The article describes the molecular cloning and sequencing of the full-length viral genome of hepatitis E virus (HEV), a nonenveloped, single-stranded, positive-sense RNA virus. The HEV genome consists of approximately 7.5 kb, with a polyadenylated 3' end. The authors report on the cloning and nucleotide sequencing of an overlapping set of cDNA clones representing the entire genome of the HEV Burma strain (HEV(B)). The largest open reading frame extends approximately 5 kb from the 5' end and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 bp downstream of the first and extends approximately 2 kb to the termination codon. ORF2 contains a consensus signal peptide sequence and a capsid-like region with a high content of basic amino acids. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver. The genomic organization of the virus is consistent with the 5' end encoding nonstructural genes and the 3' end encoding the viral structural genes. The expression strategy involves the use of three different open reading frames and at least three different transcripts. The findings suggest that HEV may represent a new class of RNA virus or a separate genus within the Caliciviridae family.The article describes the molecular cloning and sequencing of the full-length viral genome of hepatitis E virus (HEV), a nonenveloped, single-stranded, positive-sense RNA virus. The HEV genome consists of approximately 7.5 kb, with a polyadenylated 3' end. The authors report on the cloning and nucleotide sequencing of an overlapping set of cDNA clones representing the entire genome of the HEV Burma strain (HEV(B)). The largest open reading frame extends approximately 5 kb from the 5' end and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 bp downstream of the first and extends approximately 2 kb to the termination codon. ORF2 contains a consensus signal peptide sequence and a capsid-like region with a high content of basic amino acids. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver. The genomic organization of the virus is consistent with the 5' end encoding nonstructural genes and the 3' end encoding the viral structural genes. The expression strategy involves the use of three different open reading frames and at least three different transcripts. The findings suggest that HEV may represent a new class of RNA virus or a separate genus within the Caliciviridae family.