Genomic conservation and CNS-associated intra-host variation of Nipah virus during the 1998–1999 Malaysian outbreak

Objectives Nipah virus (NiV) is a highly pathogenic henipavirus responsible for recurrent outbreaks of severe encephalitis. This study aimed to address gaps in historical genomic data by characterizing NiV genomes recovered from archived central nervous system (CNS) tissues from the 1998–1999 Malaysian outbreak and evaluating viral evolution and intra-host genetic variation. Methods We analyzed 18 NiV genomes obtained from archived CNS tissues. Whole-genome sequencing was performed, followed by phylogenetic reconstruction, comparative genomic analysis, and intra-host single nucleotide variant (iSNV) profiling. Paired CNS tissue samples from the individual patients were included to assess regional viral heterogeneity across distinct CNS tissues. Results All Malaysian NiV sequences formed a highly conserved, monophyletic clade (NiV-MY), distinct from the Bangladesh-India lineage (NiV-BD). Overall genomic diversity was low, with high amino acid conservation across the genome. Although intra-host variation was limited, however, paired CNS tissue samples from two patients contained closely related but non-identical genomes, differing by a single nucleotide in the attachment glycoprotein (G) gene, indicating localized genetic variation across distinct CNS tissues. Conclusions NiV from the Malaysian outbreak demonstrated remarkable genomic conservation consistent with a single dominant introduction or a highly restricted set of closely related introductions. Nevertheless, the presence of limited intra-host variation highlights ongoing micro-evolution within the CNS and underscores the importance of continued genomic surveillance to better understand NiV evolutionary dynamics.