JOURNAL OF VIROLOGY, Nov. 2009, p. 11808–11818
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 22
The Roles of Chemokines in Rabies Virus Infection: Overexpression
May Not Always Be Beneficial?
Ling Zhao,1,2Harufusa Toriumi,1† Yi Kuang,1Huanchun Chen,2and Zhen F. Fu1,2,3*
Departments of Pathology1and Infectious Diseases,3College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602,
and State-Key Laboratory of Agricultural Microbiology, Department of Preventive Veterinary, College of Veterinary Medicine,
Huazhong Agricultural University, Wuhan 430070, China2
Received 1 July 2009/Accepted 31 August 2009
It was found previously that induction of innate immunity, particularly chemokines, is an important
mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on
RABV infection, chemokines macrophage inflammatory protein 1? (MIP-1?), RANTES, and IP-10 were
individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were
characterized in vitro for growth properties and expression of chemokines. It was found that all the recom-
binant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in
a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was
found that overexpression of MIP-1? further decreased RABV pathogenicity by inducing a transient innate
immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing
neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration
and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain
barrier permeability. These studies indicate that overexpression of chemokines, although important in con-
trolling virus infection, may not always be beneficial to the host.
Rabies virus (RABV) is a negative-strand RNA virus belong-
ing to the Rhabidoviridae family, genus Lyssavirus, which
causes rabies (fatal encephalomyelitis) in many species of
mammals (5). More than 55,000 humans die of rabies each
year worldwide (26). Once clinical signs develop, rabies is
always fatal (12, 53). Despite the lethality of rabies, only mild
inflammation and little neuronal destruction were observed in
the central nervous system (CNS) of rabies patients (31, 32).
Adaptation of wild-type (wt) RABV in laboratory animals
and/or cell culture leads to attenuation in phenotype, and lab-
oratory-adapted RABVs have been used for vaccine develop-
ment (1, 10). To delineate the mechanism(s) of RABV atten-
uation, previous studies compared the host responses to
infection with either laboratory-attenuated or wt RABV (52).
It was found that laboratory-attenuated RABV induced exten-
sive inflammation, apoptosis, and neuronal degeneration, as
well as induction of expression of innate immune genes in the
CNS; however, wt RABV caused little or no neuronal damage
and avoided the activation of expression of innate molecule
genes. Other investigators also reported the induction of in-
nate immunity in mice or neuronal cells infected with labora-
tory-attenuated viruses (20, 33, 37). The mostly upregulated
genes in the innate immune responses after infection with
attenuated RABV include genes encoding for inflammatory
chemokines and type I interferon (IFN) as well as IFN-related
proteins (20, 33, 378). Further studies have shown that the
expression of chemokines (mRNA and proteins), particularly
macrophage inflammatory protein 1? (MIP-1?; CCL3), RAN-
TES (CCL5), and IP-10 (CXCL10), correlates with the infil-
tration of inflammatory cells and enhancement of blood-brain
barrier (BBB) permeability (23).
Chemokines are a group of small (?8- to 14-kDa), basic,
structurally related molecules that can attract inflammatory
cells along concentration gradients and enhance leukocyte-
endothelial cell interactions (55). The tertiary structure of che-
mokines is highly conserved; they contain at least four cysteine
residues that form two disulfide bonds (50). Chemokines have
been divided into major subfamilies on the basis of the ar-
rangement of the two N-terminal cysteine residues, CC and
CXC. CC chemokines act primarily upon monocytes, whereas
CXC family members are specific for neutrophils and lympho-
cytes (14). Chemokines regulate cell trafficking of various types
of leukocytes through interactions with G-protein-coupled re-
ceptors with seven transmembrane regions (55). Most chemo-
kine receptors are stimulated by more than one chemokine,
and one ligand can bind to more than one receptor (50). This
combination of redundancy and promiscuity might act as a
safety factor to ensure adequate host defenses (15, 30). Che-
mokines have direct antiviral activities and/or recruit inflam-
matory cells to the site of infection to kill virus or virus-infected
cells (30, 34). However, due to their ability to direct migration
of inflammatory cells, overexpression of chemokines may have
detrimental effects, especially in the process of autoimmune
inflammation. In an experimental autoimmune encephalomy-
elitis model, IP-10, monocyte chemoattractant protein 1
(MCP-1), and MIP-1? were strongly upregulated (14). Admin-
istration of anti-IP-10 antibody decreased disease incidence
and severity and the infiltration of mononuclear cells into the
* Corresponding author. Mailing address: Department of Pathology,
College of Veterinary Medicine, University of Georgia, 501 D.W.
Brooks Drive, Athens, GA 30602. Phone: (706) 542-7021. Fax: (706)
542-5828. E-mail: firstname.lastname@example.org.
† Present address: Research Institute for Production Development,
15, Shimogamo Morimoto-CHO, Sakyo-ku, 606-0805, Kyoto, Japan.
?Published ahead of print on 9 September 2009.
In the present study, the roles of chemokines in RABV
infection were further investigated by cloning and expressing
MIP-1?, RANTES, and IP-10 in the genome of the RABV
HEP-Flury strain. It was found that overexpression of MIP-1?
decreased the pathogenicity by inducing transient expression
of chemokines and infiltration of inflammatory cells into the
CNS. In contrast, recombinant RABV expressing RANTES
and IP-10 induced persistent and high-level expression of che-
mokines and extensive infiltration of inflammatory cells into
the CNS, causing neurological diseases and death.
MATERIALS AND METHODS
Cells, viruses, antibodies, and animals. Mouse neuroblastoma cells (NA) were
maintained in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented
with 10% fetal bovine serum (Gibco, Grand Island, NY). BSR cells, a cloned cell
line derived from BHK-21 cells, were maintained in Dulbecco’s modified Eagle’s
medium (Mediatech) containing 10% fetal bovine serum. Recombinant RABV
strains were propagated in BSR cells. CVS-11 was propagated in mouse neuro-
blastoma cells. CVS-24 was propagated in suckling mouse brains as described
previously (54). Fluorescein isothiocyanate (FITC)-conjugated antibody against
the RABV N protein was purchased from FujiRab (Melvin, PA). Anti-RABV
nucleoprotein (N) monoclonal antibody 802-2 was obtained from Charles Rup-
precht, Centers for Disease Control and Prevention. Antibodies used for flow
cytometric analysis, such as CD3 (17A2), Ly6G (RB6-8C5), CD45 (30-F11), and
CD11b (M1/70), were purchased from BD Pharmingen (San Jose, CA). Anti-
CD3 polyclonal antibody was purchased from Abcam (England). Biotinylated
secondary antibodies were purchased from Vector Laboratories (Burlingame,
CA). Female BALB/c mice at the age of 6 to 8 weeks were purchased from
Harlan and housed in temperature- and light-controlled quarters in the Animal
Facility, College of Veterinary Medicine, University of Georgia. All animal
experiments were carried out as approved by the Institutional Animal Care and
Construction of recombinant RABV clones. Mouse MIP-1?, RANTES, and
IP-10 cDNAs were amplified from RNA extracted from RABV-infected mouse
brain using the SuperScript III One-Step reverse transcription (RT)-PCR system
with Platinum Taq DNA polymerase (Invitrogen-Life Technology). The primer
sets used for PCR were designed by Primer3 (http://primer3.sourceforge.net/)
(Table 1). The PCR products were digested with BsiWI and NheI (New England
Biolabs, Berverly, MA) and then ligated into RABV vector pHEP-3.0 (18) that
had been previously digested with BsiWI and NheI. The resulting plasmids had
each of the chemokine genes cloned between RABV glycoprotein (G) and the
polymerase (L) genes and were designated pHEP-MIP1?, pHEP-RANTES, and
pHEP-IP10, respectively (Fig. 1).
Rescue of recombinant RABV. Recombinant RABVs were rescued as de-
scribed previously (18). Briefly, BSR cells were transfected with 2.0 ?g of full
infectious clone, 0.5 ?g of pH-N, 0.25 ?g of pH-P, 0.1 ?g of pH-L, and 0.15 ?g
of pH-G using SuperFect transfection reagent (Qiagen, Valencia, CA) according
to the manufacturer’s protocol. After incubation for 4 days, the culture medium
was removed and fresh medium added to the cells. After incubation for another
3 days, the culture medium was transferred into NA cells and examined for the
presence of rescued virus by using FITC-conjugated antibody against the RABV
Virus titration. Viruses were titrated by direct fluorescent antibody assay in
NA cells. NA cells in 96-well plates were inoculated with serial 10-fold dilutions
of virus and incubated at 34°C for 2 days. The culture supernatant was removed
and the cells were fixed with 80% ice-cold acetone for 30 min. The cells were
then stained with FITC-conjugated anti-RABV N antibodies. Antigen-positive
foci were counted under a fluorescence microscope (Zeiss, Germany), and viral
titers were calculated as fluorescent focus units (FFU) per milliliter. All titrations
were carried out in quadruplicate.
ELISA and multiplex ELISA. Brains were homogenized in a ninefold volume
of phosphate-buffered saline (PBS) containing 0.1% NP-40 and Complete pro-
tease inhibitor (Roche Applied Science, Indianapolis, IN). The homogenates
were centrifuged at 11,000 ? g for 30 min to remove debris, and the supernatants
were taken out carefully and aliquoted into microtubes at 0.5 ml/tube. The
supernatant was subjected to an enzyme-linked immunosorbent assay (ELISA)
to quantify the amount of MIP-1?, RANTES, and IP-10 individually in cell
culture supernatants or mouse brain suspensions by using the murine MIP-1?,
RANTES, and IP-10 ELISA kit (R&D Systems, Minneapolis, MN) according to
the manufacturer’s protocol. A multiplex ELISA kit (Quansys Biosciences, Lo-
gan, UT) was used to quantify a panel of 16 cytokines (interleukin-1? [IL-1?],
IL-1?, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MCP-1, IFN-?, tumor
necrosis factor alpha [TNF-?], MIP-1?, granulocyte-macrophage colony-stimu-
lating factor, and RANTES) in brain extracts according to the manufacturer’s
Quantitative real-time RT-PCR. To determine viral load, real-time RT-PCR
was performed on the RNA samples using G gene-specific primers (5?-
CCATCTGGATGCCTGAGAAT-3? and 5?-GGCACCATTTGGTCTCATCT-
3?) in an Mx3000P apparatus (Stratagene, La Jolla, CA). With a 100-ng sample
RNA or no-template control, PCR was performed in two steps; only one primer
was used for cDNA synthesis at 50°C for 30 min, and both primers were used in
PCR amplification. Each reaction was carried out in duplicate. The reverse
transcriptase and DNA polymerase were from a One-Step Brilliant II SYBR
green QRT-PCR master mix kit (Stratagene). For absolute quantitation, a stan-
dard curve was generated from serial diluted RABV G RNAs of known copy
numbers, and the copy numbers of samples were normalized to 1 ?g of total
RNA. The RNA standard was prepared from pH-G by using a reverse transcrip-
tion system (Promega) according to the manufacturer’s protocol.
Histopathology and immunohistochemistry. For histopathology and immuno-
histochemistry, animals were anesthetized with ketamine-xylazine and perfused
by intracardiac injection of PBS followed by 10% neutral buffered formalin as
described previously (23). Brain tissues were removed and embedded with par-
affin. Histopathology was performed by staining the paraffin-embedded sections
with hematoxylin and eosin. For immunohistochemistry, paraffin-embedded
brain sections were heated at 70°C for 10 min and then dipped in CitriSolv
(Fisher Scientific) three times for 5 min and dried until chalky white. Slides were
incubated with proteinase K (20 ?g/ml) in 10 mM Tris-HCl (pH 7.4 to 8.0) for
15 min at 37°C and rinsed three times with PBS. The primary antibodies and then
secondary antibodies were used for immunological reactions. Finally, diamino-
benzidine was used as a substrate for color development.
Leukocyte isolation from the CNS. Mouse brains infected with different re-
combinant viruses were harvested on days 3, 6, and 9 postinfection (p.i.) and
digested with 2 ?g/?l collagenase D (Worthington Biochemical Corporation,
Lakewood, NJ), 1 ?g/?l DNase I (Sigma-Aldrich) in Hanks balanced salt solu-
tion (with Ca2?and Mg2?) for 1 h to disperse the tissue into single-cell suspen-
sion. Viable cells were separated by discontinuous Percoll gradient (70/30%)
centrifugation for 25 min (650 ?g at room temperature, without brake). After
being washed once with Hanks balanced salt solution (without Ca2?or Mg2?;
Invitrogen) and counted, cells were stained for CD3 (17A2), Ly6G (RB6-8C5),
CD45 (30-F11), and CD11b (M1/70) with directly conjugated antibodies (BD
Pharmingen) for 30 min at 4°C and then fixed with 1% paraformaldehyde. Data
collection and analysis were performed with a BD LSR-II flow cytometer and BD
FACSDiva software (BD Pharmingen).
Measurement of BBB permeability. BBB permeability was assessed using a
modification of a previously described technique (35, 49) with the following
markers: sodium fluorescein (NaF; 100 ?l of 100 mg/ml, intravenous [i.v.]);
fluorescein-dextran (FITC-dextran) of molecular mass 10,000 Da (FITC-dextran-
10,000; 200 ?l of 100 mg/ml; i.v.); fluorescein-dextran (FITC-dextran) of molec-
ular mass 150,000 Da (FITC-dextran-150,000; 200 ?l of 37.5 mg/ml, i.v.). Mice
TABLE 1. Primers used for amplification of chemokines
Forward primer (5?–3?)Reverse primer (3?–5?)
VOL. 83, 2009 EXPRESSION OF CHEMOKINES IN THE RV GENOME11809
received these markers intravenously under anesthesia. After 10-min circulation
for NaF and FITC-dextran-10,000 and 4-h circulation for FITC-dextran-150,000,
peripheral blood was collected. Serum (50 ?l) was recovered and mixed with an
equal volume of 15% trichloroacetic acid (TCA). After centrifugation for 10 min
at 10,000 ? g, the supernatant was recovered and made up to 150 ?l by adding
30 ?l 5 M NaOH and 7.5% TCA. The brain was perfused with PBS injected
through the left ventricle to flush out intravascular fluorescein. Then the brain
tissues were homogenized in cold 7.5% TCA and centrifuged for 10 min at
10,000 ? g to remove insoluble precipitates. After addition of 30 ?l 5 M NaOH
to 120 ?l supernatant, the fluorescence was determined using a BioTek spectro-
photometer (Bio-Tek Instruments) with excitation at 485 nm and emission at 530
nm. Markers taken up into tissue are expressed as the micrograms of fluores-
cence in cerebrum per mg of brain tissue divided by the micrograms of fluores-
cence per ?l of serum to normalize uptake values of the dye for blood levels of
the dye at the time of tissue collection (49).
Statistical analyses. All experiments were repeated at least three times. Sta-
tistical significance of the differences between different treatment groups was
analyzed with SigmaStat software (Systat Software Inc., San Jose, California).
One-way analysis of variance with the Holm-Sidak method was used to analyze
clinical score, body weight, chemokine/cytokine concentration, and immune cell
infiltration into the CNS.
In vitro characterization of recombinant RABVs. Our pre-
vious studies indicated that induction of chemokines, particu-
larly MIP-1?, RANTES, and IP-10, is important for RABV
attenuation (23, 52). To further investigate the roles of che-
mokines in RABV infection, the genes encoding murine MIP-
1?, RANTES, and IP-10 were amplified from virus-infected
mouse brain and cloned into rHEP (18) between the G and the
L genes (Fig. 1A). Recombinant viruses were rescued in BSR
cells as described previously (18), and these viruses are desig-
nated as HEP-MIP1?, HEP-RANTES, and HEP-IP10, respec-
tively. To characterize these viruses in vitro, the growth kinet-
ics of these viruses were examined in NA cells. As shown in
Fig. 1B, no significant difference in growth kinetics was ob-
served between each of the recombinant viruses and the pa-
rental virus, indicating that viral growth was not affected by the
insertion of chemokines. The ability of the recombinant RABV
to produce chemokines was determined by measuring chemo-
kine production in virus-infected cells. As shown in Fig. 1C,
production of the intended chemokine was detected in NA
cells infected with each recombinant RABV in a dose-depen-
dent manner. No chemokine was detected in NA cells infected
with parent virus rHEP.
Pathogenicity of recombinant RABVs in mice. To determine
the effect of chemokine expression on RABV infection,
BALB/c mice (10 per group) at 6 to 8 weeks of age were
infected with 105FFU of recombinant viruses by the intrace-
rebral (i.c.) route. Infected mice were monitored twice daily for
2 weeks. Body weight was measured, and the development of
diseases and death was recorded. The animals were scored for
clinical signs as follows: 0, normal mouse; 1, disorder move-
ment; 2, ruffled fur; 3, trembling and shaking; 4, paralysis; 5,
As shown in Fig. 2A, mice infected with HEP-MIP1? were
similar to sham-infected mice during the observation period.
Neither obvious weight loss nor clinical signs were observed in
these two groups of mice. Mice infected with parent virus
rHEP lost about 7% of their body weight compared with sham-
infected mice (P ? 0.05), and one mouse developed mild
symptoms including rough fur and slow movement at days 6 to
9 p.i. and then recovered. Mice infected with HEP-RANTES
FIG. 1. Construction and characterization of recombinant RABVs
expressing different chemokins. (A) Construction of full-length recom-
binant RABVs. Chemokine genes MIP-1?, RANTES, and IP-10 were
individually inserted between BsiWI and NheI sites of the pHEP-3.0
vector. (B) Growth curves of the recombinant and parental rabies
viruses in NA cells. NA cells were infected with different recombinant
RABVs at a multiplicity of infection (MOI) of 0.01. At days 1, 2, 3, 4,
and 5 p.i., culture supernatants were recovered and virus titers were
determined in NA cells. (C) Chemokine production in NA cells by
recombinant viruses. NA cells were infected with different recombi-
nant RABVs at MOIs of 0.001, 0.01, 0.1 and 1. After 24 h of incubation
at 34°C, the culture supernatants were recovered and the concentra-
tion of the indicated chemokine was determined by ELISA.
11810 ZHAO ET AL.J. VIROL.
lost about 14% of their body weight and 30% of the mice
developed severe symptoms such as rough fur and emaciation,
but no paralysis. One mouse in this group died at day 12 p.i.
(Fig. 2C). Mice infected with HEP-IP10 lost about 21% of
their body weight. Seventy percent of the mice in this group
developed severe symptoms, and 30% of the mice succumbed
to infection at day 10 or 11 p.i. (Fig. 2C). The observed symp-
toms in HEP-IP10-infected mice occurred significantly more
frequently (P ? 0.01) than in mice infected with the parental
virus (Fig. 2B). Mice infected with HEP-IP10 lost more body
weight (P ? 0.05) than those infected with the parent virus
(Fig. 2A). These results indicate that recombinant RABV ex-
pressing MIP-1? is more attenuated while viruses expressing
RANTES or IP-10 enhanced RABV pathogenicity compared
to the parental virus.
To determine if the effects of chemokines on pathogenicity
are associated with virus replication, the virus titers, viral an-
tigen, and viral genomic RNA in the mouse brain were deter-
mined at days 3, 6, and 9 p.i. Consistent with the previous study
(47), virus titer was not detected in the brains of mice infected
with any of the viruses during the period (data not shown). By
immunohistochemical analysis, viral antigen (N) was only spo-
radically detected in the region of hippocampus at only day
3 p.i. in mice infected with each of the viruses, but not in
sham-infected mice (data not shown). Quantification of viral
genomic RNA by real-time PCR revealed that the copy num-
ber of viral genomic RNA in mouse brains was highest at day
3 p.i. Although the lowest copy number of viral genomic RNA
was detected in mice infected with HEP-MIP-1? among all the
groups, particularly at days 3 and 6 p.i., there is no significant
difference for the quantity of the genomic RNA among mice
infected with the parent or the recombinant viruses (Fig. 2D).
The data indicate that the rate of viral replication is low, and
overexpression of chemokines has no apparent effect on viral
replication in adult mice. Thus, the rate of replication is not a
determinant for the pathogenicity of different recombinant
viruses expressing different chemokines.
Expression of chemokines and cytokines in mouse brain
after infection with recombinant RABVs. To investigate the
mechanism of HEP-MIP1? attenuation relative to rHEP and
of exacerbated disease associated with HEP-RANTES and
HEP-IP10 viruses, the expression of these chemokines was
determined by ELISA. A multiplex ELISA was also performed
to measure the expression of other inflammatory chemokines
and cytokines. As shown in Fig. 3, all the recombinant viruses
induced the expression of the intended chemokine to high
levels at day 3 p.i. except HEP-IP10, which induced high-level
IP-10 expression at day 6 p.i. Interestingly, expression of one
chemokine led to the expression of other chemokines. Virus
expressing MIP-1? induced only a transient high level of
MIP-1? at day 3 p.i. (P ? 0.01), and its expression declined
quickly by days 6 and 9 p.i. Expression of MIP-1? did induce
the expression of other chemokines compared to sham-in-
fected mice. However, the level of expression was mostly the
lowest among the mice infected with all the recombinant vi-
ruses. In contrast, viruses expressing RANTES or IP-10 not
only induced high and persistent expression of the respective
chemokines but also induced high expression of other chemo-
kines. HEP-RANTES induced significantly higher expression
of RANTES at day 3 (P ? 0.01) and 6 p.i. (P ? 0.05) than the
FIG. 2. Effects of overexpression of chemokines on virus pathoge-
nicity and virus replication. Body weight (A), clinical score (B), survi-
vorship (C), and viral genomic RNA (D) were monitored in BALB/c
mice (n ? 10) after i.c. infection with 105FFU of different recombinant
RABV or medium (sham infection) as described in Materials and
Methods. Data were obtained from 10 mice (three mice for genomic
RNA) in each group and are given as mean values ? standard errors.
Asterisks indicate significant differences between the indicated exper-
imental groups:*, P ? 0.05;**, P ? 0.01;***, P ? 0.001.
VOL. 83, 2009 EXPRESSION OF CHEMOKINES IN THE RV GENOME11811
parent virus, and the level persisted with a slight reduction by
day 9 p.i. This virus also induced a higher level of IP-10 ex-
pression (P ? 0.001) at day 6 p.i. than the parent virus and
HEP-MIP1?. HEP-IP10 induced a significantly higher level of
IP-10 expression at day 6 p.i. than the parent virus and HEP-
MIP1? (P ? 0.001). The IP-10 expression was slightly reduced
by day 9 p.i. in HEP-IP-10-infected mice, but still significantly
higher than in mice infected with other viruses (P ? 0.05). In
addition, HEP-IP10 induced higher expression of other che-
mokines and cytokines as well. It induced the highest expres-
sion of MIP-1? at day 6 and RANTES at day 9 p.i. It also
induced the highest expression of MCP-1, TNF-?, and IL-6 at
days 6 and 9 p.i. The parent virus rHEP induced the highest
expression of RANTES at day 6 p.i. and only low expression of
all other chemokines or cytokines. Overall, parent rHEP and
HEP-MIP1? induced low-level expression of chemokines and
cytokines while HEP-RANTES, particularly HEP-IP10, in-
duced high expression of not only the intended chemokine but
also other chemokines and cytokines. Expression of IP-10,
MCP-1, and TNF-? at high levels correlates well with the
development of diseases in the animals.
Induction of inflammation in mouse brain by recombinant
RABVs. Chemokines and cytokines produced in large quanti-
ties may cause the huge influx of inflammatory cells into the
brains (50). To investigate if expression of chemokines and
cytokines induces inflammation in the CNS of mice infected
with each of the recombinant RABVs, histopathology was per-
formed to analyze the inflammatory cells present in brain tis-
sue. Less infiltration of inflammatory cells was observed in the
brain of mice infected with HEP-MIP1? than that in rHEP-
infected mouse brain at days 3, 6, and 9 p.i. At day 6 p.i.,
HEP-RANTES and HEP-IP10 induced much more inflamma-
tory infiltration than rHEP. By day 9 p.i., infiltration of inflam-
matory cells decreased in rHEP-infected mice, while infiltra-
tion of inflammatory cells continued to persist or increase at
day 9 p.i. in HEP-RANTES- and HEP-IP10-infected mice. No
inflammatory cells were seen in brains of sham-infected mice
(Fig. 4A). To quantify the infiltration of inflammatory cells,
immunohistochemical analysis revealed that slightly fewer
CD3-positive cells were detected in the HEP-MIP1?-infected
mouse brains than in those infected with rHEP at days 3, 6, and
9 p.i., while significantly more CD3-positive cells were detected
in the brains of mice infected with HEP-RANTES and HEP-
IP10 at 6 and 9 day p.i. (P ? 0.001). By day 9 p.i., the number
of CD3-positive cells decreased in mice infected with rHEP but
continued to increase in mice infected with HEP-RANTES
and HEP-IP10 (Fig. 4B and C).
Differentiation of inflammatory cells infiltrated into or ac-
tivated in mouse brain after infection with recombinant
RABVs. To differentiate the inflammatory cells infiltrated into
FIG. 3. Concentration of chemokines and cytokines in mouse brains after infection with recombinant RABVs. BALB/c mice were infected i.c.
with 105FFU different recombinant RABVs. At days 3, 6, and 9 p.i., brains were harvested and homogenized. After centrifugation, the suspension
was used to measure the concentration of indicated chemokines and cytokines by using multiplex ELISA kits. Experiments were performed with
three mice for each time point and condition. Asterisks indicate significant differences between experimental groups:*, P ? 0.05;**, P ? 0.01;
***, P ? 0.001.
11812 ZHAO ET AL. J. VIROL.
or activated in the CNS after infection with different RABVs,
leukocytes were recovered from mouse brains and analyzed
by flow cytometry. The populations of activated microglia/
microphage, neutrophils, and T cells were differentiated using
cell surface markers CD11b, Ly6G, and CD3, respectively (Fig.
5A). CD45 was used as a maker for all the inflammatory cells.
At day 3 p.i., the cells of each type were found to be less than
3 ? 103/brain (Fig. 5A), and no significant difference was
detected among these groups of mice infected with each re-
combinant virus. By day 6 p.i., inflammatory cells increased
quickly to more than 104/brain (Fig. 5A). HEP-MIP1a induced
equal or less while HEP-RANTES induced more infiltration of
all the cell types compared with the parent virus. However, no
significant differences were observed. On the other hand,
HEP-IP10 induced significantly more infiltration of activated
microglia/macrophages (P ? 0.0042), neutrophils (P ?
0.0056), and CD3?T cells (P ? 0.0041) compared with the
parent virus. By day 9 p.i., the number of CD11bhi/CD45hi
activated microglia/macrophages decreased in all groups (Fig.
5B), whereas the number of neutrophils and CD3?T cells
remained the same or continued to increase in HEP-RANTES-
and especially in HEP-IP10-infected mouse brains. Taken to-
gether, the histopathological and flow cytometric analyses
suggested that the increased neutrophils and CD3?T cells
trafficking to and accumulating in the mouse brain correlate
with the pathogenicity of HEP-RANTES and HEP-IP10.
Enhancement of BBB permeability after infection with re-
combinant RABV. To investigate if infection with each of the
recombinant viruses induces changes in BBB permeability, the
leakage of sodium fluorescein from the circulation into CNS
tissues was measured in the cerebrum, cerebellum, and spinal
cord. No significant change in BBB permeability was observed
in the cerebellum or the spinal cord (data not shown). BBB
permeability was significantly enhanced in the cerebrum of
mice infected with all the viruses by 6 days p.i compared to
sham-infected mouse brain. BBB permeability in mice infected
with HEP-RANTES or HEP-IP10 was significantly higher than
that of rHEP- or HEP-MIP1?-infected mice. By day 9 p.i.,
BBB permeability in mice infected with HEP-IP10 was signif-
icantly higher than that in rHEP-infected mice (P ? 0.01) or
HEP-RANTES-infected mice (P ? 0.05) (Fig. 6A). These data
indicate that infection with all the viruses enhanced BBB per-
meability at day 6 p.i. compared to sham infection. Further-
more, HEP-IP10 induced significantly higher and more persis-
tent BBB permeability than parent virus or HEP-MIP1?.
To investigate if overexpression of different chemokines can
induce BBB changes so that large molecules can easily enter
the CNS, different-sized markers such as NaF (376 Da), FITC-
dextran-10,000 (10 kDa), and FITC-dextran-150,000 (150 kDa)
were used to measure changes of BBB permeability at day
6 p.i. As shown in Fig. 6B, molecules of 150 kDa or larger did
not infiltrate into the cerebrum for any mice in any of the
groups. Only HEP-IP10 induced significantly higher perme-
ability to a 10-kDa marker (P ? 0.001) than other viruses.
This indicates that overexpression of chemokines MIP-1? or
RANTES can induce enhancement of BBB permeability to
allow small molecules (NaF, 376 Da) to enter into the CNS,
while overexpression of IP-10 can significantly enhance the
permeability to allow not only small but also large molecules
(10 kDa) across the BBB.
To investigate if the enhancement of BBB permeability is
associated with chemokine expression in the brain or in the
serum, the concentrations of chemokines (MIP-1?, RANTES,
and IP-10) were determined at day 6 p.i. As shown in Fig. 6C,
the concentrations of chemokines (MIP-1?, RANTES, and
FIG. 4. Inflammatory responses induced by recombinant RABVs.
BALB/c mice were infected i.c. with 105FFU different recombinant
RABVs, and brains were harvested after extensive perfusion at days 3,
6, or 9 p.i. (A) Pathological changes were observed in paraffin sections
after hematoxylin and eosin staining. (B) Quantification of CD3-pos-
itive T lymphocytes in the hippocampus sections was performed with
anti-CD3 antibody. (C) CD3-positive cell numbers were quantified and
are expressed as mean values ? standard errors obtained from three
mice at each time point. Asterisks indicate significant differences be-
tween the indicated experimental groups:*, P ? 0.05;**, P ? 0.01;
***, P ? 0.001.
VOL. 83, 2009 EXPRESSION OF CHEMOKINES IN THE RV GENOME11813
IP-10) in the brain were much higher than that in the serum.
Overall, infection with different viruses by the i.c. route did not
significantly affect the chemokine concentration in the serum.
In the mouse brain, HEP-RANTES induced a significantly
higher production of RANTES and IP-10, while HEP-IP10
induced a significantly higher production of IP-10. Only the
IP-10 level correlated well with the enhancement of BBB per-
The RABV genome has been used to express antigens from
other viruses (7, 28, 45), host proteins (7, 29, 38), or an extra
copy of the RABV G gene (8). Expression of these proteins in
the RABV genome invariably results in virus attenuation in the
mouse model. For example, expression of host proteins, such
as cytochrome c, TNF-?, or IL-2, has led to attenuated patho-
genicity (7, 29, 38). In the present study, chemokines MIP-1?,
RANTES, and IP-10 were cloned into the genome of the
RABV HEP-Flurry strain. It was found that although expres-
sion of MIP-1? further reduced RABV pathogenicity, expres-
sion of RANTES, or IP-10 enhanced RABV pathogenicity in
the mouse model. It has been reported previously that expres-
sion of host immune proteins, notably IL-4, resulted in en-
hanced pathogenicity in other viral expression systems. It is
believed that expression of IL-4 may downregulate immune
responses, thus exacerbating diseases. Ectromelia virus ex-
pressing IL-4 developed symptoms of acute mousepox with
high mortality by suppressing cytolytic responses of NK and
cytotoxic T lymphocytes (CTL) and the expression of IFN-? by
the latter (19). The clearance of recombinant vaccinia virus
expressing IL-4 was delayed compared with control recombi-
nant vaccinia virus because the expression of IL-4 suppresses
antiviral CTL responses and production of nitric oxide (44).
Expression of IL-4 by recombinant respiratory syncytial virus
resulted in an accelerated pulmonary inflammatory responses,
yet the CTL response was deficient in the production of IFN-?
and was nonfunctional for in vitro cell killing (4).
The rationale to clone chemokines into the RABV genome
was to further investigate the role of chemokines in RABV
infection, because our previous studies showed that expression
of chemokines, particularly MIP-1?, RANTES, and IP-10, cor-
relates with infiltration of inflammatory cells into the CNS,
enhancement of BBB permeability, and attenuation of RABV
FIG. 5. Differentiation of inflammatory cells that infiltrated into
the CNS by using flow cytometric analysis. BALB/c mice were infected
i.c. with 105FFU of different recombinant RABVs, and brains were
harvested after extensive perfusion at days 3, 6, and 9 p.i. CNS leuko-
cytes were isolated by Percoll centrifugation and analyzed by flow
cytometry with the indicated antibodies. (A) Representative flow cy-
tometric plots of inflammatory cell infiltration in the mouse brain at
days 3, 6, and 9 p.i. with HEP-IP10. Resting microglia (CD45int,
CD11bint) appear in region 1 (R1), and activated microglia/macro-
phages (CD45hi, CD11bhi) appear in R2. CD45hiLy6Ghicells that
appear in R3 were defined as neutrophils. CD3?T cells appear in R4.
(B) The absolute numbers of specific inflammatory cells in brains were
calculated (three mice per group for each time point). Asterisks indi-
cate significant differences between the indicated experimental groups:
*, P ? 0.05;**, P ? 0.01;***, P ? 0.001.
11814ZHAO ET AL. J. VIROL.
(23). Inflammatory cells and other immune effectors attracted
by chemokines enter the CNS and help clear RABV-infected
cells, thus attenuating RABV pathogenicity. Yet, infection
with the recombinant RABV expressing these chemokines re-
sulted in a very different outcome. One possibility is that ex-
pression of these chemokines affects virus replication. To this
end, virus titers, viral antigen, and viral genomic RNA were
measured in the mouse brain at various time points after in-
fection. No virus was detected, and viral antigen was detected
only sparsely in mice infected with each of the viruses. Quan-
titative RT-PCR revealed no significant difference in the copy
numbers of viral genomic RNA. Thus, expression of chemo-
kines did not change the rate of RABV replication in the
context of HEP-Flurry strain and thus could not account for
the difference of pathogenicity induced by these recombinant
Chemokines are redundant and multifunctional. Expression
of one chemokine in the brain could induce the expression of
other chemokines or cytokines and thus would have profound
effects in recruiting different subsets of inflammatory cells into
FIG. 6. Determination of changes in BBB permeability and chemokine levels in the brain versus serum. BALB/c mice were infected i.c. with
105FFU of recombinant RABVs. At days 3, 6, and 9 p.i., BBB permeability was determined by uptake of NaF (A). The extent of BBB permeability
to different-sized markers was compared at day 6 p.i. (B). The concentrations of chemokines in both serum and brains were assayed by ELISA (C).
Each set of data has at least triplicates. Data are given as mean values ? standard errors. Asterisks indicate significant differences between the
indicated experimental groups:*, P ? 0.05;**, P ? 0.01;***, P ? 0.001.
VOL. 83, 2009 EXPRESSION OF CHEMOKINES IN THE RV GENOME 11815
the CNS (30). To determine if expression of a particular che-
mokine in the RABV genome leads to the expression of other
chemokines and infiltration of a particular set(s) of inflamma-
tory cells, the expression of chemokine/cytokine was monitored
using a multiplex ELISA, and inflammatory cells infiltrating
into the CNS were differentiated by flow cytometry. Each of
the recombinant viruses expressed high levels of the intended
chemokine at day 3 or 6 p.i. However the level of MIP-1? in
mice infected with HEP-MIP1? subsided quickly. In addition,
only low to moderate levels of other chemokines are induced in
these mice. Likewise, only low and transient infiltration of
inflammatory cells at day 6 p.i., and by day 9 p.i. infiltration of
inflammatory cells returned to the level found in sham-infected
animals. In contrast, HEP-RANTES and particularly HEP-
IP10 not only induced high and persistent expression of the
intended chemokines but also induced high expression of other
chemokines. High and persistent infiltration of inflammatory
cells, particularly neutrophils and T cells, was also observed in
the CNS, which can produce neurotoxins, free radicals, and
proinflammatory cytokines, causing CNS destruction (13).
Overall our studies indicate that transient expression of che-
mokines may help attenuation, while the high and persistent
expression of these chemokines, particularly IP-10, may be
responsible for the enhanced pathogenicity. It is known that
MIP-1? is a monocyte chemokine and may activate resident
microglia within the brain (27, 39). Microglia are constantly
moving and analyzing the CNS and are able to recognize and
swallow foreign antigens and act as antigen-presenting cells
(39). It is conceivable that the high level of MIP-1? expression
in the earlier stage of infection results in activation of residen-
tial microglia and inhibition of virus replication. As a conse-
quence, less virus replication leads to less expression of other
chemokines/cytokines and less infiltration of inflammatory
cells. Indeed, the least amount of viral genomic RNA was
detected in mice infected with recombinant RABV expressing
MIP-1? among all the infected groups. Likewise, the expres-
sion levels of chemokines/cytokines are the lowest in this group
of mice, except for the high level of MIP-1? expression at day
3 p.i. Consequently, the least infiltration of inflammatory cells
into the CNS was observed in this group of mice when com-
pared with the groups of mice infected with other RABVs. In
contrast, the expression of RANTES or IP-10 attracted large
numbers of neutrophils and T cells into the CNS (6, 43, 48),
which resulted in the induction of a high level of IFN-? and
TNF-? expression (14), leading to severe diseases and deaths.
It seems that the results from this present study contradict
our previous findings, particularly with regard to IP-10 expres-
sion. In our previous study, expression of IP-10 correlated with
RABV attenuation (23, 52). In those studies, attenuated
RABV strain B2C was used. In the present study, overexpres-
sion of IP-10 induced immune-mediated diseases. This could
be related to the stage of disease or the amount and the
duration of chemokine expression. Earlier and transient ex-
pression of chemokines including IP-10 is important in clearing
RABV from the CNS; however, high and persistent expression
may cause excessive damage. A high level of IP-10 expression
was detected at the stages of diseases when animals were
infected with B2C at high doses, and extensive inflammation
and apoptosis were found in these animals (42, 52). Thus, the
findings in the present study are not contradictory to our pre-
vious studies; rather, they support our previous hypothesis that
laboratory-attenuated RABV induces neurological diseases by
immune-mediated pathogenesis (52). Beneficial and detrimen-
tal effects of chemokine expression, particularly IP-10, have
also been reported in other viral infections. For example, ex-
pression of IP-10 in CNS following infection with mouse hep-
atitis virus (MHV) (25), lymphocytic choriomeningitis virus
(3), and Theiler’s virus (16) is important in initiating and main-
taining protective Th1 immune responses. Overexpression of
IP-10 from the MHV genome promoted protection from coro-
navirus-induced neurological and liver diseases (51). Neutral-
ization or genetic deficiency of CXCL10 in mice leads to in-
creased viral burden and delayed virus clearance upon
infection with West Nile virus (22) or MHV (6). However,
persistent expression of IP-10 following MHV infection may be
detrimental to the host by recruiting excessive CD4?T cells
into the CNS. CD4?T cells release additional chemokines,
such as RANTES, which enhances the infiltration of macro-
phages and increase the severity of demyelination (24, 25). The
precise role of chemokines, particularly IP-10, in RABV pro-
tection and pathogenicity is not currently known and will be
addressed by controlling (with small interfering RNA) or ab-
lating (knocking out) the expression of each chemokine.
Recently it has been reported that enhancement of BBB
permeability is one of the important mechanisms for RABV
attenuation (35, 40, 41). Pathogenic strains of RABV, such as
SHBRV, are deficient in BBB opening and prevent immune
effectors from entering the CNS. On the other hand, attenu-
ated RABV induces enhancement of BBB permeability, thus
allowing small molecules (presumably immune effectors) to
enter the CNS (9). In our study, we found that all the recom-
binant RABVs enhanced the BBB permeability; however,
HEP-RANTES and HEP-IP10 induced more extensive and
prolonged enhancement of BBB permeability than HEP-
MIP1? or rHEP. Furthermore, HEP-IP10 induced BBB per-
meability to the extent that allowed large molecules (10 kDa)
to enter the CNS. Although the consequence is not entirely
clear, this may have allowed more inflammatory cells or other
toxic substances to enter into the CNS, resulting in severe
diseases and deaths. In other virus models of CNS autoimmu-
nity and virus-induced neuroinflammation, such as experimen-
tal autoimmune encephalomyelitis (21), multiple sclerosis (46),
lymphocytic choriomeningitis virus infection (2), and Borna
disease (17), CNS inflammation was generally associated with
increased BBB permeability and CNS damage and disease.
CNS inflammation can be prevented by decreasing or inhibit-
ing BBB permeability (17, 21, 36). Thus, precaution should be
taken if strategies to enhance BBB permeability are to be
developed to treat clinical rabies or other CNS diseases.
This work was supported partially by Public Health Service grant
AI-051560 from the National Institute of Allergy and Infectious Dis-
eases (Z.F.F.) and by a grant from The Natural Science Foundation of
China (30928020 to Z.F.F. and H.C.).
We thank Charles Rupprecht from the Centers for Disease Control
and Prevention for supplying monoclonal antibody 802-2. We also
thank Yongjun Wen and Hualei Wang for help with some of the
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