Detection of hepatitis E virus (HEV) in Italian pig herds

Hepatitis E is a public health concern in many developing countries, where it is primarily transmitted by the faecal-oral route through contaminated water and food . The disease is caused by a small, non-enveloped ssRNA virus classified as the separate genus Hepevirus. Although hepatitis E disease occurs only sporadically in countries with good health care systems, the seroprevalence in healthy individuals can be high. Porcine hepatitis E virus (HEV) is not pathogenic to general pig populations, but there is evidence that the virus may be a zoonotic agent and that animal reservoirs may exist. Experimental interspecies transmission of HEV between non-human primates and pigs has been demonstrated , and seroepidemiological studies have shown that pig handlers are at higher risk of HEV infection than the general population. In Japan, studies have supported the possibility of zoonotic transmission, as consumption of undercooked pig organs or meat and, in one case, of deer meat, was closely linked to cases of hepatitis E in human beings. The first porcine strain of HEV was characterised in the USA in 1997. Since then, several other porcine strains have been described worldwide. In the past few years, sporadic cases of autochthonous hepatitis E in human beings have been reported in several European countries, including Italy. In many of these cases, the infecting HEV strain showed a high degree of homology with porcine strains of HEV detected in the same country. In recent years in Europe, HEV in pig herds has been reported only in Spain, the UK and the Netherlands. Thirty-four faecal and 22 serum samples were collected from five different farrow-to-finish farms located in northcentral Italy. Samples were collected from healthy pigs between 2 and 5 months of age. Faecal samples represented pools of faeces from animals of the same age group. Total RNA was extracted from 140 µl of faecal suspension or serum. Template cDNA was reverse transcribed using random hexamers, according to standard protocols. A 145 base pair (bp) fragment of the ORF2 of HEV was amplified from the prepared cDNA by nested PCR. A faecal suspension from a human patient with hepatitis E was used as a positive control. Nested RT-PCR products were visualised on 2% agarose gel, and bands of the correct size were excised and purified. Nucleotide sequencing was performed using the ABI PRISM BigDye Terminator kit. Sequences were assembled with SEQMAN, and alignment was performed using the ClustalX algorithm. The HEV genome was detected in 2 faecal pools (5,9 %) collected at 2 different farms, but all the serum samples were negative. The positive faecal pools were obtained from groups of pigs aged 4,5 and 2,5 months, respectively. Phylogenetic analysis of the two viral sequences (113 bp), was performed by the neighbour-joining method. Bootstrap confidence values were calculated by using the Seqboot and Consense programs. A phylogenetic tree was created with the Treeview software using an avian HEV isolate as out group. Phylogenetic analysis of the viral sequences showed that the 2 Italian strains, HEVBO/01 and HEVPI/01, belonged to genotype 3, as did other porcine and human HEV strains indigenous to Europe. However, they differed significantly from each other, being only 84% identical (18 nucleotide changes). The 2 Italian strains clustered with strains from countries where HEV is considered non-endemic. In particular, HEVPI/01 was related (with 90% identity) to a human strain (AY540113) detected in a sporadic case of acute autochthonous hepatitis E in Spain. This report represents the first description of HEV in Italian pig herds, and confirms the presence of the virus in apparently healthy pigs. These findings are important, because of the potential risk of transmission of porcine HEV to human beings, either by contact with infected pigs or by ingestion of contaminated undercooked meat.