Journal of Bacteriology and Virology 2015. Vol. 45, No. 3 p.250 – 255 http://dx.doi.org/10.4167/jbv.2015.45.3.250
Communication
Safety and Immunogenicity of a Recombinant Rabies Virus Strain (ERAG3G) in Korean Raccoon Dogs *
Dong-Kun Yang1 , Ha-Hyun Kim1, Hyun-Ye Jo1, Hee-Won Kim2, Sung-Suk Choi1 and In-Soo Cho1 1
Viral Disease Division, Animal and Plant Quarantine Agency, MAFRA, Anyang; 2Wild Life Center, Gyeonggi-do Veterinary Service Laboratory, Pyeongtack, Korea
A new alternative rabies bait vaccine strain named ERAG3G, which is applicable to wild animals, was developed to eliminate rabies in South Korea. In this study, the safety and immunogenicity of the strain was evaluated in Korean raccoon dogs. The ERAG3G was propagated in BHK/T7-9 cells. Korean raccoon dogs were administered ERAG3G (1 ml, 108.0 FAID50/ml) orally or intramuscularly to evaluate its safety and immunogenicity. The raccoon dogs were observed for 70 days after administration, and immunogenicity was measured using a fluorescent antibody virus neutralization test. The ERAG3G strain was not pathogenic to Korean raccoon dogs immunized via the intramuscular or oral route. Raccoon dogs administered the candidate vaccine via the oral route developed high virus neutralizing antibody (VNA) titers ranging from 13.7 to 41.6 IU/ ml 70 days post administration. Raccoon dogs inoculated intramuscularly with the ERAG3G strain developed moderate VNA titers ranging from 0.5 to 13.7 IU/ ml. These findings suggest that the ERAG3G strain is safe and induces a protective immune response in raccoon dogs. Key Words: Recombinant rabies virus, Immunity, Raccoon dogs
Eastern Europe and Eastern North America since the late
INTRODUCTION
1990s. Raccoon dogs (Nyctereutes procynoide koresis) remain the main wildlife vector in Korea (2). The estimated
Rabies causes about 55,000 human deaths per year,
Korean raccoon dog and stray dog population is 260,000,
indicating that it is a dangerous public health problem and
and the wild animal bite incidence, including bites from
one of the most important zoonoses in the world (1). Most
raccoon dogs, has been 4.5% among 2,310 bites reported
human rabies cases are related to a bite from a rabid dog,
since 2011. Raccoon dog rabies has affected high-risk rabies
and dogs are the main vector in many countries, including
regions in two Korean provinces, and the epizootic disease
those in Asia. However, wild animals such as raccoons,
has recently extended into southern areas of the Han River
raccoon dogs, badger, foxes, and mongoose have been
(3). Although the eradication of sylvatic rabies by a trap-
associated with the rabies virus (RABV) circulating in
vaccination-release program is optimum, wild animals, in-
Received: May 9, 2015/ Revised: August 17, 2015/ Accepted: August 19, 2015 Corresponding author: Dong-Kun Yang. Animal and Plant Quarantine Agency, 175 Anyang-ro, Anyang-si, Gyeonggi-do 14089, Korea. Phone: +82-31-467-1783, Fax: +82-31-467-1797, e-mail:
[email protected] ** This work was supported financially by a grant (B-AD14-2011-13-01) from Animal, and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea. *
CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/). ○
250
ERAG3G Strain is Safe and Immunogenic in Raccoon Dogs
251
cluding the raccoon dog, are difficult to trap (4). In addition,
propagate the recombinant ERAG3G strain, which was
most raccoon dogs are too aggressive to administer a par-
constructed in 2011 using a reverse genetic system (2). The
enteral vaccination; intramuscular vaccination is not usually
ERAG3G strain was deposited in the Korean Veterinary
possible, as they are stray, free-ranging, and fierce dogs.
Culture Collection (Accession number: KVCC-VR1500046).
Oral rabies vaccination (ORV) with a modified live virus
After propagation, the ERAG3G strain was titrated in 96-
via the oral route has been successful in field trials to
well microplates using 10-fold dilutions. The microplates
prevent rabies in wild animals and has contributed to the
were incubated for 3 days, after discarding supernatant, and
development of a commercial bait vaccine for the SAG2
then fixed the cells in cold acetone (-20℃) for 20 min. After
strain in France (5, 6). Several types of vector vaccines,
three successive washes with phosphate-buffered saline
including a canarypox-rabies glycoprotein recombinant
(PBS; pH 7.2), the microplates were reacted with a specific
vaccine, a recombinant adenovirus-vectored vaccine, and a
monoclonal antibody against RABV for 45 min at 37℃ and
vaccinia-recombinant glycoprotein (V-RG) virus for wild
then stained with fluorescence isothiocyanate conjugated
animals such as raccoon dogs and foxes, have been com-
goat-anti mouse IgG + IgM. After rinsing with PBS, the
mercialized. The distribution of ORV has contributed to a
microplates were air-dried and examined at 400× under a
rapid decrease in animal rabies in many countries (7~10).
fluorescence microscope (Nikon, Tokyo, Japan). The viral
As ORV has helped prevent the spread of wild animal rabies
titer determined by a fluorescent antibody test was calculated
in European countries and the USA, the Korean Veterinary
according to the Reed and Muench method (13). The RABV
Authority has distributed a bait vaccine (V-RG) to high-
challenge virus standard (CVS)-11 was widely used in deter-
risk rabies regions. The result of ORV has been a drastic
mination of virus neutralizing antibody (VNA). The CVS-11
reduction in rabies cases in Korea (11). Nevertheless, human
strain purchased from American Type Culture Collection
vaccinia infection cases have been reported: two women
(ATCC, VR-959) was propagated in baby hamster kidney
with a chronic skin disease were exposed to V-RG (12). The
(BHK)-21 cells and was also titrated in the method men-
women developed vaccinia virus infection on skin of their
tioned above.
hand. One of the patients was treated with human vaccinia immune globuline intravenous and antiviral agent. Oral rabies vaccines have not been approved for human use.
Safety and immunogenicity of the ERAG3G strain in raccoon dogs
Therefore, new alternative rabies bait vaccines applicable to
This experimental design was submitted to the laboratory
pet dogs and wild animals are needed to eradicate rabies. A
animal ethics committee (QIA, Seoul, Korea) and was
recombinant RABV strain called ERAG3G was constructed,
approved (QIA2013-668). The wounded raccoon dogs were
and efficacy testing of the strain has been conducted in mice.
bred for treatment before releasing to where they were
The ERAG3G strain has been considered as oral rabies
caught. Six-month-old raccoon dogs, which were RABV
vaccine candidate for wild raccoon dogs. In this study, the
sero-negative, were divided into three groups. Groups 1 and
safety and immunogenicity of the ERAG3G strain were
2 included four raccoon dogs each that were inoculated with
evaluated in wild raccoon dogs.
the ERAG3G strain (1 ml, 108.0 fluorescent assay infectious dose, FAID50/ ml) via the oral route using a syringe without
MATERIALS AND METHODS Cells and viruses
a needle and via the intramuscular route, respectively. Two raccoon dogs in the control group remained untreated except for taking blood. The control group was bred with Group 1.
Murine neuroblastoma (NG108-15) cells were maintained
After administering the vaccine, all raccoon dogs were kept
in DMEM supplemented with 5% fetal bovine serum at
in a designated place to watch their behavior. All raccoon
37℃ in a 5% CO2 incubator. NG108-15 cells were used to
dogs were monitored daily for adverse effects, including
252
D-K Yang, et al.
anorexia, prostration, anxiety, agitation, aggression, and
washes with PBS, the microplates were reacted with a
paralysis. Blood was collected from all raccoon dogs, in-
specific monoclonal antibody against RABV for 45 min at
cluding the control group, 5 and 10 weeks after inoculation.
37℃ and then stained with fluorescence isothiocyanate conjugated goat-anti mouse IgG + IgM. After rinsing with
Serological assay
PBS, the microplates were air-dried and examined at 400×
The VNA titer was determined by a fluorescent antibody
under a fluorescence microscope. The comparison of the
virus neutralization (FAVN) test (14). In brief, a positive
measured titer of the tested sera with that of the OIE positive
WHO reference serum adjusted to 0.5 IU/ ml was used as
standard serum of a known VN titer allowed determination
the positive control. Serum samples and the positive and
of the VN titer of the tested sera in IU/ ml. The comparison
negative controls were distributed in four consecutive wells
to IU/ ml was made by using the mean value of the OIE
and serially diluted. Then, RABV (CVS-11 strain) containing
standard serum (14).
about 100 FAID50/50 μl was added to each well. A 50 μl aliquot of BHK-21 cells containing 4 × 105 cells/ ml was added to each well after 60 min of incubation at 37℃, and the microplates were incubated in a humidified incubator with 5% CO2 at 37℃ for 72 h. The microplates were then fixed in cold acetone for 20 min. After three successive
RESULTS Safety and immunogenicity of the ERAG3G strain in raccoon dogs All raccoon dogs were sero-negative for RABV VNA
Table 1. Virus neutralizing antibody (VNA) titers of raccoon dogs administrated with ERAG3G strain via oral or intramuscular route VNA titer (IU/ ml) Group Group 1
Animal No.
Route of inoculation 0 week
5 weeks
10 weeks
0.07
41.6
41.6
0.07
13.7
13.7
3
0.07
13.7
13.7
4
0.07
1 2
Oral
7.90
13.7
GM*
0.07
19.2
20.6
SD**
0.00
15.2
14.0
Group 2
5
0.07
0.87
0.50
0.07
0.87
0.5
7
0.07
1.51
13.7
8
0.07
1.51
13.7
GM
0.07
1.19
7.10
SD
0.00
0.40
7.60
0.07
0.07
0.07
0.07
0.07
0.07
GM
0.07
0.07
0.07
SD
0.00
0.00
0.00
6
Group 3 (control)
9 10
IM
No treatment
*GM: Geometric mean, **SD: standard deviation. All raccoon dogs immunized with the ERAG3G strain induced high neutralizing antibody titer >0.5 IU/ ml against RABV at 10 weeks after administration.
ERAG3G Strain is Safe and Immunogenic in Raccoon Dogs
253
Figure 1. Example of fluorescent assay virus neutralization (FAVN) test performed in BHK-21 cells. Fluorescent in the BHK-21 cells was not found in the A neutralized by serum antibody against RABV, but fluorescent appeared in the B due to absence of serum antibody against RABV.
before vaccine administration, but sero-converted 35 days
vaccination coverage is required for complete eradication
after oral inoculation (Table 1). None of the vaccinated or
(18). Therefore, a new more effective approach is desired
unvaccinated raccoon dogs exhibited clinical signs of rabies
to overcome this limitation.
during the experiment. All raccoon dogs in Group 1 that
ORV has been developed to replace parenteral vaccination
were administered the ERAG3G strain orally developed
because of its practical advantages, including no need to
RABV VNA titers of 7.9~41.6 IU/ ml (geometric mean,
restrain dogs or risk handling aggressive dogs. ORV can also
19.2 IU/ ml) 35 days after inoculation and had a geometric
be applied to wild animals. Large numbers of bait vaccines
mean VNA titer of 20.6 IU/ ml after 70 days. All raccoon
have been distributed to control and eliminate RABV in wild
dogs in Group 2 that were inoculated intramuscularly with
animals. Many countries have carried out ORV programs
the ERAG3G strain had RABV VNA titers of 0.5~13.7
with SAG2, a recombinant adenovirus expressing the rabies
IU/ ml 70 days after inoculation (Fig. 1). Two raccoon dogs
protein, or V-RG bait (8~10, 19). These programs have
in Group 3 remained RABV sero-negative throughout the
contributed to the elimination of rabies in several countries,
test, confirming that no contact transmission had occurred
including Finland, the Netherlands, Italy, Canada, and the
between the vaccinated and control animals.
USA (20, 21). Previously, we found that the ERAG3G strain has a
DISCUSSION
glutamic acid (Glu) coding sequence (GAA) at position 333 of the glycoprotein, demonstrating that the ERAG3G
Raccoon dogs play a key role in transmitting the disease
strain is non-pathogenic to mice, cats, and dogs (2, 22).
in Northeast Asia, including Korea, and the ferret badger is
However, other researcher reported that amino acid 333 of
the major vector species in Taiwan (14~16). Each country
the G protein is not entirely responsible for the pathogenicity
has made efforts to control RABV through the mass vacci-
of RABV. For example, RC-HL, a non-pathogenic RABV
nation of pets and domestic animals. Government policy
fixed strain, has an Arg (R) at this position. The RABV G
has led to a significant decrease in human and animal rabies
protein is the major determinant of viral pathogenicity and
cases in many countries (17). Nevertheless, the ability to
the major protective antigen. It is reported that attenuated
completely eliminate rabies is limited because 80% dog
RABV strains express higher levels of G protein in the
254
D-K Yang, et al.
infected neurons than highly pathogenic street strains (23).
in raccoon dogs administered a bait vaccine containing the
Therefore, we assume that the ERAG3G strain may increase
ERAG3G strain are needed.
the expression of G protein in the infected cells. We also
In conclusion, based on the clinical observations the
demonstrated that 4- and 6-week-old mice administered the
ERAG3G strain was safe and immunogenic in Korean
ERA strain at a titer of 107.5 FAID50/ ml via the intramuscular
raccoon dogs. A single oral or intramuscular immunization
or intracranial route were pathogenic 6 days after inocu-
with the ERAG3G strain induced high VNA titers against
8.5
lation, whereas mice inoculated with 10 FAID50/ ml of the
RABV in raccoon dogs. Therefore, the ERAG3G strain
ERAG3G strain did not display any clinical signs after a
should be sufficient for use as a live vaccine or oral bait
15-day observation period (24). Another study reported that
vaccine in wild animals.
the ERAG333 strain containing Glu at position 333 was not pathogenic to mice that were > 7 days old (25). Furthermore,
REFERENCES
orally immunizing mice with the ERAG3G strain induced complete protection (24). In our study, the safety and immunogenicity of the ERAG3G strain were evaluated in Korean raccoon dogs administered via the oral or intramuscular route. As carnivores are primarily responsible for transmitting rabies, the safety and immunogenicity of an oral vaccine candidate should be evaluated in the target animals. The SAG2 strain is safe based on the absence of adverse clinical signs, salivation, and the absence of replication of the vaccine strain in the brains of vaccinated dogs (25). Our results demonstrate that none of the raccoon dogs inoculated with 108.0 FAID50/ ml intramuscularly or orally showed any adverse effects associated with disease behavior, and that all raccoon dogs administered the ERAG3G strain orally developed high VNA titers of 13.7~41.6 IU/ ml 10 weeks after administration. This suggests that raccoon dogs vaccinated orally with the ERAG3G strain are protected from a challenge by virulent RABV, which provides a basis for using a rabies bait vaccine strain. Similar results were reported for raccoon dogs vaccinated with the SAG2 strain; they developed VNA titers of 6.59~19.9 IU/ ml 60 days after vaccination (21). In addition, all raccoon dogs inoculated intramuscularly with the ERAG3G strain developed moderate VNA titers of 0.5~ 13.7 IU/ ml after 10 weeks. Raccoon dogs administrated with the ERAG3G strain via oral route showed higher level of VNA titer, indicating that direct contact to target organ, tonsil, enable the inoculated animals to induce better immune response. However, we did not conduct an efficacy test in the target animals. Therefore, further study and a challenge
1) Yousaf MZ, Qasim M, Zia S, Khan Mu, Ashfaq UA, Khan S. Rabies molecular virology, diagnosis, prevention and treatment. Virol J 2012;21:9:50. 2) Yang DK, Nakagawa K, Ito N, Kim HH, Hyun BH, Nah JJ, et al. A single immunization with recombinant rabies virus (ERAG3G) confers complete protection against rabies in mice. Clin Exp Vaccine Res 2014;3:176-84. 3) Oem JK, Kim SH, Kim YH, Lee MH, Lee KK. Reemergence of rabies in the southern Han river region, Korea. J Wildl Dis 2014;50:681-8. 4) Sobey KG, Rosatte R, Bachmann P, Buchanan T, Bruce L, Donovan D, et al. Field evaluation of an inactivated vaccine to control raccoon rabies in Ontario, Canada. J Wildl Dis 2010;46:818-31. 5) Hanlon CA, Niezgoda M, Morrill P, Rupprecht CE. Oral efficacy of an attenuated rabies virus vaccine in skunks and raccoons. J Wildl Dis 2002;38:420-7. 6) Orciari LA, Niezgoda M, Hanlon CA, Shaddock JH, Sanderlin DW, Yager PA, et al. Rapid clearance of SAG-2 rabies virus from dogs after oral vaccination. Vaccine 2001;19:4511-8. 7) Baer GM, Abelseth MK, Debbie JG. Oral vaccination of foxes against rabies. Am J Epidemiol 1971;93:487 -90. 8) Brown LJ, Rosatte RC, Fehlner-Gardiner C, Bachmann P, Ellison JA, Jackson FR, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB, an adenovirus-rabies recombinant vaccine. Vaccine 2014;32:984-9. 9) Faber M, Dietzschold B, Li J. Immunogenicity and safety of recombinant rabies viruses used for oral vaccination
ERAG3G Strain is Safe and Immunogenic in Raccoon Dogs
255
of stray dogs and wildlife. Zoonoses Public Health 2009; 56:262-9. 10) Taylor J, Meignier B, Tartaglia J, Languet B, VanderHoeven J, Franchini G, et al. Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species. Vaccine 1995; 13:539-49. 11) Joo YS, Lee JH, Lee KK, Bang HA, Lee WC. Retrospective study of extensive vaccination programs for canine rabies control and public health in Korea. Jpn J Infect Dis 2011;64:513-5. 12) Rupprecht CE, Blass L, Smith K, Orciari LA, Niezgoda M, Whitfield SG, et al. Human infection due to recombinant vaccinia-rabies glycoprotein virus. N Engl J Med 2001;345:582-6. 13) Howard DR. Rabies virus titer from tissues of naturally infected skunks (Mephitis mephitis). Am J Vet Res 1981; 42:1595-7. 14) Cliquet F, Aubert M, Sagné L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods 1998;212:79-87. 15) Chiou HY, Hsieh CH, Jeng CR, Chan FT, Wang HY, Pang VF. Molecular characterization of cryptically circulating rabies virus from ferret badgers, Taiwan. Emerg Infect Dis 2014;20:790-8. 16) Yang DK, Kim SY, Oh YI, Lee JA, Cho SD, Lee KW, et al. Epidemiological Characteristics of Rabies in South Korea from January 2004 to March 2011. J Bacteriol Virol 2011;41:165-171. 17) Franka R, Wu X, Jackson FR, Velasco-Villa A, Palmer DP, Henderson H, et al. Rabies virus pathogenesis in relationship to intervention with inactivated and attenu-
ated rabies vaccines. Vaccine 2009;27:7149-55. 18) Follmann EH, Ritter DG, Baer GM. Evaluation of the safety of two attenuated oral rabies vaccines, SAG1 and SAG2, in six Arctic mammals. Vaccine 1996;14:270-3. 19) Fehlner-Gardiner C, Rudd R, Donovan D, Slate D, Kempf L, Badcock J. Comparing ONRAB® AND RABORAL V-RG® oral rabies vaccine field performance in raccoons and striped skunks, New Brunswick, Canada, and Maine, USA. J Wildl Dis 2012;48:157-67. 20) Cliquet F, Aubert M. Elimination of terrestrial rabies in Western European countries. Dev Biol (Basel) 2004; 119:185-204. 21) Cliquet F, Gurbuxani JP, Pradhan HK, Pattnaik B, Patil SS, Regnault A, et al. The safety and efficacy of the oral rabies vaccine SAG2 in Indian stray dogs. Vaccine 2007;25:3409-18. 22) Tuffereau C, Leblois H, Bénéjean J, Coulon P, Lafay F, Flamand A. Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 1989;172:206-12. 23) Morimoto K, Hooper DC, Spitsin S, Koprowski H, Dietzschold B. Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol 1999;73:510-8. 24) Yang DK, Kim HH, Choi SS, Kim JT, Jeong WH, Song JY. Oral immunization of mice with recombinant rabies vaccine strain (ERAG3G) induces complete protection. Clin Exp Vaccine Res 2015;4:107-13. 25) Bankovskiy D, Safonov G, Kurilchuk Y. Immunogenicity of the ERA G333 rabies virus strain in foxes and raccoon dogs. Dev Biol (Basel) 2008;131:461-6.
