Biology subjects

van Leeuwen, S.

Publications and source records attributed to van Leeuwen, S..

12 recordsLinked to original sources

bioRxiv2026

Long-term experimental evolution of seasonal influenza A/H3N2 virus reveals diverse evolutionary potential

The repeatability of evolutionary outcomes is a fundamental question with implications for predicting the evolution pathogens like influenza viruses. Here, we tested whether seasonal A/H3N2 influenza virus evolution follows reproducible paths in a simplified setting where the natural cellular environment is maintained but adaptive immune selection is removed. We serially passaged replicate populations of an A/H3N2 virus for 1,200 generations across 12 independent differentiated human airway epithelium culture lines (three replicates of four donors) and performed deep sequencing to reconstruct the evolutionary dynamics. Rates of variant accumulation differed significantly among lines but not among donors, and parallel evolution was minimal. Populations generated polymorphisms not observed in the global human population, reversions to historical sequences, and changes at positions held near-invariant globally since 1968. These findings suggest A/H3N2 evolutionary trajectories are shaped less by intrinsic functional constraint than by selective context and help explain why predicting influenza virus evolution remains difficult.

evolutionary biology↗

bioRxiv2025

Extreme GC3 Codon Bias in a Novel Brown Seaweed Virus Results in Pseudoambigrammatic Characteristics

Although viruses are often studied in relation to disease, they can also offer valuable insights into RNA functionality. Due to their vast range of hosts and high mutation rates, viruses can quickly adapt to changing environments. It is therefore expected that viruses exploit all possible RNA functionalities to develop strategies for host infection and propagation. Our aim is to discover RNA viruses with survival strategies that reveal unknown RNA characteristics and functionalities. We have chosen seaweed as host organism because relatively little is known about RNA viruses in seaweed. Additionally, seaweeds are evolutionary distant from terrestrial plants and inhabit environments with entirely different conditions, increasing the likelihood of discovering new RNA viruses with unknown survival strategies. Here we report a new, unclassified negative-stranded single-strand RNA virus from seaweed Saccharina latissima that showed a unique genome characteristic. The novel virus, named SalaUV-NL1, contains a large segment of 7.065 nucleotides coding for a 2.296 amino acids protein and a small segment of 2.120 nucleotides coding for a 328 plus a potential 315+ amino acids protein. The large segment also appeared to have an additional large open reading frame (ORF) on the negative strand, suggesting it could be an ambigrammatic virus. However, the virus displayed an extreme GC3 codon bias (98%) in the (+)ORF, which differs from the typical avoidance of just the reverse-complement stop codons seen in ambigrammatic viruses. Consequently, the (-)ORF appears to occur accidental and is unlikely to encode a functional protein. Therefore, we have termed our virus to be pseudoambigrammatic. Several other viruses with the same pseudogrammatic high GC3 codon bias were identified in Genbank, most without a (-)ORF. The SalaUV-NL1 virus codon bias surpasses the high GC3 codon bias of the S. latissima host genes, however other species that host pseudoambigrammatic viruses do not display a high GC3 codon bias. Comparison of several variants of SalaUV-NL1 showed that the relative nucleotide change rate, particularly for silent changes from A/T to G/C, was extremely high (up to 81%). This novel virus demonstrates a new survival strategy of RNA viruses that is not yet understood.

genomics↗

bioRxiv2024

Discovery of new and variant Potyviruses in Asparagales plants from a Dutch urban botanic garden

Although viruses play an important role in human health and plant health, most viruses are still undetected. One of the reasons is that they may evolve in distinct natural habitats that are not yet extensively sampled for virus analysis. An example of such a habitat is urban botanic gardens. We analyzed 25 Asparagales plants with a mild to severe disease phenotype from a Dutch urban botanic garden for the presence of known an unknown virus types and virus variants by smallRNA-seq and RNA-seq. We found in all samples evidence for (past) Potyviridae presence, mostly Ornithogalum virus (OV) and Ornithogalum mosaic virus (OrMV), as well as a new Iris mild mosaic virus variant (IMMV) and a yet unknown species of Potyvirus. Also, presence of a new Betaflexivirus, a new Polerovirus and a new Phenuivirus were detected. Most analyzed plants harbored multiple viruses, 18 out of 25 plants showed evidence for three to seven viruses and 12 out of 13 viruses were present in four to 11 samples. In this study, we describe the characteristics of a newly discovered Potyvirus and identify several variants of known potyviruses. We place these findings in the context of known viruses. However, we were unable to link these potyviruses to any specific disease phenotype.

genomics↗

bioRxiv2024

A new Polerovirus species in plants from the Asparagaceae family defined by its RNA-silencing repressor protein P0

To contribute to the discovery of the global virome, we explored plants from an urban botanic garden in the Netherlands for new and variant plant viruses. Analyzing RNA from 25 plants from the Asparagaceae family with both RNA-seq, as well as smallRNA-seq revealed in six plants the presence of a variant Polerovirus from the Solemoviridae family that shows an overall RNA-sequence identity of 93% to the known Ornithogalum Virus 5 (OV-5). Amino acid sequence comparison of the complex set of proteins produced by the new virus revealed that all but Protein-0, showed high similarity (> 91%) with those of the OV-5 virus. The similarity between the new P0 protein and the OV-5 P0 protein, however, was for all variants less than 83%, well below the ICTV species protein-based demarcation criterium. Hence, we named the new virus Asparagaceae Polerovirus 1 (AspPolV-1).

genomics↗

bioRxiv2024

Discovery of a new Phenuivirus species in Lachenalia plants reveals possible co-evolution between 5' and 3' RNA sequence motifs

This study reports the discovery of a new Phenuivirus species, named Lachenalia Phenuivirus-1 (LacPhV-1), from Lachenalia plants in an urban botanic garden in Amsterdam. Using a combination of smallRNA-seq, RNA-seq, and advanced bioinformatics, we identified a segmented negative-strand RNA virus in the Phenuiviridae family. Our findings show significant divergence between this new virus and known Phenuiviruses, such as Tulip Streak Virus (TuSV) and Lactuca Big Vein associated Phlebovirus (LBVaPV), supporting its classification as a distinct species. Notably, the sequence differences found in the conserved 5 and 3 ends of these segments suggest potential co-evolution. Despite the observed genomic distances, there is significant conservation in the RdRp subdomain, underscoring evolutionary relationships among LacPhV-1, TuSV, and LBVaPV. Our findings expand the known global virome and highlight the importance of exploring plant viromes in diverse ecological settings to better understand virus evolution and diversity.

genomics↗

bioRxiv2024

A new Betaflexivirus species in Ferraria crispa plants

The global virome is still largely unknown. In this study we describe the discovery of a new Betaflexivirus species in Ferraria crispa plants. The plant samples were collected in an experiment of 25 Asparagales plants, obtained from an urban botanic garden in the Netherlands, and analyzed by smallRNA-seq as well as RNA-seq. The new Betaflexivirus only occurred in four Ferraria cripsa plants and was tentatively named Ferraria Betaflexivirus 1 (FerBfV-1). This Betaflexivirus showed an RNA genome structure characteristic for the genus Capillovirus, a single-stranded (+) RNA virus. The closest known Capilloviruses in GenBank showed a protein similarity less than 49%, which is well below the proposed demarcation value of species in this genus. Further comparison analysis of the Capillovirus genus revealed the possible presence of two groups.

genomics↗

bioRxiv2024

Discovery of novel RNA viruses in commercially relevant seaweeds Alaria esculenta and Saccharina latissima

Seaweeds are increasingly recognized as sustainable food sources; however, their large-scale cultivation faces challenges similar to land crops, including susceptibility to pathogens. Plant viruses pose a significant threat to global food security, yet little is known about the diversity of viruses in seaweeds. This study investigates virus-associated small interfering RNA (siRNA) responses in commercially relevant seaweed species to understand RNA virus diversity, particularly in edible varieties. Through small RNA sequencing of 16 samples from Saccharina latissima and Alaria esculenta, we identified three new RNA viruses Aev-NL1, Slv-NL2 and Slv-NL3, and one new DNA virus (phaeovirus). The partial genome of the new DNA virus was discovered in the A. esculenta samples and shared 67% DNA sequence identity with the major coat protein of the large double-stranded DNA phaeovirus Feldmannia irregularis virus a. In four out of five A. esculenta samples, a new bisegmented ormycovirus-like RNA virus (Aev-NL1) was identified. A similar new virus, Slv-NL1, was found in previously published S. latissima RNA-seq data, sharing 87% sequence identity with Aev-NL1. Lastly, two novel RNA viruses, Slv-NL2 and Slv-NL3, were discovered in all eight S. latissima samples sharing limited similarity at the genome level but high sequence identity at protein level of both ORFs (>94%). Further investigation of the novel viruses presence across our limited set of samples revealed no conclusive associations with diseased seaweed phenotypes. The discovery of four new viruses in only a limited set of samples highlights the presence of previously unrecognized viral diversity in seaweed, thereby underscoring the importance of understanding viral diversity in seaweed as its virome is currently understudied.

genomics↗

bioRxiv2022

Single-cell transcriptomics reveals subset-specific metabolic profiles underpinning the bronchial epithelial response to flagellin

Respiratory epithelial cells line the airways and represent the first line of defense against respiratory pathogens. The cellular heterogeneity of the airway wall has only recently been recognized by single-cell analyses. Here, we leveraged single-cell RNA sequencing of primary human bronchial epithelial cells growing in air-liquid interface to determine cell-specific changes evoked by flagellin, a protein driving the motility of many mucosal pathogens. We detected seven cell clusters in the human epithelium, including ciliated cells, ionocytes and several states of basal and secretory cells, of which only inflammatory basal cells and inflammatory secretory cells showed a proportional increase in response to flagellin. Only inflammatory secretory cells showed evidence of metabolic reprogramming toward aerobic glycolysis, and inhibition of the mTOR pathway specifically reduced this subset, prevented this metabolic shift, and reduced inflammatory gene transcription in these cells. This study expands our knowledge of the airways immune response to flagellated pathogens to single cell resolution and defines a novel target to modulate mucosal immunity during bacterial infections.

immunology↗

bioRxiv2020

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test

To battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy. We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as "2019-nCoV not detected", whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as "Invalid Result" and the procedure should be repeated. In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

microbiology↗

bioRxiv2019

New observations on non-coding RNAs involved in the dual translation system in zebrafish development.

Cellular translation relies heavily on the involvements of several types of non-coding RNAs. In previous studies we have identified a dual translation system in zebrafish development, involving maternal-type and somatic-type rRNAs, snoRNAs, and snRNAs. In this study we focused on several remaining non-coding RNAs involved in the translation system; tRNAs, RNase P, and SRP RNA. Even though our studies have been limited in extent, for all three types of non-coding RNA we were able to identify a maternal-specific type, with substantial sequence differences as compared to the somatic-type variant. Hence, these RNA types complement the previously discovered RNA types in the unique dual translation system in zebrafish development.

developmental biology↗

bioRxiv2019

Maternal- and Somatic-type snoRNA Expression and Processing in Zebrafish Development

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that play an important role in the complex maturation process of ribosomal RNAs (rRNAs). SnoRNAs are categorized in classes, with each class member having several variants present in a genome. Similar to our finding of specific rRNA expression types in zebrafish embryogenesis, we discovered preferential maternal- and somatic-expression for snoRNAs. Most snoRNAs and their variants have higher expression levels in somatic tissues than in eggs, yet we identified three snoRNAs; U3, U8 and snoZ30 of which specific variants show maternal- or somatic-type expression. For U3 and U8 we also found small-derived snoRNAs that lack their 5 rRNA recognition part and are essentially Domain II hairpin structures (U-DII). These U-DII snoRNAs from variants showed similar preferential expression, in which maternal-type variants are prominently expressed in eggs and subsequently replaced by a somatic-type variants during embryogenesis. This differential expression is related to the organization in tandem repeats (maternal type) or solitary (somatic-type) genes of the involved U snoRNA loci. The collective data showed convincingly that the preferential expression of snoRNAs is achieved by transcription regulation, as well as through RNA processing. Finally, we observed small-RNAs derived from internal transcribed spacers (ITSs) of a U3 snoRNA loci that via complementarity binding, may be involved in the biosynthesis of U3-DII snoRNAs. Altogether, the here described maternal- and somatic-type snoRNAs are the latest addition to the developing story about the dual ribosome system in zebrafish development.

developmental biology↗

bioRxiv2019

An alternative spliceosome defined by distinct snRNAs in early zebrafish embryogenesis

Splicing removes intronic RNA sequences from pre-mRNA molecules and enables, by alternative splicing, the generation of multiple unique RNA molecules from a single gene. As such, splicing is an essential part of the whole translation system of a cell. The spliceosome is a ribonucleoprotein complex in which five small nuclear RNAs (snRNAs) are involved; U1, U2, U4, U5, and U6. For each of these snRNAs there are variant gene copies present in a genome. Furthermore, in many eukaryotic species there is an alternative, minor spliceosome that can splice a small number of specific introns. As we previously discovered an embryogenesis-specific ribosomal system in zebrafish early embryogenesis based on variant rRNA and snoRNA expression, we hypothesized that there may also be an embryogenesis-specific spliceosome. An inventory of zebrafish snRNA genes revealed clustered and dispersed loci for all but U2 major snRNAs. For each minor spliceosome snRNA, just one gene locus was found. Since complete snRNA molecules are hard to sequence, we employed a combined PCR-sequencing approach to measure the individual snRNA-variant presence. Analysis of egg and male-adult samples revealed embryogenesis-specific and somatic-specific variants for each major snRNA. These variants have substantial sequence differences, yet none in their mRNA binding sites. Given that many of the sequence differences are found in loop structures indicate possible alternative protein binding. Altogether, with this study we established that the spliceosome is also an element of the embryogenesis-specific translation system in zebrafish.

developmental biology↗