Hepatitis Central

In rapid succession, two research teams have independently isolated an infectious clone of the Hepatitis C virus (HCV), proving that the virus alone is sufficient to cause disease, and broadly expanding the research horizon for HCV investigators.

Charles M. Rice, Ph.D., of Washington University of St. Louis, supported in part through an NIAIDHepatitis C Cooperative Research Center grant, led one team and Robert H. Purcell, M.D., head of the Hepatitis Viruses Section in NIAID’s Laboratory of Infectious Diseases (LID) in Bethesda, led the other.

"This is a major development that no doubt will profoundly influence the field of Hepatitis C research," says NIAID Director Anthony S. Fauci, M.D. "Having an infectious HCV clone available will greatly facilitate studies on virus evolution, pathogenesis, and host immune response. It will enable scientists to better understand the factors and mechanisms that determine whether virus is cleared from the body or produces a chronic infection."

When the Hepatitis C virus was first discovered in 1989, scientists believed they had found the cause of the many cases of hepatitis, often acquired through blood transfusions, that could not be attributed to either the Hepatitis A or B viruses. Since then, researchers have detected HCV genetic sequences and antibodies in patients with classic non-A, non-B hepatitis, and screening the blood supply for HCV has significantly lowered the incidence of this disease.

Nevertheless, scientists have been unable to grow the virus efficiently in tissue culture or to purify it using other laboratory procedures, steps necessary to prove conclusively that a suspected infectious agent can cause disease. Therefore, questions have remained about whether the HCV genetic sequences found in patients truly represent the infectious agent, or are defective or noninfectious versions of the virus.

Drs. Rice and Purcell and their colleagues have now answered these questions. As reported recently in Science, Dr. Rice and his colleagues at Washington University and at the Food and Drug Administration in Bethesda extracted HCV RNA, the virus’s genetic material, from the serum of an individual infected with the virus. They then made thousands of DNA "clones" of the HCV RNA which, in turn, were transcribed back into RNA. They injected the 34 most promising RNA transcripts into the livers of chimpanzees—the only known non-human host for HCV—to see if the transcripts could cause infection.

When none of these RNA transcripts caused infection in the chimpanzees, Dr. Rice and his colleagues analyzed the sequences of numerous HCV clones to find a "consensus sequence" of highly conserved regions within the HCV genome. The researchers then constructed a full-length "consensus clone," reflecting the consensus sequence. RNA transcripts of the consensus clone, which had been tagged with a unique molecular identification marker, were then injected directly into different regions of the livers of two healthy chimpanzees.

In the weeks following injection, the researchers detected signs of infection in the chimpanzees, including enzymes produced in response to liver damage, antibodies to HCV, and the presence of HCV RNA. Liver biopsies of each animal also showed tissue damage typical of that seen in HCV-infected chimpanzees. The identification marker on the HCV RNA revealed that the consensus clone had caused infection.

"The RNA levels in the chimps increased steadily in the weeks following inoculation," says Dr. Rice. "If the RNA had come from the original inoculum, instead of from new virus particles, its concentration would have decreased. The RNA also was resistant to RNase, an enzyme that breaks down naked RNA. The enzyme does not degrade RNA that is inside viral particles."

Reporting in the Proceedings of the National Academy of Sciences, Dr. Purcell and his LID colleagues recently confirmed these findings. Using slightly different techniques, the NIAID group also constructed a consensus clone of the same HCV strain as that used by the St. Louis team. RNAtranscripts of the consensus clone injected into the liver of one chimpanzee caused the chimpanzee’s HCV RNA levels to rise over time. The genetic sequence of the HCV recovered from the chimpanzee matched that of the consensus clone, indicating that the clone had caused infection.

The new finding should be a boon to HCV research. "Viral clones can now be used to establish infections and study the genetics of HCV replication in laboratory animals," says John La Montagne, Ph.D., director of NIAID’s Division of Microbiology and Infectious Diseases. "Importantly," he adds, "large quantities of infectious HCV RNAs can be produced and used to find ways to grow the virus in cell culture."

—by John Bowersox

References: AA Kolykhalov et al. Science 277:570-74 (1997); M Yanagi, RHPurcell, SUEmerson, and J Bukh

PNAS94:8738-43 (1997)

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July 1997