Avian hepatitis E virus, vaccines and methods of protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis E

#The material on a single compact disc containing a Sequence Listing file provided in this application is incorporated by reference. The date of creation is Sep. 9, 2002 and the size is approximately 21.4 KB.

#1. Field of the Invention

#The present invention concerns a novel avian hepatitis E virus, immunogenic compositions, diagnostic reagents, vaccines and methods of detecting or protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis E.

#The background of the invention and its departure from the art will be further described hereinbelow with reference to the accompanying drawings, wherein:

#FIG. 1 shows the amplification of the 3′ half of the avian HEV genome by RT-PCR: Lane M, 1 kb ladder; Lanes 1 and 2, PCR with ampliTaq gold polymerase; Lanes 3 and 4, PCR with ampliTaq gold polymerase in the presence of 5% v/v dimethyl sulfoxide (hereinafter referred to as “DMSO”); Lane 5 and 6, PCR amplification with a mixture of Taq polymerase and pfu containing in an eLONGase® Kit (GIBCO-BRL, Gaithersburg, Md.).

#FIGS. 2A and 2B represent the amino acid sequence alignment of the putative RNA-dependent RNA polymerase (RdRp) gene of avian HEV (which corresponds to SEQ ID NO:4) with that of known HEV strains. The conserved GDD motif is underlined. The sequence of the prototype Burmese strain is shown on top, and only differences are indicated. Deletions are indicated by hyphens (-).

#In accordance with the present invention, there is provided a novel avian hepatitis E virus (hereinafter referred to as “avian HEV”). The new animal strain of HEV, avian HEV, has been identified and genetically characterized from chickens with HS syndrome in the United States. Like swine HEV, the avian HEV identified in this invention is genetically related to human HEV strains. Unlike swine HEV that causes only subclinical infection and mild microscopic liver lesions in pigs, avian HEV is associated with a disease (HS syndrome) in chickens. Advantageously, therefore, avian HEV infection in chickens provides a superior, viable animal model to study human HEV replication and pathogenesis.

#Electron microscopy examination of bile samples of chickens with HS syndrome revealed virus-like particles. The virus was biologically amplified in embryonated chicken eggs, and a novel virus genetically related to human HEV was identified from bile samples. The 3′ half of the viral genome of approximately 4 kb was amplified by reverse-transcription polymerase chain reaction (RT-PCR) and sequenced. Sequence analyses of this genomic region revealed that it contains the complete 3′ noncoding region, the complete ORFs 2 and 3 genes, the complete RNA-dependent RNA polymerase (RdRp) gene and a partial helicase gene of the ORF1. The helicase gene is most conserved between avian HEV and other HEV strains, displaying 58 to 60% amino acid sequence identities.

#By comparing the ORF2 sequence of avian HEV with that of known HEV strains, a major deletion of 54 amino acid residues between the putative signal peptide sequence and the conserved tetrapeptide APLT of ORF2 was identified in the avian HEV. As described herein, phylogenetic analysis indicated that avian HEV is related to known HEV strains such as the well-characterized human and swine HEV. Conserved regions of amino acid sequences exist among the ORF2 capsid proteins of avian HEV, swine HEV and human HEV. The close genetic-relatedness of avian HEV with human and swine strains of HEV suggests avian, swine and human HEV all belong to the same virus family. The avian HEV of the present invention is the most divergent strain of HEV identified thus far. This discovery has important implications for HEV animal model, nomenclature and epidemiology, and for vaccine development against chicken HS, swine hepatitis E and human hepatitis E.