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Journal of Virology, November 1999, p. 9521-9531, Vol. 73, No. 11
Department of Physical Therapy, Taegu
University, Taegu, South Korea1;
Department of Molecular Biology, Princeton University,
Princeton, New Jersey 085442; and
Departments of Neuroscience and Psychiatry, University of
Pittsburgh, Pittsburgh, Pennsylvania 152603
Received 23 March 1999/Accepted 21 July 1999
Neurotropic alphaherpesviruses have become popular tools for
transynaptic analysis of neural circuitry. It has also been
demonstrated that coinfection with two viruses expressing unique
reporters can be used to define more complicated circuitry. However,
the coinfection studies reported to date have employed nonisogenic strains that differ in their invasive properties. In the present investigation we used two antigenically distinct recombinants of the
swine pathogen pseudorabies virus (PRV) in single and
double infections of the rat central nervous system. Both viruses are derivatives of PRV-Bartha, a strain with reduced virulence that is
widely used for circuit analysis. PRV-BaBlu expresses
Neurotropic alphaherpesviruses can
replicate within postmitotic neurons and produce infectious progeny
that pass transneuronally to infect other synaptically linked neurons
(20, 25). This self-amplifying spread in neurons has been
exploited by a number of investigators to gain further insight into the
organization of neuronal circuitry in the mammalian brain (9, 18,
27, 28, 40, 44). However, the use of a variety of different strains of the human and swine pathogens has also revealed
strain-dependent differences in the invasiveness, replication, and
transport of these viruses through the central nervous system (CNS)
(the brain and the spinal cord). For example, selective tropism of
different strains of virus has been demonstrated in a variety of
systems (3, 17, 30, 32), and strain-dependent differences in the direction of transport of viruses have been reported (4, 13,
41, 47). Considerable insights into factors that influence viral
virulence have also emerged from this experimental approach (1, 2,
16, 19, 21, 31, 45). However, the molecular mechanisms that
direct these processes in vivo remain largely undefined.
Recombinant viruses that express unique gene products as reporters of
infection are useful tools for defining connections among neurons
(23, 26). In a notable application of this experimental approach, Jansen and colleagues injected two genetically modified forms
of the Bartha strain of pseudorabies virus (PRV-Bartha) into peripheral targets innervated by separate populations of spinal
cord neurons (23). Transynaptic infection of CNS neurons by
both strains of virus was demonstrated, but the percentage of animals
that exhibited dual-infected neurons was remarkably small. For example,
only 20 of 256 animals exhibited productive replication of both
viruses, and of those, only 8 were viewed as containing a specific
pattern of infection worthy of analysis. It is likely that the low
infection rate in this experiment is due, at least in part, to the use
of titers of virus that were considerably below the 50% lethal dose
(LD50) for PRV-Bartha in rats. However, other factors may
have contributed to the low frequency of neuronal coinfection.
In the present investigation we compared the abilities of two
antigenically distinct recombinants of PRV-Bartha to invade and
replicate within visual and autonomic circuitry after single and double
inoculations (see references 9 and
20 for reviews of the circuitry paradigms). The data
demonstrate noteworthy differences in the virulence and invasiveness of
these strains that influence their ability to coinfect neurons in the
rat CNS.
Animals and facilities.
Adult male Sprague-Dawley rats
(n = 67) weighing 200 to 260 g at the time of
inoculation were used in this analysis. All inoculations were done in a
laboratory approved for use of class 2 (BSL-2) infectious agents, and
the animals were housed in this facility throughout the experiment. The
laboratory met the specifications of the U.S. Department of Health and
Human Services (44a), and the experiments were approved by
the University of Pittsburgh Institutional Animal Care and Use
Committee. Details regarding the application of these safety procedures
in our laboratories have been published previously (12, 18).
Viral strains.
PRV-Bartha, an attenuated vaccine strain,
served as the parental strain for both of the recombinants used in this
investigation (5). Figure 1
illustrates the genome organizations of all of the strains used in this
analysis. PRV-BaBlu contains the lacZ gene inserted into the
gG gene of the unique short (Us) region of the viral genome and
expresses
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Circuit-Specific Coinfection of Neurons in the Rat Central
Nervous System with Two Pseudorabies Virus Recombinants
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-galactosidase, and PRV-D expresses the PRV membrane protein gI, the
gene for which is deleted in PRV-BaBlu. Antibodies to -galactosidase
identify neurons infected with PRV-BaBlu, and antibodies monospecific
for PRV gI identify neurons infected with PRV-D. The ability of these strains to establish coinfections in neurons was evaluated in visual
and autonomic circuitry in which the parental virus has previously been
characterized. The following conclusions can be drawn from these
experiments. First, PRV-D is significantly more neuroinvasive than
PRV-Bartha or PRV-BaBlu in the same circuitry. Second, PRV-D is more
virulent than either PRV-Bartha or PRV-BaBlu, and PRV-BaBlu is less
virulent than PRV-Bartha. Third, in every model examined, PRV-D and
PRV-BaBlu coinfect some neurons, but single infections predominate.
Fourth, prior infection with one virus renders neurons less permissive
to infection by another virus. Fifth, prior infection by PRV-D is more
effective than PRV-BaBlu in reducing invasion and spread of the second
virus. Collectively, the data define important variables that must be considered in coinfection experiments and suggest that the most successful application of this approach would be accomplished by using
isogenic strains of virus with equivalent virulence.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-galactosidase under the control of the gG promoter.
Construction of this mutant was by the methods of Mettenleiter and Rauh
(29) and has been described in a prior analysis of cardiac
circuitry (39). PRV-D was provided by Tamar Ben-Porat and
was described previously (16). PRV-D was constructed in two
steps, as follows. First, the Us deletion of PRV-Bartha was repaired
with wild-type PRV DNA to restore the gI, gE, Us9, and Us2 genes. The
gE gene was then deleted, to reduce the virulence of this strain
(16, 45). The gI protein, which is expressed by PRV-D but
not by PRV-BaBlu, was used as the unique marker of neurons infected
with this virus.
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FIG. 1.
Schematic representation of genomic organizations of PRV
mutants. Four strains of virus were used in this study. PRV-Becker
(6), a wild-type laboratory strain, was used in a small
group of animals to verify prior assessments of PRV virulence in visual
circuitry (16). PRV-Bartha (5), an attenuated
vaccine strain, was used for similar purposes and was the parental
strain used for construction of PRV-BaBlu and PRV-D. Construction of
PRV-BaBlu and PRV-D is described in Materials and Methods and has been
reported previously (16, 38).
80°C over the duration of these experiments.
Aliquots were thawed immediately prior to injection, and unused
portions were inactivated with Clorox bleach and discarded.
Primary antibodies.
The following reagents were used to
localize infected neurons in fixed sections of brain tissue. A rabbit
polyclonal antiserum (Rb133) produced against acetone-inactivated virus
reacted with cells infected by all strains of virus used in this
analysis (15). PRV-D-infected cells were identified with a
polyvalent rabbit antiserum (Rb1544) that recognizes the glycosylated
precursor of gI (formerly designated gp63). This antiserum was produced by immunizing rabbits with a polypeptide corresponding to amino acids
60 to 268 of gI expressed in Escherichia coli
(45). PRV-BaBlu-infected cells were identified with mouse
monoclonal antibodies specific for -galactosidase, which were
purchased from Sigma Chemical Company (St. Louis, Mo.) or 5 Prime-3
Prime, Inc. (Bolder, Colo.).
Experimental paradigms. Two well-characterized models of PRV invasiveness were used in this analysis (Fig. 2). Each model has the advantage of peripheral inoculation and addresses different aspects of viral invasiveness and cell-to-cell transmission. The eye model provides a measure of spread to the brain by anterograde routes. Virus infects ganglion cell neurons in the retina (17) and spreads through axons of these neurons in the optic nerve to invade second-order CNS neurons through synaptic contacts formed between the axon terminals and target neurons (Fig. 2, left panel). Prior analysis has shown that PRV-Bartha produces a selective infection of components of this circuitry involved in the regulation of biological timing, while other functionally distinct subdivisions of the visual system involved in visual perception and reflex movement of the eyes (dorsal geniculate nucleus and tectum) are not infected (17). The caudal brain stem model provides a measure of invasiveness of the brain by retrograde transport of virus. This model involves injection of virions into the stomach wall, where they invade the peripherally projecting axons of caudal brain stem neurons in the dorsal motor vagal nucleus (DMV) that control the stomach musculature (14, 15, 46). Thus, in this model, virus particles are retrogradely transported to DMV neurons in the brain stem, where first-order replication occurs. The number of brain stem neurons infected is roughly proportional to the amount of virus injected into the stomach muscles. After replicating in the DMV neurons, virus spreads to infect other CNS neurons synaptically connected to the DMV (Fig. 2, right panel). In this report we have focused on the transynaptic spread of virus to the immediately adjacent nucleus of the solitary tract (NST) and the spatially distant paraventricular hypothalamic nucleus (PVN). CNS neurons are also infected by viral transport through sympathetic ganglia and the spinal cord in this model, but infection via these routes is delayed relative to the DMV infection (14, 15, 34).
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Eye injections. Two microliters of virus suspension was injected into the vitreous body of one eye (approximately 5 × 105 PFU for PRV-D and 9 × 105 PFU for PRV-BaBlu). When a mixture of both viruses was injected, a 1:1 solution of virus stock was prepared, and 2 µl of the mixture was injected into the vitreous body of one eye. For some experiments, 2 µl of one virus was injected into one eye, and 2 µl of the other virus was injected in the other eye.
Stomach injections.
A total of 4 µl of virus was injected
into three sites on the ventral wall of the stomach. Three types of
double-inoculation paradigms were used. In the first, a 1:1 mixture of
PRV-D and PRV-BaBlu was prepared, and 4 µl of the mixture was
injected into the stomach as described above. In the second, 4 µl of
PRV-BaBlu was injected into three sites on the ventral stomach,
followed by injection of 4 µl of PRV-D into the same sites 24 h
later. In the third, the order of virus injections used in the second experiment was reversed (PRV-D followed by PRV-BaBlu 24 h later). Prior to injections, all animals were anesthetized with ketamine and
xylazine as described previously (18). Further details
regarding the experiments are provided in Table
1.
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Tissue processing.
At specific times following infection,
animals were anesthetized with an overdose of ketamine-xylazine and
killed by transcardiac infusion of buffered aldehyde solutions by
previously described procedures (12, 18). The brain was
removed and postfixed for 1 h at 4°C prior to cryoprotection in
20% phosphate-buffered sucrose at the same temperature. Tissue was
then cut in the coronal plane at 35 µm per section with a freezing
microtome and stored in cryopreservant (45) at 20°C
prior to immunohistochemical analysis. Immunohistochemical localizations were conducted by using immunoperoxidase or
immunofluorescence procedures. In both instances, sections at a
frequency of 210 µm through the brain were washed to remove
cryopreservant and then transferred to primary antibodies for a 24- to
48-h incubation at 4°C. The immunoperoxidase localizations were
accomplished with the avidin-biotin modification (22) of the
peroxidase-antiperoxidase method with affinity-purified secondary
antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.)
and Vectastain Elite reagents (Vector Laboratories, Burlingame,
Calif.). The immunofluorescence localizations used secondary antibodies
conjugated to fluorescein isothiocyanate (FITC) or CY2 or CY3 (Jackson
ImmunoResearch Laboratories, Inc.) to produce green (FITC and CY2) or
red (CY3) fluorescence. These fluorescent secondary antibodies were
used at a dilution of 1:500. Sections from tissue processed with
FITC-conjugated immunoglobulin G were mounted on slides, and coverslips
were applied with Fluoromount G (Southern Biotechnology Associates,
Inc., Birmingham, Ala.). Tissues processed with the CY2- and
CY3-conjugated secondary antibodies were dehydrated and cleared, and
coverslips were applied with Cytoseal 60 (Stevens Scientific, River
Dale, N.J.). Specific details of all of the aforementioned procedures
have been published previously (12, 18).
Experimental analysis. The virulence of each strain was estimated by observing and documenting symptoms of viral infection in all animals. We did not conduct a quantitative analysis of mean time to death as previously done with PRV-Becker and PRV-Bartha (10). However, a subset of animals infected with each strain succumbed naturally to infection, and these animals gave natural terminal end points. We also carefully monitored animals for overt signs of viral infection (lethargy, weight loss, or spiked coat). We combined this information with that obtained in our prior analyses of virulence (10, 16) to produce the graphic representations shown in Fig. 3.
The extent of infection in the CNS produced by each strain of virus was initially assessed with tissue processed for immunoperoxidase localization of viral antigens with the rabbit anti-PRV polyclonal antiserum that recognizes all strains of PRV used in this study. Immunoperoxidase localizations were then conducted with tissue from each animal by using the gI and-galactosidase monoclonal antibodies. Coinfection of neurons in animals inoculated with PRV-D and
PRV-BaBlu was determined by using dual-label immunofluorescence localizations. The gI glycoprotein was identified with the secondary antibody conjugated to FITC or CY2 to produce green fluorescence, and
-galactosidase was identified with the CY3-conjugated secondary antibody to produce red fluorescence. Fluorophors in each sample were
excited with the appropriate filters, and infected neurons were
photographed with Kodak Ektachrome 160 color film or images were
digitized and analyzed with a Simple 32 image analysis system (C-Imaging Systems). Dual-labeled images were collected by dual exposure of the same frame of color slide film and by digitizing images
of sections illuminated with a filter cube that excites both CY2 and
CY3 (Omega). Examination of tissue with high-magnification objectives
(20× to 40×) provided assurance that yellow fluorescence was due to
colocalization of both fluorophors rather than to overlap of cells
differentially infected with different viruses.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our goal was to define basic parameters that would facilitate the use of two recombinant viruses expressing unique reporters for the definition of complex synaptic arrangements in neuronal circuitry. Accordingly, we sought to determine if prior infection of neurons with one strain of virus rendered neurons resistant to infection by a second strain. We found that differences in virulence and rate of invasion of visual and autonomic circuits by the two recombinant viruses markedly affected the ability of the two viruses to establish a double infection.
Virulence parameters. Clear differences in the virulence of infections produced by PRV-Bartha and by the recombinant viruses were apparent in both visual and autonomic circuitry (Fig. 3). Although PRV-D and PRV-BaBlu are derived from PRV-Bartha, PRV-D was more virulent than PRV-Bartha, which, in turn, was more virulent than PRV-BaBlu. This rank order of virulence was the same for both experimental models even though they involve infection of different populations of neurons.
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-galactosidase gene at the gG gene locus of PRV-Bartha had the
effect of delaying the onset of symptoms and extending survival by
approximately 20 h. Whether this phenotype is due to the lack of
gG, to effects of the lacZ insertion on adjacent genes (Us3
or gD gene), or to a direct effect of -galactosidase expression is
not clear at this time.
PRV-D was more virulent than the parental strain or PRV-BaBlu in both
circuits (Fig. 3). Animals exhibited symptoms at approximately 80 h after inoculation, and no animal survived longer than 95 h,
irrespective of whether virus was injected into the eye or stomach
wall. Additionally, the symptoms exhibited by PRV-D-infected rats were
quite similar to those produced by the virulent Becker strain of PRV.
For example, oral and nasal excretions were much more pronounced in
these animals than in rats inoculated with PRV-BaBlu or PRV-Bartha. It
is likely that the increased virulence of PRV-D results from the
expression of gI, Us9, or Us2, the genes for which are absent in
PRV-Bartha and PRV-BaBlu.
As expected, when animals were coinfected with two viruses that
differed in virulence, the animal exhibited the virulence parameters of
the most virulent strain. This was true in every model tested (Fig. 3).
Single infections. In single-injection paradigms, the time course of anterograde or retrograde transynaptic spread of PRV-D into the CNS was at least 24 h faster than that for PRV-Bartha or PRV-BaBlu. PRV-D-infected animals also died sooner. However, it is important to point out that recent data have shown that PRV virulence does not always correlate directly with the extent of viral invasion (46).
(i) Anterograde transneuronal infection. Infection of the suprachiasmatic nuclei (SCN) in the hypothalamus is dependent on the establishment of a productive infection in retinal ganglion cells in the eye, followed by anterograde transneuronal infection of a subset of SCN neurons that receive direct synaptic input from these infected ganglion cells (Fig. 2). Other SCN neurons that are not contacted by retinal axons become infected through local connections within the SCN. Therefore, three orders of viral replication and transynaptic passage can be confidently assessed in this model. Clear differences in the progression of infection in the SCN were observed following intravitreal injection of PRV-Bartha, PRV-BaBlu, and PRV-D. After injection of the parental virus (PRV-Bartha), infected neurons were first visible in the retinorecipient portion of the SCN at 73 h postinoculation (Fig. 4A) and became increasingly prevalent in this portion of the nucleus through 80 h (Fig. 4B). By 93 h, transynaptic passage of virus led to infection of neurons throughout the SCN (Fig. 4C). Replication and transynaptic passage of PRV-BaBlu was delayed relative to those produced by PRV-Bartha. Scattered infected neurons were observed in the retinorecipient SCN by 73 h, but subsequent replication and transynaptic passage of virus in the SCN were substantially delayed. For example, it took PRV-BaBlu 94 h to achieve the same extent of infection in the retinorecipient SCN that was achieved within 80 h of injection of PRV-Bartha (compare Fig. 4B and E). Furthermore, the extent of infection in the SCN at 119 h after intravitreal injection of PRV-BaBlu was less than that produced by PRV-Bartha at 93 h (compare Fig. 4C and F). This contrasted with the more rapid progression of infection produced in the same circuitry by PRV-D. Animals infected with this virus exhibited occasional infected neurons in the retinorecipient SCN as early as 48 h after injection of PRV-D (Fig. 4G), an onset of infection that was approximately a day earlier than that after injection of either PRV-Bartha or PRV-BaBlu. Thereafter, the number of infected neurons in the retinorecipient SCN increased rapidly, and there was transynaptic spread of virus throughout the SCN within 77 h of inoculation (Fig. 4I).
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(ii) Retrograde transneuronal infection. At 70 h following injection of PRV-BaBlu into the stomach, first-order replication of virus was apparent in a large number of neurons in the DMV of the caudal brain stem, and there was slight retrograde transynaptic infection of neurons in the immediately adjacent NST (Fig. 5C). By 99 h the transynaptic passage of virus had infected neurons throughout the NST (Fig. 5D) and had also spread into synaptically linked areas of the caudal brain stem, such as the area postrema and the portion of the brain stem tegmentum containing the A1 catecholamine cell group (data not shown). This temporal progression of infection was in dramatic contrast to that produced by identical inoculation of PRV-D. The same magnitude of retrograde transynaptic infection of the DMV and NST observed at 70 h after injection of PRV-D required approximately 100 h to be achieved after injection of PRV-BaBlu (compare Fig. 5B and D).
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Coinfection studies. (i) PRV-D infection suppresses PRV-BaBlu invasion of visual circuitry. By injecting PRV-D into one eye and PRV-BaBlu into the other eye, we were able to compare the efficiencies of anterograde transynaptic infection of the two viruses under circumstances in which they did not compete with one another for the initial infection of retinal ganglion cells. Under these circumstances, PRV-D rapidly invaded second-order neurons in the SCN and then passed transynaptically to infect other neurons in the SCN by 77 h (Fig. 6D). By 95 h the virus had moved transynaptically to infect neurons throughout the SCN (Fig. 6F). Similarly, PRV-D-infected neurons were prevalent within the intergeniculate leaflet (IGL) of the thalamus (Fig. 6E), a circumscribed cell group in the geniculate complex that receives synaptic input from collaterals of the retinal axons that also synapse in the SCN (Fig. 6A). The extensive PRV-D infection in both the SCN and IGL contrasted with the restricted distribution of neurons infected with PRV-BaBlu. In both regions, the number of neurons replicating PRV-BaBlu was a small subset of the total number of infected neurons (Fig. 6D to F). Furthermore, the relative number and distribution of PRV-BaBlu-infected neurons in the longest-surviving animals were reduced from those observed at the same time when PRV-BaBlu was injected alone. Neurons replicating both viruses were observed, but single infections predominated. There are several potential explanations for this finding. PRV-BaBlu may be less efficient than PRV-D at infecting the primary retinal ganglion cells, it may be transported to the axon terminals at a lower rate, or it may be less efficient at transynaptic spread to the second-order neurons. Additionally, neurons previously infected with PRV-D may exclude PRV-BaBlu from replicating, a phenomenon that is well documented for tissue culture infections (7, 8, 24). In any case, anterograde transynaptic infection of retinorecipient neurons and subsequent spread of virus through synaptically linked neurons are dominated by PRV-D even though both viruses are injected simultaneously and do not have to compete with one another for the initial infection of retinal ganglion cells.
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(ii) PRV-D suppresses PRV-BaBlu retrograde invasion of autonomic circuitry. PRV-D also reduced the invasiveness of PRV-BaBlu in coinjection paradigms involving autonomic circuitry. These experiments introduce the virus into the caudal brain stem through viral invasion of axon terminals and retrograde transport to the parent cell bodies (14, 15, 34, 46). Three experiments were conducted to assess neuroinvasiveness in this paradigm. The first involved simultaneous inoculation of equivalent amounts of the two strains in question. In the other two experiments, infection by each virus was separated by 24 h. One set of animals were inoculated with PRV-BaBlu followed by PRV-D, and for the other set the sequence was reversed. These manipulations allowed us to evaluate the influence of one strain on the replication and invasiveness of the other. In all three experimental models, PRV-D infected a larger population of neurons earlier in the course of infection than did PRV-BaBlu.
After injection of a mixture of both viruses, PRV-D infected a large number of DMV (first-order) and NST (second-order) neurons within 67 h (green fluorescence in Fig. 6H). A relatively small percentage of these PRV-D infected neurons were also replicating PRV-BaBlu (yellow fluorescence), while only scattered neurons were infected solely with PRV-BaBlu (red fluorescence). At longer postinoculation intervals, the number of infected neurons in both the DMV and NST increased substantially. Neurons infected with both viruses were common but were substantially reduced compared to the number of neurons replicating PRV-D. Neurons infected with only PRV-BaBlu were very rare (Fig. 6I). Often, neurons containing dense staining for-galactosidase also exhibited more restricted staining for the gI
marker of PRV-D. In some neurons the gI immunoreactivity was
restricted to the cell nucleus, while in others it filled both the
nucleus and the cytoplasm. It should be emphasized that the
invasiveness of PRV-BaBlu in this coinjection paradigm was less than
that observed at comparable survival intervals when PRV-BaBlu was
injected alone. This is apparent in comparisons of the extent of
PRV-BaBlu infection 70 h after individual inoculation (Fig. 5C)
with that produced in the dual-inoculation paradigm at 67 and 72 h
(Fig. 6H and 6I). As we observed in the eye model, the rapid invasion
and subsequent spread of PRV-D interfered with infection and spread of
PRV-BaBlu. Interestingly, the opposite does not appear to be true;
prior infection by PRV-BaBlu does not suppress the replication of PRV-D
in the same neurons. We deduced this by comparing the localizations of
-galactosidase and gI immunoreactivities in neurons replicating both
viruses. We have shown previously that at early stages of infection,
viral proteins are found predominantly in membranes in and around the nucleus, with little antigen present in neuronal processes. Neurons in
late stages of infection exhibit robust staining of viral antigens in
the cell body and processes (9, 39). The presence of gI immunoreactivity in perinuclear membranes of neurons in advanced stages
of PRV-BaBlu replication (judged on the basis of the extensive distribution of -galactosidase immunoreactivity throughout the infected neurons and their processes) indicated that prior infection with PRV-BaBlu did not suppress replication of PRV-D.
The ability of PRV-D to replicate and spread through synaptically
linked neurons previously infected with PRV-BaBlu was illustrated vividly in the experiment in which PRV-BaBlu was injected into the
stomach wall at 24 h prior to injection of PRV-D. When animals were killed at 94 h after the initial inoculation (an effective postinoculation interval of 70 h for PRV-D), the distribution of
antigens unique to each virus in the caudal brain stem was quite
similar to that observed 72 h after simultaneous injection of both
strains (compare Fig. 6I and K). Thus, prior replication of PRV-BaBlu
had little effect on the ability of PRV-D to replicate in the DMV and
pass transynaptically to infect NST neurons.
Although prior infection with PRV-BaBlu had no apparent effect on PRV-D
replication and transynaptic passage in the caudal brain stem,
examination of the forebrain regions connected to the DMV and NST
indicated that PRV-D replication had been delayed in these neurons. For
example, the PVN projects densely to the DMV-NST complex (Fig. 6G), and
neurons in this area have been shown to be infected by transynaptic
passage of PRV injected into the stomach (46) and other
visceral targets (see reference 27 for a review). At
72 h following simultaneous inoculation of PRV-D and PRV-BaBlu
into the stomach wall, infected neurons that contained one or both
viruses were observed in the PVN (Fig. 6J). As in the caudal brain
stem, PRV-D infected the largest number of neurons, consistent with the
previously demonstrated higher rate of replication and invasiveness.
When PRV-BaBlu was injected 24 h prior to PRV-D and the animals
were sacrificed 94 h after the PRV-BaBlu injection (70 h after the
PRV-D injection), the extent of infection produced by PRV-D was
reduced. A substantial number of PRV-BaBlu-infected PVN neurons were
observed (Fig. 6L), and the relative number was essentially the same as
that observed at equivalent survival in single-injection paradigms
involving this virus (data not shown). However, many of the
PRV-BaBlu-infected cells also exhibited gI immunoreactivity in their
nuclei, indicating early stages of coinjection with PRV-D (Fig. 6J).
The number of these cells was substantially reduced from the numbers
observed at equivalent survival (approximately 70 h) after
simultaneous injection of the two viruses (Fig. 6J) or single injection
of PRV-D (data not shown). These data indicate that while invasion and
transynaptic passage of PRV-D in the caudal brain stem is not
compromised by prior infection with PRV-BaBlu, infection of more
spatially distant regions of the brain synaptically linked to the DMV
and NST is delayed.
When the temporal sequence of injection was reversed (PRV-D infection
followed 24 h later by PRV-BaBlu infection), PRV-BaBlu barely
invaded the CNS before the animals died. This most likely reflects the
increased virulence of PRV-D and the inefficient neuroinvasiveness of
PRV-BaBlu. Two animals that were sacrificed approximately 72 h
after the PRV-D inoculation exhibited a pattern of PRV-D-infected
neurons equivalent to that seen after single injection of this virus
(data not shown). Thus, the only conclusion that we made from these
data was that subsequent inoculation of PRV-BaBlu did not alter the
temporal dynamics of PRV-D invasion within this time frame.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The following conclusions can be drawn from these experiments. First, the progression of invasion and spread of PRV-D in the CNS is significantly faster than that for infection by PRV-Bartha or PRV-BaBlu in the same circuitry. Second, PRV-D is more virulent than either parental PRV-Bartha or PRV-BaBlu, but PRV-BaBlu is less virulent than PRV-Bartha. Third, neurons have the capacity to replicate both recombinants under the proper circumstances. Fourth, prior infection with one recombinant reduces the ability of neurons to replicate the other recombinant strain. Fifth, prior infection by PRV-D is more effective than infection with PRV-BaBlu in reducing invasion and spread by the second virus. These findings have important implications for the design and interpretation of investigations involving the use of multiple strains of virus to define neural circuits and indicate that interference of one strain with the replication of a second strain may produce false negatives. The data also suggest that the use of isogenic strains of virus may produce more reliable co-infection.
Neurovirulence in coinfection paradigms. The influence of virulence upon coinfection was readily apparent in all of our experimental models. The more virulent PRV-D tended to suppress the replication and spread of the less virulent PRV-BaBlu, but this attenuated strain was less efficient at reducing the replication of PRV-D. The effect could not be attributed to differences in infectious dose, since the injected concentrations of the two strains were very similar. In fact, the titer of PRV-BaBlu was slightly higher than that of the more virulent PRV-D (4.75 × 108 versus 2.5 × 108). The difference in virulence of the two PRV strains most likely reflects the presence or absence of the gI, gE, Us9, and Us2 genes in the Us region of the PRV genome. Prior analysis of PRV in visual circuitry has shown that deletion of one or both of the gE and gI envelope glycoprotein genes from this region of the wild-type genome reduces the virulence produced by intravitreal inoculation of wild-type PRV (16, 45). Similarly, selective mutations in the terminal cytoplasmic domain of gE produce a similar reduction of virulence but do not compromise viral invasiveness of visual or autonomic circuits (42, 46). We recently found that deleting the Us9 gene from PRV-Becker also resulted in a reduction of virulence (unpublished observations). The insertion of lacZ in the gG gene (PRV-BaBlu) in the present study further attenuated the virulence of PRV-Bartha and also slowed the invasiveness of the virus. The differences in virulence and invasiveness between the two strains biased the temporal course of viral replication and spread in favor of PRV-D, an aspect of invasiveness with an important influence upon the number of neurons that were coinfected with PRV-BaBlu.
Neuroinvasiveness in coinfection paradigms. The double-infection approach has been employed with strains of herpes simplex virus and PRV engineered to express unique gene products and is dependent on the ability of two strains of virus to replicate in the same neuron. In studies of neuronal circuits that modulate the activity of the viscera and homeostatic function, Jansen and colleagues (23) and Levatte et al. (26) demonstrated that recombinant viruses expressing unique reporters can be used to dissect the synaptic organization of polysynaptic circuits. Such experiments are a powerful exploitation of the neurotropism and invasiveness of alphaherpesviruses to define principles of synaptic organization that simply cannot be defined with other techniques. Our experiments underscore the usefulness of this experimental approach but also reveal the importance of considering the biology of mixed infections by similar viruses in the nervous system of a living animal. From tissue culture experiments, we know that attachment and entry of herpesvirus particles can be blocked if the cell is already infected or if it expresses the viral membrane protein gD (7, 8, 24, 43). We do not know if such gD-mediated exclusion functions in animals. For PRV, gD is not required for cell-cell spread of infection, and thus gD-mediated exclusion may be possible only during primary infection where particles attach to infected cells. Coinjection experiments should not be subject to this type of exclusion if the rates of attachment, entry, and replication of both viruses are similar. However, any experiment in which replication of one virus takes place in a neuron prior to the attachment of the second virus particle may well be subject to gD-mediated exclusion.
The number of neurons infected by retrograde transport of PRV-D from the stomach far exceeded the number of those that were coinfected or were only replicating PRV-BaBlu. It is not clear why this should be, but one possibility is that PRV-BaBlu infects axon terminals inefficiently compared to PRV-D. Comparison of the rates of attachment, fusion, and entry for each virus has not been done. The diffusely ramifying axonal processes of DMV neurons in the stomach musculature and the environment of the inner surface of the retina provide a nonuniform surface for viral infection that we cannot mimic in the tissue culture dish. It is also possible that the presence of the gI and Us9 genes in PRV-D improves the efficiency of invasion of permissive neurons in both stomach and retina. Attachment and entry of PRV-BaBlu might be reduced by simple competition with PRV-D if the number of receptors is limiting. This possibility is supported by the predominance of PRV-D infection in both models. As we do not know the multiplicity of infection (number of virus particles per number of susceptible cells or axon terminals), we cannot say with confidence that every permissive cell is infected with both viruses. We know that while coinfection does occur, we likely are not saturating permissive cells with both viruses, because we observe an exclusive infection by PRV-BaBlu in a small number of neurons after simultaneous injection of both strains. The differential abilities of two strains of virus with differing virulence to efficiently coinfect neurons depend on the timing of the second infection with respect to the first, the effect of the first virus on neuronal function, and the defense that the brain mounts against the infection. Even with isogenic strains where every gene is identical with the exception of a reporter, prior replication of one virus will certainly influence any subsequent replication by another virus, if only through the marked effect of herpesvirus infection on cell macromolecular metabolism (36). In addition, infection of the CNS with PRV and herpes simplex virus elicits a complex and predictable nonneuronal response that isolates infected neurons and may suppress viral replication (see reference 11 for a review). Importantly, the magnitude of this nonneuronal response is directly dependent on the virulence of the infecting virus (14, 35) and the cells that contribute to it. These cells express potent molecules such as nitric oxide in response to PRV infection (37), which have documented antiviral effects (see reference 33 for a review). Thus, the failure of PRV-BaBlu to coinfect neurons previously infected by PRV-D may be related to the nonneuronal response elicited by prior replication of PRV-D.| |
ACKNOWLEDGMENTS |
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We gratefully acknowledge the technical assistance of Marlies Eldridge, Jen Shew Yen, Tariq Syed, and Sadiq Syed.
The contribution of J.-S. Kim was supported by a fellowship awarded by the Korea Science and Engineering Foundation. This work was supported by NIH grants RO1s MH53574 (to J. P. Card) and NINDS33506 (to L. W. Enquist).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Neuroscience, 446 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260. Phone: (412) 624-6995. Fax: (412) 624-9198. E-mail: Card{at}bns.pitt.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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