ANIMAL MODELS TO STUDY VIRAL PATHOGENESIS
The great German clinical microbiologist Robert Koch formulated a set of rules for demonstrating that a specific microorganism is the causative agent of a specific disease. These rules are very much in force today. In essence, Koch's rules are as follows:
1 The same pathogen must be able to be cultured from every individual displaying the symptoms of the disease in question.
2 The pathogen must be cultivated in pure form.
3 The pathogen must be able to cause the disease in question when inoculated into a suitable host.
While these rules can be applied (with caution) to virus‐mediated human diseases, it is clearly not ethical to inoculate a human host with an agent suspected to cause a serious or life‐threatening disease (criterion 3). Regrettably, this ethical point has been missed more than once in the history of medicine. Examples of the excesses of uncontrolled human experimentation stand as a striking indictment of Nazi Germany, but excesses are not confined to totalitarian forms of government. The infamous Tuskegee syphilis studies are an example of a medical experiment gone wrong. These studies, ostensibly to evaluate new methods to treat syphilis, were carried out on a large group of infected African‐American men in the rural southern United States by physicians of the US Public Health Service in the 1930s and 1940s. Even though effective treatments were known, a number of men were treated with placebos (essentially sugar pills) to serve as “controls” and to allow the physicians to accumulate data on progression of the disease in untreated individuals. Other examples of potentially life‐threatening experiments in the United States with little effort to explain the dangers or potential benefits (the criteria for informed consent) using volunteer prisoners as test subjects are also well documented.
This discussion should not lead to the conclusion that it is never appropriate to use human subjects to study a disease or its therapy. Human experimentation (such as in clinical trials) is critical to ensuring treatment safety and effectiveness, but to do such studies in an ethical manner, the risks and benefits must be fully understood by all those involved.
One extremely effective way to obtain reliable data on the dynamics of disease and its course in an individual is to develop an accurate animal model. A researcher's need to experimentally test variable factors during infection in order to build a detailed molecular and physiological picture of the disease in question can only be accomplished with a well‐chosen model. The lack of a suitable animal model for a viral disease is almost always a great impediment to understanding its disease processes and developing treatments.
Another important reason for using an animal model to study virus infection is that useful information can often be obtained with very defined and controlled experimental approaches. The ability of a virus to cause specific symptoms can be determined by careful control of the viral genotype and site of inoculation in the animal, followed by observation of the symptoms as they develop. The passage of a virus throughout the body during infection can be studied by dissection of specific organs, careful gross and microscopic observation, and simple measurement (assay) of virus levels in those organs. The host response to infection can be determined (in part, at least) by measuring the animal's production of antibodies and other immune factors directed against the infecting virus.
Much information concerning the interaction between a virus and its animal host can be obtained by using a combination of sophisticated molecular analyses on animals with defined genetic properties. Some examples are outlined in Parts III and IV. For example, transcription of a portion of the HSV genome in latently infected neurons can be observed by use of sophisticated methods to detect viral RNA in tissue in situ. Methods for introduction, mutation, and inactivation of specific genes controlling one or another aspect of the immune response can be introduced into mice (and potentially other animals) using methods described in Part V. These and many other techniques provide detail and richness to the “picture” of the virus–host interaction, and all are required for a full understanding of the interaction between virus and cell and virus and host. However, the basic outline of the course of viral infection in animals can be obtained by using the most simple and readily applied experimental tools: observation, dissection, and measurement of virus.
The use and sacrifice of animals raise significant ethical questions, and the suffering caused must be thoroughly considered in the design of appropriate experimental protocols. For example, an experimental study that establishes important aspects of a disease may be too devastating to repeat as a casual laboratory exercise. Appropriate treatment of animals, limiting pain and suffering and maximizing comfort to the animal, is a practical as well as ethical requirement for animal study.
It is very important to realize that animal models, by their very nature, can only approximate the course and nature of the disease occurring in humans. In order to carry out a valid and reproducible scientific study, the mode of infection, the amount of infecting virus, the age and sex of the animal, its nutritional background, and many other factors must be carefully and reproducibly controlled. Thus, any experimental animal model for a human disease is a compromise with the real world. For example, the amounts of virus inoculated into the animal and the site of inoculation (e.g., mouth, eye, subcutaneous, or intracerebral) must be the same for all test subjects, a situation very different from the “real” world. Also, the model disease in the animal may well be different, in whole or in part, from the actual disease seen in a human population. Genetic makeup of the animal (inbred or outbred, specific genetic markers present or absent), age, and sex of the infected host must be controlled to generate interpretable and reproducible results. Obviously, while certain diseases favor certain age groups, an infected population will evidence a wide range of variation in genetic and physical details.
Another complication is that the viral pathogen sometimes must be specifically adapted to the test animal. Virus directly isolated from an infected human may not cause disease in an animal unless it is first passaged in cells from that animal, or a mouse strain that is deficient in certain immune responses is used. In addition, safety considerations must be taken into account. Working with virus characterized by a very high mortality rate, such as Ebola virus, requires heroic and expensive precautions and containment facilities for study.
Despite very real problems with the use of animals to study virus‐caused disease, it often is the only way to proceed. Careful and accurate clinical observations of infected individuals, animals, or plants provide many details concerning the course of viral infection. But only in a complete plant or animal model can the full course of disease and recovery as a function of controlled variations of infection and physiological state of the host be studied. This is true even when many aspects of virus infection can be studied in cultured cells and with cloned fragments of the viral genome.
The animal models for viral disease described in this and later chapters demonstrate some of the methods, successes, and limitations involved in the use of animals. Despite the problems associated with working with experimental animals, key basic data could not have been and cannot be obtained any other way.
A mouse model for studying poxvirus infection and spread
Many of the models developed for the study of viral pathogenesis involve the use of mice. These animals have an excellent immune system, can be infected with many viruses adapted from human diseases, and are relatively inexpensive to use. Frank Fenner's studies on the pathogenesis of mousepox carried out in the 1950s provided what is still a classic model for experimental study of viral pathogenesis.
Although