Effect of increased CpG and UpA dinucleotides in the West Nile virus genome on virus transmission by 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 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 vector mosquitoes and 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 with wild-type WNV, although 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 effects of manipulating these frequencies across the complete transmission cycle remained unexplored. Our study provides comprehensive insights into 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, whereas 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.