Basic Virology. Martinez J. Hewlett

smallpox virus is extinct in the wild, the recent realization that smallpox has been extensively studied as a weapon, and fears that it may be in the possession of terrorists, bring these classic studies into sharp focus. Further, other animal poxviruses such as monkeypox can infect humans, and human encroachment of tropical habitats has led to significant occurrence of this disease in tropical Africa. Another poxvirus, myxoma virus, is endemic in rabbit populations in South America, and was used in a temporarily successful attempt to control the ecological threat posed by the high rate of rabbit multiplication in Australia. While touted at the time as an example of successful biological control, numerous complications occurred with its use. Thus, this “experiment” is a valuable example of the benefits and problems involved with biological control.

In Fenner's classic study of mousepox pathogenesis, virus was introduced by subcutaneous injection of the footpad, and virus yields in various organs, antibody titer, and rash were scored. As noted, the basic experiment thus required only careful dissection of the infected animal, measurement of virus titers, and careful observation, but provided a detailed picture of the virus’s pattern of spread within the animal. The patterns of virus spread and the occurrence of disease symptoms are illustrated in Figure 3.4.

      Of course, the model is just that; it does not completely describe virus infection in the wild. An example of a significant deviation from one “natural” mode of infection is when poxvirus is transmitted as an aerosol, leading to primary infection in the lungs. This is a difficult infection route to standardize and is only rarely utilized. Also, examining experimental infection of animals in the laboratory ignores the dynamics of infection and the interactions between virus and the population. As a consequence, genetic changes in virus and the host, both of which are the result of the disease progressing in the wild, are ignored.

Figure 3.4 The course of experimental poxvirus infection in laboratory mice. Virus is inoculated at day 0 in the footpad of each member of a large group of genetically equivalent mice. Mice are observed daily, and antibody titers in their serum are measured. Selected individuals are then killed, and various organ systems assayed for the appearance and presence of virus. Note that symptoms of the disease (rash and swollen foot) only become noticeable after a week.

Rabies: where is the virus during its long incubation period?

      Rabies and its transmission by the bite of infected animals to other animals and humans are well known in almost all human cultures. The disease and its transmission were carefully described in Arabic medical books dating to the Middle Ages, and there is evidence of the disease in classical times. One of the puzzles of rabies virus infection is the very long incubation period of the disease. This long period plays an important role in the mechanism of spread, and it is clear that animals (or humans) infected with the virus can be vaccinated weeks to months after infection and still mount an effective immune response.

The pathogenesis of rabies has been studied for over a century, and our current understanding is well founded in numerous careful studies made at varying levels of sensitivity using a number of approaches. An example of the use of immunological methods is shown in Figure 3.5. The basic course of infection starts with inoculation of virus at a wound caused by an infected animal, followed by limited virus replication at the site of primary infection. For the disease to develop, the virus must enter a neuron at a sensory nerve ending. These sensory nerve endings exist in all sites where the virus is known to enter an animal. Following this, the virus spreads passively to the nerve cell body in a dorsal root ganglion, where it replicates to a high level. Either this replicated virus, or other virus moving directly, passes into neurons of the cerebellum and cerebral cortex, where it replicates to high levels. Such replication leads to distinct behavioral changes associated with virus transmission. The virus also moves away from the central nervous system (CNS) to sensory neurons and salivary glands of the oral mucosa, where it replicates and is available for injection into another animal.

      

Figure 3.5 Visualization of rabies virus–infected neurons in experimentally infected animals. (a) A schematic representation of the pathogenesis of rabies in an experimentally infected laboratory animal. (b) Immunofluorescent detection of rabies virus proteins in neurons of infected animals. As described in Chapters 7 and 12, the ability of an antibody molecule to specifically combine with an antigenic protein can be visualized in the cell using the technique of immunofluorescence. The cell and the antibody bound to it are then visualized in the microscope under ultraviolet light, which causes the dye to fluoresce (a yellow‐green color). The top left panel shows replication of rabies virus in a sensory nerve body in a dorsal root ganglion along the spine of an animal infected in the footpad. The bottom left panel shows the virus replicating in a neuron of the cerebellum, while the top right panel shows infected neurons in the cerebral medulla. Infection of the brain leads to the behavior changes so characteristic of rabies infections. Finally, the sensory nerve endings in the soft palate of a hamster infected with rabies virus at a peripheral site contain virus, as shown by the fluorescence in the bottom right panel. This virus can move to the saliva, where it can be spread to another animal. The arrows point to selected cells showing the variation in signal intensity that is typical of infections in tissues.

      As early as 1887, CNS involvement was shown to result from direct spread of the virus from the site of infection into the CNS, as experimental animals that had their sciatic nerve severed prior to injection of the footpad with rabies virus did not develop the disease. The following experiment showed that the virus can remain localized at the site of infection for long periods of time: The footpad of several experimental animals was injected with virus at day 0 and then the inoculated foot was surgically removed from different groups at days 1, 2, 3, and so on after infection. Mice whose foot was removed as long as three weeks after infection survived without rabies, but once neurological symptoms appeared, the mice invariably died. Since removal of the foot saved the mice, it is clear that the virus remained localized there until it invaded the nervous system.

      Finally, a similar experiment showed that rabies virus virulence for a specific host could be increased by multiple virus passages (rounds of virus replication) in that host. Virus isolated from a rabid wild animal takes as long as a week to 10 days to spread to the CNS of an experimentally infected laboratory animal. In contrast, isolation of virus from animals developing disease and re‐inoculation into the footpad of new animals several times result in a virus stock that can invade the test animal's CNS in as little as 12–24 hours. Further, the virus stock that has been adapted to the laboratory animal is no longer able to efficiently cause disease in the original host. As described in Chapter 8, this is one way of isolating strains of virus that are avirulent for their natural host and have potential value as vaccines.

Herpes simplex virus latency

      There are two closely related types of herpes simplex virus: type 1 (facial, HSV‐1) and type 2 (genital, HSV‐2). Both establish latent infections in humans, and reactivation from such infections is important to virus spread. Some details concerning latent infection by HSV are discussed in Chapter 18 in Part IV. Different animal models demonstrate both general similarities and specific