All images are courtesy of the National Cancer Institute
Researchers at Chiron made virology history in 1987 when they discovered the hepatitis C virus (HCV), not by isolating viral particles, but by cloning and sequencing its genome. Subsequently, scientists developed tests for HCV infection and deciphered aspects of its lifecycle. But the virus has stubbornly resisted every attempt to grow it in vitro, leaving major parts of its biology inscrutable.
But not for long: Researchers have finally found a way to produce HCV in the lab, along with two other notoriously difficult-to-grow human pathogens – human papillomavirus (HPV) and Norwalk virus (NV). The developments should ease basic research into these three viruses and aid historically hampered efforts to develop diagnostics, vaccines, and drugs. Yet researchers say chances are slim the methods will directly aid research into other recalcitrant viruses in the future.
"Progress is almost linearly connected with the ability to grow a virus in the lab," says Peter Palese, professor of microbiology at Mount Sinai School of Medicine in New York. Researchers credit Nobel laureates John Enders and Renato Delbucco with developing, in the 1940s and 1950s, techniques to grow viruses in culture. Before that, researchers could only grow viruses by inoculating material from infected patients into animals.
Today, literally hundreds of cell lines are available; yet researchers still run up against viruses they cannot culture. In such cases, the clever application of molecular biology tools combined with perceptive clinical and laboratory observations can break the block.
A TRIO OF BREAKTHROUGHS
For HCV, a clinical oddity provided the key. Four years ago, Takaji Wakita, at the Tokyo Metropolitan Institute for Neurosciences, isolated a strain of HCV genotype 2a from a patient with fulminant hepatitis – an extremely rare complication of HCV infection.1 Every previous attempt to introduce the HCV genome into cell lines had failed to result in viral replication. But working with Ralf Bartenschlager at the University of Heidelberg, Wakita showed that this strain, dubbed JFH1 (for Japanese fulminant hepatitis-1), replicated efficiently when transfected into hepatoma cells.2 Several tests proved the resulting virions are HCV, including biophysical analysis, immunoelectron microscopy visualization, and successful infection of both hepatoma cells and chimpanzees.
Within a month Charles Rice at Rockefeller University in New York City, and Francis Chisari at The Scripps Research Institute in La Jolla, Calif., reported similar findings with related, yet more efficient, cell lines.34
The new system will allow researchers to tackle aspects of the viral lifecycle that were previously inaccessible, such as entry and assembly. "People are pretty excited," notes Rice. Both he and Chisari say they have already received dozens of requests for the system.
Rice's group has begun constructing chimeric viruses using the RNA replication machinery genes from JFH1 and the structural proteins from other strains, particularly types 1a and 1b, the cause of most HCV infections. "I suspect that it won't be long before we're able to replicate other [HCV] strains in the lab," says Stan Lemon, an HCV researcher at the University of Texas Medical Branch in Galveston.
Noroviruses, particularly NV (the "cruise-ship virus"), have also resisted all previous efforts to grow in vitro. In July, Mary Estes at Baylor College of Medicine cleared that hurdle when she reported the first replication and packaging of viral RNA into virus particles by using the MVA/T7 system in human embryonic kidney cells.5
The resulting virions appear to be true NV particles, but researchers aren't certain because no animal model or cell culture system currently exists that permits infection. "This is a first step," Estes says. "In the past we couldn't even show that the genome would replicate; now we can go out and look for a cell line that might support replication." In the meantime, the packaging system, used successfully before to produce infectious particles of related animal viruses, should advance the analysis of NV replication and aid the testing of antiviral drugs, Estes says.
The final viral breakthrough involved HPV. In contrast to HCV and NV, HPV has been produced in the lab for more than a decade, but only via time-consuming, low-yield techniques such as mouse xenografts and organotypic raft culture systems. Dohun Pyeon, an assistant scientist working in Paul Ahlquist's lab at the University of Wisconsin-Madison, used a viral packaging system developed at the National Cancer Institute to efficiently produce 1,000-times more infectious virus per culture dish than raft culture.6 The resulting viral particles successfully infected an immortalized human keratinocyte cell line.
Michelle Ozbun, at the University of New Mexico School of Medicine, plans to compare the packaged virus to HPV produced in raft culture to look for differences. But regardless of any limitations they may find, she says, "I think it's going to be a really big deal." The system will allow researchers to prepare viral types they haven't been able to grow in raft culture, as well as study mutations that might affect replication.
TOOLS OF THE TRADE
According to Brian Mahy, senior scientific advisor at the National Center for Infectious Diseases, HCV, HPV, and NV were the only significant human viruses scientists could not really grow in the lab. But the methods used to produce them are specific and therefore unlikely to be generally useful to researchers who encounter new difficult-to-grow viruses, says virologist Donald Ganem of the University of California, San Francisco. "There's no uniform strategy here," says Ganem. "It all depends on the architecture of the genome you clone." Virologists nevertheless have a rich set of tools available to them to circumvent future problems.
"Tissue culture remains important, but it has a lot of friends today," says Robert Gallo, director of the Institute of Human Virology at the University of Maryland Biotechnology Institute. Prior to HCV's recent breakthrough, for instance, researchers studied its replication cycle using a replicon system, developed in Barten-schlager's lab in 1999.
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The group derived synthetic RNA "replicons" from a cloned HCV genome by deleting the structural region; when transfected into human hepatoma cells, the molecules produced large quantities of HCV RNAs and proteins. Though the replicons reproduced inefficiently without specific cell culture-adaptive mutations, the system was a boon to researchers previously unable to access this phase of the virus' lifecycle. Ganem believes the replicon system might be generalized to study similar RNA viruses.
More standard techniques play important roles, as well. With even a fragment of viral nucleic acid, scientists can develop antibodies, clone viral proteins, and hunt for the complete genome. PCR probes and microarrays that represent genomes from a range of viruses can help virologists identify and classify new pathogens – information that can help them determine which culture techniques might be effective. And increasingly sensitive molecular assays allow scientists to detect viruses (like retroviruses) that don't display cytopathic effects in cell culture, and thus develop culture systems that might previously have been written off as useless.
MEETING THE NEXT THREAT
As such tools are applied not only to viral research but also to discovery, researchers may come up against more and more "unculturable viruses," say Rice and Ganem. "This is the way most new viruses are going to be discovered in the future," Ganem predicts. "The next viral genome we come up with, we're going to be faced with the same problem."
In some cases, as with HCV and HIV, clinical hints may provide the clues needed to develop culture systems. But such clues are of little help if the target cell itself cannot be cultured. Some viruses, such as poliovirus and herpes simplex virus-1, ignore their natural tropisms and replicate readily in several different cell lines. Others, however – HCV, HPV, and NV among them – seem to infect only highly specialized cells that themselves are difficult to grow in the lab.
Hepatotropic viruses are notoriously difficult. Relatively few hepatic cell lines exist, and none accurately represents tissue in vivo. Even primary hepatocytes, when cultured, begin to lose liver-specific gene expression within just a few days, limiting their utility. As a result, though culture systems exist for, say, hepatitis B virus, researchers still lack the means to infect cell lines with the virus they produce, restricting their ability to study early events in the viral lifecycle such as infection, entry, and uncoating.
Similarly, the HPV lifecycle is tightly linked to skin cell differentiation, and researchers believe NV grows in the highly differentiated cells at the ends of intestinal villi, called enterocytes. "Part of the problem with virus cultivation is that we don't have highly differentiated culture systems for a lot of different cell types," Ganem says. "One of the things that I think would benefit virus cultivation is a more determined effort at developing procedures for cultivating many different types of primary cells."
Tissue engineers may help in that quest. Linda Griffith, professor of biological and mechanical engineering at the Massachusetts Institute of Technology, has been developing a "liver on a chip" – a microfabricated bioreactor designed to mimic three-dimensional tissue structure in vivo. She began the project because of US Department of Defense interest in methods to culture viruses that couldn't be grown any other way.
Griffith says her system already maintains primary rat hepatocytes better than other culture methods, preserving liver-specific transcription and drug metabolic rates for up to three weeks. She plans to begin using human cells soon. She has a long-standing collaboration with Jack Wands, pathology professor at Brown Medical School, to develop a model of hepatitis B infection using the bioreactor. "Viral culture is an area where I think tissue engineering could have a big impact," Griffith says.
While such tools will no doubt help virologists in their efforts to better produce recalcitrant viruses, experts say, in the end, growing a new virus comes down to three things: effort, time, and money. "The important technique in all these cases is persistence," says Ahlquist.