Construction of an infectious cDNA clone of a swine genotype 3 HEV strain isolated in Shanghai, China.
OBJECTIVE
Infectious cDNA clones are important tools for studying molecular mechanisms in RNA viruses. The aim of this study was to construct an infectious cDNA clone for SAAS-JDY5, which is a genotype 3 HEV strain of swine origin.


METHODS
Construction employed overlapping PCR and restriction analysis to ligate nine cDNA fragments into a full-length cDNA clone containing 14 mutations compared to the consensus HEV genome sequence. Megaprimer PCR-directed mutagenesis restored nine non-silent mutations back to the consensus sequence while the other five silent mutations were maintained as genetic markers.


RESULTS
HEV proteins were identified by an immunoﬂuorescence assay in Huh7 cells infected with capped RNA transcripts of the full-length cDNA clone, while HEV viremia, fecal HEV RNA and seroconversion were recorded in inoculated Sprague-Dawley rats.


CONCLUSIONS
Our data confirmed the successful construction of an infectious cDNA clone of swine HEV strain pGEM4z-SAAS-JDY5, and support the use of rats as an HEV infectious model.
Institute of Animal Science and Veterinary Medicine and Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai, PR China.
Fusheng
F
Si
Biao
B
Shi
Xi
X
Wang
Yumin
Y
Zhu
Xia
X
Liu
Qian
Q
Yang
Zhen
Z
Li
D000818:Animals; D002681:China; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004279:DNA, Viral; D004195:Disease Models, Animal; D005260:Female; D005838:Genotype; D016751:Hepatitis E / Q000662:veterinary* / Q000821:virology; D016752:Hepatitis E virus / Q000235:genetics* / Q000302:isolation & purification*; D009154:Mutation; D017207:Rats, Sprague-Dawley; D059386:Reverse Genetics / Q000379:methods; D013552:Swine; D013553:Swine Diseases / Q000821:virology*
D018076:DNA, Complementary; D004279:DNA, Viral