Hepatitis E virus (HEV) is the causative agent of hepatitis E, and is a positive sense single stranded RNAvirus. The HEV genome is approximately 7.2 kb and contains three open reading frames. ORF2 encodes the major capsid protein. HEV causes large epidemics in developing countries, and is emerging in inchstrialized areas, which accounts for an increasing number of sporadic cases. Genetic similarity between human and swine HEV strains from the same area suggests zoonotic transmission, which might play an important role in spreading infection in Europe and other industrialized areas. Recently, hepatitis E cases have been linked to eating raw or undercooked meat from deer, wild boar or pigs. In 2006, we evaluated the presenceof HEV in swine farms in Northern Italy. The 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. Adeletion fragment lacking the first 111 aa at the N-terminal 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 the Italian strain, presenting an 85% identity with the genotype 3 prototype swine HEV strain described by Meng et al. in the US in 1997. The bacmide with the HEVA1110RF2 was transfected into Sf9 and High Tn5 insect cells, and the BacHEVA lllORF2virus stock obtained was used to express the HEV capsid protein. Similar protein expression was obtained in Sf9 and Tn5 cells under serum-free culture conditions. One protein band of 55 kDa was detected by western blotting using a pig serum in either cell lysate and supernatant. Self-assembly of capsid protein into virus-like particles was investigated by electron microscopy.

Cloning and expression of a swine Hepatitis E virus capsid protein in insect cells

MARTELLI, FRANCESCA;OSTANELLO, FABIO;
2008

Abstract

Hepatitis E virus (HEV) is the causative agent of hepatitis E, and is a positive sense single stranded RNAvirus. The HEV genome is approximately 7.2 kb and contains three open reading frames. ORF2 encodes the major capsid protein. HEV causes large epidemics in developing countries, and is emerging in inchstrialized areas, which accounts for an increasing number of sporadic cases. Genetic similarity between human and swine HEV strains from the same area suggests zoonotic transmission, which might play an important role in spreading infection in Europe and other industrialized areas. Recently, hepatitis E cases have been linked to eating raw or undercooked meat from deer, wild boar or pigs. In 2006, we evaluated the presenceof HEV in swine farms in Northern Italy. The 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. Adeletion fragment lacking the first 111 aa at the N-terminal 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 the Italian strain, presenting an 85% identity with the genotype 3 prototype swine HEV strain described by Meng et al. in the US in 1997. The bacmide with the HEVA1110RF2 was transfected into Sf9 and High Tn5 insect cells, and the BacHEVA lllORF2virus stock obtained was used to express the HEV capsid protein. Similar protein expression was obtained in Sf9 and Tn5 cells under serum-free culture conditions. One protein band of 55 kDa was detected by western blotting using a pig serum in either cell lysate and supernatant. Self-assembly of capsid protein into virus-like particles was investigated by electron microscopy.
Proceeding of the 4th Med-Vet-Net Annual Scientific Meeting
38
38
Di Bartolo I.; Ponterio E.; Martelli F.; Ostanello F.; Ruggeri F.M.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/61467
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