Viromic analysis of cerebrospinal fluid (CSF) samples identified an unclassified RNA virus exhibiting an atypical genomic architecture.

The central nervous system (CNS) is highly susceptible to infections that can lead to severe neurological morbidity and mortality. Despite advances in diagnostic technologies, a significant proportion of encephalitis and meningitis cases remain etiologically undiagnosed, underscoring the need for novel pathogen discovery approaches. Here, we report the identification of a novel unclassified RNA virus, provisionally named hucaurvirus ("human CSF-associated unclassified RNA virus"), in the cerebrospinal fluid (CSF) of a pediatric patient with unexplained meningoencephalitis, using metagenomic next-generation sequencing (mNGS). The hucaurvirus exhibits a monopartite, positive-sense RNA genome of 6.5 kb which contains two overlapping open reading frames (ORFs), which was confirmed by conventional PCR amplification and Sanger sequencing. The large ORF encodes a polyprotein containing a capsid domain, while the small ORF overlaps within the large ORF and encodes the RNA-dependent RNA polymerase (RdRp) protein. BLASTp search based on the amino acid sequence of RdRp showed that hucaurvirus had the highest sequence identity and query coverage of 39.2% and 90%, respectively, to a noda-like virus (GenBank no. MT138110). Phylogenetic analysis showed that hucaurvirus was closely related to four viruses annotated as noda-like viruses, forming a cluster that is adjacent to the cluster of the Carmotetraviridae family and appears to form a new viral family within the order Tolivirales. PCR screening confirmed the presence of hucaurvirus in one of ten CSF samples, validating its detection. Our findings emphasize the utility of mNGS in uncovering novel pathogens and expanding our understanding of viral diversity in CNS infections.