Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing

Simple Summary Astroviruses are important human pathogens, associated with gastro-enteric disease in children and recently with encephalitis in immunocompromised patients. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates in association with enteric and extra-intestinal diseases or, in some cases, as components of the enteric virome without a clear link with specific clinical signs. As a proof of concept, we conjugated the versatility and broad reactivity of a commonly used consensus primer set, able to amplify in a nested RT-PCR protocol the RNA-dependent RNA polymerase (RdRp) of most members of the Astroviridae family, with a nanopore sequencing platform, in order to assess the potential of this approach to generate astroviromic data in complex matrices. Amplicons generated from mussels were used to generate libraries, either alone or in pools, and subjected to deep sequencing. Overall, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. Avian-origin astrovirus sequences were predominant, likely due to contamination of surface water. Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.