Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing

**Background:** Astroviruses (AstVs) are important human enteric pathogens transmitted via contaminated food and water, and they have been identified across a wide range of animal hosts including mammals, birds, reptiles, amphibians, fish, and invertebrates. The genetic diversity of AstVs poses significant challenges for diagnostics and taxonomy. Filter-feeding mollusks concentrate viruses from water and are often consumed raw, making them valuable bio-indicators for exploring viral diversity. This study aimed to assess the potential of coupling a broadly reactive panastrovirus consensus RT-PCR protocol with Oxford Nanopore Technologies (ONT) deep sequencing to generate astroviromic data from shellfish matrices.

**Methods:** Samples were collected in two campaigns: 48 oyster (Crassostrea gigas) samples from four Italian production areas (November 2016–October 2018) and 86 bivalve samples (Mytilus galloprovincialis, Crassostrea gigas, Ostrea edulis) from the Gulf of La Spezia (December 2020–December 2021). Viral RNA was extracted from digestive tissues using the ISO 15216-1:2017 method with proteinase K digestion and NucliSENS magnetic extraction. AstV screening used a nested RT-PCR protocol with panastrovirus primers targeting a ~420-nt fragment of the RNA-dependent RNA polymerase (RdRp). Amplicons were sequenced using the ONT MinION platform with Rapid Barcoding Kit 24. Reads were analyzed via the Genome Detective Virus Tool (GDVT) v2.48, which included trimming, DIAMOND protein-based alignment, SPAdes de novo assembly, and BLASTx/BLASTn against NCBI RefSeq. Phylogenetic analysis was performed using maximum-likelihood (Tamura-Nei 4-parameter model, gamma distribution, 1000 bootstrap replicates) in MEGA X.

**Key Results:** RT-PCR screening yielded amplification in 3/48 (6.3%) oyster samples from the 2016–2018 campaign (all from the Gulf of La Spezia class B area) and 18/86 (20.9%) bivalve samples from the 2020–2021 campaign (9 mussels, 9 oysters, across all nine production areas). Sequencing produced 340.370 Mb of data (491,410 reads), of which 219,564 (44.6%) were AstV-specific. AstV reads per sample ranged from 845 to 64,214 (median 8,533). A total of 37 AstV RdRp sequence contigs were generated (mean 2.8 contigs per barcode, range 1–5). Only 5/37 (13.5%) sequences had >80.1% nucleotide identity to database sequences; 2/37 (5.4%) scored 72.2–80.0%; and 30/37 (81.1%) scored 57.6–69.3%. Avian-like AstV sequences predominated (23/37, 62.1%), with top BLAST matches including AAstV/HKG/2015/white eye bird/D11-148.2 (63.5–67.5% nt identity) and AAstV/SWE/2014/mallard/313 (80.1–92.5% nt identity). Two bat-like sequences (5.4%) showed 68.4–72.2% identity to bat AstVs. Two reptilian-like sequences (5.4%) showed 67.9–69.3% identity to gecko AstV. Ten sequences (27.1%) were distantly related to AstVs from aquatic animals (shrimp, fish), with 57.6–60.2% identity to FAstV/CHN/2014/shrimp/BHWZXX13371. No human astrovirus sequences were detected, although two mussel samples had very low-level human AstV by qRT-PCR (Cq > 38.5).

**Clinical Implications:** This study demonstrates that targeted panastrovirus amplicon sequencing with Nanopore technology can effectively characterize diverse astrovirus populations in environmental samples, revealing a predominance of avian and aquatic animal AstVs in shellfish from Italian coastal waters. The absence of human AstV sequences, despite low-level detection by qRT-PCR in two samples, suggests that avian and aquatic AstVs may outcompete human strains in broadly reactive amplification. The high proportion (86.5%) of sequences with <69.3% nucleotide identity to known AstVs underscores the vast undiscovered genetic diversity within the Astroviridae family. While the risk of human infection from non-human AstVs in shellfish appears low, the detection of avian nephritis virus-related sequences and serological evidence of AAstV antibodies in poultry workers suggests potential for cross-species transmission. The authors propose that astroviromic data could serve as a proxy for microbiological water quality monitoring, though further validation in areas with lower sanitary standards is needed.