JOURNAL OF VIROLOGY, Feb. 2009, p. 1173–1183
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 3
Repair of the UL21 Locus in Pseudorabies Virus Bartha Enhances
the Kinetics of Retrograde, Transneuronal Infection
In Vitro and In Vivo?†
D. Curanovic ´,1M. G. Lyman,1C. Bou-Abboud,2J. P. Card,2and L. W. Enquist1*
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544,1and Department of Neuroscience,
University of Pittsburgh, Pittsburgh, Pennsylvania 152172
Received 6 October 2008/Accepted 13 November 2008
The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect
its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that
abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous
studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We
used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of
the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among
neurons is diminished. Repair of the UL21 locus with wild-type sequence restored efficient transneuronal
spread both in vitro and in vivo. It is likely that mutations in the Bartha UL21 gene confer defects that affect
infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection.
These events manifest as slower kinetics of retrograde viral spread in a neural circuit.
Pseudorabies virus (PRV) is the causative agent of Aujesz-
ky’s disease in swine. It is a member of the Alphaherpesvirus
subfamily of the Herpesviridae family, which includes the hu-
man pathogens herpes simplex virus and varicella-zoster virus.
One facet of the alphaherpesvirus infectious cycle is condi-
tional neuroinvasiveness. Upon initial inoculation of mucosal
epithelium, infection spreads to the peripheral neurons inner-
vating the mucosa via retrograde axonal transport. Here, viral
latency is established that persists for the lifetime of the host
(19). During occasional reactivation from latency, newly rep-
licated viral particles are transported to the original site of
infection via anterograde axonal transport, causing a recurring
epithelial lesion. Transneuronal spread of infection from the
peripheral nervous system to the central nervous system (CNS)
is rare in the natural host. Remarkably, in susceptible nonnat-
ural hosts, the spread of alphaherpesviruses almost invariably
proceeds to the CNS, with lethal consequences (18, 31).
Because of their broad host range, self-amplifying character,
and ability to spread directionally through synaptically con-
nected circuits, alphaherpesviruses have been used successfully
as neural circuit tracers (16). The most widely used tracing
strains are PRV Bartha and various recombinants expressing
reporter genes. The PRV Bartha strain was originally devel-
oped as a live vaccine against Aujeszky’s disease in swine by
serial passage in culture (1). PRV Bartha elicits protective
immunity in swine without causing disease (25). Studies to
understand the genetic basis of its attenuation and restricted
pattern of nervous system infection have yielded considerable
information regarding the mechanisms of alphaherpesvirus
pathogenicity (20, 22, 27).
Several features make PRV Bartha particularly appealing
for neural tracing studies. (i) It is attenuated, which allows the
inoculated animals to live longer than those infected with a
wild-type PRV strain; the extended survival time, in turn, en-
ables extensive viral spread and labeling of the nervous system
(2). (ii) Despite its attenuation, the virus replicates well in
tissue culture cells (23). (iii) In vivo and in vitro studies have
demonstrated that a small deletion in the unique short region
of the genome, encompassing glycoprotein E (gE), gI, and US9
genes, renders the strain incapable of spread from an infected
presynaptic cell to a postsynaptic cell (anterograde spread),
with little effect on spread from a postsynaptic cell to a pre-
synaptic cell (retrograde spread) (17, 30). Thus, infecting ani-
mals with PRV Bartha allows unambiguous interpretation of
neural circuit architecture.
While studies performed with PRV Bartha have produced
insight into the mechanisms of anterograde spread, no alpha-
herpesvirus mutants defective in retrograde transport and
spread have been identified that do not affect virus replication.
Previous in vivo studies have suggested that the kinetics of
PRV Bartha retrograde spread through neural circuits are
slower than those of the wild-type PRV Becker strain (8, 40).
Therefore, we sought to characterize this defect and, by
genomic repair, to improve the efficiency of retrograde-
directed infection by PRV Bartha.
MATERIALS AND METHODS
Virus strains and cells. PRV Becker is a laboratory wild-type strain; PRV
GS443 encodes green fluorescent protein-tagged VP26 in the PRV Becker back-
ground (37). PRV Bartha is an attenuated vaccine strain (1); PRV 765 encodes
red fluorescent protein-tagged VP26 in the PRV Bartha background (Ann Ral-
dow, unpublished data). PRV 158 contains the unique long (UL) region of
Bartha and the unique short (US) region of Becker (24). PRV BaBe is Becker
containing the USdeletion of Bartha (10). PRV 43/25 aB4 is Bartha with the
* Corresponding author. Mailing address: Department of Molecular
Biology, Princeton University, 314 Schultz Laboratory, Princeton, NJ
08544. Phone: (609) 258-2415. Fax: (609) 258-1035. E-mail: lenquist
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 19 November 2008.
wild-type PRV Kaplan sequence restoring BamHI fragment 4 and the USregion
(22). PRV 326 was constructed for this study and is PRV 43/25 aB4 with the US
deletion of Bartha reintroduced. The strain was created by cotransfection of
PRV 43/25 aB4 DNA and linearized pGS277, which contains a 9-kb PstI frag-
ment from the Bartha USregion, into PK15 cells. The black plaque assay was
used to screen for and pick gE nonimmunoreactive plaques. Normal expression
of genes upstream and downstream of the deletion was verified by Western
blotting (not shown). All strains were propagated in PK15 (porcine kidney) cells,
which were purchased from the American Type Culture Collection, and the virus
titers were determined in PK15 cells. The cells were maintained in Dulbecco’s
modified Eagle medium supplemented with 10% fetal bovine serum and peni-
cillin and streptomycin. Viral infections of PK15 cells were performed in Dul-
becco’s modified Eagle medium supplemented with 2% fetal bovine serum and
penicillin and streptomycin.
Neuronal cultures. Embryonic rat superior cervical ganglia (SCG) were iso-
lated from Sprague-Dawley rats (Hilltop Labs, Inc., Scottdale, PA) on embryonic
day 16 and cultured in compartmentalized cultures as described before (11, 12).
The neuronal medium was changed every 3 days. All animal work pertaining to
SCG dissection was done in accordance with the Institutional Animal Care and
Use Committee of the Princeton University Research Board under protocol
Antibodies and fluorescent dye. The hybridoma producing the monoclonal anti-
body specific for VP5, the major capsid protein, was made by Alex Flood and the
Princeton Molecular Biology Department monoclonal antibody facility. The
lypophilic dye DiI was purchased from Molecular Probes and used to stain neurons.
The dye was added to medium in the neurite compartment (N-compartment) fol-
lowing infections and incubated for 24 h. Alexa secondary fluorophore (Molecular
Probes) was used at a dilution of 1:400.
Viral infection of compartmented neurons. Infections of neurons were per-
formed as described previously (11). To determine the efficiency of retrograde-
directed infection of neurons, viral inoculum containing 105PFU was added to
the neurite compartment and adsorbed for 1 h in a humidified incubator at 37°C
and 5% CO2. Inoculum was removed, and conditioned neuronal medium was
returned to the neurite compartment following the adsorption period. At the
appropriate time point, the contents of the soma compartment (S-compartment)
were collected by scraping the surface of the dish with a gel-loading tip. To study
the efficiency of neuron-to-cell spread of infection, PK15 cells were plated in the
neurite compartment of 2-week-old compartmentalized neuronal cultures, and
the medium of this compartment was supplemented with 1% fetal bovine serum.
PK15 cells formed a confluent monolayer in the N-compartment 24 h after
plating. At this point, viral inoculum was added to the soma compartment for 1
hour. Following adsorption, conditioned neuronal medium was returned to the
S-compartment. The contents of the neurite and soma compartments were col-
lected separately 24 h after infection.
Immunofluorescence. Teflon trichambers (Tyler Research) were assembled on
UV-sterilized Aclar (EM Sciences) strips; neurons were cultured and infected as
described above. At 24 hours postinfection (hpi), all compartments were washed
twice with phosphate-buffered saline (PBS) containing 3% bovine serum albumin
(BSA) (PBS-BSA), chambers were gently lifted, and silicone grease was scraped
off the Aclar strips. Samples were then fixed with 4% paraformaldehyde in PBS
for 10 min. Fixative was washed away with three PBS-BSA rinses, after which the
samples were permeabilized using 0.5% saponin and 3% BSA in PBS (PBS-
BSA-SAP). Incubations with primary and secondary antibodies were performed
for 1 hour in PBS-BSA-SAP. Following two rinses with PBS-BSA-SAP and one
rinse with distilled water, the samples were mounted on glass slides using Aqua
Poly/Mount (Polysciences). Images were taken on an inverted epifluorescence
microscope (Nikon Eclipse TE300).
Live-cell imaging. Dissociated SCG were cultured in glass-bottom dishes
(MatTek Corporation, Ashland, MA) and infected with 105PFU approximately
2 weeks postplating. At 16 hpi, the samples were placed in a humidified live-cell
imaging chamber that provides 5% CO2and constant temperature at 37°C (Live
Cell Systems). Movies were captured on a Perkin-Elmer R30 spinning disk
confocal microscope using ImageView software. Capsid movement was tracked
and analyzed with ImageJ software (National Institutes of Health, Bethesda)
using the MTrackJ plugin (created by E. Meijering).
In vivo experiments. Fourteen adult male Sprague-Dawley rats (Harlan)
weighing 300 to 350 g at the time of virus injection were used in the analysis. The
experiments were performed in a laboratory dedicated to and approved for
biosafety level 2? experiments. Animals were moved to the facility 2 days prior
to the virus injections and lived within the facility throughout the experiment.
Photoperiod (12 h light; light on at 0700) and temperature (22 to 25°C) were
standardized, and food and water were available ad libitum. The experiments
conformed to the regulations mandated in the Guide for the Care and Use of
Laboratory Animals (28a) and Biosafety in Microbiological and Biomedical Lab-
oratories (37a). The experimental protocols were approved by the University of
Pittsburgh IACUC, the Recombinant DNA Committee, and the Division of
Environmental Health and Safety.
Each animal was anesthetized with isoflurane. The abdomen was shaved, and
an incision was made through the skin and subjacent musculature. The stomach
was gently removed from the peritoneal cavity, and the ventral wall was injected
with PRV Bartha (1.7 ? 109PFU/ml) or PRV 326 (2 ? 109PFU/ml). A total of
two microliters of virus was injected through three penetrations of the ventral
wall of the stomach using a 10-?l Hamilton syringe with a beveled needle. At
each of the three sites, the needle was inserted into the stomach wall at the
greater curvature, and the tip of the needle was pushed to the hilus under visual
guidance. Following injection of virus, the needle was left in place for a minimum
of 2 min prior to removal to prevent reflux along the needle tract. We observed
no reflux from any of the injection sites. The stomach was then returned to the
peritoneal cavity, and the abdominal wall and skin were sutured using 4.0 silk
suture. The animals recovered on a heating pad and were then returned to their
The experiments were terminated by transcardiac perfusion of anesthetized
animals 48 (n ? 7) or 72 (n ? 7) hours following virus injection. Following deep
sodium pentobarbital-induced anesthesia, the heart was exposed through a tho-
racotomy, a canula was inserted into the ascending aorta through the left ven-
tricle, and the right atrium was slit. Approximately 100 ml of physiological saline
was then infused under controlled pressure using a peristaltic pump to clear the
vasculature of red blood cells. This was followed by infusion of approximately 400
ml of paraformaldehyde-lysine-periodate fixative (26). The brain and spinal cord
of each animal were removed and postfixed in paraformaldehyde-lysine-perio-
date fixative for 2 or 3 days at 4°C, cryoprotected by immersion in 20% phos-
phate-buffered sucrose, and sectioned using a freezing microtome. The brain was
sectioned serially in the coronal plane into six wells of cryoprotectant (39) at 35
?m/section, and the spinal cord was sectioned serially in the horizontal plane into
four wells of cryoprotectant at 40 ?m/section. Tissue was stored in cryoprotectant
at ?20°C until immunohistochemical processing to preserve antigenicity.
A minimum of one bin of brain and spinal cord tissue from each animal was
processed for immunohistochemical localization of infected neurons using a
rabbit polyclonal antiserum raised against acetone-inactivated PRV (Rb133).
The specificity of this antiserum for localization of PRV-infected neurons in vivo
was documented previously (6). A second bin of brain and spinal cord tissue from
each animal was processed for immunocytochemical localization of immune cells
of monocytic lineage using a mouse monoclonal antibody generated against the
antigen ED1 (14). All antigens were localized using avidin-biotin immunoper-
oxidase procedures previously described (4). Essential reagents employed for
these localizations included affinity-purified secondary antibodies (Jackson
ImmunoResearch Laboratories) and the Vectastain Elite ABC kit (Vector Lab-
Quantification of viral spread in vivo. The temporal kinetics of viral invasion
of central autonomic circuitry were determined through a quantitative analysis of
central cell groups previously shown to be synaptically linked to the parasympa-
thetic (dorsal motor vagal nucleus [DMV]) and sympathetic (intermediolateral
[IML] cell column) outflow to the stomach (6, 9, 33, 40). The organization of this
circuitry is illustrated schematically (see Fig. 6). Cells were counted in sections
through 25 cell groups that contribute to this circuit (see Table S1 in the
supplemental material). The sections through each cell group selected for anal-
ysis were standardized to ensure comparable comparisons between animals. Cells
within each cell group were counted using an image analysis system (Stereo-
Investigator version 7; Microbrightfield, Inc.) attached to a Nikon Optiphot 2
photomicroscope. The boundaries of each tissue section were traced, the entire
section was systematically scanned using the 40? objective, and the position of
each infected neuron within each section was recorded. Thus, both the distribu-
tion and number of neurons within each cell group were determined for each
animal. For each brain region at each survival time (48 and 72 h), Student’s t test
was used to determine whether statistical differences in the number of infected
neurons were produced by infection with PRV Bartha and PRV 326. Differences
between groups were considered significant when the P value was ?0.05. Rep-
resentative examples of infected regions were photographed using an Olympus
photomicroscope and assembled into figures using Adobe Illustrator and Pho-
PRV Bartha undergoes retrograde-directed axonal trans-
port with wild-type PRV kinetics. Our original hypothesis was
1174 CURANOVIC´ET AL.J. VIROL.
that the observed delay in retrograde transneuronal spread of
PRV Bartha resulted from inefficient axonal transport from
neuronal termini toward the cell bodies. Possible defects lead-
ing to this phenotype might affect the processivity or rate of
dynein-mediated retrograde transport of viral particles. There-
fore, we used live-cell microscopy to characterize the move-
ment of Bartha capsids in axons of dissociated embryonic rat
SCG. We infected neurons with either PRV 765, which con-
tains red fluorescent protein-tagged VP26 in the Bartha back-
ground, or PRV GS443, which contains green fluorescent pro-
tein-tagged VP26 in the PRV Becker background. We infected
the cultures at a high multiplicity of infection (MOI) and
imaged at 16 hpi (see the movies in the supplemental mate-
rial). At this point, newly replicated and strongly fluorescing
capsid puncta are easily detectable. Because the dissociated
neurons establish synaptic connections in culture (32), retro-
grade-directed spread of infection can occur from an infected
postsynaptic cell to a presynaptic cell; this feature enabled us
to observe trafficking of capsids toward neuronal cell bodies.
We tracked retrograde movement of fluorescent puncta and
measured the length of each run, which we define as a period
of uninterrupted movement; in addition, we calculated the
average velocity of the runs. Figure 1 shows the distribution
and mean values of the measurements obtained. The average
length of 124 runs by PRV 765 capsids was 5.11 ?m, while the
average length determined for 120 runs by PRV GS443 capsids
was 4.47 ?m; this difference was not significant by a two-
sample T/P test (Student’s t test, P ? 0.475). The average
velocity of PRV 765 capsids was 1.07 ?m/s, which was compa-
rable to the 1.09 ?m/s measured for PRV GS443 capsids (P ?
0.680). These data suggest that the retrograde intracellular
trafficking of Bartha capsids occurs with wild-type PRV kinet-
ics and cannot account for the observed delay in retrograde
In vitro time course of retrograde-directed neuronal infec-
tion. We assessed whether the kinetic defect in neuronal in-
fection by PRV Bartha can be recapitulated in vitro by per-
forming infections of compartmented neuronal cultures. In this
system, neuronal soma and axons are maintained in separate
fluid environments. Therefore, inoculum can selectively be ap-
plied to axons, and the efficiency of retrograde-directed infec-
tion of cell bodies was ascertained by determining the titer of
infectious virus in the soma (11). The sections of the tricham-
ber are designated soma compartment (S-compartment, where
the neurons are plated), methocel compartment (M-compart-
ment, where viscous medium is placed), or neurite compart-
ment (N-compartment, where axons emerge) (Fig. 2A).
We applied viral inoculum to the N-compartment and har-
vested the contents of the S-compartment at several time
points. Twelve hours after the infection of axons, PRV Bartha
titers in the soma compartment were comparable to the wild-
type PRV Becker levels (Fig. 2B and C). A 2-log-unit differ-
ence in the mean titers of PRV Becker and Bartha, previously
observed by Ch’ng and Enquist (11), was apparent at 24 hpi but
decreased significantly by 48 hpi. These in vitro experiments
recapitulate the kinetic delay of PRV Bartha infection in ani-
mal models. It is noteworthy that the range of titers of Bartha-
infected samples increased over time, while titers of Becker
samples remained closely clustered. This pattern indicates ef-
ficient primary infection with asynchrony in subsequent viral
spread (secondary infection) among neurons in the S-compart-
ment. Accordingly, we examined the efficiency of secondary
transneuronal infection by PRV Bartha using immunofluores-
PRV Bartha does not spread efficiently to second-order neu-
rons in vitro. We developed an immunofluorescence assay to
test the hypothesis that PRV Bartha does not undergo efficient
spread among S-compartment neurons. Not all neurons extend
axons that reach the N-compartment; instead, axons often
form connections with other neurons in the S-compartment.
These cells cannot become directly infected by viral inoculum
applied to the N-compartment but become infected only by
secondary spread of infection from neurons that undergo pri-
mary infection (retrograde spread). We labeled the cells that
extend axons across the full length of the trichamber by adding
the lypophilic dye DiI to only the N-compartment. The dye
diffuses laterally in the axonal membrane and reaches the cog-
nate cell body, thereby labeling all neurons that can undergo
primary infection. Approximately 15% of soma in the S-com-
partment of each sample exhibited DiI fluorescence (data not
Twenty-four hours after infection of DiI-labeled axons, viral
capsids were detected in the S-compartment via immunofluo-
rescence. The number of cells exhibiting both DiI and capsid
fluorescence was scored as cells that have undergone primary
infection. Cells exhibiting capsid fluorescence only are a result
of viral spread within the soma compartment and were scored
as having undergone secondary infection. We counted all cells
FIG. 1. Axonal retrograde transport kinetics. Dissociated SCG
neurons were cultured on glass-bottom MatTek dishes for 2 weeks.
Cultures were infected with PRV GS443 or PRV 765. The movies in
the supplemental material were captured 16 h postinfection on a Per-
kin-Elmer R30 spinning disk confocal microscope. (A) Color-coded
arrowheads track the movement of individual capsid puncta in 5-s
intervals. (B) Distribution of capsid puncta run lengths is shown in box
plots (n ? 120). (C) Distribution of average run velocities of capsid
puncta (n ? 85). The open squares in box plots designate the minimum
value, first quartile, median, and third quartile of the data set; filled
squares are mean values.
VOL. 83, 2009REPAIR OF UL21 IN PRV BARTHA ENHANCES RETROGRADE SPREAD 1175
in randomly selected fields among three independent samples
infected with either PRV Becker or PRV Bartha and calcu-
lated the percentage of cells with primary or secondary infec-
tion. PRV Bartha and PRV Becker infect equivalent numbers
of cells via primary infection: of the 200 cells counted, 27%
exhibited both DiI and capsid fluorescence in Bartha-infected
samples, while 31% of cells were dually labeled in Becker-
infected samples. However, of the 200 cells counted in Bartha-
infected chambers, 16% exhibited capsid labeling and no DiI
fluorescence, compared to 40% in Becker-infected samples
(Fig. 3). These data indicate inefficient secondary infection by
Bartha and support the hypothesis that the spread of infection
in the soma compartment, but not the primary infection of
neurons, is impaired in Bartha-infected neuronal cultures.
While PRV Bartha exhibits titers 100 times lower than PRV
Becker upon retrograde infection, its efficiency of secondary
spread is only 2 times lower than that of wild-type PRV (Fig.
2 and 3). This discrepancy is explained by the nature of our
secondary spread assay, which measures the number of in-
fected cells, and not infectious virions; immunofluorescence
against the major capsid protein VP5 enables quantification of
cells containing the viral antigen but does not distinguish be-
tween unincorporated VP5 protein, an empty capsid, or an
infectious, fully assembled virion.
PRV Bartha repair strain PRV 43/25 aB4 is restored for
retrograde spread. To locate the mutation in the PRV Bartha
genome that is responsible for the observed retrograde infec-
tion phenotype, we performed infections with several Bartha
repair strains. PRV 158 contains the unique long region of
Bartha and a repair of the Bartha USdeletion with Becker
sequence. Conversely, PRV BaBe contains the unique long
region of Becker and harbors the same deletion in the US
region that is found in Bartha. The mean titer of PRV 158
upon retrograde infection was equivalent to that of Bartha
(4.77 ? 104PFU), while the mean titer of BaBe was equivalent
to that of Becker (1.93 ? 103PFU). Therefore, the mutation
responsible for the retrograde infection phenotype of PRV
Bartha is located in its unique long region.
Several mutations in this region of the PRV Bartha genome
have been characterized, namely, point mutations in glycopro-
tein C (36), glycoprotein M (15), Us3, and the intergenic UL20-
UL21 region and three amino acid substitutions in the UL21
gene product (21). UL21 is a nonessential capsid-associated
protein shown to play a role in genome processing and/or
FIG. 2. Retrograde infection time course in vitro. (A) Trichamber
culture system for study of directional infection of neurons. Dissoci-
ated SCG neurons are plated in the S-compartment of the trichamber.
Axonal growth is guided into the N-compartment by a series of grooves
etched in the dish surface. Inoculum is applied to the N-compartment,
and contents of the S-compartment are harvested, and the virus titers
were determined. (B and C) Time course of retrograde neuronal
infection by PRV Becker (B) or PRV Bartha (C). Filled squares
indicate mean values at each time point.
FIG. 3. PRV Bartha does not spread efficiently to second-order
neurons in vitro. DiI was added to the N-compartment immediately
following infection with PRV Becker (A), PRV Bartha (B), or PRV
43/25 aB4 (C). Samples were processed for immunofluorescence
against the major capsid protein VP5 at 24 hpi using a green secondary
antibody. Arrows mark dually fluorescent cells. Arrowheads mark cells
exhibiting VP5 fluorescence only. Bars ? 40 ?m. (D) The percentage
of cells exhibiting both DiI and VP5 fluorescence is represented by
filled bars; empty bars are cells exhibiting VP5 fluorescence only (n ?
200). The chi-square test was used on raw data to determine signifi-
cance. *, P ? 0.05.
1176 CURANOVIC´ET AL.J. VIROL.
packaging (13). We hypothesized that the UL21 mutations
found in Bartha may result in a delay of genome packaging,
which may affect the rate of second-order spread of infection
among neurons and amplification of infection. We therefore
tested PRV 43/25 aB4, a strain derived from PRV Bartha via
virulence rescue experiments (23). In this strain, the BamHI
fragment 4 of the unique long region, encompassing UL21, has
been repaired with wild-type PRV Kaplan sequence. Upon
retrograde infection, the mean of PRV 43/25 aB4 titers was
5.60 ? 104, equivalent to wild-type PRV levels. In addition, the
efficiency of viral spread among neurons in the S-compart-
ment, as assessed by immunofluorescence, improved signifi-
cantly over that of PRV Bartha: of the 200 neurons counted,
32% had undergone primary infection, and 33% had under-
gone secondary infection (Fig. 3). PRV 43/25 aB4 also contains
a repair of the Bartha USdeletion; however, our data on
retrograde infection with BaBe and individual analysis of gE,
gI, and Us9 deletion mutants indicate that the gene products of
the unique short region do not play a role in retrograde spread
(Fig. 4) (11). Therefore, mutations in the UL21 locus are re-
sponsible for the reduced retrograde infection defect by
PRV 326 is a more efficient retrograde tracing strain in vitro
and in vivo. Our studies suggest that repair of the UL21 locus
in PRV Bartha would result in a faster retrograde-restricted
neural circuit tracer. We therefore constructed PRV 326 by
reintroducing the USdeletion of Bartha into the genome of
PRV 43/25 aB4. In vitro analysis revealed that PRV 326 titers
upon retrograde-directed infection of neurons exceed wild-
type PRV levels, reaching 2.42 ? 106PFU (Fig. 5B). The
strain’s ability to undergo anterograde-directed, neuron-to-cell
spread of infection was tested by infecting the neuronal soma
and determining the titer of the contents of the neurite com-
partment, in which epithelial PK15 detector cells were plated
and allowed to establish contact with the resident axons. PRV
326 was incapable of anterograde-directed, neuron-to-cell
spread of infection (Fig. 5A), demonstrating its potential as a
retrograde-restricted neural circuit tracer with rapid spread
To characterize the temporal kinetics of PRV 326 spread
through neural circuitry, we compared the invasiveness of PRV
326 and PRV Bartha after inoculation of the ventral wall of the
stomach in rats. This model system has been used extensively
in prior analyses of PRV neuroinvasiveness, including PRV
Bartha (6, 7, 9, 33–35, 40). Each virus produced the pattern of
transport predicted by prior investigations that have employed
this experimental model to evaluate the invasiveness of PRV.
However, the temporal kinetics of invasiveness differed sub-
stantially between strains, with PRV 326 invading the central
autonomic circuits at a significantly faster rate than PRV Bar-
tha. The data supporting these assertions are presented below.
Injection of PRV into the ventral wall of the stomach pro-
duces retrograde transneuronal infection of preautonomic cir-
cuits through the sympathetic and parasympathetic branches of
the autonomic nervous system (Fig. 6). Infection of the brain
through sympathetic pathways is delayed relative to parasym-
pathetic pathways due to circuit architecture and the number
of neurons that constitute the circuit. Virus is directly trans-
ported into the caudal brain stem through parasympathetic
circuits and then passes transneuronally to infect other cell
groups in the brain stem and forebrain. In contrast, brain stem
and forebrain neurons antecedent to the sympathetic outflow
become infected only after replication of virus in peripheral
sympathetic ganglia and transneuronal infection of pregangli-
onic neurons in the intermediolateral cell column of the tho-
racic and lumbar spinal cord. We designed our analysis to
determine the progression of infection through both divisions
of the autonomic nervous system and incorporated quantita-
tive measures and statistical analysis. It is also important to
note that circuits on the left side of the brain principally in-
nervate parasympathetic outflow to the ventral surface of the
FIG. 4. PRV 43/25 aB4 restores wild-type PRV retrograde titers.
(A) Diagram of genomes used to map the PRV Bartha retrograde
defect. (B) Viral inoculum was added to the N-compartment of neu-
ronal cultures. The contents of the S-compartment were harvested,
and virus titers were determined at 24 hpi. Retrograde titers achieved
by each strain are shown by filled symbols (n ? 4). Empty squares
denote the mean values for each data set.
FIG. 5. PRV 326 is defective for neuron-to-cell spread, but it un-
dergoes efficient retrograde infection of neurons. (A) The epithelial
PK15 detector cells were plated in the N-compartment of neuronal
cultures prior to infection. Viral inoculum was applied to the S-com-
partment, the contents of the S- and N-compartments were harvested
at 24 hpi (n ? 7), and virus titers were determined. (B) Viral inoculum
was adsorbed to axons in the N-compartment. The contents of the
S-compartment were harvested at 24 hpi (n ? 7), and virus titers were
determined. Filled squares represent the mean value of each data set.
VOL. 83, 2009 REPAIR OF UL21 IN PRV BARTHA ENHANCES RETROGRADE SPREAD1177
stomach and that the inverse is true of polysynaptic circuits
innervating the dorsal surface.
Analysis of the entire brain and spinal cord 48 hours follow-
ing injection of PRV Bartha into the stomach revealed an
infection of caudal brain stem parasympathetic neurons con-
fined to the dorsal motor vagal nucleus and a total absence of
infection of preganglionic sympathetic neurons in the spinal
cord (see Table S1 in the supplemental material). In contrast,
injection of the same volume and concentration of PRV 326
revealed a statistically significant increase in the number of
DMV (parasympathetic) neurons and transneuronal infection
of synaptically linked neurons in the immediately adjacent
nucleus of the solitary tract (see Table S1 in the supplemental
material). Similarly, the total absence of PRV Bartha infection
in spinal cord contrasted with the presence of PRV 326-in-
fected sympathetic preganglionic neurons in multiple IML seg-
ments of thoracic and lumbar segments of the cord.
The increased invasion of central circuits by PRV 326 com-
pared to PRV Bartha observed 48 h postinoculation was even
more apparent 72 h postinoculation (see Table S1 in the sup-
plemental material). Statistically significant increases in the
number of PRV 326-infected neurons were observed in the
DMV (parasympathetic) and IML (sympathetic) cell groups,
and there were also statistically significant increases in retro-
grade transneuronal infection of neurons synaptically linked to
these cell groups. Quantitative comparisons of the numbers of
infected neurons in the IML, DMV, and in selected synapti-
cally linked populations of neurons are presented graphically
in Fig. 7. Figure 8 illustrates maps of all these regions, and Fig.
9 shows photomicrographs of representative cases. The results
FIG. 6. PRV 326 invades sympathetic and parasympathetic circuits at a faster rate than PRV Bartha does. The organization of polysynaptic
circuits innervating the stomach is illustrated in the midsagittal schematic diagram of the rat brain. Virus injected into the ventral wall of the
stomach invades the central nervous system through sympathetic and parasympathetic circuits. Virus injected into the stomach wall is retrogradely
transported to the dorsal motor vagal complex (DVC) in the caudal brain stem through parasympathetic circuits. Infection of sympathetic neurons
in the intermediolateral cell column (IML) of the thoracic spinal cord is temporally delayed compared to that resulting from invasion of
parasympathetic circuits due to the need for first-order replication in sympathetic ganglia. First-order parasympathetic and sympathetic neurons
are represented in black in the diagram. Neurons presynaptic to parasympathetic circuits are denoted in red, and those linked to the IML are
represented in blue. Note that the paraventricular nucleus (PVN) of hypothalamus is synaptically linked to both. (A) The synaptic organization
of circuits in the DVC is illustrated in coronal section. Neurons of the dorsal motor vagal nucleus (DMV) receive synaptic contact from neurons
in the immediately adjacent nucleus of the solitary tract (nts) and the area postrema (AP). (B to E) The magnitude of retrograde transneuronal
passage of PRV Bartha (B and D) and PRV 326 (C and E) through the DVC (B and C) and IML (D and E) 72 h following inoculation of the
stomach is illustrated in the photomicrographs. PRV 326 exhibits more extensive retrograde transneuronal infection of the DVC than PRV Bartha
does, and larger numbers of IML neurons are infected in the IML of PRV 326-infected animals. Abbreviations: A5, midbrain catecholamine cell
group; BNST, bed nucleus of stria terminalis; CeA, central nucleus of the amygdala; IC, insular cortex; LHA, lateral hypothalamic area; PFC,
prefrontal cortex; R, raphe; RVLM, rostroventrolateral medulla; VMM, ventromedial medulla.
1178 CURANOVIC´ET AL.J. VIROL.
were striking and unambiguous. For example, an average of
146 neurons were observed in multiple IML segments of the
left thoracic and lumber spinal cord after infection with PRV
Bartha compared to an average of 1,491 neurons in animals
infected with PRV 326. Similarly, there was almost a twofold
increase (122 versus 234) in the number of PRV 326-infected
DMV neurons in animals compared to animals infected with
PRV Bartha. The increased infection of the DMV and IML by
PRV 326 was mirrored by statistically significant increases in
the number of infected neurons in brain stem and forebrain
cell groups known to project to one or both of these cell
groups. For example, the paraventricular hypothalamic nu-
cleus that projects to both the DMV and IML contained an
average of 226 PRV 326-infected neurons compared to an
average of only 6 neurons infected by PRV Bartha. Similarly,
the central nucleus of the amygdala, which projects to the
DMV but not to the IML contained an average of 64 neurons
after PRV 326 inoculation compared to an average of 2 fol-
lowing equivalent inoculation with PRV Bartha. These in-
creases and others documented in Table S1 in the supplemen-
tal material were consistent among animals and were
We have previously demonstrated that immune cells of
monocytic lineage (ED1?) invade areas of viral replication at
advanced stages of infection (33). That analysis demonstrated
that ED1? cells invade the DMV approximately 70 h after
injection of PRV Bartha into the ventral stomach wall and an
increase in number with advancing survival. Staining of tissue
for the ED1 antigen in the present study confirmed this finding
for PRV Bartha-infected animals and revealed more extensive
extravasation of ED1? cells from the vasculature at this sur-
vival interval (72 h) in PRV 326-infected animals (Fig. 10). The
extent of invasion of ED1? cells in the PRV 326-infected
animals was more consistent with that observed 90 to 96 h after
injection of PRV Bartha (33). This finding is consistent with
the increased invasiveness of PRV 326 relative to PRV Bartha.
In vivo retrograde infection by PRV Bartha of neural cir-
cuitry innervating the eye and stomach is slow compared to
infection by wild-type PRV Becker (6, 40). We employed live-
cell imaging and single particle tracking techniques to charac-
terize this phenotype in vitro. Our analysis revealed that the
average intra-axonal retrograde-directed run length and veloc-
ity 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 pri-
mary infection in compartmented neuronal cultures, we mea-
sured 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
UL21 locus, which contains seven point mutations (21). Mi-
chael 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 UL21 has been implicated in the pro-
duction of encapsidated infectious particles. Mutagenesis of
the gene in PRV leads to impaired cleavage of the concate-
meric viral genome into single-unit lengths (13). Furthermore,
the complete absence of UL21 protein in PRV results in an
increase in the number of empty capsids (38). As genome
cleavage and encapsidation are linked processes in alphaher-
pesviruses, inefficient DNA processing may lead to a delay in
nucleocapsid assembly, which in turn may lead to delayed
transneuronal transmission of infectious particles. Our prelim-
inary 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 ob-
servations 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 UL21 is not required
for efficient infectious particle assembly in epithelial cells, and
its function is cell type specific. This hypothesis has been sug-
gested previously (23, 38). 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
FIG. 7. Quantitative analysis reveals a statistically significant in-
crease of PRV 326 transport through preautonomic circuits compared
to controls. The number of infected neurons observed in selected areas
of the CNS 72 h following injection of PRV Bartha or PRV 326 into
the stomach is illustrated. In each area, the number of PRV 326-
infected neurons shows a statistically significant increase relative to
PRV Bartha. The dramatic increase in the number of infected neurons
in the PVN is likely related to the fact that this nucleus is synaptically
linked to both the dorsal motor vagal complex (DVC) and IML,
whereas the rostroventrolateral medulla (RVLM), midbrain catechol-
amine cell group (A5), central nucleus of the amygdala (CeA), and bed
nucleus of stria terminalis (BNST) are selectively linked to the IML.
Values that are statistically significantly different for PRV Bartha and
PRV 326 are indicated with an asterisk (Student’s t test, P ? 0.05).
VOL. 83, 2009REPAIR OF UL21 IN PRV BARTHA ENHANCES RETROGRADE SPREAD 1179
24 h postinfection (11). However, simultaneous infection of all
neurons precludes detection of spread delays, and input inoc-
ulum applied to the cell bodies obscures subsequent measure-
ments 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 un-
dergo primary infection (unpublished observations), which ef-
fectively 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 repli-
cation and transneuronal passage of virus through the nervous
system. Our findings clearly demonstrate that repair of the
mutations present in the UL21 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 syn-
aptic connections between neurons (3, 5). 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 neu-
rons were shown to be permissive to infection by PRV in other
studies, the authors hypothesized that the observed lack of
FIG. 8. Distribution of infected neurons at three comparable levels of the neuraxis 72 h following injection of PRV Bartha or PRV 326 into
the ventral wall of the stomach. The gray bars on the sagittal section at the top of the figure illustrate the positions of the coronal sections shown
immediately below. The coronal sections sample the caudal brain stem at the level of the dorsal motor vagal complex (DMV) (nucleus of the
solitary tract [nts] and area postrema [AP]), through the cardiovascular regulatory cell group linked to the sympathetic outflow (rostroventrolateral
medulla [RVLM]), and through a level of the forebrain through the paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA).
Infected neurons were mapped by systematic examination of the section with a 40? objective. The position of each cell was recorded using an
image analysis system. Each red dot represents an infected neuron. The maps demonstrate the dramatic increase in transport of PRV 326 relative
to PRV Bartha in preautonomic circuits synaptically linked to the sympathetic and parasympathetic outflow.
1180 CURANOVIC´ET AL.J. VIROL.
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
The potential influence of repairing the mutations of the
UL21 gene upon the virulence and cytotoxicity of PRV 326 also
merits attention. Several studies suggest that UL21 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, 21). We did not observe any increased cyto-
pathogenicity compared to PRV Bartha following infection of
central autonomic circuits with PRV 326. The pattern of trans-
port of this virus recapitulated that observed with PRV Bartha
and other deletion mutants that are transported only retro-
gradely through this circuitry. However, we did note that ani-
FIG. 9. The increase in the magnitude of infection of preautonomic circuits resulting from infection with PRV 326 compared to PRV Bartha
is striking and unambiguous. The extent of infection of four cell groups following identical inoculation of PRV Bartha (A, C, E, and G) or PRV
326 (B, D, F, and H) is illustrated. Comparisons of infection in the rostral portion of the nucleus of the solitary tract (A and B), rostroventrolateral
medulla (C and D), midbrain catecholamine cell group (E and F), and paraventricular nucleus (G and H) are illustrated. Each comparison reveals
larger numbers of infected neurons, a finding that was confirmed by statistical analysis. The yellow areas in the schematic insets illustrate the
locations of the areas that were photographed. The third ventricle of the hypothalamus (3V) is indicated. The magnification of all images in this
figure is the same. Bar ? 100 ?m.
VOL. 83, 2009 REPAIR OF UL21 IN PRV BARTHA ENHANCES RETROGRADE SPREAD1181
mals infected with PRV 326 exhibited more pronounced symp-
toms of infection (e.g., oronasal secretions) indicative of stress
than PRV Bartha-infected animals at the same time postin-
oculation 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 UL21 locus in viral invasiveness and confirm the findings
of Klupp and colleagues regarding its role in virulence (21).
We acknowledge support from the National Institutes of Health
(grant R01 33506 [to L.W.E.] and grant NCRR P40 RR0118604 [to
J.P.C., P. L. Strick, and L.W.E.]). L.W.E. acknowledges support from
the Center for Behavioral Neuroscience Viral Tract Tracing Core at
Georgia State University through the STC Program of the National
Science Foundation under agreement IBN-9876754 to L.W.E. and Tim
Bartness. M.G.L. was supported by The American Cancer Society
Eastern Division–Mercer Board Postdoctoral Fellowship (PF-08-264-
We acknowledge Peggy Bisher for help with electron microscopy.
1. Bartha, A. 1961. Experimental reduction of virulence of Aujeszky’s disease
virus. Magy. Allatorv. Lapja 16:42–45.
2. Brittle, E. E., A. E. Reynolds, and L. W. Enquist. 2004. Two modes of
pseudorabies virus neuroinvasion and lethality in mice. J. Virol. 78:12951–
3. Card, J. P., J. R. Dubin, M. E. Whealy, and L. W. Enquist. 1995. Influence
of infectious dose upon productive replication and transynaptic passage of
pseudorabies virus in rat central nervous system. J. Neurovirol. 1:349–358.
4. Card, J. P., and L. W. Enquist. 1999. Transneuronal circuit analysis with
pseudorabies viruses, vol. 1. John Wiley & Sons, San Diego, CA.
5. Card, J. P., L. W. Enquist, and R. Y. Moore. 1999. Neuroinvasiveness of
pseudorabies virus injected intracerebrally is dependent on viral concentra-
tion and terminal field density. J. Comp. Neurol. 407:438–452.
6. Card, J. P., L. Rinaman, J. S. Schwaber, R. R. Miselis, M. E. Whealy, A. K.
Robbins, and L. W. Enquist. 1990. Neurotropic properties of pseudorabies
virus: uptake and transneuronal passage in the rat central nervous system.
J. Neurosci. 10:1974–1994.
7. Card, J. P., P. Levitt, M. Y. Gluhovsky, and L. Rinaman. 2005. Early expe-
rience modifies the postnatal assembly of autonomic emotional motor cir-
cuits in rats. J. Neurosci. 25:9102–9111.
8. Card, J. P., M. E. Whealy, A. K. Robbins, R. Y. Moore, and L. W. Enquist.
1991. Two alphaherpesvirus strains are transported differentially in the ro-
dent visual system. Neuron 6:957–969.
9. Card, J. P., L. Rinaman, R. B. Lynn, B.-H. Lee, R. P. Meade, R. R. Miselis,
and L. W. Enquist. 1993. Pseudorabies virus infection of the rat central
nervous system: ultrastructural characterization of viral replication, trans-
port, and pathogenesis. J. Neurosci. 13:2515–2539.
10. Card, J. P., M. E. Whealy, A. K. Robbins, and L. W. Enquist. 1992. Pseu-
dorabies virus envelope glycoprotein gI influences both neurotropism and
virulence during infection of the rat visual system. J. Virol. 66:3032–3041.
11. Ch’ng, T. H., and L. W. Enquist. 2005. Neuron-to-cell spread of pseudora-
bies virus in a compartmented neuronal culture system. J. Virol. 79:10875–
12. Ch’ng, T. H., E. A. Flood, and L. W. Enquist. 2005. Culturing primary and
transformed neuronal cells for studying pseudorabies virus infection. Meth-
ods Mol. Biol. 292:299–316.
13. de Wind, N., F. Wagenaar, J. Pol, T. Kimman, and A. Berns. 1992. The
pseudorabies virus homology of the herpes simplex virus UL21 gene product
is a capsid protein which is involved in capsid maturation. J. Virol. 66:7096–
14. Dijkstra, C. D., E. A. Dopp, P. Joling, and G. Kraal. 1985. The heterogeneity
of mononuclear phagocytes in lymphoid organs: distinct macrophage sub-
populations in the rat recognized by monoclonal antibodies ED1, ED2 and
ED3. Immunology 54:589–599.
15. Dijkstra, J. M., T. C. Mettenleiter, and B. G. Klupp. 1997. Intracellular
processing of pseudorabies virus glycoprotein M (gM): gM of strain Bartha
lacks N-glycosylation. Virology 237:113–122.
16. Ekstrand, M. I., L. W. Enquist, and L. E. Pomeranz. 2008. The alpha-
herpesviruses: molecular pathfinders in nervous system circuits. Trends Mol.
17. Enquist, L. W. 2002. Exploiting circuit-specific spread of pseudorabies virus
in the central nervous system: insights to pathogenesis and circuit tracers.
J. Infect. Dis. 186(Suppl. 2):S209–S214.
18. Goodpasture, E. W., and O. Teague. 1923. Transmission of the virus of
herpes fibrils along nerves in experimentally infected rabbits. J. Med. Res.
19. Hill, T. J., and H. J. Field. 1973. The interaction of herpes simplex virus with
cultures of peripheral nervous tissue: an electron microscopic study. J. Gen.
20. Klupp, B. G., H. Kern, and T. C. Mettenleiter. 1992. The virulence-deter-
mining genomic BamHI fragment 4 of pseudorabies virus contains genes
corresponding to the UL15 (partial), UL18, UL19, UL20, and UL21 genes
of herpes simplex virus and a putative origin of replication. Virology 191:
21. Klupp, B. G., B. Lomniczi, N. Visser, W. Fuchs, and T. C. Mettenleiter. 1995.
Mutations affecting the UL21 gene contribute to avirulence of pseudorabies
virus vaccine strain Bartha. Virology 212:466–473.
22. Lomniczi, B., S. Watanabe, T. Ben-Porat, and A. S. Kaplan. 1984. Genetic
basis of the neurovirulence of pseudorabies virus. J. Virol. 52:198–205.
23. Lomniczi, B., S. Watanabe, T. Ben-Porat, and A. S. Kaplan. 1987. Genome
location and identification of functions defective in the Bartha vaccine strain
of pseudorabies virus. J. Virol. 61:796–801.
24. Lyman, M. G., G. L. Demmin, and B. W. Banfield. 2003. The attenuated
pseudorabies virus strain Bartha fails to package the tegument proteins Us3
and VP22. J. Virol. 77:1403–1414.
25. McFerran, J. B., and C. Dow. 1975. Studies on immunisation of pigs with the
Bartha strain of Aujeszky’s disease virus. Res. Vet. Sci. 19:17–22.
26. McLean, I. W., and P. K. Nakane. 1974. Periodate-lysine-paraformaldehyde
fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cy-
FIG. 10. Virus-induced recruitment of immune cells into the brain is increased in PRV 326-infected animals. The extent of ED1? immune cell
recruitment into the dorsal motor vagal complex 72 h after infection with PRV Bartha (A) or PRV 326 (B) is illustrated. Note the larger number
of immunopositive cells within the DMV and immediately adjacent nucleus of the solitary tract (nts) of PRV 326-infected animals. The area
postrema (AP) is indicated. The yellow area in the schematic inset illustrates the location of the area illustrated in the photomicrographs. The
magnification of both images is the same. Bar ? 100 ?m.
1182 CURANOVIC´ET AL.J. VIROL.
27. Mettenleiter, T. C., L. Zsak, A. S. Kaplan, T. Ben-Porat, and B. Lomniczi. Download full-text
1987. Role of a structural glycoprotein of pseudorabies in virus virulence.
J. Virol. 61:4030–4032.
28. Michael, K., B. G. Klupp, A. Karger, and T. C. Mettenleiter. 2007. Efficient
incorporation of tegument proteins pUL46, pUL49, and pUS3 into pseudo-
rabies virus particles depends on the presence of pUL21. J. Virol. 81:1048–
28a.National Resource Council. 1996. Guide for the care and use of laboratory
animals. National Academy Press, Washington, DC.
29. O’Donnell, P., A. Lavin, L. W. Enquist, A. A. Grace, and J. P. Card. 1997.
Interconnected parallel circuits between rat nucleus accumbens and thala-
mus revealed by retrograde transynaptic transport of pseudorabies virus.
J. Neurosci. 17:2143–2167.
30. Pickard, G. E., C. A. Smeraski, C. C. Tomlinson, B. W. Banfield, J. Kaufman,
C. L. Wilcox, L. W. Enquist, and P. J. Sollars. 2002. Intravitreal injection of
the attenuated pseudorabies virus PRV Bartha results in infection of the
hamster suprachiasmatic nucleus only by retrograde transsynaptic transport
via autonomic circuits. J. Neurosci. 22:2701–2710.
31. Pomeranz, L. E., A. E. Reynolds, and C. J. Hengartner. 2005. Molecular
biology of pseudorabies virus: impact on neurovirology and veterinary med-
icine. Microbiol. Mol. Biol. Rev. 69:462–500.
32. Potter, D. D., S. C. Landis, S. G. Matsumoto, and E. J. Furshpan. 1986.
Synaptic functions in rat sympathetic neurons in microcultures. II. Adren-
ergic/cholinergic dual status and plasticity. J. Neurosci. 6:1080–1098.
33. Rinaman, L., J. P. Card, and L. W. Enquist. 1993. Spatiotemporal responses
of astrocytes, ramified microglia, and brain macrophages to central neuronal
infection with pseudorabies virus. J. Neurosci. 13:685–702.
34. Rinaman, L., P. Levitt, and J. P. Card. 2000. Progressive postnatal assembly
of limbic-autonomic circuits revealed by central transneuronal transport of
pseudorabies virus. J. Neurosci. 20:2731–2741.
35. Rinaman, L., M. R. Roesch, and J. P. Card. 1999. Retrograde transynaptic
pseudorabies virus infection of central autonomic circuits in neonatal rats.
Dev. Brain Res. 114:207–216.
36. Robbins, A. K., J. P. Ryan, M. E. Whealy, and L. W. Enquist. 1989. The gene
encoding the gIII envelope protein of pseudorabies virus vaccine strain
Bartha contains a mutation affecting protein localization. J. Virol. 63:250–
37. Smith, G. A., S. P. Gross, and L. W. Enquist. 2001. Herpesviruses use
bidirectional fast-axonal transport to spread in sensory neurons. Proc. Natl.
Acad. Sci. USA 13:3466–3470.
37a.U.S. Department of Health and Human Services. 1999. Biosafety in micro-
biological and biomedical laboratories. U.S. Department of Health and
Human Services publication no. (CDC) 88-8395. U.S. Department of Health
and Human Services, Washington, DC.
38. Wagenaar, F., J. M. Pol, N. de Wind, and T. G. Kimman. 2001. Deletion of
the UL21 gene in pseudorabies virus results in the formation of DNA-
deprived capsids: an electron microscopy study. Vet. Res. 32:47–54.
39. Watson, R. E., S. T. Wiegand, R. W. Clough, and G. E. Hoffman. 1986. Use
of cryoprotectant to maintain long-term peptide immunoreactivity and tissue
morphology. Peptides 7:155–159.
40. Yang, M., J. P. Card, R. S. Tirabassi, R. R. Miselis, and L. W. Enquist. 1999.
Retrograde, transneuronal spread of pseudorabies virus in defined neuronal
circuitry of the rat brain is facilitated by gE mutations that reduce virulence.
J. Virol. 73:4350–4359.
VOL. 83, 2009 REPAIR OF UL21 IN PRV BARTHA ENHANCES RETROGRADE SPREAD 1183