Assessment of Future Scientific Needs for Live Variola Virus (1999)

As noted earlier, smallpox was eradicated prior to the modem age of cell and molecular biology, virology, and immunology. Therefore, the basics of viral replication, determinants of viral virulence, and pathogenesis of the disease are not as well understood as they are for other pathogens.

Since variola virus is a pathogen that is uniquely adapted to cause severe, widespread human illness, it is highly likely that it has evolved to specifically thwart an effective immune response to infection. Poxviruses are the largest of the viruses and produce many proteins that are not necessary for virus replication, but presumably enhance the ability of the virus to cause disease. The multiple mechanisms used by poxviruses to evade host immune responses, the unique proteins these viruses produce, and their interactions with the host are just beginning to be identified. As the database expands, questions about the interactions of variola virus with human cells and immune responses and about the functions of these disease-producing variola proteins will become more obvious and pressing. The ability to identify the interactions between variola virus and host proteins would likely provide new insights into important aspects of the human immune system that would not be apparent from studies of other viruses.

Viruses adapt to their hosts in large part by evolving to interact efficiently with host cells in initiating infection and producing large amounts of virus. The vires spreads to different organs of the host and in this process causes tissue damage. Strains of a virus (e.g., variola major and variola minor) differ in their vim-

lence or ability to cause fatal disease. The differences in virulence may be due to changes in the rapidity of virus replication and spread, the amounts of vires produced, the ability to damage the cells in which the vires replicates, or the ability to evade the immune response of the host. In addition, orthopoxvirus tissue tropism genes have been identified in vaccinia vires and cowpox (C7L, K1L, and CHOhr), and the morphogenesis of the multiple forms of orthopoxvirus particles is coming better understood [43, 44]. The genetic basis of orthopoxvirus infections may thereby be revealed. Infection of human cells grown in tissue culture could begin to provide answers to some of the following questions:

• Is there a unique molecule or series of molecules on the surface of human cells that makes them distinctly susceptible to infection with variola virus? What is the normal function of this molecule?
• How and in what order are the many genes of the virus expressed to produce viral proteins? Do these proteins affect the infected cell by stimulating growth, by causing death, or by inhibiting death so the virus can grow for a longer period of time? Does this vary between variola major and variola minor?
• Do any of the viral proteins provide new potential targets for antiviral drugs that can block virus replication without harming host cells? These targets may suggest new types of drugs that can be developed to treat other infections.

Finally, judging from what is known about other poxviruses, modulation of host immune responses is highly likely to contribute to the virulence of the virus. Infection of immune system cells could make it possible to assess direct effects on such cells, and incubation of human immune system cells with proteins secreted by infected cells could allow identification of potentially unique interactions between viral proteins and mediators of the antiviral immune response. These interactions could be used to identify important and potentially unique aspects of the human response to virus infections.

Replication of variola virus in different types of cell cultures could provide valuable information on how this virus distinctly infects and affects human cells. It could not, however, provide information on how the virus spreads through the host or how it counteracts the host immune response.

Cultures that involve a number of cells organized into tissues and organs can currently be studied in bioreactors, SCID-hu mice, and raft cultures. These systems could allow investigators to answer some of the following questions:

• How does the virus spread from one cell to another in infected human tissue, such as the lung, lymph nodes, and skin? Is this different for variola major and variola minor?
• Does the infection of one cell induce damage or dysfunction in nearby cells, particularly cells of the immune system, without infecting them? What are the viral products and human cell targets for such effects? How does this virus cause such severe damage to human tissues, including inducing encephalitis?

Some questions regarding how the virus spreads and causes immune suppression can be answered only by examining the results of infection in an intact host. Since human infection is not possible, answers to these questions would require developing new animal models (e.g., nonhuman primates, transgenic mice, SCID mice reconstituted with the relevant human cells; see also Chapter 8). With such model systems, the following questions could be addressed:

• How and why does the virus spread so efficiently in human organs?
• How does the virus damage host tissues and cells to cause severe disease? Is this the direct result of virus replication in spleen, lymphoid, and bone marrow cells; virus production of mediators that damage even uninfected human cells; or induction of a harmful antiviral immune response?
• What kinds of effective interventions (antiviral drugs, antibodies, immune modulators) can be developed to treat smallpox or smallpox-like diseases?
• How effective are new types of vaccines?