New Molecular Tools Enable Researchers To Correlate Viruses, Diseases

Viruses, Diseases Author: Karen Young Kreeger Sidebar: Professional Resources for Viral Disease Researchers In the mid- to late 1980s, numerous correlations were discovered between viruses and various types of cancers. For example, Epstein-Barr virus was associated with nasopharyngeal carcinoma and B-cell lymphoma, hepatitis B virus with liver cancer, and human papillomavirus with cervical cancer. Now, a decade later, basic and clinical scientists are finding out that viruses may also play a role in an array of other diseases, like atherosclerosis and diabetes, as well as such mental disorders as schizophrenia. "There are going to be more connections made between viruses and many types of diseases in the future because virologists are turning their attention more and more to diseases to which there is no known cause, like atherosclerosis," says Joseph Melnick, Distinguished Service Professor of virology at Baylor College of Medicine. And, with the advent and now ubiquitous use of an array of molecular-biological tools, scientists are getting better at recognizing viral genomes, and thereby their association with a number of diseases. "We have much better tools with which to find them," remarks Laura Manuelidis, professor and head of the neuropathology section at the Yale University School of Medicine. "For example, PCR [polymerase chain reaction] is very sensitive for detection. Cloning, sequencing, and subtractive techniques have expanded our vision of the viral world. " The emergence and reemergence of infectious agents worldwide have captured the attention of both the popular and scientific press, most recently dozens of international medical journals. Last month the Journal of the American Medical Association featured the causes and consequences of such agents in a special issue (Vol. 275, Jan. 17, 1996). Almost 40 other journals-Lancet and the Indian Journal of Medical Research, for example-also focused on global, emerging microbial threats in their January issues. Virologists cite many reasons for the increasing discovery of relationships between viruses and diseases. Better tools-in the lab, such as PCR, and in the office, such as bioinformatic computer software-are chiefly responsible. These tools have empowered scientists to analyze these relationships "in terms of more complex interactions," according to Baruch Blumberg, Fox Chase Distinguished Scientist and scientific adviser to the president of the Fox Chase Cancer Center in Philadelphia. He specifically points to the "accumulation of enormous amounts of data" from the Human Genome Project. For instance, better data-management computer programs have enabled scientists to archive a great amount of epidemiological, genetic, and biomedical data and to simultaneously consider the multiple environmental and genetic variables influencing the etiology of a disease. "I think it's likely that for any given disease there will be many contributing causative factors, and very often they will be viruses," surmises Blumberg. "That pertains to cancers and many other diseases. What you really want to understand is the interaction of these many factors, because they're the ones where intervention can take place." He cites the example of Korean patients with chronic liver disease who are also carriers of hepatitis B (B. Blumberg, Journal of the Royal College of Physicians of London, 29:31-40, 1995). "People with elevated iron stores are significantly more likely to develop liver cancer than those with lower levels," he explains. Nerve Center: Viruses can play a role in neurodegenerative disorders, according to the research of, from left, Kamel Khalili, Laura Manuelidis, and Royce Waltrip. Neurovirologist Kamel Khalili, director of the molecular neurovirology section at Jefferson Medical College in Philadelphia, adds that advances in the technologies used in cell biology, immunology, and virology, as well as an increasing appreciation by scientists about their interconnection, have also aided in the development of his discipline. Giant technological leaps forward have occurred in the laboratory over the last 40 years: the introduction of tissue culture in the early 1950s; the development of immunocytochemistry years later; and, more recently, the widespread and growing use of PCR and other modern molecular biological techniques (see Tools). "Years ago, with a known disease we'd go to the patient, get specimens, and try to isolate the virus," recalls Melnick. "It was done first in animals. Then tissue culture came along, expanding our horizons enormously. Most of the enteroviruses were discovered because we had tissue culture. If it weren't for this, poliovirus would be in this class by itself. Now it's one of 70 viruses [J.L. Melnick, Biologicals, 21:305-9, 1993]." In the late 1940s, virologists demonstrated that poliovirus could be grown in cultures of human cells. This opened up the way for all sorts of viruses-starting with the enteroviruses-to be purified, visualized with an electron microscope (EM), and analyzed biochemically. One area of research that traces a major step in its evolution to improved cellular techniques is neurovirology, the study and treatment of viral infections of the nervous system (R.T. Richard, Journal of NeuroVirology, 1:2-4, 1995). But the development of this field owes much to research veterinarians and their subjects. In the late 1950s and early 1960s, virologists and veterinary scientists began seeing similarities within the etiology of a seemingly disparate group of neurodegenerative disorders in animals and humans. Yale's Manuelidis traces the association back to Björn Sigurdsson, an Icelandic veterinarian who attributed two relatively obscure diseases of sheep-scrapie and Visna-to what he called slow viruses, because they took so long to act. "We really got into this story because of the late Lewis Thomas, who was chairman of pathology at Yale in 1971," she notes. "He said: 'Why don't you work on scrapie? It's sort of magical, nobody knows what it is.' So we transmitted Creutzfeldt-Jakob disease [CJD]-which is related to scrapie-to rodents, providing the needed models." But it seems the jury is still out on exactly what causes CJD, a rare brain disease marked by premature dementia and loss of motor control. "The bottom line is that there are two basic views about the disease," asserts Manuelidis. "One is that it's caused by a host protein-a prion-that is somehow modified to change and replicate itself. Then there are others who think it's a virus that hasn't yet been identified." In 1982, Stanley B. Prusiner, a professor of neurology and biochemistry at the University of California, San Francisco, suggested that a prion could be a new type of infectious agent that caused scrapie and CJD. His controversial theory eventually won him several awards, most recently the 1995-96 Wolf Prize for medicine (Notebook, The Scientist, Jan. 22, 1996, page 30). Within the last year, Manuelidis's group has published evidence implicating a virus in CJD (L. Manuelidis et al., Proceedings of the National Academy of Sciences, 92:5124-8, 1995). In their experiments, CJD brain fractions that were rich in prion proteins, but low in nucleic acids, were barely infectious. In contrast, samples with proteins attached to nucleic acids-a viral structure-were very infectious, but lost their infectivity when these structures were disrupted. "Neither theory is proven, no matter what anyone tells you," states Manuelidis. "The protein doesn't transmit the disease, and nobody's identified a specific viral nucleic acid or an EM viral particle. "The viral implication of CJD is why I got interested in following work on the Borna disease virus, a natural slow infection of horses, sheep, cats, and ostriches," adds Manuelidis. "The infectious agent was always called a virus, but no one was able to identify it, although they could transmit it to many species. Several years ago two groups-one from Scripps Institute in La Jolla and another from Johns Hopkins-identified a viral sequence, and a year ago a group in Germany finally identified a Borna virus particle by EM." "The idea that Borna might be infecting humans is not new," remarks Royce Waltrip, an assistant professor of psychiatry at the University of Maryland School of Medicine, who is also trained in virology. "I was interested in the possibility of viruses stimulating the immune system to cause disease as a model for schizophrenia." Today there are few solid leads for identifying the causes of schizophrenia. Borna disease virus (BVD) has a predilection to infect the hippocampus and frontal cortex of the brain, which are thought to be associated with schizophrenia. "But no one has looked at the relationship between BVD and schizophrenia systematically," maintains Waltrip. Using the western blot technique to detect the virus, Waltrip and colleagues looked at serum in patients from the National Institute of Mental Health Twin Study unit. In a presentation at the Society for Neuroscience meeting last December in San Diego, Waltrip reported finding antibodies to the virus in blood samples from nine of 25 schizophrenic sets of twins and in patients with the greatest diffuse neurological abnormalities. The need to combine clinical and basic biomedical research characteristic of Waltrip's work is what fertilized the birth of the Journal of NeuroVirology in March 1995. The publication was started by a group of scientists who hope to form a professional society in the next two to three years. "Several years ago at an NIH-sponsored meeting, the Workshop on NeuroAIDS, it was clear to the two distinct groups of attendees that a more accessible and consistent forum was needed to facilitate communication between clinicians and basic scientists with a common goal," recalls Jefferson's Khalili, editor-in-chief of the journal. Melnick, along with celebrated heart surgeon Michael DeBakey and Ervin Adam, a professor of virology-his Baylor colleagues-are particularly interested in cytomegalovirus (a human herpesvirus that often causes opportunistic infections in immune-suppressed heart-transplant and AIDS patients) and human atherosclerosis (J.L. Melnick, Bioessays, 17:899-903,1995). "A virus in the herpes family produces atherosclerosis in chickens, in addition to leukemia, which everyone knew it caused," says Melnick. "The lesions that our veterinary medical colleagues at Cornell University described were like the lesions we see in people with atherosclerosis. We decided to take a look." One immediate impetus for this line of questioning is that atherosclerosis accounts for half the deaths in the United States. "We're still working on tying the connection together. We've never observed an infectious cytomegalovirus, but do find nucleic acid sequences of the virus from arterial lesions in about 80 percent of the patients with atherosclerosis. One thing that's fascinating to me is that clinicians, by means of echograms, have found that people who have developed thick arterial walls are those with cytomegalovirus in these lesions [J.L. Melnick et al., Journal of Medical Virology, 42:170-4, 1994; P.D. Sorlie et al., J. Med. Virol., 42:33-7, 1994]." Over the last 20 years, viruses have been implicated in a range of cancers, especially lymphomas. Today, cancer biologists and virologists estimate that about one in seven cancers worldwide is strongly linked to viruses. About 80 percent of these are cervical and liver cancers, caused by human papillomavirus and hepatitis B virus, respectively. The Epstein-Barr virus (EBV) accounts for most of the remainder. Recently, two intertwined pieces of epidemiological evidence led a British team to look for EBV in breast tumors: EBV's long-suspected involvement in Burkitt's lymphoma, a cancer seen mostly in the sub-Sahara, and the region's ominous nickname-the breast-cancer belt. Last year virologist Beverly E. Griffin of Hammersmith Hospital in London and colleagues discovered that 21 percent of samples taken from cancerous breast tissue contained EBV DNA. Proliferation: Harvard University's Elliott Kieff and coworkers recently discovered that an Epstein-Barr virus protein alters B-cell production. Photo: Barbara Steiner Elliott Kieff, the Albee Professor of Microbiology and Molecular Genetics and a professor of medicine at Harvard University, studies how EBV induces B-cells to proliferate. "Recently, we've learned how an EBV gene for an integral membrane protein actually alters cell growth. In the last two years that protein was shown to be absolutely essential for the virus to cause B-cell proliferation. Last year, my colleagues [G. Mosialos et al., Cell, 80:389-99, 1995] found out how it works." Essentially, the EBV protein enters a cell by mimicking an always-activated receptor on the cell's surface. "In fact, research on this receptor has long paralleled EBV research in showing similar effects on B lymphocyte activition and proliferation," Kieff says. Sidebar: Professional Resources for Viral Disease Researchers

New Molecular Tools Enable Researchers To Correlate Viruses, Diseases

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