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New Hepatitis C Culture System for Study of Virus
  Scientists a step closer to hepatitis C vaccine
Jan 24, 2007
The Seattle Times
SEATTLE - University of Washington scientists have devised a unique way to grow the hepatitis C virus in the laboratory -- an important step in the quest for a vaccine and improved treatment for what has become one of the most widespread infectious diseases in the world.
The researchers for the first time were able to keep the virus reproducing for at least two months, enabling it to infect liver cells, where it does its devastating work.
"We'll be able to better see what damage is done to cells, and it will provide a way to test antiviral agents... and help develop a vaccine," said Dr. Nelson Fausto, chairman of the UW department of pathology, who directed the research.
Hepatitis C infects about 170 million people worldwide, including more than 4 million in the U.S. The virus is carried in the blood and is usually spread by contaminated needles during drug abuse. It is also spread, rarely, by sex, by an infected mother to her child, or by other contact with infected blood.
The disease becomes chronic in the majority of patients. After 20 to 30 years, about one-fifth of them have liver scarring that can lead to cancer. Hepatitis C liver disease is the leading reason for patients needing a liver transplant.
Fausto and his UW colleagues made headlines last June as the first scientists to isolate human liver stem cells, which are the ancestors of all liver cells. The cells were taken from aborted fetuses donated to research, then grown in the lab and infused in mice, where they replaced dead liver cells.
The team now has used the liver stem cells in developing the new lab culture for hepatitis C, which is to be reported in the February edition of The American Journal of Pathology.
It was a tedious process, taking about four years to perfect, Fausto said. But the researchers proved in several ways that the culture is a nourishing home for the virus.
Scientists injected the cells into a culture with genetic material from the virus. The viruses then reproduced into the culture, the culture itself was mixed with more cells, and those cells, too, became infected.
The team also mixed the blood of patients with different strains of hepatitis C into the culture of liver stem cells. Again, the virus thrived and reproduced.
"I think we finally have it," said Fausto. "We can grow this virus in normal (liver cells)."
Other scientists have grown the virus using cancerous liver cells or using a virus from a patient with a rare case of rapidly escalating hepatitis. But neither is typical of real-life infections, Fausto said.
Fausto said the next step in the research is to see whether laboratory animals can be infected with the laboratory-grown viruses. That would further establish the viability of the culture method as a way to study the virus.
Universities and pharmaceutical companies alike have "tremendous interest" in developing a vaccine and more treatments, because hepatitis C affects so many people, Fausto said.
It is difficult research, he said, but he predicted that a vaccine could be developed in about five years, and testing of it completed in a few more years.
New culture method for hepatitis C virus uses primary hepatocytes and patient serum
Researchers open the way for improved study of hepatitis C virus by devising a novel virus culture system that allows replication of patient-isolated virus in nontransformed hepatocytes, instead of culture-adapted virus strains in transformed cell lines. The related report by Lazaro et al, "Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes," appears in the February issue of The American Journal of Pathology.
Hepatitis C virus (HCV) infection affects approximately 170,000,000 people worldwide. HCV liver disease, which may induce liver inflammation, cirrhosis, and/or hepatocellular carcinoma, represents the foremost reason for liver transplantation in much of the U.S.
Study of HCV replication within liver cells, or hepatocytes, has been hampered by a lack of adequate virus culture systems. Some systems allow the virus to infect cells but do not permit prolonged replication and production of virus, while other systems rely on derivatives of permissive virus isolates for efficient replication in transformed (mutated) cell lines. Still lacking has been a system to sustain replication of novel virus isolates from patients using nontransformed hepatocytes.
Nelson Fausto of the University of Washington School of Medicine has crossed this hurdle using a human fetal hepatocyte culture system that was previously developed in his lab. Using this system, his group has demonstrated sustained replication and production of virus particles for at least 2 months, with these virus particles able to infect new cells.
In their first experiments, Fausto and colleagues transfected hepatocyte cultures with HCV genomic RNA and found replication of HCV RNA genomes and production of core protein (for virus particle formation). Release of infectious virus particles was confirmed, as media from these cells were able to infect naive hepatocytes. Finally, virus particles were examined by electron microscopy and shown to possess the expected size and shape of HCV virus particles.
Once the system was established, the group examined whether sera from patients carrying HCV could infect the human fetal hepatocytes. When sera from patients infected with different HCV strains were added to the hepatocyte culture system, viral replication occurred and new virus particles were produced.
In both transfection and infection models, virus particles were released in a cyclical manner, with bursts of virus produced every 10-14 days. This is similar to what has been reported during clinical HCV infection, possibly due to the host's natural defenses. Interestingly, cultured hepatocytes responded to viral replication by displaying signs of distress and cell death and by expressing interferon-beta, a cellular antiviral, in an effort to control the infection.
This culture system provides a breakthrough in studying HCV replication in nontransformed hepatocytes, the natural target of the virus. By allowing infection by patient serum containing a wide array of virus strains, this system may allow better understanding of the differences between different strains, further improving treatment strategies. Source : American Journal of Pathology
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