Detection of Norovirus from Berries in Serbia by Digital PCR and NGS

Human norovirus (HuNoV), a primary cause of foodborne illness, is frequently transmitted through contaminated berries. Serbia is a global producer of raspberries and other berries, yet data on HuNoV prevalence and genogroup diversity are limited. This study aimed to assess the prevalence, viral load, and genotypes of HuNoV circulating in raspberries and blackberries marketed in Serbia. A total of 450 berry samples were collected in 2023 from orchards, cold storage facilities, local markets, and roadside vending stands. Norovirus RNA was extracted using a modified ISO 15216-2 protocol. RT-qPCR positive samples were subsequently quantified by digital RT-PCR (RT-dPCR). Genotyping employed next-generation sequencing (NGS) of genome encoding major and minor capsid proteins, supplemented by Sanger sequencing. Nineteen samples (4.2%) tested positive for HuNoV, including four GI and fifteen GII strains, with higher prevalence in frozen (11.1%) than fresh berries (2.0%). Viral loads ranged from 34-105 gc/g for GI and 23-658 gc/g for GII, with medians of 72 and 153 gc/g, respectively. Genotypes GI.6, GII.2, GII.4, and GII.7 were identified, each represented by more than two sublineages and multiple introduction events detected by phylogenetic analysis. RT-dPCR provided precise quantification, while NGS enabled genotype characterization, together supporting enhanced surveillance, risk assessment, and management of norovirus contamination in berries destined for domestic and international markets.