Download Innate immune responses in raccoons after raccoon rabies virus

Journal of General Virology (2014), 95, 16–25
DOI 10.1099/vir.0.053942-0
Innate immune responses in raccoons after raccoon
rabies virus infection
Vythegi Srithayakumar,1,2 Hariharan Sribalachandran,3 Rick Rosatte,4
Susan A. Nadin-Davis5 and Christopher J. Kyle2,6
Correspondence
Vythegi Srithayakumar
[email protected]
1
Environmental and Life Sciences Graduate Program, Trent University, 1600 West Bank Drive
Peterborough, ON, Canada
2
Natural Resources DNA Profiling and Forensics Centre, DNA Building, Trent University,
2140 East Bank Drive, Peterborough, ON, Canada
3
Biology Department, Trent University, 1600 West Bank Drive Peterborough, ON, Canada
4
Ontario Ministry of Natural Resources, Wildlife Research and Development Section, Trent
University, DNA Building, 2140 East Bank Drive, Peterborough, ON, Canada
5
Centre of Expertise for Rabies, Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency,
3851 Fallowfield Road, Ottawa, ON, Canada
6
Forensic Science Department, Trent University, 2140 East Bank Drive, Peterborough, ON, Canada
Received 28 March 2013
Accepted 30 September 2013
Zoonotic wildlife diseases pose significant health risks not only to their primary vectors but also to
humans and domestic animals. Rabies is a lethal encephalitis caused by rabies virus (RV). This
RNA virus can infect a range of terrestrial mammals but each viral variant persists in a particular
reservoir host. Active management of these host vectors is needed to minimize the negative
impacts of this disease, and an understanding of the immune response to RV infection aids
strategies for host vaccination. Current knowledge of immune responses to RV infection comes
primarily from rodent models in which an innate immune response triggers activation of several
genes and signalling pathways. It is unclear, however, how well rodent models represent the
immune response of natural hosts. This study investigates the innate immune response of a
primary host, the raccoon, to a peripheral challenge using the raccoon rabies virus (RRV). The
extent and temporal course of this response during RRV infection was analysed using genes
predicted to be upregulated during infection (IFNs; IFN regulatory factors; IL-6; Toll like receptor3; TNF receptor). We found that RRV activated components of the innate immune system, with
changes in levels of transcripts correlated with presence of viral RNA. Our results suggest that
natural reservoirs of rabies may not mimic the immune response triggered in rodent models,
highlighting the need for further studies of infection in primary hosts.
INTRODUCTION
Unprecedented and rapid landscape and environmental
changes are altering the distributional and dispersal
patterns of many wildlife species and their associated
pathogens (Harvell et al., 2002; Daszak et al., 2000). Recent
examples include the expansion of several infectious
diseases such as avian influenza, severe acute respiratory
syndrome, West Nile fever/encephalitis, chronic wasting
disease, tuberculosis and Lyme disease (Daszak et al., 2000,
2001). These diseases pose a significant health risk not only
to their primary host, but also non-target species including
humans, companion animals, livestock and species of
Two supplementary tables are available with the online version of this
paper.
16
conservation concern (Daszak et al., 2000). An in-depth
understanding of the interaction between pathogen and host
can help in the development of tools to mitigate such
threats. However, due to the many challenges associated
with studying mechanisms of host–pathogen interactions in
natural populations, model systems are often employed as
surrogates. Although vital information can be gained from
surrogate studies, model systems often do not incorporate
the effects of co-evolutionary pressures as found in natural
hosts and their pathogens, and thus may not provide
information indicative of the disease’s true pathology.
Rabies virus is the type species of the genus Lyssavirus, family
Rhabdoviridae. Rabies virus (RV) is a highly neurotropic
virus that infects mammals and almost invariably causes a
fatal encephalopathy (Jackson, 2007). Although significant
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Rabies virus activates innate response in vector species
advances have been made in rabies prevention and control,
it continues to pose significant public health risks. A
conservative estimate of 55 000 human deaths are annually
attributed to this disease (World Health Organization,
2005), but mortality numbers may be higher than reported
because of limited reporting and diagnosis in developing
countries (Knobel et al., 2005). Dogs are the most important
rabies reservoirs in Asia and Africa, where the majority of
human rabies cases occur (Fu, 1997; Knobel et al., 2005). In
North America, canine rabies has been controlled through
pet vaccination and other control measures but terrestrial
reservoirs (raccoons, foxes and skunks) and bats continue to
present serious threats given the high densities of these
species in urban areas (Totton et al., 2002; Rosatte & Allan,
2009; Rosatte et al., 2010, 2011).
Rabies is typically the result of a bite from an infected
animal with the deposition of virus-laden saliva in the
wound. The virus infects local sensory and motor neurons
and then ascends to the brain by retrograde transport
(Tsiang et al., 1986; Shankar et al., 1991; Prosniak et al.,
2001). Uncontrolled replication of virus in the central
nervous system (CNS) leads to disease and ultimately
death. Host defence against early stages of infection is
provided by an innate immune response, which may
prevent viral invasion and replication before the adaptive
immune response is established (Haller et al., 2006;
Johnson et al., 2006; Zhao et al., 2011). RV often evades
the host’s immune system, and establishes the disease in
the brain, explaining the near 100 % mortality rate in
humans if timely post-exposure treatment is not
administered (Jackson, 2007; Wang et al., 2005).
Therefore, it is important to understand the innate
immune response following rabies infection; such data
not only aids in developing and refining post-exposure
treatments, but also effective treatment once clinical
disease has established.
Immune pathways and associated genes are well characterized in mice and have been utilized as primary model
systems to expand our knowledge of immune responses to
rabies infection. Several studies using rodent models have
reported upregulation of several cytokines and receptors
associated with innate immune responses following
infection with fixed laboratory RV strains (Marquette
et al., 1996; Prosniak et al., 2001; Baloul & Lafon, 2003;
Saha & Rangarajan, 2003; McKimmie et al., 2005; Wang
et al., 2005; Johnson et al., 2006; Mansfield et al., 2008;
Zhao et al., 2011). While these studies have yielded a
significant understanding of the mechanisms associated
with rabies pathogenesis and the host’s response, mice are
not reservoirs for rabies, so events observed in this species
may not closely resemble the disease under natural
conditions. In this study, we focused on the interaction
between the raccoon strain of rabies virus (RRV) and its
natural reservoir host, the raccoon (Procyon lotor), to gain
a better understanding of innate immune responses
http://vir.sgmjournals.org
triggered by virus infection. RRV was first noted in
Florida around the 1940s where it remain localized until
the late 1970s, when a second outbreak facilitated rapid
spread along the eastern seaboard of North America (Biek
et al., 2007). High densities of raccoons in urban and
suburban areas (Totton et al., 2002) and the concomitant
increase in potential human and pet exposures present a
serious threat to human health. Contrary to the common
belief that exposure to RV is always lethal, empirical
studies have suggested that raccoon exposure to RRV does
not always result in clinical disease, perhaps due to interindividual differences in innate immune responses (Carey
& Mclean, 1983; Szanto, 2009). For example, 5–40 % of
raccoons survive RRV challenge (K. Knowles, personal
communication; Carey & Mclean, 1983), and the incubation period can vary among raccoons and vaccinated
animals that have not demonstrated seroconversion
(Rupprecht et al., 1988). The culmination of these factors
illustrates that there is variability in the immune response
of raccoons to RRV exposure that may result in abortive
infections.
We investigated the host innate immune response to a
peripheral challenge with RRV by determining the extent
and temporal course of changes in transcript levels of a
limited but crucial set of genes that are known to be
generally upregulated during viral infection (Wang et al.,
2005; Johnson et al., 2006; Mansfield et al., 2008). These
included: (1) two IFNs, IFN-a, which is involved in the
production of an antiviral state, and IFN-c, which is
involved in inflammatory responses; (2) IFN regulatory
factors (IRF-1 and IRF-7), selected for their important role
in the development of antiviral state and inflammation
response respectively (Haller et al., 2006); (3) IL-6, as it is
known to be involved in inflammatory response to viral
infection (Frei et al., 1989); (4) Toll-like receptor 3 (TLR3), chosen for its role in recognizing dsRNA (an
intermediary of viral replication), and activating the
production of IFNs (Alexopoulou et al., 2001); and (5)
TNF receptor (TNF-R), which binds TNF involved in
cytolysis of infected cells and in inducing IFNs in response
to viral infections (Camelo et al., 2000; Faber et al., 2005).
We examined changes in transcript levels of these
components in several different individuals inoculated
with the same strain of virus to explore the extent of host
response variation. Further, we evaluated cytokine expression levels from a temporal perspective to determine the
earliest time point to detect the immune response
following peripheral inoculation, which closely mimics
the natural route of transmission. From a spatial
perspective, we also investigated where, in the route of
transmission, the immune response was first detected. In
addition, differences between individuals’ genes responsible
for the innate immune response were assessed to explore
possible effects on disease outcome. Information gained
from this study aims to not only provide information on
host response to this deadly virus, but also insight into how
some animals may be able to resist fatal infection.
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V. Srithayakumar and others
RESULTS
and were euthanized at 533 days p.i. Although these
samples did not exhibit clinical symptoms of RRV, low
levels of the virus was detected in the brains of two
raccoons (533+2 and 533+3; Szanto, 2009).
Variable response in raccoons following RRV
challenge
The course of infection in raccoons was variable and
peripheral inoculation did not always result in disease or
CNS invasion, as determined by the detection of viral RNA
in the brain (Table 1). Viral RNA was detected in the brain
and salivary glands using real-time PCR, with RRV-specific
primers and probes (Szanto, 2009). The two raccoons that
were euthanized 4 days prior to the experiment had no
detectable virus in any of the tissues. In the test group,
where two raccoons were euthanized at 2, 4, 8, 15 and
32 days post-inoculation (p.i.), viral RNA was only
detected in the brain of the sample that was euthanized
at 8 days p.i. (Szanto, 2009). Virus was not detected in any
of the other samples from this group in the tissues tested,
nor did they exhibit any clinical symptoms (Szanto, 2009).
The remaining 10 animals (challenge group) were kept
under observation until they exhibited clinical symptoms
at which point they were euthanized. Six of the 10 animals
showed clinical symptoms at 20, 21 and 23 days p.i. Viral
RNA was detected in the brains of all these samples, and in
the salivary glands of two of these samples (21+1 and
22+1; Szanto, 2009). Four of the 10 raccoons showed no
clinical symptoms for the duration of the challenge study
Temporal expression of immune genes during
RRV challenge
Following peripheral infection with RRV, a number of
transcriptional changes were observed in the brain. The
earliest point that we were able to detect an indication of
an innate immune response was at 8 days p.i. (Fig. 1),
however, it was not statistically significant (P.0.05). Once
the clinical signs of rabies were apparent (20–24 days),
expression of all genes under investigation increased
significantly (Fig. 1). IFN-a transcripts increased fivefold
in expression (P50.002), and this was much lower
compared with the other IFN genes that were examined.
The expression of IFN-a was lower in than other genes, at
earlier time points and different tissues that were
examined in this study. IFN-c demonstrated a 130-fold
increase in the brain once clinical signs were apparent
(P50.002), and this increase was much higher than that
noted for other genes. Transcripts for IFN regulatory
factors (IRF-1 and IRF-7) increased by 50- and 120-fold,
respectively (P50.001 and P,0.001). Viral RNA was
Table 1. Time course of RRV infection in raccoons
Viral detection data was obtained from the doctoral thesis of Szanto (2009).
Sample*
Days p.i.
Clinical signs
Viral RNA in brainD
Viral RNA in salivary glands
Control 1
Control 2
2+1
2+2
4+1
4+2
8+1
8+2
15+1
15+2
20+1
20+2
21+1
22+1
23+1
23+2
32+1
32+2
533+1
533+2
533+3
533+4
24
24
2
2
4
4
8
8
15
15
20
20
21
22
23
23
32
32
533
533
533
533
–
–
–
–
–
–
–
–
–
–
+
+
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
+
–
–
–
+
+
+
+
+
+
–
–
–
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
+
+
–
–
–
–
–
–
–
–
*Sample shows days p.i. and the sample numbers that were euthanized that day.
DViral RNA detected by quantitative PCR ,45 Ct.
18
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Journal of General Virology 95
Rabies virus activates innate response in vector species
180
*
160
IFN-α
IFN-γ
IRF-1
IRF-7
140
120
100
mRNA transcript fold change
80
60
40
20
0
2
4
8
30
15
20_24
32
533
*
IL-6
TLR-3
TNF-R
25
20
15
10
5
0
2
4
8
15
20_24
Time (days p.i.)
32
533
Fig. 1. Host transcriptional response to infection with RRV within
raccoons in the brain. Two raccoons were included in each group
except for the 20–24 days group, which had six, and the 533 days
group, which had four. Results are normalized against b-actin and
are presented as fold change relative to the control. Each bar
shows the mean±SEM of the group; significantly different values
(unpaired t-test, P,0.05) are indicated by asterisk (*).
detected in all samples that showed upregulation of IFNassociated genes.
Levels of mRNA for IL-6, a cytokine involved in
inflammation, increased 20-fold (P50.009), while expression of TLR-3, which detects intermediate viral products,
increased 16-fold (P50.001) in the brain following clinical
signs. The expression of the receptor for TNF, which is
involved in cytolysis and induction of IFNs, increased 14fold (P50.003). Virus was also detected in samples that
showed a significant increase (P,0.05) in the expression of
genes associated with an innate immune response. In the
animals that did not show clinical symptoms, traces of the
immune gene transcripts were present in the brain at
earlier time points; however they were not statistically
significant.
Spatial expression of immune genes during RRV
challenge
A spatial examination of gene expression associated with
the immune response revealed a number of transcriptional
http://vir.sgmjournals.org
changes in the challenge study animals. Innate immune
gene transcripts were not detected at sites of inoculation
in any of the samples (Fig. 2). However, samples from
individuals that displayed clinical symptoms of RRV
showed a significant increase in the expression of the
genes investigated in the spinal cord (Fig. 2). IFN-a
demonstrated a seven-, five- and twofold increase in the
spinal cord, brain and salivary glands, respectively.
Transcripts of IFN-c increased by 116-fold, 130-fold and
eightfold in the spinal cord, brain and salivary glands,
respectively (Fig. 2). The expression of IRF-1 increased by
70-fold, 53-fold and twofold and IRF-7 increased by 160fold, 120-fold and twofold in the spinal cord, brain and
salivary glands, respectively (Fig. 2). Transcripts of IL-6
showed a 16-fold increase in the spinal cord and 20-fold
increase in the brain, and a twofold increase in the salivary
glands of the samples that showed clinical symptoms (Fig.
2). The expression of TLR-3 increased by 20-fold, 16-fold
and 2.5-fold, in spinal cord, brain and salivary glands,
respectively (Fig. 2). TNF-R showed a 16-fold increase in
the brain followed by 14-fold increase in the brain and 1.5fold increase in the salivary glands (Fig. 2). The increases
noted in spinal cord and brains of the samples that showed
clinical symptoms were statistically significant (P,0.05)
(Fig. 2). Virus was also detected in all the samples and
tissues that showed a significant increase in the immune
gene transcripts.
Genetic variation
We examined variation in the coding regions of the gene
amplicons selected for expression analysis to see if any
underlying genetic variation influenced expression of these
genes. We found no variation within the IFNs (IFN-a, IFNc, IRF-1 and IRF-7), IL-6 or TNF-R. The TLR-3 fragment
had one nucleotide difference (CAT) in four samples;
however, it did not result in an amino acid change (data
not shown).
DISCUSSION
Living organisms are continuously exposed to a stream of
pathogens that have the potential to disrupt biological
processes. Humans and other vertebrates rely on an array
of defensive measures produced by the immune system for
constant and continued protection. The clinical outcome
of viral infections depends on the balance between viral
replication and host response. Innate immune responses
are the host’s first line of defence against infections. During
pathogen–host co-evolution, many viruses have developed
strategies to successfully evade the host’s innate immune
system (Samuel, 2001). The majority of previous studies
investigating immune response to RV infection, which
have focused on murine models and laboratory strains of
rabies, have shown that the expression of several genes
involved in the innate immune response increased (Wang
et al., 2005; Johnson et al., 2006; Mansfield et al., 2008).
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V. Srithayakumar and others
30
30
IL-6
25
20
20
15
15
10
10
5
5
0
0
2
4
8
15
20–24
32
533
30
2
30
TNF-R
mRNA transcript fold change
TLR-3
25
25
25
20
20
15
15
10
10
5
5
0
8
15
20–24
32
533
4
8
15
20–24
32
533
4
8
15
IFN-α
0
2
180
160
140
120
100
80
60
40
20
0
4
4
8
15
20–24
32
533
100
90
80
70
60
50
40
30
20
10
0
IFN-γ
2
2
4
8
15
20–24
32
533
IRF-1
2
20–24
32
533
300
IRF-7
250
Muscle
Spinal cord
Brain
Salivary gland
200
150
100
50
0
2
4
8
15
20–24
32
533
Time (days p.i.)
Fig. 2. Host transcriptional response to infection with RRV in different tissues of raccoons. Each group contained two raccoons,
except for the 20–24 days group, which had six, and the 533 days group, which had four. Results are normalized against bactin and are presented as fold change relative to the control. Each bar shows the mean±SEM of the group; significantly
different values (unpaired t-test, P,0.05) were found only in samples obtained at 20–24 days p.i.
20
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Journal of General Virology 95
Rabies virus activates innate response in vector species
While these studies are valuable, providing insight into
mechanisms triggered by RV, it is important to note that
the response elicited in a natural reservoir of the virus may
be different than in surrogate model systems. Accordingly,
we examined host response triggered by RRV in its primary
vector, the raccoon. Our findings indicate that the timing
of the immune response plays a crucial role in the
pathogenesis of RRV, highlighting the need for further
studies in primary hosts of rabies.
This study has shown that raccoons infected with RRV and
exhibiting clinical signs displayed statistically significant
upregulation of transcripts of several genes associated with
the innate immune response in the brain. Samples from
exposed animals not exhibiting clinical symptoms of viral
infection did not show such a response. In the brain, the
earliest time point at which activation of innate immune
response was detected was 8 days p.i., however the increase
noted was not statistically significant. Previous studies
using mouse models have detected the immune response in
the brain as early as 4 days after peripheral inoculation
(Johnson et al., 2006; Mansfield et al., 2008; Zhao et al.,
2011). The distance from the site of inoculation to the
brain is greater in racoons than mice, which may explain
the delay noted in raccoons, as there is a time-dependent
movement of virus along the peripheral nerves from the
site of inoculation to the CNS (Kelly & Strick, 2000). There
may also be variation in the length of time the virus
remains localized at or near the site of inoculation before
entry into the CNS (Charlton et al., 1997; Shankar
et al., 1991).
Components critical to innate immunity include the IFNs,
which induce chemo-attractive and inflammatory responses
in the infected cells and play a crucial role in setting up an
antiviral environment (Wang et al., 2005; Chelbi-Alix et al.,
2006; Faul et al., 2010; Rieder et al., 2011). Similar to
previous studies (Wang et al., 2005; Johnson et al., 2006;
Mansfield et al., 2008; Zhao et al., 2011), this study found
type I and II IFNs (IFN-a and IFN-c) as well as IFN
regulatory factors (IRF-1 and IRF-7) to be upregulated in
RV-infected tissues. Compared with the expression of other
genes involved in the IFN pathway, the expression of IFN-a
was weak, however, similar results were found in other
studies examining IFN-a in response to rabies infections
(Wang et al., 2005; Johnson et al., 2006). Similar results with
weaker and delayed IFN-a expression compared with other
IFN genes were found in other viruses including influenza A
virus and Sendai virus (Marié et al., 1998; Osterlund et al.,
2005). The importance of IFNs in response to rabies is
illustrated by previous studies that found that the administration of IFN-inducing poly(I-C) resulted in various
degrees of protection against RV in mice, hamsters, rabbits
and monkeys (Harmon et al., 1974; Hilfenhaus et al., 1975).
Similarly, it has been noted that IFN-a/b receptor knockout
mice had a higher virus titre than immunologically intact
mice after infection with RV (Hooper et al., 1998). These
data suggest that the IFN pathway plays a crucial role in RV
resistance through innate immune response.
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Apart from the IFN signalling pathway, increased expression of other genes encoding inflammatory responses, as
well as viral recognition receptors, was observed.
Inflammatory cytokine IL-6 and TNF-R, which is involved
in recruiting lymphocytes to the infection, were both
upregulated in the samples from animals with clinical
symptoms of RRV. Similar results have been found in other
studies, which suggest inflammatory response and infiltration of lymphocytes play a major role in response against
RV (Wang et al., 2005; Johnson et al., 2006; Mansfield et al.,
2008; Solanki et al., 2009). RRV also induced the
expression of TLR-3 in the CNS of the raccoons, which
was correlated with the detection of the viral RNA. In
mouse models, TLR-3 was found to be expressed earlier
than other genes, suggesting that activation of this receptor
is an early marker of the innate immune response to RV
(McKimmie et al., 2005; Mansfield et al., 2008). We did not
detect expression of TLR-3 at earlier time points suggesting
that RRV may evade early detection by this receptor.
Analysis of samples taken in close proximity to the site of
inoculation did not reveal upregulation of any of these
gene transcripts. Under natural conditions animals are
known to experience long and variable incubation periods
(Baer & Cleary, 1972). There is uncertainty about the
events that occur during the incubation period (Jackson,
2007), but based on an experimental study using skunks,
the virus was found to remain localized at or near the site
of inoculation (Charlton et al., 1997). Charlton et al.
(1997) did not detect an inflammatory response at the site
of inoculation, which suggests that an immune response
was not triggered by the low dosage of virus used in that
study. In this study, no innate immune response was
detected, either because this response was highly transitory
and localized or non-existent. In the present study, an
increase in the expression of the suite of genes at the spinal
cord in individuals that showed clinical symptoms was
detected. It was interesting to note that in some of these
animals, the level of expression in the spinal cord was
higher than in the brain, whereas for the remainder, the
opposite was true. This suggests that immune response
triggered once the virus reaches the CNS may not be
enough to control the disease. There was a slight increase in
the transcripts in the salivary glands of two of the animals
that showed clinical symptoms. The decrease noted in
the expression of the transcripts with the progression of the
virus suggests failure of the immune system to control the
infection. Similar results were found in a previous study,
where slight increase of gene expression is indicative of
innate immune response was noted in the salivary glands,
with a decrease after the onset of clinical symptoms
(Mansfield et al., 2008).
Although chemo-attractive and inflammatory responses are
often present in the RV-infected brain (Wang et al., 2005;
Chelbi-Alix et al., 2006; Johnson et al., 2006; Mansfield et al.,
2008; Lafon et al., 2008; Zhao et al., 2011), like most viruses,
RV has developed strategies to evade the immune system.
Specifically, the RV phosphoprotein (P) can impair the IFN
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V. Srithayakumar and others
signalling pathway by both interfering with transcriptional
activation of IFN and by disrupting the function of STAT-1,
involved in transcriptional activation of many secondary
products that contribute to an antiviral state (Conzelmann,
2005; Brzózka et al., 2006; Chelbi-Alix et al., 2006; Vidy et al.,
2007). The findings in this study and others show that
transcripts from a range of genes involved in the IFN
signalling pathway increase upon RV infection, which
suggests that this inhibition by RV P is not absolute, but it
may contribute to a reduced antiviral response over time.
Future proteomics studies to examine regulation of IFNrelated genes in RV-infected tissues may provide insight into
the kinetics of this process and the role of RV P in impairing
the signalling cascade. Such studies can also provide insight
into the kinetics of this process and the role of RV P in
impairing the signalling cascade.
While we found evidence of induction of the innate immune
response following RRV infection in raccoons, there was
variability in response between different individuals. The
genes examined in this study were upregulated in the brains
of all individuals that showed clinical symptoms; however,
the expression of these genes in the spinal cord and salivary
glands differed. Similarly, in previous studies using murine
models, variation was noted in immune responses and
histopathological changes caused by the virus (Jackson &
Reimer, 1989; Johnson et al., 2006; Mansfield et al., 2008;
Zhao et al., 2011). Although several factors, such as the
strain of RV and differences in the route of infection used by
the virus may contribute to these differences, host genetics
may also play a crucial role in disease outcome. We found no
variation within the coding regions of the genes examined;
however, more comprehensive analysis of the non-coding
regions that affect the translation and transcription of these
genes may provide insight into variations that influence
disease outcome. Several studies examining promoter
regions, introns and transcription binding sites of innate
immune genes have found variation that is linked to various
disease outcomes as a result of infection with smallpox virus
and hepatitis C virus (Ovsyannikova et al., 2012; Cussigh
et al., 2011).
Although the expression levels of innate immune genes in
raccoons were similar to previous published studies using
mouse models, some differences were noted in both timing
and level of expression, highlighting the need to replicate
these studies in natural reservoirs of rabies. While this
study attempted to examine host response to rabies
infection in natural reservoirs, it is important to highlight
that the virus was propagated in murine cells, and the effect
that this has on the infection is not known. Future studies
using virus propagated in a cell line of the host will
provide a more comprehensive understanding of host–
pathogen interaction. While evidence of the initiation of
the immune response was noted in raccoons, it was
variable among individuals suggesting that host genetic
factors may have a crucial role in the pathogenesis of RV.
The genes upregulated in this study may have a critical role
in response against RV, however, the timing of the
22
upregulation of these genes and nature of these genes
(inflammatory response) suggests that they may also
contribute to the pathogenesis, resulting in encephalopathy. In all the animals that succumbed to RRV in the
challenge study, genes related to innate immune response
were initially upregulated, followed by a decrease in
expression levels with viral propagation and disease
progression. This suggests that immune response needs
to be initiated before entry of the virus into the brain in
order for the host to successfully clear it.
Our findings enhance the understanding of the pathways
triggered by RRV in their primary vector, raccoons. We
found several genes indicative of the innate immune
response to be upregulated in the brains of raccoons
following RRV infection. However, further knowledge of
the factors contributing to the pathogenesis is needed to
gain insight into which host factors may provide immunity
to this virus. These results provide a basic framework for
further studies investigating genes associated with both the
innate and the adaptive immune response. In the samples
from animals that showed clinical symptoms of RRV, we
found the presence of RRV to be strongly correlated with
the innate immune response; which suggests that the
immune response in the host may be more effective if the
pathogen is detected early in the infection. Therefore indepth analysis of the roles of receptors responsible for
detecting viruses and mechanisms that RRV employs to
bypass these receptors will also prove valuable. Greater
understanding of the interaction between a host’s immune
response and the rabies virus variant associated with it may
aid in the development of antiviral therapies to augment
innate immune system function and prevent fatal disease.
METHODS
The initial premise of the challenge experiment was to examine viral
progression in raccoons after inoculation with RRV (Szanto, 2009);
therefore, the challenge study, experimental design and virus
preparation were performed by Canadian Food Inspection Agency
(CFIA) in conjunction with Ontario Ministry of Natural Resources
(OMNR). This study started with RNA extraction from previously
collected tissues.
Challenge study. Twenty-two raccoons were captured in southern
Ontario from regions that had not been exposed to RRV, by staff of
the OMNR (lack of prior exposure to rabies was confirmed through
serology tests). These animals were transported to a biocontainment
level 3 animal care facility at the CFIA, in Nepean Ontario where the
challenge study was conducted. All animal experimentation was
performed according to the guidelines of the Canadian Council on
Animal Care with the approval of the institute’s animal care
committee. Additional details of the challenge study can be found
in Szanto (2009).
Virus preparation and experimental design. An RRV stock was
propagated in murine neuroblastoma cells as described previously
(Szanto, 2009). Two raccoons were euthanized 4 days prior to the
start of the experiment to serve as negative controls. Twenty raccoons
were inoculated into the right hind leg muscle with 0.5 ml virus stock
(106.7 TCID50). Raccoons were housed individually and monitored
daily for clinical signs of rabies. Two raccoons were chosen at random
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Journal of General Virology 95
Rabies virus activates innate response in vector species
on days 2, 4, 8, 12 and 32 p.i. and euthanized (test group of 10
animals). The remaining 10 animals were kept under observation
until they exhibited clinical signs of rabies (challenge group), at which
point they were euthanized. Animals that did not show clinical
symptoms during the duration of the challenge study were euthanized
at 533 days p.i. Tissues collected from all animals included: muscle
tissue from the inoculated right leg muscle, a section of lumbar spinal
cord, cerebral cortex and salivary gland. Samples were immediately
frozen and stored at 280 uC until use.
manufacturer’s protocol; the gene-specific primers listed in Table S2
were used to prime cDNA synthesis. TaqMan PCR was performed on
a StepOnePlus instrument, using TaqMan Fast Universal PCR Master
Mix (Applied Biosystems) according to the manufacturer’s conditions. All experimental samples contained at least three technical
replicates with no template controls. All transcripts were normalized
against b-actin and expression levels were calculated using the 2–DDCt
method (Schmittgen & Livak, 2008). Statistical significance was
determined by Student’s t-test, with P values of ,0.05 considered
statistically significant. Results are presented as mean±SEM.
RNA extraction and reverse transcription. Total RNA was
extracted from all samples using TRIzol (Invitrogen) following the
manufacturer’s standard protocol. Precipitated RNA was dissolved in
50 ml DEPC-treated water and quantified using a NanoDrop 8000
spectrophotometer (Thermo Scientific). DNA was removed by
treatment with Turbo DNase enzyme (Applied Biosystems) following
the manufacturer’s protocol. RNA quality and quantity was assessed
again using gel electrophoresis and the NanoDrop 8000 spectrophotometer. DNA contamination was assessed through a mitochondrial
DNA cytochrome b gene fragment amplification (primer sequence).
PCR was prepared with the components in the following concentrations: 16 PCR buffer (Invitrogen), 0.2 mM each dNTP, 1.5 mM
MgCl2, 0.2 mM forward primer, 0.2 mM reverse primer, 0.05 U Taq
DNA polymerase (Invitrogen) ml21, 10 ng RNA and ddH2O as
needed for a total volume of 12 ml. Amplification conditions were as
follows: initial denaturation at 94 uC for 5 min; 35 cycles of
denaturation at 94 uC for 30 s, annealing at 55 uC for 1 min and
extension at 72 uC for 1 min; final extension at 60 uC for 45 min; and
4 uC hold. Two microlitres of the reaction was subjected to agarose
gel electrophoresis to assess contamination. Without prior cDNA
generation using a Thermoscript reverse transcription system
(Invitrogen), amplicons were not detected in the samples, thereby
showing that the expression levels were not artificially influenced by
contaminating DNA.
Gene assay design. Since raccoon genomic information is limited,
primer pairs for the initial amplification of the raccoon genes of
interest (IFN-a. IFN-c, IL-6, TLR-3, IRF-1 and -7, and TNF-R) were
designed based on the sequences of conserved regions in phylogenetically related carnivores available from GenBank (Table S1 available
in JGV Online). DNA extracted from a raccoon from Ontario using a
Qiagen kit was used as template. Amplicons were generated using
each primer pair as follows: PCR components were in the following
concentrations: 1.56 PCR buffer (Invitrogen), 0.2 mM each dNTP,
1.5 mM MgCl2, 0.6 mM forward primer, 0.6 mM reverse primer,
0.05 U Taq DNA polymerase ml21, 10 ng DNA and ddH2O for a total
volume of 12 ml. Two microlitres of the amplified product was
subjected to agarose gel electrophoresis to confirm gene amplification. PCR products were purified using ExoSAP (New England
BioLabs) following the manufacturer’s instructions. BigDye
Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and the
forward primer were used to sequence the fragments. When
comparing the gene sequences obtained from raccoons using the
Basic Local Alignment Search Tool (BLAST), with respective genes in
phylogenetically related carnivores, the high degree of similarity
(.84 % at the nucleotide level; .89 % at the amino acid level; data
not shown) strongly implied the identity of the obtained sequences.
Once sequences of these raccoon-specific amplicons were determined,
primers and Taqman probes to target them were designed using
Custom TaqMan Expression Assays (Applied Biosystems; Table S2).
Genetic variation. Fragments of the genes selected were amplified in
all the experimental samples using primers listed in Table S1
following the PCR conditions described above with cDNA as the
template. The amplicons were sequenced from both directions and
PCR products were ethanol precipitated, then electrophoresed
and visualized on an ABI 3730 DNA Analyzer. Sequences were edited
and aligned to the similar gene of other species using MEGA4.1
(Tamura et al., 2007).
ACKNOWLEDGEMENTS
We would like to thank Drs B. White and B. Saville for their helpful
suggestions and support of this project. We acknowledge the
invaluable help of staff of the Canadian Food Inspection Agency
Centre of Expertise for Rabies for performance of the challenge study,
the collection of tissues from the animals and the permission to use
these samples for the current study. We thank Dr Annamaria Szanto
for RNA preparation from all tissues. We would also like to thank
members of the Saville laboratory for their assistance with RNA work.
This project occurred in collaboration with the Rabies Research Unit
of the Ontario Ministry of Natural Resources (OMNR). This research
was funded through a NSERC grant to C. J. K. (355850), and funds
from the OMNR, from Ontario Graduate Scholarship (201203145) to
V. S. and from the OMNR Summer Student Program to H. S.
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