Gemykibivirus detection in acute encephalitis patients from Nepal

Abstract

Acute encephalitis syndrome (AES) causes significant morbidity and mortality worldwide. In Nepal, Japanese encephalitis virus (JEV) accounts for ~5-20% of AES cases, but ~75% of AES cases are of unknown etiology. We identified a gemykibivirus in CSF collected in 2020 from an 8-year-old male patient with AES using metagenomic next-generation sequencing. Gemykibiviruses are single stranded, circular DNA viruses in the family Genomoviridae. The complete genome of 2,211 nucleotides was sequenced, which shared 98.69% nucleotide identity to its closest relative, Human associated gemykibivirus 2 isolate SAfia-449D. Two real-time PCR assays were designed, and screening of 337 cerebrospinal fluid (CSF) and 164 serum samples from AES patients in Nepal collected in 2020 and 2022 yielded 11 CSF and 1 serum sample that were positive in both PCR assays. Complete genomes of seven of the positives were sequenced. These results identify a potential candidate etiologic agent of encephalitis in Nepal.

Importance: Viral encephalitis is a devastating disease, but unfortunately, worldwide, the causative virus in many cases is unknown. Therefore, it is important to identify viruses that could be responsible for cases of human encephalitis. Here, using metagenomic sequencing of CSF, we identified a gemykibivirus in a male child from Nepal with acute encephalitis syndrome (AES). We subsequently detected gemykibivirus DNA in CSF or serum of 12 more encephalitis patients by real-time PCR. The virus genomes we identified are highly similar to gemykibiviruses previously detected in CSF of three encephalitis patients from Sri Lanka. These results raise the possibility that gemykibivirus could be an underrecognized human pathogen.

Keywords: acute encephalitis syndrome; emerging infection; gemykibivirus; metagenomic analysis.

Fig 1

Phylogenetic trees of (A) amino acid sequences of the index case REP protein and representatives of each genus in the family Genomoviridae; (B) whole genome nucleotide sequences of the positive samples from Nepal compared with other genomes within the genus Gemykibivirus.

Fig 2

Schematic of the genome of Human gemykibivirus 2 Nepal/N0000051/2020.

Fig 3

Map of Nepal and locations of the gemykibivirus positive cases (shaded). The map was adapted from https://github.com/itsbohara/nepal-gis.git under Apache License 2.0.