Stanford Scientists Help Bring Study Of Smallpox Virus Into 'Molecular Age'

In one of two companion articles published in this week’s advance online issue of the Proceedings of the National Academy of Sciences, David A. Relman, MD, associate professor of medicine and of microbiology and immunology, and his colleagues identify a number of distinctive molecular events that occur in monkeys following infection with the smallpox virus that do not occur in monkeys infected with a different virus. They hope their findings will lead to better strategies for combating the highly contagious and frequently fatal disease. Their work provides a foundation for future testing of better vaccines, potential drugs for treatment, and new diagnostic tools, especially during the early stages of smallpox infection. The study was funded by the National Institutes of Health and the Howard Hughes Medical Institute.

“The amazing thing about smallpox is that it is a virus that killed more humans than any other infectious agent in history, yet we know very little about the mechanisms by which it caused disease,” Relman said.

The Stanford team launched its project after learning that a study of smallpox infection in monkeys was under way at the Centers for Disease Control and Prevention. That project is being conducted jointly by the CDC and the U.S. Army Medical Research Institute of Infectious Diseases. Stanford scientists worked with blood samples at the CDC to obtain data about changes in the animals’ immune systems.

It was only five years ago that scientists were prepared to destroy the last two official remaining collections of live smallpox virus. In that year, however, the U.S. National Academy of Science’s Institute of Medicine recommended that work with live virus be pursued for the primary purpose of antiviral drug development. The following year, the World Health Assembly agreed to this plan, postponing the destruction of the virus. Today, with fears that terrorists could develop the virus, researchers are using the surviving samples of virus to learn more about the disease.

Relman, along with biochemistry professor Patrick Brown, MD, PhD, and graduate student Kate Rubins, looked for the genes that were activated or repressed in the circulating blood cells of monkeys infected with smallpox. They identified these genes by comparing blood samples obtained repeatedly during the course of infection with those obtained from the same monkeys prior to infection. Their goal was to understand better the strategy used by smallpox virus, as well as the defense strategy of the primate host.

Additionally, they compared smallpox infection with Ebola infection to tease apart the changes that might be relatively specific to smallpox rather than those that generally occur in overwhelming viral infections.

To see which genes were activated during infection and which ones were repressed, the team used DNA microarrays – glass slides containing pieces of DNA corresponding to each of approximately 18,000 different genes. They found several key components of the immune system that appeared to respond differently to smallpox than to Ebola.

One of the most intriguing aspects of their results, said Rubins, the first author of the paper analyzing the host response to smallpox, was the glimpse they provide of the highly orchestrated interaction between the infected host animal’s immune system and the virus. Most striking, she said, was that certain critical molecules that control immune response were shut down in smallpox infection but not Ebola, indicating that smallpox may be producing inhibitors of these molecules.

Of particular interest in future studies, said Relman, is to identify the events that happen soon after infection – the “early molecular signatures” associated with the virus. Such markers could be useful in guiding interventions in the event of a smallpox outbreak.

Smallpox transmits only among humans. Since the disease no longer exists in humans, Army researcher Peter Jahrling, PhD, set out to create an animal model that mimicked as many aspects of human smallpox as possible, using some of the preserved strains from the CDC and the maximum containment laboratory at CDC that is sanctioned for use with smallpox. He tried infecting cynomolgus macaques, monkeys that are used frequently for infectious disease studies. He found that although the monkeys did not get sick through inhalation of the virus – the way people usually become infected – they developed many of the features of later stage smallpox, as well as features of the hemorrhagic form of this disease, from injected virus.

When Relman first learned about Jahrling’s plans, he realized that the model would be a rich opportunity for exploring what goes on in the host animal’s immune system during infection. “No prior information about the molecular features of the host immune response to smallpox was available,” he said. “Smallpox disappeared from nature before the molecular age.”

Given the devastating mortality rates of smallpox infection only a generation ago, there are some who are reluctant to revive the virus even for research purposes, said Relman.

However, he added, the potential of using smallpox as an agent of bioterrorism tips the scales in favor of preparedness. To build defenses against a disease agent, its tactics must be understood. “Smallpox is known as one of the most sophisticated viruses in outsmarting the defenses of its natural host – humans,” he said. “It can teach us in general about the strategies used by viruses to evade and subvert host defenses, as well as provide new insights into the workings of the human immune system.”