# First Report of Hepatitisis E Virus in Shellfish in Southeast Italy
## 2 Materials and Methods  
A total of 225 samples of different species of shellfish commercially sold in the Apulia region were collected from January to December 2018. These samples included mussels (_Mytilus galloproucialis_, \(n\) = 182), oysters (_Crassostrea gigsas_, \(n\) = 32), and other species, including _Modiolus barbatus_ (_n_ = 5), _Tapes decussates_ (_n_ = 2), _Solen margintatus_ (_n_ = 2), _Aequipeten opercularis_ (_n_ = 1), _Callista chione_ (_n_ = 1). The geographic origins of these shellfish samples are shown in Table 1.  
All samples were collected by local health services, in accordance with national official monitoring activities for microbiological criteria of shellfish (e.g., _Salmonella_ and _Escherichia coli_). All the collected samples were transported to the Istituto Zooprofialtico Sperimentale della Puglia e della Basilicata (Foggia, Italy) and stored at 4 \({}^{\circ}\)C until analyzed. The presence of HEV was determined as described [13], using a method that follows the structure and quality criteria of the standardized ISO 15216-2:2019 \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Species**} & \multicolumn{6}{c}{**Origin**} & \multirow{2}{*}{**Total**} \\ \cline{2-2} \cline{4-6}  & \multirow{2}{*}{**Italy**} & \multirow{2}{*}{**Spain**} & \multirow{2}{*}{**Greece**} & \multicolumn{2}{c}{**The Netherlands**} & \multirow{2}{*}{**France**} \\ \cline{2-2} \cline{5-6}  & & & & & & \\ \hline _Mytilus_ & & & & & & \\ _galloprovicinalis_ & & & & & & \\ _Crassostrea gigsas_ & 12 & & & & 14 & 6 & 32 \\ _Modiolus barbatus_ & 5 & & & & & 5 \\ _Tapes decussates_ & 2 & & & & & 2 \\ _Solen margintatus_ & 2 & & & & & 2 \\ _Aequipeten_ & & & & & & 1 \\ _opercularis_ & & & & & & 1 \\ _Callista chione_ & 1 & & & & & 1 \\ Total & 180 & 16 & 9 & 14 & 6 & 225 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Geographic origins of the tested shellfish samples. 15-60 specimens of each sample were casually chosen, and their digestive tissue was cut up, cleaned, and shredded with a sterile blade. Following digestion, 2 mL proteinase K (0.1 mg/mL) and 10 mL of megovirus clone MC\({}_{0}\) as process control (supplied by the Istituto Superiore di Sanita, Rome, Italy) were added to an aliquot of 2.0 g of each sample, and incubated at 37 \({}^{\circ}\)C for 60 min with shaking, and then at 60 \({}^{\circ}\)C for 15 min to deactivate the enzyme. After centrifugation at 3000\(\times\)\(g\) for 5 min, the supernatants were recovered. Viral nucleic acids were extracted employing the Nuclisens extraction kit (BioMerieux, Paris, France) according to the manufacturer's protocol, and the eluted RNA (100 mL) was immediately analyzed or stocked at \(-\)80 \({}^{\circ}\)C until use. Real-time RT-PCR for HEV was carried out on a 7500 Fast Real-Time PCR system thermocyler (Applied Biosystems, CA, USA) using an RNA UltraSense One-Step Quantitative RT-PCR System (Life Technologies, Carlsbad, CA, USA), and primers and probe targeting the ORF3 region of HEV [20,21]. The thermal cycling conditions were 50 \({}^{\circ}\)C for 60 min and 95 \({}^{\circ}\)C for 5 min, followed by 45 cycles of 95 \({}^{\circ}\)C for 15 s, 60 \({}^{\circ}\)C for 1 min, and 65 \({}^{\circ}\)C for 1 min. An external amplification control (in vitro synthesized RNA supplied by the Istituto Superiore di Sanita, Rome, Italy) was added to the samples to evaluate the presence of PCR inhibitors. Samples found to be HEV positive were retested (another aliquot of digestive tissue from the same pooled samples) at an independent laboratory as described [22], both for confirmation (i.e., to rule out false-positive results) and for quantification, according to [PERSON] et al. 2019 [22], with the results defined as the number of HEV genome copies (gc) per gram of digestive tissue, and calculated according to the standardized ISO 15216-1:2017 method [23]. In detail, the quantification was done using a linearized plasmid containing the target sequence of HEV to generate the standard curve.