The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.
Subject(s)Bivalvia , COVID-19 , Animals , Humans , SARS-CoV-2 , Shellfish , Wastewater
To date, the microbiome, as well as the virome of the Croatian populations of bats, was unknown. Here, we present the results of the first viral metagenomic analysis of guano, feces and saliva (oral swabs) of seven bat species (Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum, Eptesicus serotinus, Myotis blythii, Myotis nattereri and Myotis emarginatus) conducted in Mediterranean and continental Croatia. Viral nucleic acids were extracted from sample pools, and analyzed using Illumina sequencing. The presence of 63 different viral families representing all seven Baltimore groups were confirmed, most commonly insect viruses likely reflecting the diet of insectivorous bats. Virome compositions of our samples were largely impacted by the sample type: invertebrate-infecting viruses were most frequently found in feces, bacterial viruses in guano, whereas vertebrate-infecting viruses were most common in swabs. Most vertebrate-infecting virus sequences were assigned to retroviruses, parvoviruses, iridoviruses, and poxviruses. We further report the complete genome sequence of a novel adeno-associated virus, densovirus and a near complete length genome sequence of a novel iflavirus. Additionally, one of the most interesting findings in this study was the difference in viromes between two contrasting habitats, the continental and Mediterranean Croatia.