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.

FIG. 1.

Construction of full-length cDNA clones of swine HEV. (A) Assembly of a full-length cDNA clone (clone C). The numbers on the scale bar indicate distances from the 5′ end, in kilobases. Eight DNA fragments with unique restriction sites, represented by thick lines, were amplified by RT-PCR and assembled into a full-length cDNA clone in the pGEM-9zf(−) vector. A unique HindIII site and a T7 RNA polymerase core promoter (shaded pentagon) were engineered at the 5′ end, and a 15-nt short poly(A) tail (shaded triangle) as well as a unique XbaI site at the 3′ end of the genome are also indicated. (B) Strategy for correcting mutations in clone C and for generating three new cDNA clones derived from clone C. Three fragments containing the mutations were excised from clone C by the use of restriction enzymes and were then subcloned. After the mutations were corrected by site-directed mutagenesis, the corrected fragments (BX, XAf, and AvN) were used to replace the corresponding fragments in clone C to produce three new full-length cDNA clones, i.e., pSHEV-1, pSHEV-2, and pSHEV-3.

FIG. 2.

Immunofluorescence staining of a subclone of Huh7 cells transfected with similar amounts of capped full-length RNA transcripts. (A and a) Mock transfected; (B and b) clone C; (C and c) pSHEV-1; (D and d) pSHEV-2; (E and e) pSHEV-3. Images labeled with capital letters show the ORF2 protein, and those labeled with lowercase letters show the ORF3 protein.

FIG. 3.

Seroconversion to anti-HEV IgG of pigs inoculated with RNA transcripts from three swine HEV clones. Anti-HEV IgG was plotted as the ELISA optical density (A₄₀₅), and the ELISA cutoff value was 0.3. Pigs 286 and 293 were each inoculated with RNA transcripts from clone pSHEV-1 (A), pigs 294 and 295 were each inoculated with RNA transcripts from clone pSHEV-2 (B), and pigs 297 and 298 were each inoculated with RNA transcripts from clone pSHEV-3 (C). (D) Pigs 281 and 285 were uninoculated controls.