Bioluminescent Imaging and Histopathologic
Characterization of WEEV Neuroinvasion in Outbred CD-
Aaron T. Phillips1*, Charles B. Stauft1, Tawfik A. Aboellail1, Ann M. Toth2, Donald L. Jarvis2,
Ann M. Powers3, Ken E. Olson1
1Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America, 2Department of Molecular
Biology, University of Wyoming, Laramie, Wyoming, United States of America, 3Division of Vector-Borne Infectious Diseases, Centers for Disease Control, Fort Collins,
Colorado, United States of America
Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in
humans and equids. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan
(McM) strain result in high mortality within 4 days of infection. Here in vivo and ex vivo bioluminescence (BLM) imaging was
applied on mice intranasally infected with a recombinant McM virus expressing firefly luciferase (FLUC) to track viral
neuroinvasion by FLUC detection and determine any correlation between BLM and viral titer. Immunological markers of
disease (MCP-1 and IP-10) were measured and compared to wild type virus infection. Histopathology was guided by
corresponding BLM images, and showed that neuroinvasion occurred primarily through cranial nerves, mainly in the
olfactory tract. Olfactory bulb neurons were initially infected with subsequent spread of the infection into different regions
of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few
infected glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The
trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem.
The recombinant virus WEEV.McM.FLUC had attenuated replication kinetics and induced a weaker immunological response
than WEEV.McM but produced comparable pathologies. Immunohistochemistry staining for FLUC and WEEV antigen
showed that transgene expression was present in all areas of the CNS where virus was observed. BLM provides a quantifiable
measure of alphaviral neural disease progression and a method for evaluating antiviral strategies.
Citation: Phillips AT, Stauft CB, Aboellail TA, Toth AM, Jarvis DL, et al. (2013) Bioluminescent Imaging and Histopathologic Characterization of WEEV
Neuroinvasion in Outbred CD-1 Mice. PLoS ONE 8(1): e53462. doi:10.1371/journal.pone.0053462
Editor: Patricia V. Aguilar, University of Texas Medical Branch, United States of America
Received August 21, 2012; Accepted November 29, 2012; Published January 2, 2013
Copyright: ? 2013 Phillips et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the National Institutes of Health (NIH) (R01 AI46435) and the RMRCE (AI065357). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Of the 29 mosquito-borne viral species within the Alphavirus
genus (Togaviridae), at least 16 are known to cause disease in
humans and animals [1–4]. Although arthritis, acute flu-like
illness, and rash are attributable to many alphaviral infections,
some alphavirus species lead to CNS infection and encephalitis.
Alphaviruses most often associated with CNS infection are limited
to the Americas, and include strains of eastern equine encephalitis
virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and
western equine encephalitis virus (WEEV). WEEV is normally
maintained in a transmission cycle involving Culex tarsalis
mosquitoes and passerine birds . Equids and humans can be
infected but do not contribute to the maintenance cycle. WEEV
was first isolated from an outbreak of equine encephalitis in the
San Joaquin Valley of California that affected almost 6,000 horses
and was associated with an equine mortality rate of 50% .
Enzootic activity of WEEV is detected most summers in southern
California via sero-conversion of sentinel animals or testing pools
of primary vectors . According to the USDA, epizootics have
been reported in horses (Canada 1975), turkeys (California 1993–
1994; Nebraska 1957), and emus (Texas and Oklahoma 1992).
These findings highlight the potential for a WEEV epidemic
outbreak in humans. Naturally acquired infection of humans has
been estimated to yield fatality rates of 8% to 15% . Human
patients may present clinically with symptoms ranging from an
acute febrile illness to fulminant encephalitis. Neurologic sequelae
may be present in survivors, particularly children and infants .
Experimental evidence suggests that WEEV strains can be
categorized into high and low mortality phenotypes in mice
[10,11]. Among the high mortality phenotypes, McM induces
rapid and lethal encephalitic disease in a mouse infection model
and is the basis of the data reported here.
New World alphavirus strains readily cause encephalitis after
aerosol or intranasal exposure in animal models making these
alphaviruses potential biodefense agents requiring efficacious
therapeutic and vaccine-based responses. Previous studies have
shown that, following respiratory routes of inoculation, neuroinva-
sion occurs preferentially through the olfactory tract by initial
infection of neuroepithelia [12–14]. Responsible for sensing
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odorants, neuroepithelial tissue is in direct contact with the
environment and easily subject to initial infection by these routes.
Viral dissemination into the CNS likely occurs through the long
axonal projections of olfactory sensory neurons (OSN), which
converge upon the olfactory bulb of the CNS (Figure S1).
Histological evidence supports this proposed mechanism 
however, published characterizations are few. Notably, reports
characterizing WEEV infection in an animal model are rare .
Additionally, WEEV is a naturally-occurring recombinant virus
generated from ancestral EEEV- and Sindbis- like virus . As
EEEV and Sindbis have markedly different disease phenotypes in
humans, a better description of WEEV pathogenesis is needed to
identify infection patterns.
All alphaviruses have an enveloped nucleocapsid containing
a single-stranded, positive-sense RNA genome with a 59 methyl-
ated cap and 39 polyadenylated termini. The 59 end of the viral
genome is translated into 4 nonstructural proteins (nsP 1–4) that
form viral replication complexes. A negative-strand RNA replica-
tion intermediate is generated and contains a subgenomic pro-
moter (SGP) or internal initiation site that initiates transcription of
the 26S subgenomic RNA. The 26S subgenomic RNA encodes
the structural proteins (Capsid, E3, E2, 6K, and E1) used in the
assembly of new virions. Genomic RNA is fully infectious when
transfected into permissive cells. Consequently, alphaviruses are
readily manipulated in the laboratory using traditional cloning
techniques followed by in vitro transcription of the viral genome.
Recombinant alphaviruses have been developed in which the SGP
sequence is duplicated to drive expression of heterologous genes
during infection [17–26]. The infectious cDNA clones are
sometimes referred to as alphavirus expression systems (AES)
. Most AESs which have been developed to date have been
based on Old World alphaviruses such as Sindbis [17,18,20,22–
24,27–30], Semliki Forest , O’nyong-nyong , or chikun-
gunya viruses . Although these AESs have significantly
enhanced our understanding of virus-vector and virus-host
interactions, reports of AESs based on propagating New World
encephalitic alphavirus isolates are less prevalent in the literature
[21,33,34]. To our knowledge, there have been no reports
describing an infectious WEEV-based AES.
A more complete understanding of the alphavirus infection
patterns in vertebrates is crucial to characterizing the pathogen-
host relationship. The technology of in vivo imaging promises to
streamline the process of investigating infectious agents in an
animal model. Firefly luciferase (FLUC) is a commonly used
in vivo BLM reporter. FLUC catalyzes the oxidation of its
substrate, luciferyl adenylate (luciferin), with the net products
being light, in the form of a photon, and oxyluciferin . Firefly
luciferase (FLUC) and its substrate, luciferin, was first used to
describe the distribution of bacteria in a living host  and has
subsequently been used to describe infection in mice for
herpesvirus type-I , a neurovirulent strain of Sindbis virus
[17,23,24,38], VEEV , EEEV [33,38], and human immuno-
deficiency virus (HIV) gene expression . Research applying
in vivo imaging technology to analyze vaccinia virus infection has
shown potential for predicting lethality of virus infection based on
A well-characterized animal model is a crucial component of
antiviral research. This is especially true when considering
pathogens such as WEEV in which the target cells (neurons) are
difficult to work with in culture. Additionally, components of the
CNS, such as the blood-brain-barrier and multiple cell types are
absent from in vitro model systems. Outbred CD-1 mouse model of
McM infection has been developed and used to characterize the
infection using traditional methods . Chemokines such as
MCP-1 and interferon-gamma increased significantly in infected
brain tissue . The inflammatory process plays a role in
neuropathogenesis, but this role may be secondary to neuronal
death resulting directly from viral replication. This is supported in
studies with VEEV which have shown extended mean time to
death of mice treated with anti-thymocyte serum . In the case
of neurovirulent Sindbis virus, neuronal death is due to in-
flammatory and excitotoxic insults or apoptosis depending on the
strain of virus, age of the mouse model, and specific neuroana-
tomical location .
The goals of these studies were two-fold. First, we sought to
better characterize the process of neuroinvasion and CNS
dissemination in the mouse model and use BLM imaging to
follow infection and identify sub-anatomic regions where the virus
attains high levels of replication. The second goal of these studies
was to characterize the luciferase-expressing recombinant WEEV
for its ability to induce disease similar to the wild-type virus, and to
determine if BLM signal correlated with biological markers of
disease. Ultimately, quantitative measurements of viral BLM can
be used to evaluate the efficacy of antiviral strategies within the
intact live animal. We show that the McM-based AES is capable of
producing a conveniently measured marker of infection and, in
doing so, provides a system for evaluating therapies aimed at
preventing disease arising from inhalation of New World
alphavirus strains. Currently, there are no approved therapies
for use in humans, and the WEEV AES presented in this report
should enhance the utility of the mouse model in developing
Materials and Methods
A full-length infectious clone (IC) of the McMillan strain of
WEEV (pMcM) was a kind gift of Dr. Thomas Welte (Colorado
State University), was derived from virus obtained from the
Arbovirus Reference Collection at the Center for Disease Control
and Prevention in Fort Collins, CO, USA, and has been previously
studied . Detailed descriptions of the molecular cloning
methods used to engineer WEEV.McM.FLUC are provided in
supplemental materials and methods section (Methods S1). In
brief, SGP sequence (nucleotides 7341–7500 of viral genome) was
duplicated immediately downstream of the last nucleotide of E1.
FLUC was inserted immediately downstream of the new SGP.
The duplicated SGP was used to initiate transcription of FLUC
and the 2ndsubgenomic mRNA.
Rescue of Virus from Infectious Clone
Linearized ICs were purified by QIAprep Spin MiniPrep Kit
(Qiagen, Valencia, CA USA) and IC genomic RNA was in vitro
transcribed using a T7 RNA Polymerase and MAXIscriptTMkit
(Life Technologies, Grand Island, NY USA). BHK cells
(26107cells in 400 mL) were electroporated with 20 mL of
genomic RNA using an ECM 630 electroporator (BTX Harvard
Apparatus, Holliston, MA USA). Two pulses of 450 V, 1200 V,
and 150 mF were administered. Media was taken from electro-
porated cells and passaged once in BHK cells to make a stock
virus. Supernatant was collected at 48 hpi and stored at 280uC.
This stock was quantified using plaque titration in Vero cells and
used for subsequent experiments. We have previously observed
WEEV-based double subgenomic constructs expressing FLUC to
be acceptably stable. Substantial luciferase activity was present out
to the 3rdpassage in BHK-21 cells and out to the 4thpassage in
C6/36 cells (data not shown).
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Virus titrations were performed in duplicate and plaque assays
were performed as described by Liu et al. (1970).
Mouse Infection and Imaging
All animal protocols used in these experiments were reviewed
and approved by the Animal Care and Use Committee at
Colorado State University (Permit #11-2605A). Mice were
handled in compliance with the PHS Policy and Guide for the
Care and Use of Laboratory Animals. Female 4–5 week old CD-1
mice (Charles River Labs, Wilmington, MA USA) were used in
this study. Intranasal inoculation was conducted at a dose of
16104PFU of McM or WEEV.McM.FLUC in a volume of
20 mL delivered drop wise onto the nostrils of lightly anesthetized
animals. Imaging was performed after 150 mg/kg of luciferin
(30 mg/mL stock diluted in PBS) was injected subcutaneously
dorsal to the cervical spine of each infected animal. Administration
of luciferin via this route has been shown to result in more
consistent signal compared to intraperitoneal administration .
Each animal was imaged 10–15 minutes after injection of
substrate. Uninfected mice were used as an imaging control to
adjust for background. Mice were anesthetized by administration
of isoflurane (Minrad Inc, Bethlehem, PA USA) through an XGI-8
anesthesia system (Caliper Life Sciences) connected to the IVIS
200 camera during imaging. Exposure time was kept to 3 minutes
under standard settings for the camera. Living Image 3.0 software
(Caliper Life Science) was used to analyze and process images
taken using the IVIS 200 camera. A threshold for significant BLM
was established using negative imaging controls at 56103p/s/
cm2/sr. Total light emission from each mouse was accomplished
by creating a region of interest of standard size for each mouse and
collecting light emission data using the software.
Sagittal whole head sections of infected mice were imaged by
injecting mice with 150 mg/kg of luciferin (30 mg/mL stock
diluted in PBS) 24 h, 48 h, 60 h, and 72 h post-infection. After 10
minutes, mice were injected with another dose of luciferin, and
promptly euthanized via inhalation of a lethal dose of isoflurane.
Animals were decapitated and whole heads bisected along the
medial sagittal plane. Resulting sections were briefly rinsed with
PBS and promptly imaged.
Three animals were euthanized at each of three time points (24,
48, and 72 h.p.i.) after obtaining BLM images. Brains were
harvested and assayed as previously described . Briefly, whole
brains were harvested, homogenized in buffered media, and
clarified by centrifugation. Supernatant was collected and divided
into aliquots. Single aliquots were used to assay for immunological
markers (MCP-1 and IP-10, R&D Systems) or virus quantification
by plaque assays as described above.
Paraffin -embedded formalin fixed tissue was rehydrated,
treated with Tris-EDTA pH 9.0 at 90uC for 15 minutes, and
blocked with SuperBlock T20 (Thermo, Rockford, IL). Biotiny-
lated polyclonal rabbit anti-FLUC antibody (Abcam, Cambridge,
MA) was used at 1:1000 dilution and incubated overnight at 4uC.
Primary antibody was washed 3 times with Tris-buffered saline
containing 0.03% Tween 20(TBST). Secondary antibody was
strepavidin-horseradish peroxidase (Rockland, Gilbertsville, PA)
and was used at a 1:6000 dilution and incubated for 30 minutes at
room temperature. Slides were again washed three times with
TBST. 3,3’-diaminobenzidine (DAB) was added to the slides and
allowed to develop stain for 5 minutes. Hematoxylin was used to
counterstain. Hyperimmune horse serum generated against
WEEV Fleming strain (CDC, Fort Collins, CO) was used for
anti-WEEV IHC at 1:600 dilution. Secondary antibody was HRP-
conjugated rabbit polyclonal antibody to horse IgG heavy and
light chain (Abcam, Cambridge, MA) used at a 1:3500 dilution. All
other conditions remained unchanged.
Preparation and Administration of Vaccine
Cationic liposomes (100 mM DOTIM lipid+cholesterol) in 10%
sucrose solution were provided by Dr. Steve Dow (CSU). Antigen
was produced in baculovirus expression systems and purified
similarly to that previously described . Briefly, antigen (WEEV
E1 glycoprotein ectodomain) was produced in Sf9 cells using
a recombinant baculovirus encoding the first 408 amino acids of
WEEV McM E1 fused to a C-terminal 8XHis tag. The E1
ectodomain protein was purified from cell culture supernatant by
nickel affinity chromatography. Cationic-liposome–nucleic-acid-
complexes (CLNCs) were prepared as previously described .
Briefly, liposomes were diluted 1:5 in sterile Tris-buffered 5%
dextrose water (pH 7.4). Poly (I:C) (InvivoGen, San Diego, CA)
was then added to a final concentration of 0.1 mg/ml causing
spontaneous formation of CLNCs. The formed complexes were
mixed with WEEV E1 glycoprotein ectodomain antigen to a final
concentration of 50 mg/mL. Resulting liposome-antigen-nucleic
acid-complexes were used to vaccinate mice at 28 days (prime)
prior to challenge and again at 14 days (boost) prior to challenge.
Untreated control animals received only CLNCs and a sham
antigen preparation. Additional controls were untreated mice that
were not inoculated (to control for background luminescence).
Animals were administered luciferin as described in previous
section. There were a total of 3 mice per group.
All titration data were log10transformed and compared using
unpaired Student’s t test. In determining the correlation of PFU
with BLM, curves were analyzed using Pearson correlation with
95% confidence interval. For chemokine quantification compar-
isons, unpaired t test was used. Analysis was conducted using
statistical analysis software (SAS) version 9.2. Survival curves were
subjected to Kaplan-Meier (log rank test) analysis using Prism
version 4.00 for Windows (GraphPad). Quantitative analysis of
bioluminescence in the assessment of vaccine efficacy was
conducted using two-tailed t-test.
Recombinant FLUC-expressing WEEV Phenotype in CD-1
WEEV.McM.FLUC infection of CD-1 mice was characterized
after administering virus by the intranasal route. WEEV.McM.-
FLUC virus expressed FLUC throughout infection (Figure 1A–C)
where signal was restricted to the head. To determine if FLUC
signal in other anatomical regions was potentially masked by signal
from the head, mice showing signs of disease and strong luciferase
signal in the head region were euthanized, decapitated, and
imaged again with an opened visceral cavity. No signal was
detected outside the head region (data not shown). Exponential
increases in BLM signal (photons/second/centimeter2/steradian)
were observed from day 2 to day 3 post-inoculation (Figures 1B, C,
& E). Infection progressed from the nasal cavity toward more
caudal regions and was symmetrical with respect to the sagittal
axis. A 0% survival rate was observed for both WEEV.McM and
WEEV.McM.FLUC in animals (n=10) inoculated by the in-
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tranasal route and a comparison of mouse survival showed no
significant difference between the two viruses (Figure 1D) (P
value=0.4795). We then compared FLUC activity against
measured infectious virus titers from whole brain homogenates
(Figure 1E–G). WEEV.McM virus replicated to 100- fold PFU/
mL higher titer than WEEV.McM.FLUC within the first 24 hpi.
WEEV.McM virus titers by 72 hpi. Comparison of total flux (p/
s) for each WEEV.McM.FLUC -inoculated mouse with viral titer
measured within the whole brain (Figure 1G), showed a strong
correlation (Pearson R=0.9903 and R2=0.9807). As expected,
uninfected control animals receiving daily luciferin injections did
not show any signs of disease.
Localization of Virus by ex vivo Imaging of Medial Sagittal
Neuroinvasion and CNS dissemination in situ was detected in
CD-1 mice intranasally inoculated with WEEV.McM.FLUC and
sacrificed at various time points. These animals were euthanized,
decapitated, and whole heads were separated along the medial
sagittal plane. Representative images are presented (Figure 2A–D)
which illustrates the course of dissemination into the CNS. BLM
signal was initially observed in the nasal turbinates and olfactory
bulb. The infection proceeded along the lateral olfactory tract and
ultimately progressed through CNS regions consistent with
olfactory sense neuronal connectivity. Infection was invariably
bilateral and intensified in regions consistent with basal nuclei,
thalamus, and hypothalamus. Ultimately, FLUC expression was
detected in neocortical regions and the brainstem by day 3 PI.
Luciferase activity in the brainstem was separated into two distinct
regions. The midbrain expression of FLUC appeared continuous
with basal nuclei, thalamus, and hypothalamus and it was here
that the greatest BLM was observed. BLM signal within the pons
was discontinuous with signal from superior regions and failed to
approach levels seen within basal nuclei, thalamus, hypothalamus,
or cerebrum. Interestingly, the cerebellum was consistently spared
from infection despite high BLM activity within posterior pons
(cerebellum’s site of attachment).
Characterization of Chemokine Induction Resulting from
Infection with WEEV.McM.FLUC
Chemokines associated with severe CNS inflammation  and
previously shown to be highly induced during McM infection 
were measured in whole brain homogenates to compare the
inflammatory responses within the CNS between WEEV.McM
and WEEV.McM.FLUC (Figure 2E–F). Robust expression of
both MCP-1 and IP-10 was observed in both infected groups of
mice. Although WEEV.McM.FLUC was found to be attenuated
when compared to wild-type McM in terms of PFU/g brain tissue
(Figure 1F) and chemokine induction (Figure 2E&F), manifestation
of clinical disease was comparable. This is supported by MTD (3.0
days vs. 3.1 days) and signs of disease exhibited in both groups.
Early events indicated that inflammatory markers resulting from
WEEV.McM.FLUC infection were significantly less than those
observed in McM infected animals at 24 hpi, but levels rapidly
approached that of McM by the following day.
All infected mice (n=10) showed variably severe clinical signs
namely depression and motor deficits culminating at 48–72 hours
post infection (PI). Most affected animals developed ataxia, with
rhythmic raising and lowering of front limbs alternatively.
Reduced stride length was visually observed in affected animals
during voluntary movement. Animals in this intermediate stage of
the disease did not appear to have visual impairment as they
remained responsive to visual stimulation. In a later stage, animals
were unresponsive to visual stimuli, but were typically responsive
to touch. Mice showed unresponsiveness during handling only in
the latest stage of disease ($72 hpi). Lateral recumbency with
tachypnea was characteristic of this terminal stage of the disease.
Pathology and Immunohistochemistry
Sagittal sections of the head were selected to facilitate viewing
the nasal mucosa, olfactory nerve as it crosses the cribriform plate
and connects to the bulb to help determine anatomic locations of
the brain lesions (Figure 2 diagram). Pathologic lesions were
observed in histological specimens prepared from imaged mice
and in mice receiving WEEV.McM.
WEEV.McM.FLUC produced comparable lesions. Serial sections
were stained using immunohistochemical methods (anti-FLUC
and anti-WEEV for recombinant virus while only anti-WEEV was
used for wt virus infections) to demonstrate viral expression at the
affected sites. Lesions and luciferase immunopositivity were
observed to follow the same pattern as the imaged FLUC activity
(Shown repeatedly in Figures 3, 4, and 5). IHC staining of both
WEEV antigen and luciferase revealed that infection was almost
exclusively limited to neurons and that dissemination was likely
through the neuronal connectivity. Histopathologic alterations
encountered within the nasal cavity and brain are summarized as
Phase I: extraneural viral lesions.
luminal aggregates of moderate numbers of neutrophils and fewer
lymphocytes were detected in the nasal cavity with focal deciliation
of respiratory mucosa corresponding to the areas of inflammation.
In markedly immunopositive animals there was a focal erosion/
ulceration (full thickness necrosis) of the respiratory mucosa and
extension of the inflammatory exudate into the adjacent congested
submucosa (Figure 3A). IHC (anti-FLUC) revealed immunoreac-
tivity of variable numbers of neuroepithelium (Figure 3B). The
numbers of positive cells increased with the severity of the clinical
symptoms. The terminal end of the olfactory nerve before crossing
the cribriform plate to merge into the olfactory bulb showed a mild
degree of neuropathy with occasional digestion chambers in-
dicative of Wallerian-type degeneration or secondary demyelin-
ation (Figure 3C), indicative of impaired axonal transport.
Occasional lymphocytes were detected in the affected branches
of the olfactory, maxillary, glossopharyngeal, and hypoglossal
Phase II: Neuroinvasion.
24–48 h PI: In the olfactory bulb,
immunoreactivity (anti-FLUC or anti-WEEV) showed high degree
of neuronal specificity within CNS tissue (Figure 3D and Figure
S2). Neuronal necrosis was commonly evident in the glomerular,
granular, external plexiform and internal plexiform layers plus the
olfactory nerve layer at the ventrum of the bulb. In the areas of
microcavitation, there were infiltrations of a few neutrophils, glial
cells and lymphocytes (Figure 3E–F). Perivascular cuffing was
prominent throughout affected areas (Figure 3G–H). Also,
myeloid cavities of the head showed variable immunoreactivity
in lymphoid precursors and monoblasts. Surrounding skeletal
muscles showed inconsistent immunoreactivity.
Phase III:CNS dissemination
48–72 h PI: The severity of the lesions in the olfactory
bulb increased and the lesions started to propagate into more
caudal regions of the brain (Figure 4). Multifocal areas of necrosis
along with positive immunoreactivity were detected in the anterior
olfactory nucleus, ventral striatum and basal forebrain at the
ventrum of the brain. Dorsally, cerebral cortex was multifocally
Twenty four hours PI:
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involved along with the pia matter and Virchow-Robin space
(fluid-filled canals that surround perforating arteries and veins in
the parenchyma of the brain). Other areas that were consistently
involved were hippocampus, thalamus, hypothalamus, caudate
putamen, mid brain, cerebellar superior peduncle, and pontome-
dullary region. Cranial nerves also showed a focal to multifocal
immunoreactivity especially trigeminal nerve and its ganglia along
with optic and cochlear nerves. Trigeminal pathways indicated
significant immunopositivity and moderate to severe pathologic
alterations namely chromatolysis, vacuolation and individual
neuronal loss (Figure 5). To ensure that anti-FLUC staining was
truly representative of viral localization, IHC staining of
WEEV.McM and WEEV.McM.FLUC antigen was performed
to discern any differences in viral distribution and localization to
Figure 1. In vivo BLM imaging of infection progress using WEEV.McM.FLUC. A: 24 hpi B: 48 hpi C: 72 hpi D: Survival analysis of
WEEV.McM.FLUC and wild-type virus (WEEV.McM) from subcutaneous and intranasal virus challenge experiments. Note uniform lethality resulting
from intranasal exposure by WEEV.McM.FLUC. E: FLUC activity was quantitatively measured in each animal at 1, 2, and 3 d.p.i. time points. Results
from BLM analysis demonstrate robust FLUC activity as infection progressed with the greatest increase observed between days 2 and 3 post-
infection. F: Brains of animals infected with WEEV.McM attain a higher viral titer more rapidly when compared with WEEV.McM.FLUC. WEEV.McM.FLUC
titers approach that of McM by 72 h.p.i. G: Regression analysis of viral titer versus FLUC activity. Linear regression line appears curved due to log10
scaling of axis as required to clearly depict all data points (R2=0.9807).
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Figure 2. Schematic depiction of anatomical organization of mouse brain in medial sagittal view. AON: anterior olfactory nucleus, BF:
basal forebrain, CC: cerebral cortex (isocortex), CP: caudate putamen, F: fornix, HIP: hippocampus, OB: olfactory bulb, VS: ventral striatum. Progress of
infection with WEEV after intranasal inoculation (A–D). Whole heads were bisected along sagittal midline and imaged at 24 hpi (A), 48 hpi (B), 60 hpi
(C) and 72 hpi (D). Luciferase activity pattern is consistent with dissemination along olfactory pathways. Regions consistent with initial infection of the
nasal turbinates show pronounced FLUC activity at 24 hpi. However, nasal turbinate BLM activity is exceeded by signal from areas consistent with
infection proceeding through olfactory information processing within the CNS, including the lateral olfactory tract, anterior olfactory nucleus, basal
ganglia, thalamus, and cerebrum. Immunological markers of disease (MCP-1 and IP-10), resulting from WEEV.MCM.FLUC, are strongly induced and
comparable to WEEV.McM at 3 d.p.i. (E–F).
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Figure 3. I. Extraneural lesions 24 hpi. A) Focal erosion/necrosis of the olfactory mucosa with deciliation of the flanking epithelium and
neutrophil infiltration into the mucosa and submucosa (Bar=100 mm). B) IHC positive staining for FLUC is highlighted in a few neuropeithelial cells
subjacent to a focal loss of olfactory mucosa (Bar=200 mm). II- Neuroinvasion from olfactory nerve 48–72 hpi. C) Terminal end of olfactory
nerve shows a digestion chamber (arrow) with occasional lymphocytes infiltrating vacuolated branches (Bar=100 mm). D) Early immunoreactivity
(anti-FLUC) in the main olfactory bulb involving scattered neurons in the external plexiform and granular layers (Bar=100 mm). E) Sagittal section H&E
showing the connection between olfactory nerve (ON) and main olfactory bulb layers affected by multifocal necrotizing lesions with associated status
spongiosis and infiltration of neutrophils. Glom=glomerular layer; EPI=external plexiform layer; IPL=internal plexiform layer; and ONL=olfactory
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lesions. IHC staining with anti-WEEV polyclonal serum showed
indistinguishable staining patterns and localization of the lesions
(Figures S2 and S3) compared to anti-FLUC IHC staining.
Additionally, anti-WEEV IHC revealed that sinus hairs (vibrissae)
were affected, supporting trigeminal nerve pathway involvement
Antiviral Efficacy Testing
To demonstrate the utility of in vivo imaging technology in the
development of effective antivirals, mice 4–6 weeks old were
vaccinated with CLNCs and WEEV E1 glycoprotein ectodomain
using a prime-boost strategy. Prime-boost vaccination, with
WEEV E1 glycoprotein ectodomain, results in 100% protection
from challenge with 16104PFU of WEEV.McM.FLUC via
(Figure 6B). Quantitative analysis on resulting images was
performed and shows a significant reduction of bioluminescent
signal in treated compared to untreated mice (p,0.01) (Figure 6A).
wereimagedat 48 hpi
In this report, we have shown that a WEEV-based AES is
capable of inducing lethal encephalitic disease in a mouse model.
Optimal in vivo reporters such as firefly luciferase may be robustly
expressed from recombinant virus despite their large coding
sequence (1.6 kb). Immunohistochemistry and BLM measure-
ments have shown that WEEV.McM.FLUC retains functional
transgene expression throughout CNS dissemination when de-
livered by intranasal routes. BLM imaging of FLUC activity
provided approximation of viral titer within the brain and gross-
scale visualization of disease progression. While measurably
attenuated in both viral replication kinetics and in the induction
of immunological markers of disease, recombinant WEEV.McM.-
FLUC remained indistinguishable from wild-type virus in terms of
histopathological lesions and MTD. This finding supports the use
of AES in the assessment of antiviral strategies targeting aerosol
exposure. Beyond approximating viral titers, convenient quanti-
fication of reporter signal may provide powerful inferences
towards therapeutic efficacy and mechanism of protection.
Therapeutics targeting viral replication, for example, should be
capable of significantly decreasing reporter level and distribution.
We show that vaccination with liposome-antigen-nucleic acid-
complexes provides significant protection from challenge with
WEEV.MCM.FLUC and that quantitative analysis of BLM does
approximate prophylactic antiviral efficacy. As effective therapeu-
tic strategies become available, BLM imaging would provide an
excellent platform in which to rapidly evaluate such strategies.
Ex vivo imaging enhanced correlative histopathological exami-
nation as lesions here compared with viral expression levels
immediately prior to euthanization. In the case of WEEV, CNS
regions associated with viral expression indicated a preference for
neuroinvasion through olfactory pathways. When examining the
olfactory system, the initial connectivity can be characterized as
a tremendous convergence of many olfactory sensory neuron
(OSN) dendrites. In the rodent, an estimated 2000 olfactory bulb
glomeruli are innervated by 56106OSN. Each glomerulus
possesses an estimated 75 mitral and tufted (MT) neurons that
receive information from OSNs. This equates to roughly
1000 OSNs for every MT neuron [47,48]. Therefore, infection
of a proportion of OSNs may result in convergence of advancing
The neuronal connectivity from the glomerulus is thought to
extend into several brain regions. Unlike other sensory systems, the
olfactory bulb may send its output directly to olfactory cortex
without obligate processing through the thalamus  although
thalamic pathways are also utilized. The connectivity of MT cells
was recently detailed in an elegant study utilizing viral tracing
techniques . The authors of that study determined that MT
cells synapse with a very large set of target neurons, to include
neurons of the lateral olfactory tract, but also among other
olfactory bulb neurons, including granule cell layer neurons near
the mirror symmetric glomerulus (a feature not found in other
sensory systems). More research is needed to determine what role
the glomerular neuronal connectivity plays in WEEV dissemina-
tion. It is conceivable that this architecture would favor increased
viral titers within the olfactory bulb and thus lead to more efficient
spread into the rest of the CNS. Bilateral communication is also
available by means of the anterior commissure. The extensive
connectivity of olfactory systems, provide broad and bilateral
dissemination potential within the brain proper.
The olfactory bulb contains specialized dendrodendritic synap-
ses, in which vesicles are observed within presynaptic and
postsynaptic membranes. As synaptic plasticity is dependent in
part upon translational machinery present at the synapse, the
process of learning and memory may be intimately tied to
encephalitic alphavirus infection. Interestingly, aside from general
somatosensation the trigeminal nerve sends fibers to the
neuroepithelium (to detect caustic stimuli). It is conceivable that
the observed infection of trigeminal ganglia and brainstem could
have originated from infected neuroepithelia. Such an alternative
neuroinvasion mechanism has been reported for VEEV infected
animals after ablation of olfactory bulbs . CNS infection was
associated with trigeminal nerve involvement. The current study
shows a similar neuroinvasion for WEEV including a novel finding
of virus localization to the follicular epithelium of vibrissae.
Information from vibrissae is delivered via trigeminal nerve first
into trigeminal sensory complex of the brain stem. From there the
virus spreads to parts of the thalamus and barrel cortex, the most
studied pathways from trigeminus to the cortex. WEEV is
a naturally-occurring recombinant virus and resembles VEEV in
its ability to infect trigeminal nerve-associated neurons and
ultimately infect brainstem nuclei. Therefore, WEEV may serve
as a relevant model system for higher priority pathogens such as
VEEV or EEEV.
Studies aimed at examining WEEV neuroinvasion or CNS
dissemination in the animal model should benefit from the use of
bioluminescent imaging. There are no specific antivirals available
for alphaviral infection and treatment is limited to supportive care.
Studies have demonstrated the limitations of immunoprophylaxis
, and while modulators of innate immunity show promise
[41,53], future generations of therapeutics may benefit from
a greater understanding of the progression of alphaviral-induced
neural disease. BLM may be useful to test antivirals, prophylactic
treatments, and evaluate pathogenesis.
nerve layer at the ventrum of the olfactory bulb (Bar=400 mm). F) Neuropil of the olfactory nerve layer shows a large vacuolar lesion (demyelination)
with individual neuronal necrosis and infiltration of small numbers of neutrophils (Bar=100 mm). G) Perivascular cuffs and multifocal gliosis in the
glomerular layer 72 hpi (Bar=200 mm). H) Close-up view of the congested glomerular vessels with pleocellular perivascular cuffs comprising
moderate numbers of neutrophils, lymphocytes and glial cells (Bar=100 mm).
Bioluminescent Imaging of WEEV in Mice
PLOS ONE | www.plosone.org8 January 2013 | Volume 8 | Issue 1 | e53462
Figure 4. Later stage dissemination throughout the brain 72 hpi. A) Expanded view of the olfactory bulb showing progression of virus into
caudal regions of the brain with multifocal necrosis and secondary demyelination (arrows) (Bar=1000 mm). B) Multifocal demyelination and positive
immunoreactivity in the anterior olfactory nucleus (AON), ventral striatum (VS), basal forebrain (BF) thalamus, hypothalamus, midbrain, hippocampus
(HIP), and cerebral cortex (CC). Fornix (F) and optic tract (circled in blue) do not show any immunoreactivity (Bar=1000 mm). C) Neuronal
immunoreactivity in caudal olfactory bulb that shows multifocal demyelinating lesions (Bar=100 mm). D) Multifocal immunoreactivity in caudal
olfactory bulb and anterior olfactory nucleus (Bar=200 mm). E) Strong immunoreactivity in hippocampus (Bar=100 mm). F) Hippocampal H&E
showing focal loss of pyramidal neurons with mild gliosis (Bar=100 mm). G) Multifocal areas of necrosis and demylelination in the cerebral cortex
(Bar=200 mm). H) Strong immunoreactivity in cerebral neurons and their dendrites revealing interneuronal spread (Bar=100 mm). All IHC images
within this figure are from anti-FLUC staining. Comparison images using anti-WEEV staining may be found in the supplemental information.
Bioluminescent Imaging of WEEV in Mice
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Figure 5. Neuroinvasion from trigeminal nerve. A) Cranial nerves including a branch of trigeminal nerve show neutrophilic perineuritis with
a large glial nodule extending to the meninges of the overlying brain section (Bar=200 mm). B) Close up of epineurium of cranial nerves infiltrated by
neutrophils and lymphocytes (Bar=100 mm). C & D) Strong immunoreactivity (anti-FLUC) of maxillary nerve including Schwann cells. Note strong and
diffuse immunoreactivity of olfactory neuropeithelium, variable staining of surrounding skeletal muscles and bone marrow elements (Bar=100 mm).
E) Trigeminal ganglion IHC positivity (Bar=100 mm). F) Trigeminal positivity is associated with immunoreactivity of the overlying meninges and brain
tissue (Bar=100 mm). G) Brainstem demyelinating lesion (potential consequence of trigeminal invasion) (Bar=400 mm). H) IHC positivity (anti-FLUC)
in the brain stem with interneuronal spread and rare immunoreactivity of glial cells (astrocytes) (Bar=100 mm). All IHC images within this figure are
from anti-FLUC staining. Comparison images using anti-WEEV staining may be found in the supplemental information.
Bioluminescent Imaging of WEEV in Mice
PLOS ONE | www.plosone.org 10 January 2013 | Volume 8 | Issue 1 | e53462
mouse. The Gruenenberg ganglion is thought to be responsible
for detecting odorants involved in suckling and these neurons
synapse at the accessory olfactory bulb. The vomeronasal organ is
responsible for detecting pheromones and also synapses at the
accessory olfactory bulb. The olfactory sensory neurons within the
olfactory epithelium synapse at the main olfactory bulb.
Diagram of odorant-sensing tissues of the
immunopositivity in the olfactory bulb at 72 HPI. Red arrows
show immunopositivity in cortical and lateral olfactory tract.
Anti-WEEV in olfactory bulb. Black arrow shows
arrows showing immunopositivity at site of a large demyelinating
lesion (rarefaction of neuropil). Red arrows show additional
immunopositivity throughout brainstem.
Anti-WEEV in the brainstem at 72 HPI. Black
Red arrow shows markedly immunopositive sinus hair. Black
arrow shows adjacent sinus hair with milder immunoposivity.
WEEV antigen in the sinus hairs at 72 HPI.
WEEV.McM.FLUC recombinant virus.
Detailed description of the molecular
events leadingto the constructionof
We would like to thank Todd Bass for histological preparation of
specimens, Dr. Eric Mossel and Cori Mossel for technical assistance,
Amber Rico for imaging assistance, Lab Animal Resources at Colorado
State University for excellent care of our mice, and Dr. Anna D. Fails for
helpful neuroanatomy consultation.
Conceived and designed the experiments: ATP CBS KEO. Performed the
experiments: ATP CBS KEO TAA. Analyzed the data: ATP CBS KEO
TAA. Contributed reagents/materials/analysis tools: AMP AMT DLJ.
Wrote the paper: ATP CBS TAA KEO.
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Bioluminescent Imaging of WEEV in Mice
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