Hepatitis E virus (HEV) is an important public health concern in many developing countries. HEV is also endemic in some industrialized counties, including the United States. With our recent discovery of swine HEV in pigs that is genetically closely related to human HEV, hepatitis E is now considered a zoonotic disease. Human strains of HEV are genetically heterogenic. So far in the United States, only one strain of swine HEV has been identified and characterized from a pig. To determine the extent of genetic variations and the nature of swine HEV infections in U.S. pigs, we developed a universal reverse transcription-PCR (RT-PCR) assay that is capable of detecting genetically divergent strains of HEV. By using this universal RT-PCR assay, we tested fecal and serum samples of pigs of 2 to 4 months of age from 37 different U.S. swine farms for the presence of swine HEV RNA. Thirty-four of the 96 pigs (35%) and 20 of the 37 swine herds (54%) tested were positive for swine HEV RNA. The sequences of a 348-bp region within the ORF2 gene of 27 swine HEV isolates from different geographic regions were determined. Sequence analyses revealed that the 27 U.S. swine HEV isolates shared 88 to 100% nucleotide sequence identities with each other and 89 to 98% identities with the prototype U.S. strain of swine HEV. These U.S. swine HEV isolates are only distantly related to the Taiwanese strains of swine HEV, with about 74 to 78% nucleotide sequence identities; to most known human strains of HEV worldwide, with <79% sequence identities; and to avian HEV, with 54 to 56% sequence identities. Phylogenetic analysis showed that all the U.S. swine HEV isolates identified in this study clustered in the same genotype with the prototype U.S. swine HEV and the two U.S. strains of human HEV. The data from this study indicated that swine HEV is widespread and enzoonotic in U.S. swine herds and that, as is with human HEV, swine HEV isolates from different geographic regions of the world are also genetically heterogenic. These data further raise potential concerns for zoonosis, xenozoonosis, and food safety.

Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV Avian hepatitis E virus (avian HEV), recently identified from a chicken with hepatitis-splenomegaly syndrome in the United States, is genetically and antigenically related to human and swine HEVs. In this study, sequencing of the genome was completed and an attempt was made to infect rhesus monkeys with avian HEV. The full-length genome of avian HEV, excluding the poly(A) tail, is 6654 bp in length, which is about 600 bp shorter than that of human and swine HEVs. Similar to human and swine HEV genomes, the avian HEV genome consists of a short 59 non-coding region (NCR) followed by three partially overlapping open reading frames (ORFs) and a 39NCR. Avian HEV shares about 50 % nucleotide sequence identity over the complete genome, 48-51 % identity in ORF1, 46-48 % identity in ORF2 and only 29-34 % identity in ORF3 with human and swine HEV strains. Significant genetic variations such as deletions and insertions, particularly in ORF1 of avian HEV, were observed. However, motifs in the putative functional domains of ORF1, such as the helicase and methyltransferase, were relatively conserved between avian HEV and mammalian HEVs, supporting the conclusion that avian HEV is a member of the genus Hepevirus. Phylogenetic analysis revealed that avian HEV represents a branch distinct from human and swine HEVs. Swine HEV infects non-human primates and possibly humans and thus may be zoonotic. An attempt was made to determine whether avian HEV also infects across species by experimentally inoculating two rhesus monkeys with avian HEV. Evidence of virus infection was not observed in the inoculated monkeys as there was no seroconversion, viraemia, faecal virus shedding or serum liver enzyme elevation. The results from this study confirmed that avian HEV is related to, but distinct from, human and swine HEVs; however, unlike swine HEV, avian HEV is probably not transmissible to non-human primates.

We recently identified and characterized a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly syndrome (HS syndrome) in the United States. Avian HEV is genetically related to but distinct from human and swine HEVs. To determine the extent of genetic variation and the seroprevalence of avian HEV infection in chicken flocks, we genetically identified and characterized 11 additional avian HEV isolates from chickens with HS syndrome and assessed the prevalence of avian HEV antibodies from a total of 1,276 chickens of different ages and breeds from 76 different flocks in five states (California, Colorado, Connecticut, Virginia, and Wisconsin). An enzyme-linked immunosorbent assay using a truncated recombinant avian HEV ORF2 antigen was developed and used to determine avian HEV seroprevalence. About 71% of chicken flocks and 30% of chickens tested in the study were positive for antibodies to avian HEV. About 17% of chickens younger than 18 weeks were seropositive, whereas about 36% of adult chickens were seropositive. By using a reverse transcription-PCR (RT-PCR) assay, we tested 21 bile samples from chickens with HS syndrome in California, Connecticut, New York, and Wisconsin for the presence of avian HEV RNA. Of the 21 bile samples, 12 were positive for 30-to 35-nm HEV-like virus particles by electron microscopy (EM). A total of 11 of the 12 EM-positive bile samples and 6 of the 9 EM-negative bile samples were positive for avian HEV RNA by RT-PCR. The sequences of a 372-bp region within the helicase gene of 11 avian HEV isolates were determined. Sequence analyses revealed that the 11 field isolates of avian HEV had 78 to 100% nucleotide sequence identities to each other, 79 to 88% identities to the prototype avian HEV, 76 to 80% identities to chicken big liver and spleen disease virus, and 56 to 61% identities to other known strains of human and swine HEV. The data from this study indicated that, like swine and human HEVs, avian HEV isolates are genetically heterogenic and that avian HEV infection is enzoonotic in chicken flocks in the United States.