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1.  Oral immunization of mice with recombinant rabies vaccine strain (ERAG3G) induces complete protection 
Purpose
New rabies vaccine bait for both pets and raccoon dogs residing in Korea is needed to eradicate rabies infection among animals. In this study, we constructed a recombinant rabies virus (RABV), the ERAG3G strain, using a reverse genetics system. Then we investigated the efficacy of this strain in mice after oral administration and the safety of this strain in cats after intramuscular administration.
Materials and Methods
The ERAG3G strain was rescued in BHK/T7-9 cells using the full-length genome mutated at the amino acid position 333 of the glycoprotein gene of RABV and helper plasmids. Four-week-old mice underwent one or two oral administrations of the ERAG3G strain and were challenged with the highly virulent RABV strain CVSN2c 14 days after the second administration. Clinical symptoms were observed and body weights were measured every day after the challenge.
Results
All mice showed complete protection against virulent RABV. In addition, cats intramuscularly inoculated with the ERAG3G strain showed high antibody titers ranging from 2.62 to 23.9 IU/mL at 28-day postinoculation.
Conclusion
The oral immunization of the ERAG3G strain plays an important role in conferring complete protection in mice, and intramuscular inoculation of the ERAG3G strain induces the formation of anti-rabies neutralizing antibody in cats.
doi:10.7774/cevr.2015.4.1.107
PMCID: PMC4313102  PMID: 25648184
Recombinant rabies virus; Reverse genetics; Mouse; Animals
2.  A single immunization with recombinant rabies virus (ERAG3G) confers complete protection against rabies in mice 
Purpose
New alternative bait rabies vaccines applicable to pet dogs and wild animals are needed to eradicate rabies in Korea. In this study, recombinant rabies virus, ERAG3G strain was constructed using reverse genetic system and the safety, efficacy and immunogenicity of the ERAG3G strain was evaluated in mice and dogs.
Materials and Methods
Using the full-length genome mutated amino acid at position 333 of glycoprotein of rabies virus (RABV) and helper plasmids, the ERAG3G strain was rescued in BHK/T7-9 cells successfully. Mice were inoculated with the ERAG3G strain for safety and efficacy. Safety and immunogenicity of the dog inoculated with the ERAG3G strain (1 mL, 108.0 FAID50/mL) via intramuscular route was evaluated for 28 days after inoculation.
Results
The ERAG3G strain rescued by reverse genetic system was propagated well in the mouse neuroblastoma cells revealing titer of 108.5 FAID50/mL and was not pathogenic to 4- or 6-week-old mice that received by intramuscular or intracranical route. Immunization with the ERAG3G strain conferred complete protection from lethal RABV in mice. Dogs inoculated with the vaccine candidate via intramuscular route showed high neutralizing antibody titer ranging from 2.62 to 23.9 IU/mL at 28 days postinoculation.
Conclusion
Our findings suggest that the ERAG3G strain plays an important role in inducing protective efficacy in mice and causes to arise anti-rabies neutralizing antibody in dogs.
doi:10.7774/cevr.2014.3.2.176
PMCID: PMC4083070  PMID: 25003091
Rabies virus; Recombinant rabies virus; Vaccine; Animals
3.  Inactivated genotype 1 Japanese encephalitis vaccine for swine 
Purpose
Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Recent genotype (G) shift phenomenon (G3 to G1) in the Asia-wide has posed a challenge for proper prevention by the current vaccine strain. Thus, new kinds of JEV G1 vaccines with enhanced immunogenicity have been required for pigs.
Materials and Methods
Recombinant porcine granulocyte monocyte-colony stimulating factor (reporGM-CSF) protein was expressed in Spodoptera frugiperda (Sf-9) cells using baculovirus expression system. Two kinds of trials with inactivated JEV vaccines containing IMS1313 adjuvant (Seppic, France) were prepared with or without reporGM-CSF protein. Safety and immunogenicity of the pigs inoculated with the JEV vaccines via intramuscular route was evaluated for 28 days after inoculation.
Results
Mice, guinea pigs, and fattening pigs inoculated with the inactivated vaccine showed no signs for 14 and 21 days. Both hemagglutination inhibition and plaque reduction neutralizing antibody titers were significantly higher in pigs immunized with the vaccine containing reporGM-CSF protein after boosting. However, on the side of vaccine efficacy, most mice (87%) immunized with the inactivated JEV vaccine survived after virulent JEV challenge. Whereas the group with the vaccine containing reporGM-CSF protein showed lower protective effects than the vaccine alone for the biological activity of the GM-CSF depending on species specific.
Conclusion
Our data indicate that animals inoculated with the JEV vaccines was safe and pigs inoculated with inactivated JEV vaccine containing reporGM-CSF protein showed higher humoral immune responses than that of inactivated JEV vaccine without reporGM-CSF protein.
doi:10.7774/cevr.2014.3.2.212
PMCID: PMC4083074  PMID: 25003095
Japanese encephalitis virus; Inactivated vaccine; GM-CSF; Swine
4.  The present and future of rabies vaccine in animals 
An effective strategy for preventing rabies consists of controlling rabies in the host reservoir with vaccination. Rabies vaccine has proven to be the most effective weapon for coping with this fatal viral zoonotic disease of warm-blooded animals, including human. Natural rabies infection of an individual is always associated with exposure to rabid animals, and the duration of clinical signs can vary from days to months. The incubation period for the disease depends on the site of the bite, severity of injury, and the amount of infecting virus at the time of exposure. The mortality of untreated cases in humans is 100%. Over the last 100 years, various rabies vaccines have been developed and used to prevent or control rabies in animals, such as modified live vaccine, inactivated rabies vaccine, and oral modified live vaccine. These have proved safe and efficacious worldwide. New-generation rabies vaccines, including recombinant rabies virus-based vaccines, vectored vaccines, DNA-based vaccines, and plant vaccines, have been explored to overcome the limitations of conventional rabies vaccines. This article discusses current and next-generation rabies vaccines in animals.
doi:10.7774/cevr.2013.2.1.19
PMCID: PMC3623496  PMID: 23596586
Rabies vaccines; Bait vaccine; Vectored vaccine; Plant vaccine
5.  Comparison of four diagnostic methods for detecting rabies viruses circulating in Korea 
Journal of Veterinary Science  2012;13(1):43-48.
It is essential to rapidly and precisely diagnose rabies. In this study, we evaluated four diagnostic methods, indirect fluorescent antibody test (FAT), virus isolation (VI), reverse transcriptase polymerase chain reaction (RT-PCR), and rapid immunodiagnostic assay (RIDA), to detect rabies in animal brain homogenates. Out of the 110 animal brain samples tested, 20 (18.2%) were positive for rabies according to the FAT. Compared to the FAT, the sensitivities of VI, RT-PCR, and RIDA were 100, 100, and 95%, respectively. The specificities of VI, RT-PCR and RIDA were found to be 100, 100, and 98.9%, respectively. Rabies viruses circulating in Korea were isolated and propagated in murine neuroblastoma (NG108-15) cells with titers ranging from 101.5 to 104.5 TCID50/mL. Although the RIDA findings did not completely coincide with results obtained from FAT, VI, and RT-PCR, RIDA appears to be a fast and reliable assay that can be used to analyze brain samples. In summary, the results from our study showed that VI, RT-PCR, and RIDA can be used as supplementary diagnostic tools for detecting rabies viruses in both laboratory and field settings.
doi:10.4142/jvs.2012.13.1.43
PMCID: PMC3317456  PMID: 22437535
immunodiagnostic assay; rabies; RT-PCR; virus isolation
6.  Serosurveillance for Japanese encephalitis virus in wild birds captured in Korea 
Journal of Veterinary Science  2011;12(4):373-377.
Climate change induced by recent global warming may have a significant impact on vector-borne and zoonotic diseases. For example, the distribution of Japanese encephalitis virus (JEV) has expanded into new regions. We surveyed the levels of hemagglutination-inhibition (HI) antibodies against JEV (Family Flaviviridae, genus Flavivirus) in wild birds captured in Korea. Blood samples were collected from 1,316 wild birds including the following migratory birds: Oceanodroma castro (n = 4), Anas formosa (n = 7), Anas penelope (n = 20), Fulica atra (n = 30), Anas acuta (n = 89), Anas crecca (n = 154), Anas platyrhynchos (n = 214), Aix galericulata (n = 310), and Anas poecilorhyncha (n = 488). All were captured in 16 locations in several Korea provinces between April 2007 and December 2009. Out of the 1,316 serum samples tested, 1,141 (86.7%) were positive for JEV. Wild birds captured in 2009 had a higher seroprevalence of ant-JEV antibodies than those captured in 2007. Wild birds with an HI antibody titer of 1 : 1,280 or higher accounted for 21.2% (280/1,316) of the animals tested. These findings indicated that wild birds from the region examined in our study have been exposed to JEV and may pose a high risk for introducing a new JEV genotype into Korea.
doi:10.4142/jvs.2011.12.4.373
PMCID: PMC3232397  PMID: 22122903
Japanese encephalitis virus; serosurveillance; wild bird
7.  Molecular characterization of Korean rabies virus isolates 
Journal of Veterinary Science  2011;12(1):57-63.
The nucleoprotein (N) and glycoprotein (G) of 11 Korean rabies virus (RABV) isolates collected from animals diagnosed with rabies between 2008 and 2009 were subjected to molecular and phylogenetic analyses. Six isolates originated from domestic animals (cattle and dogs) and five were obtained from wild free-ranging raccoon dogs. The similarities in the nucleotide sequences of the N gene among all Korean isolates ranged from 98.1 to 99.8%, while those of the G gene ranged from 97.9 to 99.3%. Based on the nucleotide analysis of the N and G genes, the Korean RABV isolates were confirmed as genotype I of Lyssavirus and classified into four distinct subgroups with high similarity. Phylogenetic analysis showed that the Korean isolates were most closely related to the non-Korean NeiMeng1025B and 857r strains, which were isolated from rabid raccoon dogs in Eastern China and Russia, respectively. These findings suggest that the Korean RABV isolates originated from a rabid raccoon dog in Northeastern Asia. Genetic analysis of the Korean RABV isolates revealed no substitutions at several antigenic sites, indicating that the isolates circulating in Korea may be pathogenic in several hosts.
doi:10.4142/jvs.2011.12.1.57
PMCID: PMC3053468  PMID: 21368564
characterization; genotype I; molecular epidemiology; rabies virus
8.  Molecular identification of the vaccine strain from the inactivated oil emulsion H9N2 low pathogenic avian influenza vaccine 
Journal of Veterinary Science  2010;11(2):161-163.
In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.
doi:10.4142/jvs.2010.11.2.161
PMCID: PMC2873817  PMID: 20458158
H9N2 avian influenza; identification of vaccine strain; oil emulsion vaccine
9.  Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30T-) 
Journal of Veterinary Science  2009;10(4):331-336.
Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.
doi:10.4142/jvs.2009.10.4.331
PMCID: PMC2807270  PMID: 19934599
AGID; baculovirus expression; bovine leukemia virus; glycoproteins
10.  Serosurveillance for Japanese encephalitis, Akabane, and Aino viruses for Thoroughbred horses in Korea 
Journal of Veterinary Science  2008;9(4):381-385.
Recent global warming trends may have a significant impact on vector-borne viral diseases, possibly affecting vector population dynamics and disease transmission. This study measured levels of hemagglutination-inhibition (HI) antibodies against Japanese encephalitis virus (JEV) and neutralizing antibodies against Akabane virus (AKAV) and Aino virus (AINV) for Thoroughbred horses in Korea. Blood samples were collected from 989 racehorses in several provinces, between October 2005 and March 2007. Sera were tested using either an HI assay or a virus neutralization test. Approximately half (49.7%; 492/989) of the horses tested were antibody-positive for JEV. The HI titer against JEV was significantly correlated with racehorse age (p < 0.05). Horses with an HI antibody titer of 1:160 or higher accounted for 3.9% of the animals tested, indicating that vectors transmitting arthropod-borne viruses bit relatively few horses. In contrast, 3.8% (19/497) and 19.5% (97/497) of horse sera collected in March 2007 were positive against AKAV and AINV, respectively. The presence of antibodies against AKAV and AINV may indicate the multiplication of AKAV and AINV in these horses.
doi:10.4142/jvs.2008.9.4.381
PMCID: PMC2811779  PMID: 19043313
arbovirus; racehorse; serosurveillance
11.  The seroprevalence of Japanese encephalitis virus in goats raised in Korea 
Journal of Veterinary Science  2007;8(2):197-199.
Japanese encephalitis virus (JEV) causes a mosquito-borne viral zoonosis that is becoming increasingly important to public health in east and south Asia. Although JEV is primarily associated with reproductive failure in swine, JEV infection can cause fever and headache in humans and is associated with aseptic meningitis and encephalitis. The exact mode of transmission, including host range and possible source of viral amplification within livestock, is still not completely clear. This study consisted of a serological survey of JEV infection in goats. A total of 804 goat serum samples were collected from 144 farms in Korea between May 2005 and May 2006. The incidence of positive cases was 12.1% (97 out of 804 goats). The seroprevalence of JEV infection in the 144 farms screened was 31.3% (45/144), indicating that JEV infection is frequent in goat farms in Korea. In addition, three districts of Korea (mainly in the southern region) had a higher seroprevalence of JEV compared to other areas. The results suggest that goats could be monitored epidemiologically as a sentinel animal for JEV transmission in Korea.
doi:10.4142/jvs.2007.8.2.197
PMCID: PMC2872721  PMID: 17519576
goat; JEV; seroprevalence
12.  Sero-survey on Aino, Akabane, Chuzan, bovine ephemeral fever and Japanese encephalitis virus of cattle and swine in Korea 
Journal of Veterinary Science  2007;8(1):45-49.
Vector-borne arboviruses produce mild to severe symptoms in domestic animals. Bovine ephemeral fever (BEF), Akabane, Aino, and Chuzan virus have been primarily attributed to reproductive disorders or febrile diseases in cattle, and Japanese encephalitis virus (JEV) is mainly associated with reproductive failures in swine. We investigated antibody titers from domestic swine against four bovine arboviruses (BEF, Akabane, Aino, and Chuzan virus) and from cattle against JEV in Korea. While the positive rates for Akabane and BEF were 37.4% and 15.7%, the positive incidence of Chuzan and Aino were relatively low, with positive rates of 3.04% and 0.4%, respectively, based on a virus neutralization assay. Antibody titers against more than one virus were also frequently detected in domestic swine. The incidence of JEV was 51.3% among domestic cattle. In addition, one positive case was detected in the thoracic fluids from 35 aborted calves, based on the hemagglutination inhibition test. Our results indicate that swine are susceptible hosts of bovine arboviruses without showing clinical symptoms in a natural environment. Moreover, we confirmed that JEV could be associated with reproductive failure in pregnant cattle, as were other vector-borne bovine arboviruses assessed in this study.
doi:10.4142/jvs.2007.8.1.45
PMCID: PMC2872696  PMID: 17322773
arboviral infection; cattle; sero-incidence; swine
13.  Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine 
Journal of Veterinary Science  2006;7(3):271-275.
The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.
doi:10.4142/jvs.2006.7.3.271
PMCID: PMC3242127  PMID: 16871022
diagnostic efficiency; I-ELISA; Japanese encephalitis virus; JEV

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