Genome Segments Research Articles

Infectious Bursal Disease Virus in Algeria: Persistent Circulation of Very Virulent Strains in Spite of Control Efforts

Infectious bursal disease (IBD) is among the most impactful immunosuppressive diseases of poultry. Its agent, infectious bursal disease virus (IBDV), is prone to both mutation and reassortment, resulting in a remarkable variability. Traditionally, IBDV characterization relies on antigenicity and pathogenicity assessment, but multiple phylogenetic classifications have been recently proposed, whose implementation in molecular surveys helps generating informative and standardized epidemiological data. In the present study, the Algerian IBDV scenario was assessed based on the novel classification guidelines by sequencing portions of both genome segments. Seventy pools of bursal samples were collected in 2022–2023 in 11 districts of Northern Algeria, mostly from broiler flocks. Out of 55 (78.6%) positive flocks, 40 (57.1%) were infected by field strains, which were characterized as very virulent strains (genotype A3B2) and phylogenetically related to previously reported Algerian strains. Significant differences in the percentage of field infections were observed between vaccinated (25/52, 46.2%) and unvaccinated (14/17, 82.3%) groups, and also between birds immunized with live intermediate (13/20, 65.0%) and intermediate plus (10/28, 35.7%) vaccines. Nonetheless, the number of field strain detections suggests a high infectious pressure and the inadequacy of current vaccination efforts, demanding a reevaluation of control measures coupled with attentive monitoring activities.

Analysis of the Monophyletic Lineage of Avian Influenza H5N1 Which Circulated in Venezuelan Birds During the 2022–2023 Outbreak

Avian influenza subtype H5N1 has caused outbreaks worldwide since 1996, with the emergence of the Guandong lineage in China. The current clade 2.3.4.4b has evolved from this lineage, with increased virulence and mass mortality events in birds and mammals. The objective of this study was the analysis of 17 viral genomes of H5N1 avian influenza isolated in Venezuela during the 2022–2023 outbreak. The eight viral genomic segments were amplified using universal primers and sequenced via next-generation sequencing. The sequences were analyzed to confirm the H5 hemagglutinin clade, identify possible genetic reassortments, and perform a phylogenetic and docking analysis of the viral isolates. The viruses found in Venezuela belonged, as expected, to clade 2.3.4.4b and formed a monophyletic clade with North American influenza viruses, with no evidence of further reassortment. The introduction of the virus in South America is associated with bird migration through the Atlantic (Venezuela), Atlantic/Mississippi (Choco, Colombia), and Pacific migratory flyways, with the emergence of several viral lineages. Several mutations were found in all segments of the genome, although none of the key mutations was involved in mammalian adaptation. Moreover, in silico structural analysis suggests, as expected, that the viral hemagglutinin maintained a predilection for avian α2,3-linked sialic acid. The unprecedented pathogenic outbreak of avian influenza disease in South America was associated with the circulation of three different lineages, which maintain a lower affinity for the mammalian receptor.

Reconstructing the population history of theNicobarese.

The Nicobarese are the major tribal groups in the Nicobar district, situated south of the Andaman group of Islands. Linguistic phylogeny suggests that the linguistic ancestors of the Nicobarese settled the Nicobar archipelago in the early Holocene. So far, genetic research on them is low-resolution and restricted to the haploid DNA markers. Therefore, in the present analysis, we have used the high-resolution biparental (1554 published and 5 newly genotyped Nicobarese individuals) and uniparental genetic markers and looked at the genetic association of Nicobarese with the South and Southeast Asian populations. We report a common ancestral component shared among the Austroasiatic of South and Southeast Asia. Our analyses have suggested that the Nicobarese peoples retain this ancestral Austroasiatic predominant component in their genomes in the highest proportion. On the Southeast Asian mainland, the Htin Mal, who speak an Austroasiatic language of the Khmuic branch, represent a population that has preserved their ethnic distinctness from other groups over time and consequently shown the highest drift with the Nicobarese. The analysis based on haplotypes indicated a significant level of genomic segment sharing across linguistic groups, indicating an ancient broader distribution of Austroasiatic populations in Southeast Asia. Based on the temporal analyses of haploid DNA, it is suggested that the forebears of the Nicobarese people may have arrived on the Nicobar Islands in the last 5000 YBP. Therefore, among the modern populations, the Nicobarese peoples and the Htin Mal language community represent good genetic proxies for ancient Austroasiatics.

DeepEnhancerPPO: An Interpretable Deep Learning Approach for Enhancer Classification

Enhancers are short genomic segments located in non-coding regions of the genome that play a critical role in regulating the expression of target genes. Despite their importance in transcriptional regulation, effective methods for classifying enhancer categories and regulatory strengths remain limited. To address this challenge, we propose a novel end-to-end deep learning architecture named DeepEnhancerPPO. The model integrates ResNet and Transformer modules to extract local, hierarchical, and long-range contextual features. Following feature fusion, we employ Proximal Policy Optimization (PPO), a reinforcement learning technique, to reduce the dimensionality of the fused features, retaining the most relevant features for downstream classification tasks. We evaluate the performance of DeepEnhancerPPO from multiple perspectives, including ablation analysis, independent tests, assessment of PPO’s contribution to performance enhancement, and interpretability of the classification results. Each module positively contributes to the overall performance, with ResNet and PPO being the most significant contributors. Overall, DeepEnhancerPPO demonstrates superior performance on independent datasets compared to other models, outperforming the second-best model by 6.7% in accuracy for enhancer category classification. The model consistently ranks among the top five classifiers out of 25 for enhancer strength classification without requiring re-optimization of the hyperparameters and ranks as the second-best when the hyperparameters are refined. This indicates that the DeepEnhancerPPO framework is highly robust for enhancer classification. Additionally, the incorporation of PPO enhances the interpretability of the classification results.

Molecular characterization and comparison of tomato zonate spot virus isolated in Japan and China.

The complete genome sequence of Orthotospovirus tomatozonae (tomato zonate spot virus, TZSV) isolated in Japan was determined and compared with that of Chinese isolates. The lengths of the S, M, and L segments of the RNA genomes of the Japanese isolate (TZSV-TZ1-3) were 3194, 4675, and 8916 nucleotides, respectively, which were similar to the Chinese isolates. Moreover, the eight motifs on the RNA-dependent RNA polymerase (RdRp) gene were conserved in both TZSV-TZ1-3 and Chinese TZSV isolates (TZSV-Bidens and TZSV-Tomato-YN). The nucleotide identity of the genes among the TZSV isolates was more than 94%, indicating low diversity among viruses. The phylogenetic analysis and the prediction of the cleavage sites in the glycoprotein showed that the TZSV-TZ1-3 isolate was closely related to TZSV-Tomato-YN isolated from China. However, there were unique frameshifts and deletions on the RdRp and glycoprotein genes of the TZSV-Tomato-YN isolate, suggesting that both isolates were genetically distinct. The findings of this study indicate that the TZSV-TZ1-3 isolate originated in China and show the sequence diversity among TZSV isolates.

Accumulation of beneficial haplotypes in Huang-Huai-Hai wheat region and its application in molecular breeding

The Huang-Huai-Hai wheat region (HHHR) is characterized by the largest cultivation area and yield among all the major wheat-producing regions in China. Over the past 70 years, significant advances in wheat breeding have been achieved in this region, resulting in high and stable yields as well as improved disease resistance. However, there is a notable deficiency in the systematic molecular-level analyses of wheat breeding advantages in HHHR. To bridge this gap, we used a Wheat 55K SNP array to evaluate 384 accessions from a core collection of wheat germplasms across China to systematically analyze the distribution patterns of beneficial haplotypes associated with traits related to yield and powdery mildew resistance specific to HHHR. Our findings indicate that varieties from HHHR demonstrate significantly superior performance in terms of yield-related traits and powdery mildew resistance compared to those from other wheat regions. Using genome-wide association studies (GWAS) analysis, we identified the QTNs associated with both grain yield and powdery mildew resistance. Importantly, beneficial haplotypes were found at significantly higher frequencies in the HHHR than in other wheat-growing regions. Based on these haplotypes, the MFP-a gene was identified as potentially regulating jasmonic acid synthesis while also playing a role in grain development and conferring powdery mildew resistance. Furthermore, identity by descent (IBD) analysis revealed specific conserved genomic segments that have become fixed through selective breeding practices in HHHR, which may serve as invaluable resources for the targeted enhancement of yield and disease resistance traits in other wheat-growing areas. Finally, using the Aimengniu breeding lineage as a case study, we elucidated the genetic basis underlying the key founder parental formations utilized in breeding programs. This study not only provides essential references and guidance for future molecular breeding initiatives in China but also has implications for enhancing wheat production worldwide.

Mapping genetic susceptibility to spontaneous preterm birth: Analysis of Utah pedigrees to find inherited genetic factors.

Spontaneous preterm birth (SPTB) is the leading cause of neonatal morbidity and mortality. It is a final common pathway for multiple etiologies, some of which are well known while others likely remain to be identified. Despite recent advancements in identifying genetic risk factors, the mechanisms of many SPTBs remain poorly understood due to the phenotypic heterogeneity and complexity. Large family-based studies decrease heterogeneity and improve power to identify genetic causes of SPTB. To identify inherited genetic factors in SPTB etiology using large families. Using the Utah Population Database, which links a 4.5 million-person genealogy to state birth certificate and fetal death records, we identified large pedigrees containing multiple women with early SPTB (<34 weeks' gestation) and any SPTB (<37 weeks' gestation). We reviewed birth certificate data to exclude those with maternal and fetal diagnoses associated with iatrogenic preterm birth, resulting in 74 large multiplex pedigrees for early SPTB. Enrolled women with SPTB underwent comprehensive clinical phenotyping with review of primary medical records. Seven pedigrees, each containing at least 3 sampled women with SPTB, were the focus of this genetic study. High-density single-nucleotide polymorphism genotyping was conducted in maternal peripheral blood samples from women in the seven pedigrees. Shared genomic segments analysis was performed to identify genome-wide significant chromosomal regions shared in excess by women with SPTB. We identified two genome-wide significant chromosomal regions. In single-pedigree SGS analysis, a 1.75 Mega base region in chromosome 8 (8q24.23) was shared by 5 out of 6 women with SPTB in a single large pedigree (false positive rate=0.028). In duo-pedigree analysis, a 1.05 Mega base region in the same 8q24.23 locus was identified in a second pedigree (false-positive rate [duo]= 0.0019). The intersecting region at the 8q24.23 locus contains FAM135B (family with sequence similarity 135 member B) and KHDRBS3 (KH RNA binding domain containing, signal transduction associated 3) genes, which have previously been implicated in oncogenesis and ovarian cancer, respectively. Duo-pedigree SPTB analysis also identified a second genome-wide significant 67 kilo base locus in chromosome 12 (12q21.1-q21.2) that was shared by all women with SPTB in two independent pedigrees (false-positive rate [duo] =0.01). The intersecting region at the 12q21.1-q21.2 locus contains CAPS2 (calcyphosine 2) and KCNC2 (potassium voltage-gated channel subfamily C member 2) genes, both implicated in vascular-related complications of pregnancy and preterm labor. Using large SPTB families, we identified shared chromosomal regions (8q24.23 and 12q21.1-q21.2), providing evidence for inherited (segregating) risk loci in SPTB etiology. Further investigation into genes in SPTB etiology, including functional validation may provide avenues for novel therapeutic development and guide efforts for SPTB prevention to prolong pregnancy and improve outcomes.

Lymphocytic choriomeningitis arenavirus utilises intercellular connections for cell to cell spread

The Arenaviridae family of segmented RNA viruses contains nearly 70 species with several associated with fatal haemorrhagic fevers, including Lassa, Lujo and Junin viruses. Lymphocytic choriomeningitis arenavirus (LCMV) is associated with fatal neurologic disease in humans and additionally represents a tractable model for studying arenavirus biology. Within cultured cells, a high proportion of LCMV spread is between directly neighbouring cells, suggesting infectivity may pass through intercellular connections, bypassing the canonical extracellular route involving egress from the plasma membrane. Consistent with this, we visualized abundant actin- and tubulin-rich connections conjoining LCMV-infected and uninfected cells within cultures, resembling tunnelling nanotubes (TNTs). Within these TNT-like connections, confocal and STED microscopy identified puncta containing the major structural components of LCMV virions alongside genomic RNA, consistent with intercellular transit of assembled virions or ribonucleoprotein genome segments. Blocking the extracellular route of infection by adding potent LCMV neutralising antibody M28 to supernatants during infection revealed around 50% of LCMV transmission was via intercellular connections. These results show arenaviruses transmission is more complex than previously thought involving both extracellular and intercellular routes.

Influenza A virus transcription generates capped cRNAs that activate RIG-I.

During influenza A virus (IAV) infection, host pathogen receptor retinoic acid-inducible gene I (RIG-I) detects the partially complementary, 5'-triphosphorylated ends of the viral genome segments and non-canonical replication products. However, it has also been suggested that innate immune responses may be triggered by viral transcription. In this study, we investigated whether an immunostimulatory RNA is produced during IAV transcription. We show that the IAV RNA polymerase can read though the polyadenylation signal during transcription termination, generating a capped complementary RNA (ccRNA), which contains the 5' cap of an IAV mRNA and the 3' terminus of a cRNA instead of a poly(A) tail. ccRNAs are detectable in vitro and in both ribonucleoprotein reconstitution assays and IAV infections. Mutations that disrupt polyadenylation enhance ccRNA synthesis and increase RIG-I-dependent innate immune activation. Notably, while ccRNA itself is not immunostimulatory, it forms a RIG-I agonist by hybridizing with a complementary negative-sense viral RNA. These findings thus identify a novel non-canonical IAV RNA species and suggest an alternative mechanism for RIG-I activation during IAV infection.

EPCO-19. MULTIOMIC PROFILING OF EPIGENETIC HETEROGENEITY IN GLIOBLASTOMA AT SINGLE-CELL RESOLUTION USING MULTIOMIC SINGLE-CELL CUT&amp;TAG

Abstract INTRODUCTION Recent evidence suggests epigenetic heterogeneity may be the primary driver of cell phenotype and treatment resistance in glioblastoma (GBM). Epigenetic heterogeneity is defined by alterations of chromatin histone modifications with activating marks such as H3K27 acetylation and repressive marks such as H3K27 trimethylation. Combinations of these histone marks govern the activity of functional genomic elements such as enhancers or promoters. Single-cell (“sc-”) technologies have only recently been adapted to the study of epigenetic cell states and significant technical challenges have limited their widespread adoption, especially in complex tissues. Here, we report the development of an optimized method for performing tissue processing and simultaneous single-cell epigenetic and transcriptomic analyses in flash frozen GBM samples. METHODS Building on our prior work, we developed a new protocol, multiomic single-cell cleavage-under-targets and tagmentation, or “mscCUT&amp;TAG”. We optimized nuclei isolation, cleanup and permeabilization from flash frozen GBM and reaction conditions for the scCUT&amp;TAG portion of the assay. Critically, these changes maintained high-quality, intact nuclei in suspension while limiting well-described technical issues such as nuclei clumping and sample loss. Furthermore, we incorporated the ability to multiplex both patient samples and histone marks. RESULTS We applied 10X scRNA-seq, 10X Multiome (RNA+ATAC-seq), and mscCUT&amp;TAG for five histone marks to the same GBM sample, enabling an integrated evaluation of the tumor’s transcriptome, chromatin accessibility and epigenetic regulation at single-cell resolution. mscCUT&amp;TAG enabled the first assessment of single-cell genome segmentation in GBM. We integrated across modalities to identify specific tumor cell populations, cell-type specific coordinated changes in epigenetics and transcriptome, and define functional regulatory elements. CONCLUSIONS We developed a novel multiomic protocol, mscCUT&amp;TAG, that allows for a comprehensive analysis of epigenetic heterogeneity in GBM. We anticipate this protocol will enhance our understanding of fundamental biology and potential therapeutic avenues in GBM.

Molecular characterization of wheat dwarf virus isolates from Serbia based on complete genome sequences.

Wheat dwarf virus (WDV), the species Mastrevirus hordei of the genus Mastrevirus in the family Geminiviridae, is a cereal virus commonly detected in several European, African and Asian countries that causes economic losses. In the spring of 2019, a severe outbreak of wheat dwarfing and yellowing was observed in many winter wheat crops across Serbia. A total of 161 samples were tested for the presence of WDV and other common wheat viruses using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). To obtain the complete genome of 23 selected isolates, several overlapping segments of the WDV genome were amplified and sequenced. A phylogenetic tree was constructed using the whole genome sequences of the WDV isolates identified in this study and 40 selected sequences from GenBank. The results of DAS-ELISA indicated the presence of WDV in all samples collected from 21 sites in all nine districts surveyed. Further molecular characterization based on complete genome sequencing of 23 selected isolates showed that the Serbian WDV isolates had low nucleotide diversity and were closely related to wheat-infecting isolates from Europe, suggesting the presence of wheat-adapted forms of WDV in Serbia. The constructed phylogenetic tree revealed that Serbian isolates grouped in clade E within the wheat-adapted forms. This study provided the first insight into the genetic structure of WDV in Serbia based on its whole genome sequence. Further studies on the vector biology and population dynamics are needed to better understand the factors influencing the emergence and spread of WDV under local agroecological conditions.

KLF13 promotes SLE pathogenesis by modifying chromatin accessibility of key proinflammatory cytokine genes

Although significant progress has been achieved in elucidating the genetic architecture of systemic lupus erythematosus (SLE), identifying genes underlying the pathogenesis has been challenging. The NZM2410-derived lupus susceptibility Sle3 locus is associated with T cell hyperactivity and activated myeloid cells. However, candidate genes associated with these phenotypes have not been identified. Here, we narrow the Sle3 locus to a smaller genomic segment (Sle3k) and show that mice carrying Sle3k and Sle1 loci developed lupus nephritis. We identify Klf13 as the primary candidate gene that is associated with genome-wide transcription changes resulting in higher levels of proinflammatory cytokines, enhanced T cell activation, and hyperresponsiveness of myeloid cells. Correspondingly, Klf13–/– mice display repression of genes involved in mediating immune activation, including key proinflammatory cytokines/chemokines in T cells and dysregulation in cytokine signaling pathways in myeloid cells in response to toll receptor ligands. Klf13 upregulation is associated with increased production of RANTES, a key chemokine in lupus nephritis, in activated T cells and the kidneys of lupus-prone mice. In sum, our findings reveal Klf13 as a key gene in the Sle3 interval in mediating lupus pathogenesis that may have implications in the rational design of new therapies for SLE.

Dominant HPAIV H5N1 genotypes of Germany 2021/2022 are linked to high virulence in Pekin ducklings

Highly pathogenic avian influenza viruses (HPAIV) of H5 clade 2.3.4.4b pose an ongoing threat worldwide. It remains unclear whether this panzootic situation would favor low virulent phenotypes expected by the ‘avirulence hypothesis’ of viral evolution. Assessing virulence in Pekin ducklings in an intramuscular infection model revealed that the two genotypes that dominated the epidemiological situation in Germany during the period 2021 and 2022 (EU-RL:CH and EU-RL:AB) were of high virulence. In contrast, rare genotypes were of intermediate virulence. The genetic constellation of these reassortants pointed to an important role of the viral polymerase complex (RdRP), particularly the PB1 genome segment, in shaping virulence in ducklings. Occulo-nasal infection of ducklings confirmed the phenotypes for two representative viruses and indicated a more efficient replication for the high virulence strain. These observations would be in line with the ‘virulence-transmission trade-off’ model for describing HPAIV epidemiology in wild birds in Germany.

A history of multiple Denisovan introgression events in modern humans.

The identification of a new hominin group in the Altai mountains called Denisovans was one of the most exciting discoveries in human evolution in the last decade. Unlike Neanderthal remains, the Denisovan fossil record consists of only a finger bone, jawbone, teeth and skull fragments. Leveraging the surviving Denisovan segments in modern human genomes has uncovered evidence of at least three introgression events from distinct Denisovan populations into modern humans in the past. Each of them presents different levels of relatedness to the sequenced Altai Denisovan, indicating a complex relationship between these sister lineages. Here we review the evidence suggesting that several Denisovan populations, who likely had an extensive geographical range, were adapted to distinct environments and introgressed into modern humans multiple times. We further discuss how archaic variants have been affected by demographic history, negative and positive selection and close by proposing possible new lines of future research.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

Characterization of bi-segmented and tri-segmented recombinant Pichinde virus particles.

Mammarenaviruses include several highly virulent pathogens (e.g., Lassa virus) capable of causing severe hemorrhagic fever diseases for which there are no approved vaccines and limited treatment options. Mammarenaviruses are enveloped, bi-segmented ambisense RNA viruses. There is limited knowledge about cellular proteins incorporated into progeny virion particles and their potential biological roles in viral infection. Pichinde virus (PICV) is a prototypic arenavirus used to characterize mammarenavirus replication and pathogenesis. We have developed a recombinant PICV with a tri-segmented RNA genome as a viral vector platform. Whether the tri-segmented virion differs from the wild-type bi-segmented one in viral particle morphology and protein composition has not been addressed. In this study, recombinant PICV (rPICV) virions with a bi-segmented (rP18bi) and a tri-segmented (rP18tri) genome were purified by density-gradient ultracentrifugation and analyzed by cryo-electron microscopy and mass spectrometry. Both virion types are pleomorphic with spherical morphology and have no significant difference in size despite rP18tri having denser particles. Both virion types also contain similar sets of cellular proteins. Among the highly enriched virion-associated cellular proteins are components of the endosomal sorting complex required for transport pathway and vesicle trafficking, such as ALIX, Tsg101, VPS, CHMP, and Ras-associated binding proteins, which have known functions in virus assembly and budding. Other enriched cellular proteins include peripheral and transmembrane proteins, chaperone proteins, and ribosomal proteins; their biological roles in viral infection warrant further analysis. Our study provides important insights into mammarenavirus particle formation and aids in the future development of viral vectors and antiviral discovery.IMPORTANCEMammarenaviruses, such as Lassa virus, are enveloped RNA viruses that can cause severe hemorrhagic fever diseases (Lassa fever) with no approved vaccine and limited therapeutic options. Cellular proteins incorporated into progeny virion particles and their biological roles in mammarenavirus infection have not been well characterized. Pichinde virus (PICV) is a prototypic mammarenavirus used as a surrogate model for Lassa fever. We used cryo-electron microscopy and proteomic analysis to characterize the morphology and protein contents of the purified PICV particles that package either two (bi-segmented) or three (tri-segmented) genomic RNA segments. Our results demonstrate a similar virion morphology but different particle density for the bi- and tri-segmented viral particles and reveal major virion-associated cellular proteins. This study provides important insights into the virus-host interactions that can be used for antiviral development and optimizing arenavirus-based vaccine vectors.

Lytic promoter activity during herpes simplex virus latency is dependent on genome location.

Herpes simplex virus 1 (HSV-1) is a significant pathogen that establishes lifelong latent infections with intermittent episodes of resumed disease. In mouse models of HSV infection, sporadic low-level lytic gene expression has been detected during latency in the absence of reactivation events that lead to production of new viruses. This viral activity during latency has been reported using a sensitive Cre-marking model for several lytic gene promoters placed in one location in the HSV-1 genome. Here, we extend these findings in the same model by examining first, the activity of an ectopic lytic gene promoter in several places in the genome and second, whether any promoters might be active in their natural context. We found that Cre expression was detected during latency from ectopic and native promoters, but only in locations near the ends of the unique long genome segment. This location is significant because it is in close proximity to the region from which latency-associated transcripts (LATs) are derived. These results show that native HSV-1 lytic gene promoters can produce protein products during latency, but that this activity is only detectable when they are located close to the LAT locus.IMPORTANCEHSV is a significant human pathogen and the best studied model of mammalian virus latency. Traditionally, the active (lytic) and inactive (latent) phases of infection were considered to be distinct, but the notion of latency being entirely quiescent is evolving due to the detection of some lytic gene expression during latency. Here, we add to this literature by finding that the activity can be found for native lytic gene promoters as well as for constructs placed ectopically in the HSV genome. However, this activity was only detectable when these promoters were located close by a region known to be transcriptionally active during latency. These data have implications for our understanding of HSV gene regulation during latency and the extent to which transcriptionally active regions are insulated from adjacent parts of the viral genome.

Metagenomic discovery of microbial eukaryotes in stool microbiomes.

Host-associated microbiota form complex microbial communities that are increasingly associated with host behavior and disease. While these microbes include bacterial, archaeal, viral, and eukaryotic constituents, most studies have focused on bacteria due to their dominance in the human host and available tools for investigation. Accumulating evidence suggests microbial eukaryotes in the microbiome play pivotal roles in host health, but our understandings of these interactions are limited to a few readily identifiable taxa because of technical limitations in unbiased eukaryote exploration. Here, we combined cell sorting, optimized eukaryotic cell lysis, and shotgun sequencing to accelerate metagenomic discovery and analysis of host-associated microbial eukaryotes. Using synthetic communities with a 1% microbial eukaryote representation, the eukaryote-optimized cell lysis and DNA recovery method alone yielded a 38-fold increase in eukaryotic DNA. Automated sorting of eukaryotic cells from stool samples of healthy adults increased the number of microbial eukaryote reads in metagenomic pools by up to 28-fold compared to commercial kits. Read frequencies for identified fungi increased by 10,000× on average compared to the Human Microbiome Project and allowed for the identification of novel taxa, de novo assembly of contigs from previously unknown microbial eukaryotes, and gene prediction from recovered genomic segments. These advances pave the way for the unbiased inclusion of microbial eukaryotes in deciphering determinants of health and disease in the host-associated microbiome.IMPORTANCEMicrobial eukaryotes are common constituents of the human gut where they can contribute to local ecology and host health, but they are often overlooked in microbiome studies. The lack of attention is due to current technical limitations that are heavily biased or poorly recovered DNA from microbial eukaryotes. We developed a method to increase the representation of these eukaryotes in metagenomic sequencing of microbiome samples that allows to improve their detection compared to prior methods and allows for the identification of new species. Application of the technique to gut microbiome samples improved detection of fungi, protists, and helminths. New eukaryotic taxa and their encoded genes could be identified by sequencing a small number of samples. This approach can improve the inclusion of eukaryotes into microbiome research.

Influenza Virus Types, Subtypes and Genomic Lineage with Its Prevention and Control: A Narrative Review

Since influenza viruses continue to be a serious hazard to both humans and animals, they are still very important. Types A, B, and C influenza viruses are among the members of the Orthomyxoviridae family. A quick summary of the molecular factors is provided, which determine pathogenicity and clinical signs and symptoms of influenza. A review of host range and evolution highlights the genetic diversity of influenza A viruses and their capacity to successfully infect a variety of hosts, including avian and mammalian species. Moreover, influenza viruses may reassemble segments because of the way their segmented genome is designed. The significance of host sialic acid distribution and viral receptor-binding hemagglutinins in species-restricted virus binding is emphasized. It results in yearly outbreaks and necessitates the creation of novel vaccination formulations. This may ultimately result in the creation of a virus that can spread among humans and has unique antigenic qualities, perhaps sparking a pandemic. Current developments in our knowledge of the seasonality, transmission, and prevention of influenza viruses are outlined, along with their significance for halting the virus's spread. Journal of Monno Medical College June, 2024; 10 (1):43-51

Long range segmentation of prokaryotic genomes by gene age and functionality.

Bacterial and archaeal genomes encompass numerous operons that typically consist of two to five genes. On larger scales, however, gene order is poorly conserved through the evolution of prokaryotes. Nevertheless, non-random localization of different classes of genes on prokaryotic chromosomes could reflect important functional and evolutionary constraints. We explored the patterns of genomic localization of evolutionarily conserved (ancient) and variable (young) genes across the diversity of bacteria and archaea. Nearly all bacterial and archaeal chromosomes were found to encompass large segments of 100-300 kb that were significantly enriched in either ancient or young genes. Similar clustering of genes with lethal knockout phenotype (essential genes) was observed as well. Mathematical modeling of genome evolution suggests that this long-range gene clustering in prokaryotic chromosomes reflects perpetual genome rearrangement driven by a combination of selective and neutral processes rather than evolutionary conservation.