Isolation and Genetic Identification of Human Non-Polio Enteroviruses at the National Reference Laboratory of Enteroviruses, Bulgaria

Background: Human non-polio enteroviruses (NPEVs) are a diverse group of RNA viruses belonging to the genus Enterovirus within the subfamily Ensavirinae of the family Picornaviridae. They are responsible for a wide range of diseases, ranging from mild febrile illnesses to severe neurological conditions. Accurate isolation and genetic identification are critical for understanding their epidemiology, evolution, and pathogenic potential. Aim: This study aimed to isolate NPEVs from clinical specimens, perform molecular identification through RT-PCR, and analyze viral sequences of parts the viral genome to elucidate their molecular genotyping. Materials and methods: Clinical specimens from patients presenting with neurological, febrile illnesses, or HFMD were collected between 2021 and 2024 and were sent to NRL “Enteroviruses” for testing. All samples were tested using viral isolation and microneutralization. Direct molecular detection of enterovirus RNA was performed by real-time RT-PCR. Partial VP1 gene sequences were used for genotyping after Sanger sequencing of the target region. Results: Successful serotyping via microneutralization was obtained for 9 isolates, which resulted in the identification of 4 echovirus 11 (E11), one echovirus 2 (E2), two coxsackievirus B (CVB), one echovirus 25 (E25), and one echovirus 6 (E6) strains. Molecular genotyping revealed multiple genotypes predominantly belonging to Enterovirus alphacoxsackie species, including CVA6, CVA16, CVA4, CVA2, and EV-A71. From Enterovirus betacoxsackie species E11, E9, E3, and E2 were detected. Conclusion: Despite the incomplete enterovirus surveillance in Bulgaria and nonsystematic approach for NPEV detection, we managed to identify different virus strains in various clinical settings. Our study underscores the importance of combining virus isolation with molecular techniques for accurate identification of NPEVs, as well as the necessity of continuous monitoring of NPEVs.