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Scientists replicate hepatitis C virus in laboratory


New in vitro model system will allow study of therapeutics and virus life cycle

For the first time, scientists have replicated hepatitis C virus (HCV) in the laboratory. The ability to replicate HCV in cell culture will allow researchers to better study the life cycle and biology of this virus and to test potential antiviral compounds, which may lead to new therapies for the liver disease that results from infection with HCV. Scientists at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), one of the National Institutes of Health (NIH), conducted the study, which appears in the Feb. 15, 2005 issue of Proceedings of the National Academy of Sciences (PNAS).

"Until recently, research on this infectious disease has suffered from the lack of a robust in vitro model system," says T. Jake Liang, M.D., Chief of the Liver Diseases Branch of the NIDDK and co-author of the study. "Our model system produced viral particles that have all the properties of the whole virus. This evidence together with an analysis of the replicated viral RNA supports a conclusion of viral replication and production."

The NIDDK group used a strain of HCV that would have applications to the greatest number of people – genotype 1, the major type of HCV of human infections worldwide and the type most resistant to current therapies. They constructed an HCV replica using a DNA copy of the original HCV single-strand RNA genome. They placed the DNA copy between two ribozymes, RNA molecules that have enzymatic function and can cleave RNA sequence at specific locations. These two ribozymes were designed to generate the correct ends of the HCV genome and to act as start and stop buttons to gene activity. The construct was "naked," meaning that it contained only nucleic acids, the genetic material of the virus, and did not have the HCV viral envelope, a protective shell of lipids and proteins that surrounds the viral RNA in fully-formed HCV. The naked HCV construct was then placed into human liver cells in a cell culture medium.

The NIDDK scientists found evidence of HCV proteins and HCV RNA within the human liver cells in the culture. Electron microscopy showed evidence of high levels of viral particles resembling fully-formed HCV outside of the human liver cells in the culture medium. The researchers believe that the HCV construct contained within the human liver cells behaved like a true HCV infection by producing fully formed copies of the virus and releasing them from the host cell into the culture medium. Further testing is needed before the researchers can determine if the viral particles produced in this system are in fact infectious. Also, this system only represents the tail end of the viral life cycle – viral replication, assembly and release from host cells. Another HCV model system is needed to show the beginning stages of the viral life cycle – viral entry into host cells and viral activity in the host cell before replication.

"With this cell-based system, we can screen compounds with a cell-based assay to look for inhibitors of virus replication," says Liang. "We can also apply this technique to develop model systems for other similar viruses."

HCV is a small, enveloped, single-stranded RNA virus in the family Flaviviridae. HCV is a major cause of liver disease in the United States and the world. One in a series of hepatitis viruses, HCV accounts for about 15 percent of acute hepatitis cases, 60 to 70 percent of chronic hepatitis cases, and up to 50 percent of cases of cirrhosis, end-stage liver disease, and liver cancer. Almost 4 million Americans, or 1.8 percent of the U.S. population, have antibodies to HCV indicating ongoing or previous infection with the virus. Approximately 10,000 to 12,000 deaths each year in the United States are due to HCV.

Marcia Vital | EurekAlert!
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