miR-430 microRNA Family in Fishes: Molecular Characterization and Evolution.

The miR-430 microRNA family has been described in multiple fish species as one of the first microRNAs expressed by the zygote. It has been suggested that this family is implicated in maternal mRNA elimination, but may also play a role in steroidogenesis, sexual differentiation, and flatfish metamorphosis. The miR-430 sequences have been found in multiple-copy tandem clusters but evidence of their conservation outside of teleost fishes is scarce. In the present study, we have characterized the tandem repeats organization of these microRNAs in different fish species, both model and of interest in aquaculture. A phylogenetic analysis of this family has allowed us to identify that the miR-430 duplication, which took place before the Chondrostei and Neopterygii groups' divergence, has resulted in three variants ("a", "b", and "c"). According to our data, variant "b" is the most closely related to the ancestral sequence. Furthermore, we have detected isolated instances of the miR-430 repeat subunit in some species, which suggests that this microRNA family may be affected by DNA rearrangements. This study provides new data about the abundance, variability, and organization of the miR-430 family in fishes.

Genome wide identification and characterization of fertility associated novel CircRNAs as ceRNA reveal their regulatory roles in sheep fecundity.

Reproductive traits play a vital role in determining the production efficiency of sheep. Maximizing the production is of paramount importance for breeders worldwide due to the growing population. Circular RNAs (circRNAs) act as miRNA sponges by absorbing miRNA activity through miRNA response elements (MREs) and participate in ceRNA regulatory networks (ceRNETs) to regulate mRNA expression. Despite of extensive research on role of circRNAs as miRNA sponges in various species, their specific regulatory roles and mechanism in sheep ovarian tissue are still not well understood. In this study, we performed whole genome sequencing of circRNAs, miRNA and mRNA employing bioinformatic techniques on ovine tissues of two contrasting sheep breeds "Small tail Han (X_LC) and Dolang sheep (D_LC)", which results into identification of 9,878 circRNAs with a total length of 23,522,667 nt and an average length of 2,381.32 nt. Among them, 44 differentially expressed circRNAs (DECs) were identified. Moreover, correlation between miRNA-mRNA and lncRNA-miRNA provided us with to prediction of miRNA binding sites on nine differentially expressed circRNAs and 165 differentially expressed mRNAs using miRanda. miRNA-mRNA and lncRNA-miRNA pairs with negative correlation were selected to determine the ceRNA score along with positively correlated pairs from lncRNA and mRNA network. Integration of ceRNA score and positively correlated pairs exhibit a significant ternary relationship among circRNAs-miRNA-mRNA demonestrated by ceRNA, comprising of 50 regulatory pairs sharring common nodes and predicted potential differentially expressed circRNAs-miRNAs-mRNAs regulatory axis. Based on functional enrichment analysis shortlisted key ceRNA regulatory pairs associated with reproduction including circRNA_3257-novel579_mature-EPHA3, circRNA_8396-novel130_mature-LOC101102473, circRNA_4140- novel34_mature > novel661_mature-KCNK9, and circRNA_8312-novel339_mature-LOC101110545. Furthermore, expression profiling, functional enrichments and qRT-PCR analysis of key target genes infer their implication in reproduction and metabolism. ceRNA target mRNAs evolutionary trajectories, expression profiling, functional enrichments, subcellular localizations following genomic organizations will provide new insights underlying molecular mechanisms of reproduction, and establish a solid foundation for future research. Graphical abstract summarizing the scheme of study.

Clues on the dynamics of DNA replication in Giardia lamblia.

Genomic replication is a critical, regulated process that ensures accurate genetic information duplication. In eukaryotic cells, strategies have evolved to prevent conflicts between replication and transcription. Giardia lamblia, a binucleated protozoan, alternates between tetraploid and octaploid genomes during its cell cycle. Using single-molecule techniques like DNA combing and nanopore-based sequencing, we investigated the spatio-temporal organization of DNA replication, replication fork progression and potential head-on replication-transcription collisions in Giardia trophozoites. Our findings indicate that Giardia chromosomes are replicated from only a few active origins, which are widely spaced and exhibit faster replication rates compared to those in other protozoan parasites. Immunofluorescence assays revealed that ∼20% of trophozoites show asynchronous replication between nuclei. Forksense and gene ontology analyses disclosed that genes in regions with potential head-on collisions are linked to chromatin dynamics, cell cycle regulation and DNA replication/repair pathways, possibly explaining the observed asynchronous replication in part of the population. This study offers the first comprehensive view of replication dynamics in Giardia, which is the pathogen that causes giardiasis, a diarrheal disease impacting millions worldwide.

Genome-wide characterization of the cytosolic sulfotransferase 1B member 1 (SULT1B1) family and its expression responses to sulfide stress in the razor clam Sinonovacula constricta.

The razor clam (Sinonovacula constricta), a typical burrowing organism in the intertidal zones, is often exposed to sulfide environment and shows strong sulfide tolerance. Located downstream of the sulfur metabolism pathway, cytosolic sulfotransferase family 1B member 1 (SULT1B1) is a key enzyme catalysing the sulfonation reaction, and plays an important role in the biotransformation of endogenous substances such as thyroid hormones (THs). To investigate their roles in sulfide resistance, a systematic analysis of S. constricta SULT1B1s (ScSULT1B1s), including genomic distribution, phylogenetic relationships, gene structure, conserved motifs, and expression profiles under sulfide stress, was performed. A total of 10 ScSULT1B1 genes were found in the S. constricta genome. Sequence analysis showed that ScSULT1B1 gene family encoded 155-425 amino acids, containing four catalytic active sites (K, N, H, and S), one PAPS binding domain at the N-terminus, and one PAPS binding and dimerization domain at the C-terminus. The spatial-temporal expression patterns of ScSULT1B1s were further estimated by quantitative real-time PCR (qRT-PCR). Among them, partial ScSULT1B1s showed significantly high expression in the gill, hepatopancreas, and siphon. Furthermore, the response expression of certain ScSULT1B1s significantly fluctuated under sulfide stress. Together, our results suggest that ScSULT1B1s, by mediating the sulfonation reaction, may regulate THs levels to maintain basic metabolic and immune functions, making S. constricta highly sulfide tolerant.

Clues on the dynamics of DNA replication in Giardia lamblia Icon for the Forest of Biologists

Genomic replication is a critical, regulated process that ensures accurate genetic information duplication. In eukaryotic cells, strategies have evolved to prevent conflicts between replication and transcription. Giardia lamblia, a binucleated protozoan, alternates between tetraploid and octoploid genomes during its cell cycle. Using single-molecule techniques like DNA combing and nanopore-based sequencing, we investigated the spatio-temporal organization of DNA replication, replication fork progression, and potential head-on replication-transcription collisions in Giardia trophozoites. Our findings indicate that Giardia chromosomes are replicated from few active origins, which are widely spaced and exhibit faster replication rates compared to other protozoan parasites. Immunofluorescence assays revealed that around 20% of trophozoites show asynchronous replication between nuclei. Forksense and gene ontology analyses disclosed that genes in regions with potential head-on collisions are linked to chromatin dynamics, cell cycle regulation, and DNA replication/repair pathways, possibly explaining the observed asynchronous replication in part of the population. This study offers the first comprehensive view of replication dynamics in Giardia, the cause of giardiasis, a diarrheal disease impacting millions worldwide.

Echiuran Hox genes provide new insights into the correspondence between Hox subcluster organization and collinearity pattern.

In many bilaterians, Hox genes are generally clustered along the chromosomes and expressed in spatial and temporal order. In vertebrates, the expression of Hox genes follows a whole-cluster spatio-temporal collinearity (WSTC) pattern, whereas in some invertebrates the expression of Hox genes exhibits a subcluster-level spatio-temporal collinearity pattern. In bilaterians, the diversity of collinearity patterns and the cause of collinearity differences in Hox gene expression remain poorly understood. Here, we investigate genomic organization and expression pattern of Hox genes in the echiuran worm Urechis unicinctus (Annelida, Echiura). Urechis unicinctus has a split cluster with four subclusters divided by non-Hox genes: first subcluster (Hox1 and Hox2), second subcluster (Hox3), third subcluster (Hox4, Hox5, Lox5, Antp and Lox4), fourth subcluster (Lox2 and Post2). The expression of U. unicinctus Hox genes shows a subcluster-based whole-cluster spatio-temporal collinearity (S-WSTC) pattern: the anterior-most genes in each subcluster are activated in a spatially and temporally colinear manner (reminiscent of WSTC), with the subsequent genes in each subcluster then being very similar to their respective anterior-most subcluster gene. Combining genomic organization and expression profiles of Hox genes in different invertebrate lineages, we propose that the spatio-temporal collinearity of invertebrate Hox is subcluster-based.

Convergent gene clusters underpin hyperforin biosynthesis in St John's wort.

The meroterpenoid hyperforin is responsible for the antidepressant activity of St John's wort extracts, but the genes controlling its biosynthesis are unknown. Using genome mining and biochemical work, we characterize two biosynthetic gene clusters (BGCs) that encode the first three steps in the biosynthesis of hyperforin precursors. The findings of syntenic and phylogenetic analyses reveal the parallel assembly of the two BGCs. The syntenous BGC in Mesua ferrea indicates that the first cluster was assembled before the divergence of the Hypericaceae and Calophyllaceae families. The assembly of the second cluster is the result of a coalescence of genomic fragments after a major duplication event. The differences between the two BGCs - in terms of gene expression, response to methyl jasmonate, substrate specificity and subcellular localization of key enzymes - suggest that the presence of the two clusters could serve to generate separate pools of precursors. The parallel assembly of two BGCs with similar compositions in a single plant species is uncommon, and our work provides insights into how and when these gene clusters form. Our discovery helps to advance our understanding of the evolution of plant specialized metabolism and its genomic organization. Additionally, our results offer a foundation from which hyperforin biosynthesis can be more fully understood, and which can be used in future metabolic engineering applications.

Molecular characterization of matrix metalloproteinase gene family across primates.

Deregulation of matrix metalloproteinases (MMPs) contributes considerably to cancers, psychiatric disorders, macular degeneration and bone diseases. The use of humans in the development of MMPs as prognostic biomarkers and therapeutic targets is complicated by many factors, while primate models can be useful alternatives for this purpose. Here, we performed genome-enabled identification of putative MMPs across primate species, and comprehensively investigated the genes. Phylogenetic topology of the MMP family showed each type formulates a distinct clade, and was further clustered to classes, largely agreeing with classification based on biochemical properties and domain organization. Across primates, the excess of candidate sites of positive selection was detected for MMP-19, in addition to 1-3 sites in MMP-8, MMP-10 and MMP-26. MMP-26 showed Ka/Ks value above 1 between human and chimpanzee copies. We observed two copies of MMP-19 in the old-world monkey genomes, suggesting gene duplication at the early stage of or prior to the emergence of the lineage. Furin-activatable MMPs demonstrate the most variable properties regarding Domain organization and gene structure. During human aging, MMP-11 showed gradually decreased expression in testis, so as MMP-2, MMP-14, MMP15 and MMP-28 in ovary, while MMP-7 and MMP-21 showed elevated expression, implying their distinct roles in different reproductive organs. Co-expression clusters were formed among human MMPs both within and across classes, and expression correlation was observed in MMP genes across primates. Our results illuminate the utilization of MMPs for the discovery of prognostic biomarkers and therapeutic targets for aging-related diseases and carry new messages on MMP classification.

Monoterpene Synthase Genes and Monoterpene Profiles in Pinus nigra subsp. laricio.

In the present study, we carried out a quantitative analysis of the monoterpenes composition in different tissues of the non-model conifer Pinus nigra J.F. Arnold subsp. laricio Palib. ex Maire (P. laricio, in short). All the P. laricio tissues examined showed the presence of the same fourteen monoterpenes, among which the most abundant were ß-phellandrene, α-pinene, and ß-pinene, whose distribution was markedly tissue-specific. In parallel, from the same plant tissues, we isolated seven full-length cDNA transcripts coding for as many monoterpene synthases, each of which was found to be attributable to one of the seven phylogenetic groups in which the d1-clade of the canonical classification of plants' terpene synthases can be subdivided. The amino acid sequences deduced from the above cDNA transcripts allowed to predict their putative involvement in the biosynthesis of five of the monoterpenes identified. Transcripts profiling revealed a differential gene expression across the different tissues examined, and was found to be consistent with the corresponding metabolites profiles. The genomic organization of the seven isolated monoterpene synthase genes was also determined.

Pathogenicity and virulence of Hepatitis B virus.

Hepatitis B virus (HBV) is a hepatotropic virus and an important human pathogen. There are an estimated 296 million people in the world that are chronically infected by this virus, and many of them will develop severe liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HBV is a small DNA virus that replicates via the reverse transcription pathway. In this review, we summarize the molecular pathways that govern the replication of HBV and its interactions with host cells. We also discuss viral and non-viral factors that are associated with HBV-induced carcinogenesis and pathogenesis, as well as the role of host immune responses in HBV persistence and liver pathogenesis.

Genomic Structure of Two Kv1.3 Channel Blockers from Scorpion Mesobuthus eupeus and Sea Anemone Stichodactyla haddoni and Construction of their Chimeric Peptide as a Novel Blocker.

Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla haddoni sea anemone have been identified as two effective Kv1.3 channel blockers. In this work, we characterized the genomic organization of both toxins. MeuKTX gene contains one intron and two exons, similar to the most scorpion toxins. There are a few reports of genomic structure of sea anemone toxins acting on Kv channels. The sequence encoding mature peptide of shtx-k was located in an exon separated by an intron from the coding exon of the propeptide and signal region. In order to make a peptide with more affinity for Kv1.3 channel and greater stability, the shtx-k/ MeuKTX chimeric peptide was designed and constructed using splicing by overlap extension-PCR (SOE-PCR) method. MeuKTX, shtx-k, and shtx-k/MeuKTX were cloned and the expression of the soluble proteins in E. coli was determined. Molecular docking studies indicated more inhibitory effect of shtx-k/MeuKTX on Kv1.3 channel compared to shtx-k and MeuKTX toxins. Key amino acids binding channel from both toxins, also involved in interaction of chimeric peptide with channel. Our results showed that the fusion peptide, shtx-k/MeuKTX could be an effective agent to target Kv1.3 channel.

Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes.

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants' terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Genomic organization and expression pattern of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata.

As one of the dominant natural enemies for insect pests, the pond wolf spider, Pardosa pseudoannulata, plays important roles in pest control. Insecticide applications threaten P. pseudoannulata and consequently weaken its control effects. The roles of P450 monooxygenases in insecticide detoxifications have been richly reported in insects, but there are few reported in spiders. In this study, 120 transcripts encoding P. pseudoannulata P450s were identified based on whole genome sequencing. Compared to P450s of Aedes aegypti and Nilaparvata lugens, several novel P450 families were found, such as CYP3310. KEGG analysis of the CYP3310 family indicated that the family might be involved in the synthesis and metabolism of polyunsaturated fatty acids and hydrocarbons. The potential P450s involved in insecticide metabolism were obtained according to the high FPKM values in fat bodies based on transcriptome sequencing. However, none of the selected P450 genes was significantly upregulated by the treatments of deltamethrin or imidacloprid. The present study provides genomic and transcriptomic information of spider P450s, especially for their roles in the synthesis and metabolism of endogenous and exogenous compounds, such as polyunsaturated fatty acids, hydrocarbons and insecticides.

Structural and molecular biology of hepatitis E virus.

Hepatitis E virus (HEV) is one of the most common causes of acute viral hepatitis, mainly transmitted by fecal-oral route but has also been linked to fulminant hepatic failure, chronic hepatitis, and extrahepatic neurological and renal diseases. HEV is an emerging zoonotic pathogen with a broad host range, and strains of HEV from numerous animal species are known to cross species barriers and infect humans. HEV is a single-stranded, positive-sense RNA virus in the family Hepeviridae. The genome typically contains three open reading frames (ORFs): ORF1 encodes a nonstructural polyprotein for virus replication and transcription, ORF2 encodes the capsid protein that elicits neutralizing antibodies, and ORF3, which partially overlaps ORF2, encodes a multifunctional protein involved in virion morphogenesis and pathogenesis. HEV virions are non-enveloped spherical particles in feces but exist as quasi-enveloped particles in circulating blood. Two types of HEV virus-like particles (VLPs), small T = 1 (270 Å) and native virion-sized T = 3 (320-340 Å) have been reported. There exist two distinct forms of capsid protein, the secreted form (ORF2S) inhibits antibody neutralization, whereas the capsid-associated form (ORF2C) self-assembles to VLPs. Four cis-reactive elements (CREs) containing stem-loops from secondary RNA structures have been identified in the non-coding regions and are critical for virus replication. This mini-review discusses the current knowledge and gaps regarding the structural and molecular biology of HEV with emphasis on the virion structure, genomic organization, secondary RNA structures, viral proteins and their functions, and life cycle of HEV.

Sequence Analysis and Ontogenetic Expression Patterns of Cone Opsin Genes in the Bluefin Killifish (Lucania goodei).

Sensory systems allow for the transfer of environmental stimuli into internal cues that can alter physiology and behavior. Many studies of visual systems focus on opsins to compare spectral sensitivity among individuals, populations, and species living in different lighting environments. This requires an understanding of the cone opsins, which can be numerous. The bluefin killifish is a good model for studying the interaction between environments and visual systems as they are found in both clear springs and tannin-stained swamps. We conducted a genome-wide screening and demonstrated that the bluefin killifish has 9 cone opsins: 1 SWS1 (354 nm), 2 SWS2 (SWS2B: 359 nm, SWS2A: 448 nm), 2 RH2 (RH2-2: 476 nm, RH2-1: 537 nm), and 4 LWS (LWS-1: 569 nm, LWS-2: 524 nm, LWS-3: 569 nm, LWS-R: 560 or 569 nm). These 9 cone opsins were located on 4 scaffolds. One scaffold contained the 2 SWS2 and 3 of the 4 LWS opsins in the same syntenic order as found in other cyprinodontoid fishes. We also compared opsin expression in larval and adult killifish under clear water conditions, which mimic springs. Two of the newly discovered opsins (LWS-2 and LWS-3) were expressed at low levels (<0.2%). Whether these opsins make meaningful contributions to visual perception in other contexts (i.e., swamp conditions) is unclear. In contrast, there was an ontogenetic change from using LWS-R to LWS-1 opsin. Bluefin killifish adults may be slightly more sensitive to longer wavelengths, which might be related to sexual selection and/or foraging preferences.

Systematic Analysis of Functionally Related Gene Clusters in the Opportunistic Pathogen, Candida albicans.

The proper balance of gene expression is essential for cellular health, organismal development, and maintaining homeostasis. In response to complex internal and external signals, the cell needs to modulate gene expression to maintain proteostasis and establish cellular identity within its niche. On a genome level, single-celled prokaryotic microbes display clustering of co-expressed genes that are regulated as a polycistronic RNA. This phenomenon is largely absent from eukaryotic microbes, although there is extensive clustering of co-expressed genes as functional pairs spread throughout the genome in Saccharomyces cerevisiae. While initial analysis demonstrated conservation of clustering in divergent fungal lineages, a comprehensive analysis has yet to be performed. Here we report on the prevalence, conservation, and significance of the functional clustering of co-regulated genes within the opportunistic human pathogen, Candida albicans. Our analysis reveals that there is extensive clustering within this organism-although the identity of the gene pairs is unique compared with those found in S. cerevisiae-indicating that this genomic arrangement evolved after these microbes diverged evolutionarily, rather than being the result of an ancestral arrangement. We report a clustered arrangement in gene families that participate in diverse molecular functions and are not the result of a divergent orientation with a shared promoter. This arrangement coordinates the transcription of the clustered genes to their neighboring genes, with the clusters congregating to genomic loci that are conducive to transcriptional regulation at a distance.

Recent genome duplications facilitate the phenotypic diversity of Hb repertoire in the Cyprinidae.

Whole-genome duplications (WGDs) are an important contributor to phenotypic innovations in evolutionary history. The diversity of blood oxygen transport traits is the perfect reflection of physiological versatility for evolutionary success among vertebrates. In this study, the evolutionary changes of hemoglobin (Hb) repertoire driven by the recent genome duplications were detected in representative Cyprinidae fish, including eight diploid and four tetraploid species. Comparative genomic analysis revealed a substantial variation in both membership composition and intragenomic organization of Hb genes in these species. Phylogenetic reconstruction analyses were conducted to characterize the evolutionary history of these genes. Data were integrated with the expression profiles of the genes during ontogeny. Our results indicated that genome duplications facilitated the phenotypic diversity of the Hb gene family; each was associated with species-specific changes in gene content via gene loss and fusion after genome duplications. This led to repeated evolutionary transitions in the ontogenic regulation of Hb gene expression. Our results revealed that genome duplications helped to generate phenotypic changes in Cyprinidae Hb systems.

The macro and micro of chromosome conformation capture.

The 3D organization of the genome facilitates gene regulation, replication, and repair, making it a key feature of genomic function and one that remains to be properly understood. Over the past two decades, a variety of chromosome conformation capture (3C) methods have delineated genome folding from megabase-scale compartments and topologically associating domains (TADs) down to kilobase-scale enhancer-promoter interactions. Understanding the functional role of each layer of genome organization is a gateway to understanding cell state, development, and disease. Here, we discuss the evolution of 3C-based technologies for mapping 3D genome organization. We focus on genomics methods and provide a historical account of the development from 3C to Hi-C. We also discuss ChIP-based techniques that focus on 3D genome organization mediated by specific proteins, capture-based methods that focus on particular regions or regulatory elements, 3C-orthogonal methods that do not rely on restriction digestion and proximity ligation, and methods for mapping the DNA-RNA and RNA-RNA interactomes. We consider the biological discoveries that have come from these methods, examine the mechanistic contributions of CTCF, cohesin, and loop extrusion to genomic folding, and detail the 3D genome field's current understanding of nuclear architecture. Finally, we give special consideration to Micro-C as an emerging frontier in chromosome conformation capture and discuss recent Micro-C findings uncovering fine-scale chromatin organization in unprecedented detail. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics.

Dysregulation of HOX as a Potential Therapeutic Target in Breast Cancer.

HOX genes belong to the highly conserved homeobox superfamily, responsible for the regulation of various cellular processes that control cell homeostasis, from embryogenesis to carcinogenesis. The abnormal expression of HOX genes is observed in various cancers, including breast cancer, where they act as oncogenes or as suppressors of cancer, according to context. In this review, we analyze HOX gene expression patterns in breast cancer and examine their relationship, based on the three-dimensional genome structure of the HOX locus. The presence of non-coding RNAs embedded within the HOX cluster and the role of these molecules in breast cancer, have been reviewed. We further evaluate the characteristic activity of HOX protein in breast cancer and its therapeutic potential.

Novel enteric viruses in fatal enteritis of grey squirrels.

Astro- and kobuviruses infect both humans and animals. Here, we report on the disease history, detection and genomic characterization of novel astro- and kobuviruses from fatal diarrhoea of two juvenile grey squirrels. The virus particles had enterovirus-like morphology and a diameter of 28-32 nm. Next-generation sequencing confirmed astro- and kobuviruses and sequence analysis revealed typical astrovirus and picornavirus genome organizations. The astrovirus ORF2 sequence clustered with a clade of unassigned astroviruses, with marmot and rodent mamastroviruses as closest relatives. For the kobuvirus, divergences greater than 49.4 % for P1 and 43.5 % in the non-structural proteins indicated a novel species. However, phylogenetic analysis of the 3D polymerase showed that it clustered with that of the newly classified ludopivirus A1, suggesting a previous recombination event in the evolution of the kobuvirus. Our data provide further insights into the diversity of astro- and kobuviruses and broaden the spectrum of viruses infecting grey squirrels.