title: Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.
abstract: Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.
authors.affiliation: Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Price's Fork Rd., Blacksburg, VA 24061-0342, USA.
authors.forename: Y W
authors.initials: YW
authors.lastname: Huang
authors.forename: G
authors.initials: G
authors.lastname: Haqshenas
authors.forename: C
authors.initials: C
authors.lastname: Kasorndorkbua
authors.forename: P G
authors.initials: PG
authors.lastname: Halbur
authors.forename: S U
authors.initials: SU
authors.lastname: Emerson
authors.forename: X J
authors.initials: XJ
authors.lastname: Meng
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D016751:Hepatitis E / Q000662:veterinary*; D016752:Hepatitis E virus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D006524:Hepatitis, Viral, Animal / Q000821:virology; D006801:Humans; D008099:Liver / Q000821:virology; D008969:Molecular Sequence Data; D012315:RNA Caps / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013552:Swine / Q000821:virology*; D013553:Swine Diseases / Q000821:virology; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D012315:RNA Caps; D012367:RNA, Viral