Molecular Epidemiology of Non-polio Enterovirus: Insights From L20B Cell Line Adaptation From Children With Acute Flaccid Paralysis in Pakistan.

BACKGROUND: Non-polio enteroviruses (NPEVs) are increasingly implicated in acute flaccid paralysis (AFP), often resembling poliomyelitis and complicating eradication efforts. In Pakistan, limited molecular surveillance has hindered comprehensive characterization. The L20B cell line, designed for poliovirus detection, occasionally supports NPEV replication, challenging AFP case interpretation. METHODS: Between January 2021 and December 2022, 4615 stool samples from AFP cases in children ≤15 years were analyzed. Of these, 435 were identified as NPEVs via L20B cytopathic effects and intertypic differentiation reverse transcription-polymerase chain reaction. VP1 sequencing was performed on 218 representative isolates, yielding 153 high-quality sequences (70.2%). The 224/222 primer set showed superior amplification. Phylogenetic analysis used MUSCLE alignment and the Neighbor-Joining method in MEGA X, with statistical evaluation of epidemiological data. RESULTS: NPEVs were frequently found in L20B-positive AFP cases, highlighting the cell line's limited specificity. Most cases involved children under 5, with a slight male bias. Enterovirus B was predominant (98.0%), especially Echovirus 7 (20.3%) and Echovirus 11 (10.5%), followed by Coxsackievirus B1 and Echovirus 33 (5.9% each). Geographic clustering was noted in Punjab (45.1%), Khyber Pakhtunkhwa (30.7%) and Sindh (20.3%), with seasonal peaks in late summer and early autumn. Phylogenetic data revealed localized Enterovirus B circulation with minimal genetic variation. CONCLUSIONS: The detection of diverse NPEVs in L20B-positive AFP cases emphasizes their relevance in post-polio surveillance. Incorporating routine VP1 sequencing, optimized primer use, and targeted seasonal and regional monitoring is vital to reduce diagnostic uncertainty and inform public health strategies.