| Hepatitis E is a fecal-oral transmission, zoonotic disease, which is caused by hepatitis E virus (HEV). HEV mainly infect young adults and cause severe liver disease especially in pregnant women, even miscarriage, stillbirth and fatality. Due to the lack of an efficient cell culture system, direct genetic manipulation using infectious cDNA clones has been an indispensable tool for studying HEV replication and pathogenensis. Therefore, the main objectives of this study are (1) to develop an infectious cDNA clone of the swine genotype4HEV strain (SAAS-FX17), and (2) to characterize the infectious activity of this cDNA clone via the direct intrahepatic inoculation of RNA transcripts of the infectious cDNA clone into SD rat.To construct the infectious cDNA clone of SAAS-FX17strain, five overlapping fragments (HEV I-5) covering the full-length viral genome flanked by unique restriction enzyme sites (5’-3’, BamHI-SanDI-HindⅢ-Bg1Ⅱ) were amplified by PCR. A SpeI enzyme site and T7core promoter sequences were engineered at the5’end fragment. A XbaI site was introduced at the3’end of the viral genome. The plasmid pGEM-9Z(-) vector was first modified by replacing the multiclonal sites with the synthetic stuffer fragment generated in this study(SpeI-BamHI-SanDI-HindⅢ-Bg1Ⅱ-XbaⅠ). Subsequently, each of the independent cDNA fragments (HEV1through HEV5) was excised from the recombinant vectors containing each insert, gel purified, and ligated into pGEM-9Z-Stuffer vector after digestion of pGEM-9Z-stuffer vector with the same restriction enzymes. After a total of five overlapping fragments were individually cloned into the pGEM-9Z-Stuffer vector and the assembled final full-length cDNA clone was successively developed and designated pGEM-9Z-HEV.The full-length cDNA clone pGEM-9Z-HEV was linearized by digestion with Xbal. The linearized plasmid DNA was subsequently purified by phenol/chloroform extraction and ethanol precipitation. Capped RNA transcripts from the linearized plasmid DNA were transcribed with T7polymerase using the Mmessage mMACHINE T7kit. Ten, SD rats negative for swine HEV RNA and antibodies were randomly divided into2groups of5rats each. Intrahepatic inoculation of200ul capped RNA transcripts was performed surgically in experimental group.5rats in the negative control group were each inoculated intrahepatically with200μL sterile PBS(PH7.4)Fecal virus shedding in experimental group was detected from2/5rats on23d post-inoculation and5/5on30d and also could be detectable on45d. Only1/5rat had detective viral RNA in feces on52d. To confirm the identity of the virus recovered from infected rats, the PCR products amplified from feces from experimental group were sequences, Sequence analyses confirmed that the PCR product amplified the infected rats originated from the full-length cDNA clones with introduced genetic marker, which suggested that the virus were not from the prototype virus pollution. The HEV seroconversion firstly occurred in rat3,4#and5#on the31st day, which peaked on45d. Whereas, the seroconversion was detected firstly on38d postinfection and reached peak on45th day. The seroconversion was firstly detected on the40th day for rat1#,2#, which peaked on the45th day.None of the negative control rats inoculated intrahapetically with PBS were negative for HEV RNA in feces and seronegative throughout the entire course of the experiment. |
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