ABSTRACT
Purpose@#The aims of the present study were to evaluate the immunogenicity of an inactivated rabies vaccine based on the ERAGS strain @*Materials and Methods@#The ERAGS virus propagated in Vero cells was inactivated with 3 mM binary ethylenimine for 8 hours. Three types of inactivated rabies vaccines were prepared to determine the minimum vaccine virus titers. Four further types of inactivated rabies vaccines were prepared by blending inactivated ERAGS with four different adjuvants; each vaccine was injected into mice, guinea pigs, and dogs to identify the optimal adjuvant. The immunogenicity of a Montanide (IMS) gel-adjuvanted vaccine was evaluated in cats, dogs, and cattle. Humoral immune responses were measured via a fluorescent antibody virus neutralization method and a blocking enzyme-linked immunosorbent assay. @*Results@#The minimum virus titer of the inactivated rabies vaccine was over 107.0 50% tissue culture infectious doses (TCID50 values)/mL. Of the four kinds of adjuvants, the IMS gel-adjuvanted vaccine induced the highest mean viral neutralizing antibody (VNA) titers of 6.24 and 2.36 IU/mL in guinea pigs and dogs, respectively, and was thus selected as the vaccine for the target animals. Cats, dogs, and cattle inoculated with the IMS gel-adjuvanted vaccine developed protective VNA titers ranging from 3.5 to 1.2 IU/mL at 4 weeks post-inoculation (WPI). @*Conclusion@#Our data indicate that cats, dogs, and cattle inoculated with an inactivated rabies vaccine derived from the ERAGS strain developed protective immune responses that were maintained to 12 WPI.
ABSTRACT
Mammalian reovirus (MRV) causes respiratory and intestinal disease in mammals. Although MRV isolates have been reported to circulate in several animals, there are no reports on Korean MRV isolates from wildlife. We investigated the biological and molecular characteristics of Korean MRV isolates based on the nucleotide sequence of the segment 1 gene. In total, 144 swabs from wild animals were prepared for virus isolation. Based on virus isolation with specific cytopathic effects, indirect fluorescence assays, electron microscopy, and reverse transcription-polymerase chain reaction, one isolate was confirmed to be MRV. The isolate exhibited a hemagglutination activity level of 16 units with pig erythrocytes and had a maximum viral titer of 105.7 50% tissue culture infectious dose (TCID50)/mL in Vero cells at 5 days after inoculation. The nucleotide and amino-acid sequences of the partial segment S1 of the MReo2045 isolate were determined and compared with those of other MRV strains. The MReo2045 isolate had nucleotide sequences similar to MRV-3 and was most similar (96.1%) to the T3/Bat/Germany/342/08 strain, which was isolated in Germany in 2008. The MReo2045 isolate will be useful as an antigen for sero-epidemiological studies and developing diagnostic tools.
ABSTRACT
Feline panleukopenia virus (FPV) causes leukopenia and severe hemorrhagic diarrhea, killing 50% of naturally infected cats. Although intact FPV can serve as an antigen in the hemagglutination inhibition (HI) test, accidental laboratory-mediated infection a concern. A non-infectious diagnostic reagent is required for the HI test. Here, we expressed the viral protein 2 (VP2) gene of the FPV strain currently prevalent in South Korea in a baculovirus expression system; VP2 protein was identified by an indirect immunofluorescence assay, electron microscopy (EM), Western blotting (WB), and a hemagglutination assay (HA). EM showed that the recombinant VP2 protein self-assembled to form virus-like particles. WB revealed that the recombinant VP2 was 65 kDa in size. The HA activity of the recombinant VP2 protein was very high at 1:215. A total of 143 cat serum samples were tested using FPV (HI-FPV test) and the recombinant VP2 protein (HI-VP2 test) as HI antigens. The sensitivity, specificity, and accuracy of the HI-VP2 test were 99.3%, 88.9%, and 99.3%, respectively, compared to the HI-FPV test. The HI-VP2 and HI-FPV results correlated significantly (r = 0.978). Thus, recombinant VP2 can substitute for intact FPV as the serological diagnostic reagent of the HI test for FPV.
ABSTRACT
Background@#Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. @*Objectives@#We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). @*Methods@#A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. @*Results@#The virus propagated in VERO cells was confirmed as RABV expressing GFP.The ERAGS-GFP showed the highest titer (108.0TCID50/mL) in VERO cells at 5 days postinoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). @*Conclusions@#We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.
ABSTRACT
Mammalian reovirus (MRV) causes respiratory and intestinal disease in mammals. Although MRV isolates have been reported to circulate in several animals, there are no reports on Korean MRV isolates from wildlife. We investigated the biological and molecular characteristics of Korean MRV isolates based on the nucleotide sequence of the segment 1 gene. In total, 144 swabs from wild animals were prepared for virus isolation. Based on virus isolation with specific cytopathic effects, indirect fluorescence assays, electron microscopy, and reverse transcription-polymerase chain reaction, one isolate was confirmed to be MRV. The isolate exhibited a hemagglutination activity level of 16 units with pig erythrocytes and had a maximum viral titer of 105.7 50% tissue culture infectious dose (TCID50)/mL in Vero cells at 5 days after inoculation. The nucleotide and amino-acid sequences of the partial segment S1 of the MReo2045 isolate were determined and compared with those of other MRV strains. The MReo2045 isolate had nucleotide sequences similar to MRV-3 and was most similar (96.1%) to the T3/Bat/Germany/342/08 strain, which was isolated in Germany in 2008. The MReo2045 isolate will be useful as an antigen for sero-epidemiological studies and developing diagnostic tools.
ABSTRACT
Feline panleukopenia virus (FPV) causes leukopenia and severe hemorrhagic diarrhea, killing 50% of naturally infected cats. Although intact FPV can serve as an antigen in the hemagglutination inhibition (HI) test, accidental laboratory-mediated infection a concern. A non-infectious diagnostic reagent is required for the HI test. Here, we expressed the viral protein 2 (VP2) gene of the FPV strain currently prevalent in South Korea in a baculovirus expression system; VP2 protein was identified by an indirect immunofluorescence assay, electron microscopy (EM), Western blotting (WB), and a hemagglutination assay (HA). EM showed that the recombinant VP2 protein self-assembled to form virus-like particles. WB revealed that the recombinant VP2 was 65 kDa in size. The HA activity of the recombinant VP2 protein was very high at 1:215. A total of 143 cat serum samples were tested using FPV (HI-FPV test) and the recombinant VP2 protein (HI-VP2 test) as HI antigens. The sensitivity, specificity, and accuracy of the HI-VP2 test were 99.3%, 88.9%, and 99.3%, respectively, compared to the HI-FPV test. The HI-VP2 and HI-FPV results correlated significantly (r = 0.978). Thus, recombinant VP2 can substitute for intact FPV as the serological diagnostic reagent of the HI test for FPV.
ABSTRACT
Background@#Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. @*Objectives@#We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). @*Methods@#A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. @*Results@#The virus propagated in VERO cells was confirmed as RABV expressing GFP.The ERAGS-GFP showed the highest titer (108.0TCID50/mL) in VERO cells at 5 days postinoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). @*Conclusions@#We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.
ABSTRACT
Feline herpesvirus type 1 (FHV-1) causes respiratory and ocular disease in cats.Although isolates of FHV-1 circulating in cats have been reported worldwide, Korean FHV-1 isolates and their features have not been reported thus far. We aimed to investigate the biological and molecular characterization of two FHV-1 isolates based on the nucleotide sequence of thymidine kinase (TK) and glycoprotein B (gB) gene. In total, 48 samples from 12 cats were prepared for virus isolation.For the diagnosis, virus isolation, indirect fluorescence assay (IFA), electron microscopy (EM), and polymerase chain reaction (PCR) and for the molecular characterization, cloning and sequencing were used. Based on many methods such as virus isolation with specific cytopathic effects, IFA, EM, and PCR, two isolates were confirmed as FHV-1 and they showed the highest viral titer (108.3 to 108.5 TCID50 /mL) in the Crandell–Rees Feline Kidney cells at 48 h after inoculation, but did not grow in MDCK and Vero cells. The nucleotide and amino acid sequences of the full TK and gB gene of FHV191071 and FHV191072 isolates were determined and compared with those of other herpesvirus strains. Two isolates possessed the same nucleotide sequences belonging to FHV-1 group and had the highest similarity (99.9%) with the KANS-02 strain, which was isolated from shelter in USA in 2016. Two isolates were confirmed as FHV-1 and they will be a useful basic resource for evaluating current FHV-1 vaccine and developing diagnostic tools.
ABSTRACT
Feline calicivirus (FCV) infection results in a common upper respiratory disease associated with oral ulceration in cats.Although FCV infection has been reported in cats worldwide, the biologic and genetic features of South Korean FCV are unclear. We aimed to investigate the biological and genetic features of South Korean FCV isolates. Crandell-Rees feline kidney (CRFK) cells were used to isolate FCV from 58 organ homogenate samples. The FCV isolates were confirmed by cytopathic effects, immunofluorescence, electron microscopy, and reverse transcription polymerase chain reaction assays. Viral genetic analysis was carried out with VP2 gene and complete genomes of FCVs. Five viruses propagated in CRFK cells were confirmed to be FCVs. The FCV17D283 isolate showed the highest viral titer of 107.2TCID50 /mL at 36 h post-inoculation. Korean FCV isolates did not grow well in Vero, BHK-21, A72, or Madin-Darby canine kidney cells. The FCV17D03 and FCV17D283 isolates had the highest genetic similarity (80.1% and 86.9%) with the UTCVM-H1 and 14Q315 strains, which were isolated in the United States and South Korea in 1995 and 2014, respectively. We isolated five FCVs from cats and detected important genetic differences among them. FCV isolates did not show any virulent effects in mice.
ABSTRACT
Feline herpesvirus type 1 (FHV-1) causes respiratory and ocular disease in cats.Although isolates of FHV-1 circulating in cats have been reported worldwide, Korean FHV-1 isolates and their features have not been reported thus far. We aimed to investigate the biological and molecular characterization of two FHV-1 isolates based on the nucleotide sequence of thymidine kinase (TK) and glycoprotein B (gB) gene. In total, 48 samples from 12 cats were prepared for virus isolation.For the diagnosis, virus isolation, indirect fluorescence assay (IFA), electron microscopy (EM), and polymerase chain reaction (PCR) and for the molecular characterization, cloning and sequencing were used. Based on many methods such as virus isolation with specific cytopathic effects, IFA, EM, and PCR, two isolates were confirmed as FHV-1 and they showed the highest viral titer (108.3 to 108.5 TCID50 /mL) in the Crandell–Rees Feline Kidney cells at 48 h after inoculation, but did not grow in MDCK and Vero cells. The nucleotide and amino acid sequences of the full TK and gB gene of FHV191071 and FHV191072 isolates were determined and compared with those of other herpesvirus strains. Two isolates possessed the same nucleotide sequences belonging to FHV-1 group and had the highest similarity (99.9%) with the KANS-02 strain, which was isolated from shelter in USA in 2016. Two isolates were confirmed as FHV-1 and they will be a useful basic resource for evaluating current FHV-1 vaccine and developing diagnostic tools.
ABSTRACT
Feline calicivirus (FCV) infection results in a common upper respiratory disease associated with oral ulceration in cats.Although FCV infection has been reported in cats worldwide, the biologic and genetic features of South Korean FCV are unclear. We aimed to investigate the biological and genetic features of South Korean FCV isolates. Crandell-Rees feline kidney (CRFK) cells were used to isolate FCV from 58 organ homogenate samples. The FCV isolates were confirmed by cytopathic effects, immunofluorescence, electron microscopy, and reverse transcription polymerase chain reaction assays. Viral genetic analysis was carried out with VP2 gene and complete genomes of FCVs. Five viruses propagated in CRFK cells were confirmed to be FCVs. The FCV17D283 isolate showed the highest viral titer of 107.2TCID50 /mL at 36 h post-inoculation. Korean FCV isolates did not grow well in Vero, BHK-21, A72, or Madin-Darby canine kidney cells. The FCV17D03 and FCV17D283 isolates had the highest genetic similarity (80.1% and 86.9%) with the UTCVM-H1 and 14Q315 strains, which were isolated in the United States and South Korea in 1995 and 2014, respectively. We isolated five FCVs from cats and detected important genetic differences among them. FCV isolates did not show any virulent effects in mice.