RESUMO
The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.
RESUMO
Hepatitis E virus (HEV) is the most frequent pathogen of a fecal-oral transmitted hepatitis in humans in many developing countries. It is endemic in the Middle East, in India, in Southeast Asia, central Asia as well as in Central and South America. It can be predominantly found in young adults. The mortality rate comes up to 2% whereas in pregnant women death rate can reach about 25%. Until a few years ago, acute hepatitis E was thought to be rare in industrialized countries: most cases were found in persons with a corresponding travel history. However, after improvement of the diagnostic possibilities, an increasing number of autochthonous cases with acute hepatitis E have become evident. Besides, a relatively high seroprevalence of 1 to 5% in the population in industrialized countries is noticeable which is converse to the only sporadic occurrence of acute hepatitis E. As a reason for this an animal reservoir for HEV is being discussed. The first HEV recovering from an animal succeeded in 1997 from a pig in the USA. Genetic typing showed close relationship between the porcine HEVand human HEV types. This confirmed the suspicion on a zoonotic potential of HEV. Transmission experiments revealed that non-human primates were sensitive to the porcine HEV and pigs could be infected with human HEV. Besides, occupational groups with close contact to pigs evidently have an increased risk for HEV infections. In addition, reports of acute hepatitis E after the consumption of undercooked wild boar meat supported the zoonotic potential of HEV. In the domestic pig and wild boar population in several European countries the virus is ubiquitous. In Germany a high percentage of the wild boars was proven to be infected with HEV. In contrast, studies of HEV circulation in domestic pigs are still pending.
