Genomic characterization of the Coxsackievirus A24 variant in the Acute Hemorrhagic Conjunctivitis outbreak (2023) in Islamabad, Pakistan through metagenomic next generation sequencing

Syed Adnan Haider; Zunera Jamal; Muhammad Ammar; Rabia Hakim; Babak Afrough; Anila Kreku; Leena Inamdar; Muhammad Salman; Massab Umair

doi:10.1016/j.jviromet.2025.115213

Genomic characterization of the Coxsackievirus A24 variant in the Acute Hemorrhagic Conjunctivitis outbreak (2023) in Islamabad, Pakistan through metagenomic next generation sequencing

J Virol Methods. 2025 Jun 27:338:115213. doi: 10.1016/j.jviromet.2025.115213. Online ahead of print.

Authors

Syed Adnan Haider¹, Zunera Jamal¹, Muhammad Ammar¹, Rabia Hakim¹, Babak Afrough², Anila Kreku², Leena Inamdar², Muhammad Salman¹, Massab Umair³

Affiliations

¹ National Institutes of Health, Islamabad, Pakistan.
² New Variant Assessment Platform, UK Health Security Agency, 10 South Colonnade, E14 4PU, United Kingdom.
³ National Institutes of Health, Islamabad, Pakistan. Electronic address: massab.umair@yahoo.com.

PMID: 40581162
DOI: 10.1016/j.jviromet.2025.115213

Abstract

Pakistan experienced a significant outbreak of Acute Hemorrhagic Conjunctivitis (AHC) in 2023. To identify the cause, in the absence of targeted diagnostic tests, the National Institute of Health, Islamabad, studied 15 conjunctivitis patients from Islamabad in September 2023. Metagenomic Next Generation Sequencing (mNGS) was performed on 10 samples collected within 48 h of symptom onset. The human Coxsackievirus A24 variant (CV-A24v), genotype IV, was detected in three samples. Phylogenetic analysis showed ∼99 % similarity with recent strains from China and 94 % similarity with the 2015 France outbreak. Mutation analysis revealed mostly non-synonymous substitutions, particularly in the VP1 region (n = 14), and differences in 2 C and 3D regions of nonstructural proteins. Comparison with 2005 Pakistan outbreak sequences showed divergence in the VP1 region, with two distinct mutations ("L16I" and "L25H"), with L16I being a rare mutation observed only in strain from China and India in 2023. Structural modeling of VP1 proteins indicated conformational differences between the 2023 and 2005 strains, suggesting potential impacts on viral infectivity and immune escape. These findings indicate the reemergence of CV-A24v in Pakistan and highlight the importance of adaptable diagnostic strategies to respond to emerging infectious threats.

Keywords: Acute Hemorrhagic Conjunctivitis; Coxsackievirus A24 variant; Outbreak; Pakistan; Pink eye infection.