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

van Bree, J.; Visser, I.; Marshall, E. M.; Willemsen, W.; van de Waterbeemd, C.; Abma-Henkens, M.; van Oers, M. M.; Pijlman, G. P.; Rockx, B.; Fros, J. J.

doi:10.1101/2025.01.22.634234

bioRxiv · 10.1101/2025.01.22.634234

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

van Bree, J.·Visser, I.·Marshall, E. M.·Willemsen, W.·van de Waterbeemd, C.·Abma-Henkens, M.·van Oers, M. M.·Pijlman, G. P.·Rockx, B.·Fros, J. J.

View original source ↗Export metadata ↓

Abstract

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.

Source connections

Published article ↗

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

van Bree, J., Visser, I., Marshall, E. M., Willemsen, W., van de Waterbeemd, C., Abma-Henkens, M., van Oers, M. M., Pijlman, G. P., Rockx, B., Fros, J. J.. 2025-01-22. 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. https://doi.org/10.1101/2025.01.22.634234

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A population-scale landscape of the subgingival microbiome reveals divergent routes to periodontal dysbiosis

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

microbiology↗

bioRxiv2026

The iron-binding siderophore enterobactin is required for the response of multi-drug resistant Klebsiella pneumoniae to zinc limitation

To persist during infection Klebsiella pneumoniae must overcome nutrient iron and zinc limitation imposed by the host immune system through a process called nutritional immunity. Secreted small molecule siderophores are a major virulence determinant of Klebsiella pneumoniae pathogenesis and are presumed to overcome nutritional immunity by binding iron for bacterial acquisition. In this work, we set out to identify how a multi-drug resistant K. pneumoniae grows in zinc limited environments. Using unbiased transcriptomics, proteomics, and an arrayed transposon screen, we identified that synthesis and uptake of the siderophore enterobactin is required to allow for growth in low zinc conditions. Iron-specific chelators did not replicate this phenotype and addition of supplemental iron through heme in growth media could not complement severe growth defects of enterobactin mutant K. pneumoniae experiencing zinc limitation. Finally, zinc starvation induced enterobactin production independent of the canonical zinc uptake regulator (Zur) transcription factor suggesting an unidentified regulatory mechanism by which Gram-negative pathogens may respond to zinc stress. Together, these studies expand the role of enterobactin beyond iron regulation and highlight a previously unreported link between iron and zinc homeostasis in Klebsiella pneumoniae.

microbiology↗

bioRxiv2026

A microbiota-derived protease links phage susceptibility to host epithelial responses

Bacteriophages are major ecological drivers of gut microbial ecology, yet whether bacterial mechanisms that determine phage susceptibility have consequences for the mammalian host remains poorly understood. Here, we identify dipeptidyl peptidase 11 (Dpp11a), the predominant active serine protease of the prevalent gut commensal Phocaeicola vulgatus, as an unexpected bacterial defence factor. Dpp11a protects against environmental proteases and confers resistance to bacteriophage infection. Metatranscriptomic analyses further reveal increased expression of both dpp11a and P. vulgatus-associated phage transcripts in ulcerative colitis stool samples, indicating that both components of this interaction are transcriptionally active in disease-associated human microbiomes. Using the microfluidic gut-on-a-chip co-culture model HuMiX, we show that the absence of Dpp11 is accompanied by altered epithelial tight-junction remodelling during phage-bacterial infection. Together, our findings reveal that the consequences of bacterial phage defence can extend beyond phage-bacterium interactions to the mammalian epithelium.

microbiology↗