediction results and unnecessary expenditure.

Search for epistatically interacting genetic variants that are associated with vasovagal syncope within biallelic combinations

The most common cause of transient loss of consciousness is vasovagal syncope (VVS), which occurs due to hypoperfusion of the brain due to the interruption of vegetative blood circulation control leading to arterial hypotension. It is known that there is a genetic predisposition to VVS, but the data on the role of individual genes are quite inconsistent. Using APSampler software,which based on a Markov chain Monte Carlo technique and Bayesian nonparametric statistics, we identified biallelic combinations associated with VVS and investigated the nature of interaction between their components. We used the previously obtained results of genomic typing of single nucleotide polymorphisms (SNPs) of 5 genes, the products of which are involved in neurohumoral regulation, and 4 SNPs within locus 2q32.1, supplemented with data for new individuals included in the study. The total sample included 175 patients with a confirmed diagnosis of VVS and 200 control individuals without a history of syncope. Eleven pairwise combinations of SNPs of different genes were found to be associated with VVS. Five of these combinations were epistatic, four of which included SNPs at the 2q32.1 locus located within or near noncoding RNA genes. It is suggested that genes of noncoding RNAs localized on chromosome 2 may directly or indirectly (through cascades of interactions) participate in the regulation of the activity of genes forming epistatic combinations with them.

Next-generation sequencing-based evaluation of the actionable landscape of genomic alterations in solid tumors: the "MOZART" prospective observational study.

The identification of the most appropriate targeted therapies for advanced cancers is challenging. We performed a molecular profiling of metastatic solid tumors utilizing a comprehensive next-generation sequencing (NGS) assay to determine genomic alterations' type, frequency, actionability, and potential correlations with PD-L1 expression. A total of 304 adult patients with heavily pretreated metastatic cancers treated between January 2019 and March 2021 were recruited. The CLIA-/UKAS-accredit Oncofocus assay targeting 505 genes was used on newly obtained or archived biopsies. Chi-square, Kruskal-Wallis, and Wilcoxon rank-sum tests were used where appropriate. Results were significant for P < .05. A total of 237 tumors (78%) harbored potentially actionable genomic alterations. Tumors were positive for PD-L1 in 68.9% of cases. The median number of mutant genes/tumor was 2.0 (IQR: 1.0-3.0). Only 34.5% were actionable ESCAT Tier I-II with different prevalence according to cancer type. The DNA damage repair (14%), the PI3K/AKT/mTOR (14%), and the RAS/RAF/MAPK (12%) pathways were the most frequently altered. No association was found among PD-L1, ESCAT, age, sex, and tumor mutational status. Overall, 62 patients underwent targeted treatment, with 37.1% obtaining objective responses. The same molecular-driven treatment for different cancer types could be associated with opposite clinical outcomes. We highlight the clinical value of molecular profiling in metastatic solid tumors using comprehensive NGS-based panels to improve treatment algorithms in situations of uncertainty and facilitate clinical trial recruitment. However, interpreting genomic alterations in a tumor type-specific manner is critical.

Average Nucleotide Identity and Digital DNA-DNA Hybridization Analysis Following PromethION Nanopore-Based Whole Genome Sequencing Allows for Accurate Prokaryotic Typing.

Whole-genome sequencing (WGS) is revolutionizing clinical bacteriology. However, bacterial typing remains investigated by reference techniques with inherent limitations. This stresses the need for alternative methods providing robust and accurate sequence type (ST) classification. This study optimized and evaluated a GridION nanopore sequencing protocol, adapted for the PromethION platform. Forty-eight Escherichia coli clinical isolates with diverse STs were sequenced to assess two alternative typing methods and resistance profiling applications. Multi-locus sequence typing (MLST) was used as the reference typing method. Genomic relatedness was assessed using Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (DDH), and cut-offs for discriminative strain resolution were evaluated. WGS-based antibiotic resistance prediction was compared to reference Minimum Inhibitory Concentration (MIC) assays. We found ANI and DDH cut-offs of 99.3% and 94.1%, respectively, which correlated well with MLST classifications and demonstrated potentially higher discriminative resolution than MLST. WGS-based antibiotic resistance prediction showed categorical agreements of ≥ 93% with MIC assays for amoxicillin, ceftazidime, amikacin, tobramycin, and trimethoprim-sulfamethoxazole. Performance was suboptimal (68.8-81.3%) for amoxicillin-clavulanic acid, cefepime, aztreonam, and ciprofloxacin. A minimal sequencing coverage of 12× was required to maintain essential genomic features and typing accuracy. Our protocol allows the integration of PromethION technology in clinical laboratories, with ANI and DDH proving to be accurate and robust alternative typing methods, potentially offering superior resolution. WGS-based antibiotic resistance prediction holds promise for specific antibiotic classes.

Genomic diversity of Salmonella enterica serovar Typhimurium isolated from chicken processing facilities in New South Wales, Australia.

Contamination of poultry products by Salmonella enterica serovar Typhimurium (STm) is a major cause of foodborne infections and outbreaks. This study aimed to assess the diversity and antimicrobial resistance (AMR) carriage of STm in three chicken processing plants using genomic sequencing. It also aimed to investigate whether any particular strain types were associated with cases of human illness. Multilevel genome typing (MGT) was used to analyze 379 STm isolates from processed chicken carcasses. The diversity of chicken STm sequence types (STs) increased from MGT1 (2 STs) to MGT9 (257 STs). STs at MGT5 to MGT9 levels that were unique to one processing plant and shared among the processing plants were identified, likely reflecting the diversity of STm at their farm source. Fifteen medium resolution MGT5 STs matched those from human infections in Australia and globally. However, no STs matched between the chicken and human isolates at high resolution levels (MGT8 or MGT9), indicating the two STm populations were phylogenetically related but were unlikely to be directly epidemiologically linked. AMR genes were rare, with only a bla TEM-1 gene carried by a 95 kb IncI1 Alpha plasmid being identified in 20 isolates. In conclusion, subpopulations that were widespread in processing plants and had caused human infections were described using MGT5 STs. In this STM population, AMR was rare with only sporadic resistance to a single drug class observed. The genomic analysis of STm from chicken processing plants in this study provided insights into STm that contaminate meat chickens early in the food production chain.

Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor

Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.

Banana Classification Using Sanger Sequencing of the Ribosomal DNA Internal Transcribed Spacer (ITS) Region.

Banana (Musa spp.) is one of the most economically important horticultural crops. There are many types of banana, with differing ploidy (usually diploid, triploid, or tetraploid) and genome types (most containing the A or/and B genome). Currently, observation and genome type detection are commonly used to identify banana germplasm resources. However, observation is tedious, while genome type detection cannot distinguish categories below genome types. It is, therefore, urgent to establish a simple and effective method for identifying banana germplasm resources. This study sequenced and analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 62 banana germplasm resources and found that the sequencing peaks, especially the 20 bp region near the 420-bp position (referred to as the 420-bp region), exhibited relatively recognizable and repeatable polymorphism characteristics. Using the 420-bp region as a marker, we were able to quickly distinguish bananas belonging to different genome type groups or different subgroups in the same genome type group. Moreover, it appeared that Sanger sequencing of ITS could be used to identify hybrid banana offspring. In general, ITS sequencing simplifies the classification of banana germplasm resources and has potential application in several areas of Musa improvement.

A chromosome-level genome assembly of the Korean minipig (Sus scrofa)

Recent advancements in sequencing and genome assembly technologies have led to rapid generation of high-quality genome assemblies for various species and breeds. Despite the importance as minipigs an animal model in biomedical research, the construction of high-quality genome assemblies of minipigs still lags behind other pig breeds. To address this problem, we constructed a high-quality chromosome-level genome assembly of the Korean minipig (KMP) utilizing multiple different types of sequencing reads and reference genomes. The KMP assembly included 19 chromosome-level sequences with a total length of 2.52 Gb and N50 of 137 Mb. Comparative analyses with the pig reference genome (Sscrofa11.1) demonstrated comparable contiguity and completeness of the KMP assembly. Additionally, genome annotation analyses identified 22,666 protein-coding genes and repetitive elements occupying 40.10% of the genome. The KMP assembly and genome annotation provide valuable resources that can contribute to various future research on minipig and other pig breeds.

Phylogenomic analyses and comparative genomic studies of Thermus strains isolated from Tengchong and Tibet Hot Springs, China.

Genus Thermus is the main focus of researcher among the thermophiles. Members of this genus are the inhabitants of both natural and artificial thermal environments. We performed phylogenomic analyses and comparative genomic studies to unravel the genomic diversity among the strains belonging to the genus Thermus in geographically different thermal springs. Sixteen Thermus strains were isolated and sequenced from hot springs, Qucai hot springs in Tibet and Tengchong hot springs in Yunnan, China. 16S rRNA gene based phylogeny and phylogenomic analyses based on concatenated set of 971 Orthologous Protein Families (supermatrix and gene content methods) revealed a mixed distribution of the Thermus strains. Whole genome based phylogenetic analysis showed, all 16 Thermus strains belong to five species; Thermus oshimai (YIM QC-2-109, YIM 1640, YIM 1627, 77359, 77923, 77838), Thermus antranikianii (YIM 73052, 77412, 77311, 71206), Thermus brokianus (YIM 73518, 71318, 72351), Thermus hydrothermalis (YIM 730264 and 77927) and one potential novel species 77420 forming clade with Thermus thalpophilus SYSU G00506T. Although the genomes of different strains of Thermus of same species were highly similar in their metabolic pathways, but subtle differences were found. CRISPR loci were detected through genome-wide screening, which showed that Thermus isolates from two different thermal locations had well developed defense system against viruses and adopt similar strategy for survival. Additionally, comparative genome analysis screened competence loci across all the Thermus genomes which could be helpful to acquire DNA from environment. In the present study it was found that Thermus isolates use two mechanism of incomplete denitrification pathway, some Thermus strains produces nitric oxide while others nitrious oxide (dinitrogen oxide), which show the heterotrophic lifestyle of Thermus genus. All isolated organisms encoded complete pathways for glycolysis, tricarboxylic acid and pentose phosphate. Calvin Benson Bassham cycle genes were identified in genomes of T. oshimai and T. antranikianii strains, while genomes of all T. brokianus strains and organism 77420 were lacking. Arsenic, cadmium and cobalt-zinc-cadmium resistant genes were detected in genomes of all sequenced Thermus strains. Strains 77,420, 77,311, 73,518, 77,412 and 72,351 genomes were found harboring genes for siderophores production. Sox gene clusters were identified in all sequenced genomes, except strain YIM 730264, suggesting a mode of chemolithotrophy.Through the comparative genomic analysis, we also identified 77420 as the genome type species and its validity as novel organism was confirmed by whole genome sequences comparison. Although isolate 77420 had 99.0% 16S rRNA gene sequence similarity with T. thalpophilus SYSU G00506T but based on ANI 95.86% (Jspecies) and digital DDH 68.80% (GGDC) values differentiate it as a potential novel species. Similarly, in the phylogenomic tree, the novel isolate 77,420 forming a separate branch with their closest reference type strain T. thalpophilus SYSU G00506T.

Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Hépatocarcinomes liés à l’infection par le VHB et potentielles nouvelles stratégies thérapeutiques

AbstractHepatocellular carcinomas (HCC) are the most common primary liver tumours. They are the second leading cause of cancer-related mortality worldwide. The main etiological factors include viral hepatitis B and C, chronic alcohol intake and metabolic syndromes. We will focus this review on the impact of hepatitis B virus (HBV) infection and the progression of liver damage to HCC. Both direct and indirect mechanisms of HBV infection contribute to the emergence of HCC. We will address these two types of mechanisms by focusing on direct viral effects, explored in numerous in vivo and in vitro studies. Published results can sometimes be contradictory. They vary depending on the study model, the viral sequence, the level of HBV expression, the type of viral genome transferred (partial or complete), the transfer method, generating a stable or transient expression, and many other parameters. Nevertheless, the knowledge acquired recently on the viral cycle, and in particular on the nature of the hepatocyte receptor of HBV, allows today to reproduce in vitro a complete viral cycle in different experimental models and thus, to better appreciate the pro-tumor activities of HBV infection.This review is obviously not exhaustive and the knowledge, particularly on the clinical and therapeutic management of HCC, whether associated with the presence of HBV or not, is currently evolving with the rise of combined immunotherapies.