# First Report of Hepatitisis E Virus in Shellfish in Southeast Italy
## 3 Results and Discussion  
Of the 225 shellfish samples tested, two (0.89%) were positive for HEV RNA by real-time RT-PCR. These two samples included one (0.5%) of the 182 samples of _M. gallo-provincialis_ tested and one (3.1%) of the 32 _C. gigas_ samples tested. The levels of virus were slightly below the limit of quantification of the method (3.9 \(\times\) 10\({}^{2}\) gc/g) with estimated concentrations of 2.5 \(\times\) 10\({}^{2}\) in the mussel sample and 7.0 \(\times\) 10\({}^{1}\) gc/g in the oyster sample (Table 2). Because of the small sample size, HEV prevalence did not differ significantly among the different bivalve species ([PERSON]'s exact test; \(p\) > 0.05). The two HEV positive samples originated from two distinct locations, with the mussel sample taken in the northern part of Apulia and the oyster sample taken in the southern part of Apulia. Interestingly, both positive samples were collected during spring 2018 (Table 2). However, multi-year sampling is needed to evaluate the significance of a supposed seasonal pattern. Attempts to genotype the two positive samples were unsuccessful, as the conventional nested RT-PCR amplification yielded negative results. This may be related to the low concentration of HEV in the two samples (Table 2), roughly corresponding to <1 gc/\(\mu\)L in the tested RNA. Viral infections have been linked to shellfish consumption, often due to undercooking of the product and sewage contamination of harvesting areas [25]. Despite shellfish consumption being a risk factor for HEV infection [13], hepatitis E following shellfish consumption has been relatively rare [26]. Several studies in Europe have reported variations in the presence of HEV in mussels harvested from regulated waters, with rates of contamination of 2.9% [12], 14.8% [11], and 24.4% [27], whereas other studies have reported no evidence of HEV. In Italy, HEV has been observed in commercially harvested shellfish, with prevalence rates ranging from 0.9% in Sicily [17] to 2.6% in the Campania region [13]. The low prevalence ratio reported in this survey was comparable to that of shellfish from Sicily [17]. One study found that HEV was present in 8.1% of mussels from Italy, although that study included bivalves that were collected from areas not approved for harvesting [28]. HEV has been detected in the environment in Italy, including in sewage [29], river waters [15], and recently, in coastal waters [13]. The detected levels of HEV RNA shown in the present work were slightly below 10\({}^{2}\) genome copies/g tissue, in agreement with other European studies on shellfish. Two studies in northwestern Spain reported contamination levels varying from 6.7 \(\times\) 10\({}^{1}\) to 8.6 \(\times\) 10\({}^{4}\) gc/g [11] and from non-quantifiable to 1.1 \(\times\) 10\({}^{5}\) gc/g [24]. In the latter study, 19.5% of positive samples presented viral genome copies below the limit of quantification, and one had a concentration of 4.0 \(\times\) 10\({}^{2}\) gc/g. A study from Scotland also reported a very low virus concentration in commercial mussels with only one sample quantifiable at \(<\)10\({}^{2}\) IU/mL [12], and a study from Italy showed that concentrations of HEV in shellfish were always below 10\({}^{2}\) gc/g [13]. Although a low concentration was estimated in the present study (2.5 \(\times\) 10\({}^{2}\) and 7.0 \(\times\) 10\({}^{1}\) gc/g), this cannot be overlooked. For other foodborne viruses, several surveys on shellfish-related outbreaks showed as very low viral genome levels (10-100 gc/g) were correlated to the probability of infections with NoV or HAV [30; 31]. Contrary, for HEV there are current data gaps regarding the relationship between genome copies numbers and infective dose [32]. The existence of HEV in shellfish may be associated with the pollution of the shellfish harvesting area by swine waste and urban sewage [11; 12]. The increasing number of patients in Italy with hepatitis E over the last years [10] may support the recent reports of HEV in shellfish.