In vivo retrograde infection by PRV Bartha of neural circuitry innervating the eye and stomach is slow compared to infection by wild-type PRV Becker (6
). We employed live-cell imaging and single particle tracking techniques to characterize this phenotype in vitro. Our analysis revealed that the average intra-axonal retrograde-directed run length and velocity of fluorescently tagged Bartha capsids were equivalent to those of fluorescent Becker capsids. Using the modified Campenot neuronal culture system, we recapitulated in vitro the strain's kinetic delay observed in vivo. Furthermore, by fluorescently labeling all neurons capable of undergoing primary infection in compartmented neuronal cultures, we measured the efficiency of viral spread from an infected presynaptic neuron to an uninfected postsynaptic neuron.
We mapped the retrograde defect of PRV Bartha to the UL
21 locus, which contains seven point mutations (21
). Michael and colleagues showed that these mutations result in inefficient tegument assembly in PRV Bartha (28
). Such a defect may diminish the infectivity of transmitted virions. In addition, the product of UL
21 has been implicated in the production of encapsidated infectious particles. Mutagenesis of the gene in PRV leads to impaired cleavage of the concatemeric viral genome into single-unit lengths (13
). Furthermore, the complete absence of UL
21 protein in PRV results in an increase in the number of empty capsids (38
). As genome cleavage and encapsidation are linked processes in alphaherpesviruses, inefficient DNA processing may lead to a delay in nucleocapsid assembly, which in turn may lead to delayed transneuronal transmission of infectious particles. Our preliminary data indeed reveal an abundance of empty capsids in the nuclei of PRV Bartha-infected neurons (not shown). However, a detailed ultrastructural study is needed to determine the significance of these observations.
PRV Bartha is known to replicate well in most nonneuronal cell lines. The single step growth kinetics of the virus are similar to those of wild-type PRV Becker, except that the final titers achieved by PRV Bartha are typically 1 log unit higher than the wild-type PRV levels. One explanation for these observations is that simultaneous infection of all cells may mask the defect in nucleocapsid assembly because viral transmission from an infected cell to an uninfected cell is not required for amplification of infection. We attempted to detect any delays in replication by performing low-MOI infections of epithelial PK15 cells. Under these conditions, the efficiency of viral spread influences the rate of viral amplification. However, we detected no difference between PRV Bartha and PRV Becker, except that PRV Bartha reached titers 1 log unit higher than those of the wild-type virus (data not shown).
An alternate explanation is that PRV UL
21 is not required for efficient infectious particle assembly in epithelial cells, and its function is cell type specific. This hypothesis has been suggested previously (23
). Ch'ng and Enquist have previously reported that PRV Bartha achieves wild-type PRV levels upon high-MOI infection of dissociated S-compartment neurons at 24 h postinfection (11
). However, simultaneous infection of all neurons precludes detection of spread delays, and input inoculum applied to the cell bodies obscures subsequent measurements even after citrate inactivation, as few de novo infectious particles are produced per neuron. In our retrograde infection assay, input inoculum is confined to the N-compartment and therefore does not affect the quantification of infectious units in the S-compartment. Additionally, only 15% of the plated neurons extend axons that reach the N-compartment and undergo primary infection (unpublished observations), which effectively establishes a low MOI in the S-compartment. These conditions enabled us to detect the PRV Bartha replication defect in neurons.
The success of neural tracing studies is dependent on replication and transneuronal passage of virus through the nervous system. Our findings clearly demonstrate that repair of the mutations present in the UL
21 locus of PRV Bartha increases the temporal kinetics of viral transport through neural circuits. These data have important implications for analysis of complex neural systems. Here, efficient transport of virus is integral to the ability to define all components of circuits that may extend throughout the full extent of the brain and spinal cord and that may differ in the number of synaptic contacts between neurons. The latter feature of neural circuitry is particularly important for avoiding false-negative results (e.g., not infecting neurons that are involved in the circuit). Strong evidence supports the conclusion that the progression of infection through a circuit depends on both the infectious dose and the number of synaptic connections between neurons (3
). To illustrate the latter point, it is useful to consider the findings of O'Donnell and colleagues (29
) who used PRV to define the organization of parallel circuits between the basal forebrain and thalamus. The authors noted that a small subset of neurons known to be involved in this circuitry were not infected. Because the neurons were shown to be permissive to infection by PRV in other studies, the authors hypothesized that the observed lack of infection was due to the established sparse projections of their axons in this circuitry. It will be important to determine whether the improved transport kinetics of PRV 326 through neural circuits can resolve issues such as that noted in the O'Donnell study.
The potential influence of repairing the mutations of the UL
21 gene upon the virulence and cytotoxicity of PRV 326 also merits attention. Several studies suggest that UL
21 contributes to the virulence of wild-type virus and that the mutations in this gene in the PRV Bartha genome contribute to its attenuated phenotype (13
). We did not observe any increased cytopathogenicity compared to PRV Bartha following infection of central autonomic circuits with PRV 326. The pattern of transport of this virus recapitulated that observed with PRV Bartha and other deletion mutants that are transported only retrogradely through this circuitry. However, we did note that animals infected with PRV 326 exhibited more pronounced symptoms of infection (e.g., oronasal secretions) indicative of stress than PRV Bartha-infected animals at the same time postinoculation did. In addition, PRV 326-infected animals lost more weight (an average of 46 g versus 10 g) during the last day of the experiment than their PRV Bartha-infected counterparts did. It will be important to examine longer survival times and the transport of virus through different circuits (e.g., following intracerebral injection) to determine the full impact of these observations on the utility of PRV 326 as a neural tracer. Nevertheless, our data provide further insight into the function of the UL
21 locus in viral invasiveness and confirm the findings of Klupp and colleagues regarding its role in virulence (21