"Although HCV was strongly linked to blood-transmitted hepatitis, it was still possible that an additional, unidentified agent was required for disease," says Charles M. Rice, Ph.D., head of the research team. "Our study essentially eliminates this possibility."
The researchers constructed differing sequences of the virus's ribonucleic acid (RNA) the genetic material of HCV and inoculated them into chimpanzees. This enabled them to identify the necessary elements for infection. The availability of unlimited quantities of this well-defined RNA will permit precise studies of how the virus turns itself into more virus. This information is essential to the development of better treatments for hepatitis C, the most common cause of liver failure in the United States.
Rice is a professor of molecular microbiology at Washington University School of Medicine in St. Louis. Research associate Alexander A. Kolykhalov, Ph.D., is lead author of the paper. Scientists at the U.S. Food and Drug Administration in Bethesda, Md., also played a pivotal role in the study.
Hepatitis C is a chronic disease that inflames the liver, producing fatigue and flu-like symptoms. The current therapy interferon fails to cure 80 percent of those infected. And about 20 percent of people with chronic HCV develop cirrhosis of the liver, which often leads to liver failure and, in some cases, liver cancer.
About 1 percent of the world's population is infected by HCV, though the rate may reach more than 10 percent in some countries. In the United States, the virus was the most common cause of transfusion-associated hepatitis before the development of screening tests for blood donors. It still spreads rapidly among intravenous drug users.
Wanting to define the exact genetic sequence that causes disease, the Washington University scientists derived copies of the RNA from a clinical sample of HCV. Like someone twirling a combination lock, they assembled overlapping pieces to create thousands of different clones. Screening these clones, Kolykhalov identified 34 that might cause infection.
Since the virus replicates poorly in cell cultures, FDA researchers Stephen Feinstone, M.D., and Kathleen Mihalik injected the 34 clonal RNAs into the livers of two chimpanzees, the only animal model for HCV infection. But neither chimp showed any sign of infection.
Rice's group then compared six of these clones to see which nucleotide occurred most often at each position. This consensus sequence on paper at that point matched none of the previously sequenced clones.
The researchers therefore constructed lengths of RNA with the consensus sequence to see if these would be infectious. They also made nine variants to include potentially important differences at or near the ends of the viral RNA.
The FDA researchers inoculated each of the 10 RNA sequences into different regions of a chimpanzee's liver. They used a different technique to inoculate lower doses into a second animal.
A week after inoculation, circulating viral RNA clearly was detectable in the animals' blood. In subsequent weeks, levels rose steadily. The animals' serum also developed markers for liver damage, and biopsies revealed signs of disease in parts of the liver that had received certain of the constructs.
"The contrast between these results and those from our failed experiments provided some of the strongest evidence that at least one of the inoculated RNA sequences was infectious," Feinstone says. And several tests confirmed that the circulating RNA was inside new virus particles and was not just inoculum leaked from the liver.
Because the researchers had tagged the different variants with silent changes in the genetic letters, they could determine which ones had proved infectious. The consensus strand alone turned out to be sufficient to cause disease.
The group now wants to compare animals that get rid of the virus with those that establish a long-lasting infection. "We need to know what kinds of immune responses eliminate the virus and how HCV manipulates the immune system if we're to develop effective vaccines or immunotherapies," Rice explains. "The ability to infect chimpanzees with a defined HCV sequence also provides an unprecedented opportunity for studying HCV evolution and the immune system's response."
The availability of this infectious sequence of RNA also will enable researchers to knock out HCV genes to see which are essential for replication. The products of such viral genes are attractive targets for the development of drugs such as protease inhibitors, which are so effective against HIV. "The lack of a genetic system for studying HCV replication was a real problem previously," Rice says.
Both Feinstone's group and Rice's group are transfecting various cells with the cloned RNA in hopes of developing cell culture models and alternative animal models, such as mice. These systems would enhance studies of the infectious process and be a boon to the pharmaceutical industry. Many companies are trying to develop better treatments for hepatitis C, but the lack of experimental systems for evaluating the effects of candidate drugs on HCV replication has hampered progress.
"The fact that interferon therapy works for at least 10 to 20 percent of patients tells you that hepatitis C is a curable disease," Rice says. "With better drugs, there's a reasonable chance we could control or perhaps eliminate this virus from the majority of people who are chronically infected."
Background
How does hepatitis C differ from other forms of hepatitis?
- There are five varieties of hepatitis: A through E. Hepatitis A is an acute disease involving liver inflammation. It is acquired though close
contact with an infected person or ingestion of feces-tainted water or food. Hepatitis B also is transmitted by blood or close personal contact,
and it causes chronic disease in about 10 percent of those infected. Chronic HBV infection, like HCV, damages the liver and can lead to cirrhosis
or liver cancer. Hepatitis C produces similar symptoms to B but is transmitted mainly in blood. Hepatitis D infects only those with hepatitis B,
while hepatitis E is similar to hepatitis A but is rare in this country.
- The current risk is extremely small. Since 1990, the blood supply in the United States has been screened for HCV. But even before serological
screening for HCV, elimination of donors at high risk for HIV also has lowered the risk of acquiring HCV.
- About 25 percent of liver cancer cases in the United States are associated with HCV. In Japan, the proportion is as high as 60 percent.
- The HCV genome is a single strand of RNA. In a liver cell, this + strand functions as a messenger RNA and then is copied into a - strand, which
acts as a template for making more + strands. The new + strands are then packaged inside protein-lipid envelopes to make new virus particles. The
viral genome codes for some of the enzymes needed for this replication, but the virus probably also recruits some liver cell functions.
- Initial reports indicated that HCV RNA might have a tail of A or U bases. And in 1995, both Rice's group and a Japanese group found that the virus
initially cloned in 1989 was incomplete it lacked a terminal section (98 nucleotides) that had changed little during evolution and therefore was
likely to be an essential part of the genome. So in early attempts to assemble infectious RNA from HCV, researchers felt like someone trying to
build a stereo without knowing what all the components should be.
- The RNA levels increased steadily in the weeks following the inoculation. If the RNA had come from the original inoculum, 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.
###
Kolykhalov AA, Agapov EV, Blight KJ, Mihalik K, Feinstone SM, Rice CM. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277, 570-574, July 25, 1997. Grants from the National Institutes of Health supported this research.
Journal
Science