Emergence of Reassortant Crimean-Congo Hemorrhagic fever virus lineages, Pakistan, 2023-2024.

Crimean-Congo hemorrhagic fever virus (CCHFV) remains endemic in Pakistan, yet whole-genome data are limited. During 2023-2024 national surveillance, 151 suspected cases were screened by qRT-PCR; 23 were confirmed at the NIH, Islamabad. Shotgun metagenomic sequencing of 17 cases (15 Pakistan, 2 Afghanistan) generated high-quality genomes, with 65% classified as Asia-1 genotype, while 35% showed segment reassortment involving Asia-2-derived S and/or M segments. Reassortment involved primarily S and M segments, while L segments remained conserved. Phylogenetic analysis revealed Asia-1 sequences closely related to strains from India, Afghanistan, Iran, and the Middle East, whereas Asia-2 sequences clustered with Indian and Central Asian strains, suggesting cross-border and regional viral exchange. S-segment phylogeography indicated northern Punjab (Rawalpindi, Islamabad, Chakwal, Attock) as a region potential involved in viral connectivity. Region-specific mutations (S: G185S, D186N; M: P90L, T122I, M35L, L443S, I1597V) may reflect localized viral evolution. These findings underscore ongoing viral diversification, reassortment, and regional connectivity, highlighting the need for integrated genomic surveillance to guide public health interventions.