Hepatitis E virus (HEV) is the causative agent of hepatitis E, and is a positive sense single-stranded RNA virus. Genetic similarity between human and swine HEV strains from the same area suggests zoonotic transmission. In 2006, we evaluated the presence of HEV in swine farms in Northern Italy. Results of molecular diagnosis indicated a wide presence of genotype 3 HEV strains, predominating in pigs worldwide. Viral RNAs from positive samples were used to obtain full length ORF2 fragments. A deletion fragment lacking the first 11 1 aa at the N-termina1 portion of the capsid protein was cloned in the Baculovirus system. The sequence of the recombinant construct confirmed the proper frame of ORF2, and the genotype 3 specificity of the Italian strain. The bacmide with the HEVAl llORF2 was transfected into Sf9 and High Tn5 insect cells, and the Bac HEVAl l l ORF2 virus stock obtained was used to express the HEV capsid protein. One protein band of 50 kDa was detected by Western blotting using a pig serum (kindly provided by N. Pavio, AFSSA) in either cell lysate and supernatant. Self-assembly of capsid protein into virus-like particles (VLPs) was investigated by electron microscopy.
Di Bartolo I. , Ponterio E., Inglese N., Martelli F., Caprioli A., Ostanello F., et al. (2008). Espressione della proteina capsidica di un ceppo di epatite E suino e sviluppo di anticorpi monoclonali. PARMA : Stamperia scrl.
Espressione della proteina capsidica di un ceppo di epatite E suino e sviluppo di anticorpi monoclonali
MARTELLI, FRANCESCA;CAPRIOLI, ANDREA;OSTANELLO, FABIO;
2008
Abstract
Hepatitis E virus (HEV) is the causative agent of hepatitis E, and is a positive sense single-stranded RNA virus. Genetic similarity between human and swine HEV strains from the same area suggests zoonotic transmission. In 2006, we evaluated the presence of HEV in swine farms in Northern Italy. Results of molecular diagnosis indicated a wide presence of genotype 3 HEV strains, predominating in pigs worldwide. Viral RNAs from positive samples were used to obtain full length ORF2 fragments. A deletion fragment lacking the first 11 1 aa at the N-termina1 portion of the capsid protein was cloned in the Baculovirus system. The sequence of the recombinant construct confirmed the proper frame of ORF2, and the genotype 3 specificity of the Italian strain. The bacmide with the HEVAl llORF2 was transfected into Sf9 and High Tn5 insect cells, and the Bac HEVAl l l ORF2 virus stock obtained was used to express the HEV capsid protein. One protein band of 50 kDa was detected by Western blotting using a pig serum (kindly provided by N. Pavio, AFSSA) in either cell lysate and supernatant. Self-assembly of capsid protein into virus-like particles (VLPs) was investigated by electron microscopy.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
