Diversity of Enterovirus genotypes and recombinant strains circulating in pediatric patients with acute gastroenteritis

Abstract

Enteroviruses (EVs) have been increasingly recognized as potential causative pathogens of acute gastroenteritis (AGE) in infants and young children worldwide. In the past decade, a number of different EV genotypes have been identified in AGE patients and EV prevalence has remarkably increased. Recombination is a common phenomenon and plays a crucial role in the evolution of EVs, significantly contributing to their adaptability, spread, and increase in virulence. Monitoring the EV epidemiology and identifying the circulating EV recombinants in pediatric patients are essential for better understanding the emergence of novel EV lineages and their epidemiological significance. This study aimed to investigate the epidemiological patterns and molecular characteristics of EV infections in children admitted to the hospitals with AGE in Chiang Mai, Thailand, from 2019 to 2022. Additionally, the study characterized and identified potential EV recombinant strains in the EV-infected cases during the period 2013–2020. A total of 1,148 stool samples collected from children hospitalized with AGE were screened for the presence of EVs using reverse transcription polymerase chain reaction (RT-PCR). The overall prevalence of EV in AGE patients was 8.8% (101 out of 1148 cases). Among the EV-positive cases, 68.3% of cases were solely infected with EV, suggesting that EV plays a potential role in AGE. The highest prevalence of EV infection was observed in 2019 at 11.7%, but a significant decline occurred during the post-COVID-19 pandemic, with infection rates dropped to 9.6% in 2020 and declined further to 5.4% and 5.5% in 2021 and 2022, respectively. The seasonal distribution of EV infections showed a significant peak during the rainy season (July to October), which aligns with the typical seasonal pattern of EV transmission in tropical regions. Genotyping and phylogenetic analysis of the EV strains detected in this study revealed that the most prevalent species was EV-A (37.5%), followed by EV-B (32.3%), EV-C (29.2%), and EV-D (1.0%). Among 25 EV genotypes detected, coxsackievirus A2 (CVA2) (13.5%), CVB2 (7.3%), and CVA24 (5.2%) were the most predominant genotypes. Interestingly, in 2019, CVA2 emerged as the predominant genotype and exhibited a unique evolution pattern in the phylogenetic tree. To investigate the molecular epidemiology, evolutionary dynamics, and recombination characteristics of the detected CVA2 strains, whole genome of 19 CVA2 strains detected in AGE patients in Chiang Mai, Thailand from 2013 to 2022 were amplified and sequenced. The full-length genome sequences of these CVA2 were analyzed in comparison with those of the reference sequences available in the GenBank database. Phylogenetic analysis of full-length VP1 region demonstrated that 15 out of 19 CVA2 strains detected in this study formed a novel lineage, designated as subgenotype C5. Other 4 CVA2 strains clustered with the reference strains previously known as subgenotype C1. Whole-genome sequence recombination analysis indicated that these CVA2 subgenotype C5 were intertypic recombinant strains of CVA2 and CVA10 in the P1 structural region and P2–P3 non-structural region, respectively. The recombination breakpoints were identified in the 2B gene within the P2 region of all CVA2 recombinant strains. Four CVA2 subgenotype C1 strains were non-recombinant strains. Amino acid mutation analysis of whole genome sequence of CVA2 recombinant strains showed that mutation of A61S in the VP3 gene and R136K in the 3D (RdRp) gene were identified in all CVA2 recombinant strains detected in this study. In addition, the S45G mutation in the RdRp gene was found to be potentially associated with the emergence of CVA2 recombinant strains circulating from 2019 to 2022. Notably, CVA2 non-recombinant strains detected before 2019, except for CMH-ST077-17, did not contain the S45G mutation in their genomes. In conclusion, this study revealed high divergence of EV genotypes circulating in pediatric patients with AGE in Chiang Mai, Thailand during 2019–2022 and the emergence of novel CVA2 subgenotype C5 recombinant strains containing specific amino acid mutations. These findings enhance understanding of the epidemiology, molecular characteristics, evolution, and genetic relationship of EV strains circulating in AGE patients and emphasize the importance of continued monitoring the prevalence, diversity, and emergence of novel EV strains associated with AGE.