Biology subjects

Willemsen, W.

Publications and source records attributed to Willemsen, W..

3 recordsLinked to original sources

bioRxiv2026

sRNAMap: a lightweight, browser-based web application for small RNA mapping, analysis and visualisation

Small RNA (sRNA) sequencing is widely used to study siRNA, miRNA and piRNA pathways and to profile sRNAs derived from viruses, transposons and host transcripts. However, many sRNA workflows rely on command-line mapping and separate software for downstream analysis and visualisation. We present sRNAMap, an HTML5/JavaScript application for mapping and analysing sRNA sequencing data. sRNAMap accepts either raw FASTA/FASTQ reads or pre-aligned BAM files. For raw reads, sRNAMap uses a k-mer-seeded alignment algorithm that runs directly in the browser using parallel Web Workers where supported. It generates strand-specific per-base coverage, 5'- and 3';-end profiles, read-length distributions and nucleotide-bias summaries, with optional library-size normalisation. It also quantifies characteristic read-overlap signatures, including 5';-to-5'; overlaps associated with ping-pong-like processing. Additional modules quantify phased sRNA production, non-templated 3'- and 5'-additions, and sequence diversity. Analyses can be saved and restored as JSON or exported as self-contained HTML reports containing figures and analysis metadata. Benchmarking against Bowtie 2 under approximately matched alignment settings using a representative virus-infected arthropod small-RNA dataset showed highly concordant mapping profiles. Per-position depth showed an R^2 of approximately 0.998, while 5';- and 3';-end counts showed R^2 values of approximately 0.97. sRNAMap is available at https://github.com/rhparry/sRNAMap under the MIT licence.

bioinformatics↗

bioRxiv2026

Sequence Homology and Tissue Tropism Determine Superinfection Exclusion of Zika virus in Aedes aegypti Mediated by an insect-specific Binjari-Zika Virus Chimera

Arboviruses such as dengue, Zika, and chikungunya viruses cause widespread disease and continue to expand their geographical range due to climate change and vector spread. Insect-specific flaviviruses (ISFs) are promising biocontrol candidates of arboviruses, due to recent studies showing that prior infection with an ISF can reduce arbovirus replication in mosquitoes through superinfection exclusion (SIE). However, the route of infection, tissue tropism, pathogenesis and the mechanisms underlying SIE of ISFs in mosquitoes remain unclear. RNA interference (RNAi) is a potent antiviral response in insects, therefore it is expected that sequence homology between the ISF and the arbovirus will strengthen SIE. Here, we used ISF Binjari virus and a chimera containing the Zika virus structural proteins prME (BinJ-ZIKV) as a model system. Intrathoracic injection of BinJ-ZIKV in Aedes aegypti led to rapid systemic infection that excluded the midgut, subsequently blocking ZIKV dissemination from the midgut. SIE was strongest in tissues where primary-virus replication was highest. This spatial component of SIE was stronger when there was sequence homology between the ISF and arbovirus and displayed a strong 21nt siRNA response, suggesting RNAi contributed to the observed SIE. Upon oral inoculation, BinJ-ZIKV replicated efficiently in mosquitoes, was detected across multiple tissues, and saliva. BinJ-ZIKV also had higher infection establishment than BinJV at lower oral titres. SIE was observed for BinJ-ZIKV infection after oral exposure interfered with subsequent ZIKV midgut infection. Together, these findings support engineered ISF-chimeras as valuable experimental tools to dissect viral determinants of SIE and to optimize mosquito-based arbovirus interference strategies. ImportanceAnnually, over 400 million people are infected with mosquito-transmitted viruses. Insect-specific flaviviruses (ISFs) can interfere with the transmission of clinically important viruses through a phenomenon termed superinfection exclusion (SIE). However, the mechanisms of SIE remain poorly understood. Using a Binjari virus chimera expressing Zika virus (ZIKV) structural proteins, we show that SIE is highly tissue-specific, with exclusion of ZIKV only occurring at sites where the chimera actively replicates and induces the mosquito antiviral RNA interference pathway. We further demonstrate that incorporation of Zika virus prM and E proteins into the ISF backbone enhances infection of the mosquito midgut following oral exposure, which enables direct inhibition of ZIKV infection after a subsequent infectious blood meal. Together, these findings define a replication-dependent, tissue-specific mechanism of ISF-mediated protection and provide a framework for reducing mosquito-borne virus transmission through SIE.

microbiology↗

bioRxiv2025

The effect of increased CpG and UpA dinucleotides in the West Nile virus genome on virus transmission by Culex mosquitoes and pathogenesis in a vertebrate host

Vertebrate animals and many small DNA and single-stranded RNA viruses that infect vertebrates have evolved to suppress genomic CpG dinucleotides. All organisms and most viruses additionally suppress UpA dinucleotides in protein coding RNA. Synonymously recoding viral genomes to introduce CpG or UpA dinucleotides has emerged as an approach for viral attenuation and vaccine development. However, studies that investigate the effects of this recoding strategy on viral replication and pathogenesis in vivo are still limited. Flaviviruses including West Nile virus (WNV) are transmitted between vertebrate hosts by invertebrate vectors. In humans, WNV infection can cause flu-like symptoms and neuroinvasive disease. We investigated how alterations in WNV dinucleotide frequencies impact virus replication, transmission by vector mosquitoes, as well as pathogenesis and neuroinvasiveness in vertebrates. In Culex pipiens vector mosquitoes and Culex cell lines only WNV with elevated UpA frequencies displayed attenuated replication. In vertebrate cell lines and primary human neuro-astrocyte co-cultures both UpA and CpG enrichment reduced viral replication. In mice, the CpG-high WNV mutant demonstrated partial attenuation with delayed weight loss compared to wild-type WNV, though infection still resulted in 100% mortality. In contrast, 75% of animals survived inoculation with the UpA-high WNV mutant and were protected against wild-type WNV challenge. Notably, all animals that succumbed to infection had similar levels of virus in the brain, irrespective of the WNV mutant. Our results underscore the complex interplay between viral genome composition and host immune responses, highlighting potential safety concerns for dinucleotide manipulation as a strategy for live-attenuated vaccine development in flaviviruses. ImportanceFlaviviruses such as West Nile virus (WNV) pose significant public health concerns due to their potential to cause severe neurological disease. Synonymously recoding flavivirus genomes to introduce CpG or UpA dinucleotides has emerged as an approach for viral attenuation and vaccine development. However, the in vivo effects of manipulating these frequencies across the complete transmission cycle remained unexplored. Our study provides comprehensive insights of how CpG and UpA recoding affects WNV replication in both the mosquito vector and vertebrate hosts. We demonstrate that elevated UpA content attenuates virus replication throughout the transmission cycle, while CpG enrichment only impacts replication in the vertebrate host. Although UpA-high WNV shows significant attenuation and provides protection against wild-type infection, animals that succumb exhibit similar brain viral loads as wild-type infections. These findings have critical implications for live-attenuated vaccine development based on dinucleotide manipulation, specifically highlighting the importance of carefully evaluating the risk of neuroinvasion.

microbiology↗