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Culturing Hepatitis C for new drugs
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An unusual HCV strain opens the pathogen's entire life cycle to scrutiny.
http://www.the-scientist.com
Hepatitis C virus (HCV) has been difficult to study since its discovery in 1989; its unculturable status left researchers with a narrow view of the viral life cycle. But the discovery a few years ago of a highly infectious HCV strain (JFH-1) by Takaji Wakita's lab, then at Tokyo Metropolitan Institute of Neuroscience, raised the possibility of developing a robust cell culture system. JFH-1 was a huge find, says Frank Chisari, from Scripps Research Institute, and Wakita gladly shared it.
The race to develop an efficient cell culture system with JFH-1 was a short one, and in the Hot Papers featured here, several labs showed that JFH-1 could produce high levels of infectious particles in vitro. Chisari's lab demonstrated that JFH-1 could be cultured in human hepatoma Huh-7-dervied cell lines. 1 In a different approach, Charles Rice's group at Rockefeller University cultured chimeras of JFH-1 to high levels, showing that the resulting virus particles could be neutralized with HCV antibody. 2 In a third paper, Ralf Bartenschlager's group at University of Heidelberg, in collaboration with Wakita, now at the National Institute of Infectious Diseases in Tokyo, replicated large amounts of JFH-1, and for the first time viewed the virions by immunoelectron microscopy. 3
With HCV persistently infecting 170 million people worldwide and causing liver disease in many, the cell culture systems promised to speed HCV research. "It was like going from virtually nothing to a workable system for genetics, cell biology, and drug screening," says Rice. In the time since, the systems have spread through the field, as researchers hurry to experiment with viral entry, assembly, and release.
Wide Open Possibilities
Thomas Baumert, an HCV researcher at the University of Freiburg, says the entire field was "waiting for this long sought after cell culture system, since we could now do functional genomics, test new vaccine approaches, and analyze the whole life cycle." Such wide open possibilities spurred Baumert to set up a JFH-1-based system in his own lab, where infection rates were found to be about "100-fold higher than all previous systems."
One of the first things Bartenschlager's lab did with such high infection rates was to create luciferase reporter viruses. This allowed them to quantify early steps of the HCV life cycle with authentic viral particles and to show that different chimeric viruses seem to use a conserved mode of entry. 4 An explosion of such work is just on the horizon, says Chisari, whose lab is now working to modify the HCV culture system to produce persistent infections. "Most infections are chronic in the vast majority of patients," says Chisari, "so we want to maintain infections continuously in tissue culture to study how the virus adapts."
Rice says his lab is also using the system "24/7," in particular looking into its biological relevance. Before the discovery of JFH-1, HCV researchers could infect chimpanzees with various HCV strains, but they either didn't replicate in cell culture or they replicated but without producing infectious virus. Rice's lab recently showed that virus produced in cell culture can establish long-term infections in chimpanzees and that re-isolated virus was still highly infectious when returned to cell culture. 5 "This underpins the exceptional properties of this [JFH-1] isolate," says Bartenschlager, "since normally when isolates from patients are put into cell culture, it's hard to measure anything."
"Ideally we still want to work with natural isolates of any type at some point." - Charles Rice
An Oddity
Yet the unique properties of JFH-1 leave room for criticisms of the culture systems, says Stanley Lemon at the University of Texas Medical Branch. JFH-1 was isolated from a patient with fulminant hepatitis C - a rare form of the disease with rapid onset - and its unusual infectivity does raise questions about whether findings can be applied to other HCV isolates. Moreover, "JFH-1 is genotype 2a, and the real medical problem is with genotype 1 viruses which are more prevalent," says Lemon. Chisari, Rice, and Bartenschlager all agree. While chimeras between JFH-1 and genotype 1 viruses can help solve this problem, "ideally we still want to work with natural isolates of any type at some point," says Rice.
Despite the caveats, these culture systems do open the search for new therapeutics. An HCV replicon system, created in 1999, allowed for the study of RNA replication. "Most of the HCV drugs currently in clinical or preclinical phase are polymerase or protease inhibitors tested with the replicon system," says Chao Lin from Vertex Pharmaceuticals, whose own drug, VX-950, an inhibitor of HCV NS3-4A protease, was tested in this way. "But with the new systems you can start to test for HCV entry, fusion, or assembly inhibitors," says Lin.
Nevertheless, all the developments in HCV cell culture systems point to one crucial question: Why is JFH-1 so infectious? Answers are only speculative at this point, but "there is a big race on to find out what is special about this 2a isolate," says Rice. The answer could reveal a good deal about why some HCV strains remain uncultured.
References
1. J. Zhong et al., "Robust hepatitis C virus infection in vitro," Proc Natl Acad Sci, 102:9294-9, 2005. (Cited in 83 papers) | [PubMed]
2. B.D. Lindenbach et al., "Complete replication of hepatitis C virus in cell culture," Science, 309:623-6, 2005. (Cited in 88 papers) | [PubMed]
3. T. Wakita et al., "Production of infectious hepatitis C virus in tissue culture from a cloned viral genome," Nat Med, 11:791-6, 2005. (Cited in 97 papers) | [PubMed]
4. G. Koutsoudakis et al., "Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses," J Virol, 80:5308-20, June 2006. | [PubMed]
5. B.D. Lindenbach et al., "Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro," Proc Natl Acad Sci, 103:3805-9, March 7, 2006. | [PubMed]
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