Alphaviruses are a group of significant human and animal pathogens. The nearly 30 members of this genus are transmitted by mosquitoes to higher vertebrates that serve as amplifying hosts (54). Alphaviruses cause different diseases but have similar replication strategies and life cycles. In insect vectors, they cause persistent lifelong infections and viral replication does not critically affect the viability of the hosts (51). These viruses also often establish persistent infection in cultured mosquito cells. In contrast, vertebrate hosts usually develop acute infection that often results in disease prior to virus clearance by the immune system (27, 31). The infection of susceptible vertebrate cells typically leads to rapid cytopathic effect (CPE) (54) and cell death.Sindbis virus (SIN) is the prototype member of the Alphavirus genus. It can replicate productively in a wide variety of cell lines of insect and vertebrate origin and causes age-dependent encephalomyelitis in mice (28). As do all other alphaviruses, SIN enters the cells via receptor-mediated endocytosis. pHdependent fusion of viral and endosomal membranes leads to release of nucleocapsids into the cytoplasm (12) followed by nucleocapsid disassembly and genome replication (64).The SIN genome is a single RNA molecule of positive polarity, 11.7 kb in length. It contains a 5Ј methylguanylate cap and a 3Ј polyadenylate tract and is translated by host-cell machinery similar to cellular mRNAs (52). The 5Ј two-thirds of the genome encodes the nonstructural proteins (nsPs), which comprise the viral components of the RNA replicase/transcriptase required for replication of the viral genome and transcription of subgenomic RNA. The 4.1-kb subgenomic RNA corresponds to the 3Ј third of the genome and is translated into structural proteins that form virus particles. Replication of SIN is very rapid and leads to high-level accumulation of virusspecific RNAs and structural proteins. Finally, a large number of viral particles released by budding from the cell surface disseminate the infection. Viral replication profoundly affects cell metabolism, with downregulation of host cell protein synthesis playing the central role (31). Cells lose integrity and die within 24 to 48 h postinfection (p.i.). In many cell types, death is accompanied by apoptotic changes (37). Replication of viral RNA and/or accumulation of SIN nsPs is sufficient to cause translational shutoff and cell death. However, expression of viral structural proteins significantly accelerates the development of CPE (18).In recent studies, we selected SIN self-replicating RNAs (replicons) that were capable of persisting in some vertebrate cell lines for an unlimited number of passages without causing CPE (1, 16). These noncytopathic replicons replicated less efficiently than the parent and contained point mutations in the gene encoding one of the nsPs, nsP2. One adaptive mutation, P 726 3L, was at the same position as that found in the SIN-1 variant that also exhibits reduced cytopathogenicity (11,63).

The identification of viral determinants of virulence and host determinants of susceptibility to virus-induced disease is essential for understanding the pathogenesis of infection. Obtaining this information requires infecting large numbers of animals to assay amounts of virus in a variety of organs and to observe the onset and progression of disease. As an alternative approach, we have used a murine model of viral encephalitis and an in vivo imaging system that can detect light generated by luciferase to monitor over time the extent and location of virus replication in intact, living mice. Sindbis virus causes encephalomyelitis in mice, and the outcome of infection is determined both by the strain of virus used for infection and by the strain of mouse infected. The mode of entry into the nervous system is not known. Virulent and avirulent strains of Sindbis virus were engineered to express firefly luciferase, and the Xenogen IVIS system was used to monitor the location and extent of virus replication in susceptible and resistant mice. The amount of light generated directly reflected the amount of infectious virus in the brain. This system could distinguish virulent and avirulent strains of virus and susceptible and resistant strains of mice and suggested that virus entry into the nervous system could occur by retrograde axonal transport either from neurons innervating the initial site of replication or from the olfactory epithelium after viremic spread.Understanding the pathogenesis of a viral infection requires a knowledge of the sites of viral replication and pathways of spread throughout the body. Traditionally, this knowledge has been obtained by infecting sufficient numbers of animals to be able to evaluate several animals per time point and assaying organs for infectious virus at many times after the initiation of infection. This process requires large numbers of animals, and free virus circulating in the blood can confound the interpretation of data on the amounts of virus in a given organ. Furthermore, important sites of virus replication may be missed because appropriate samples were not taken. In vivo imaging might provide an alternative method of assaying viral infection that would eliminate these concerns.In vivo imaging was first used to detect sites of bacterial replication in intact, living animals in 1995 (3). Both green fluorescent protein (26) and firefly luciferase (3) have been used as reporters. Luciferase offers the advantages of producing an inherently low background in animals and not accumulating (25). Luciferase can therefore be used to monitor realtime activity in living animals (6). Significant technological advances continue to be made in our ability to detect small amounts of light, and luciferase imaging has been used successfully to monitor tumors (4, 17, 21), bacterial infections (3, 19), herpes simplex virus expression (12), and viral gene expression (5, 25). However, its usefulness for studies of viral pathogenesis, host susceptibility, and virus strain differences is unclear....

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.

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