Rabies neutralizing antibody titers in Korean dogs and cats intended for overseas travel

Animals imported from abroad are a cause of rabies outbreaks in many countries. Therefore, rabies serology testing for dogs and cats traveling abroad is an important measure to reduce the incidence of rabies. Rabies virus antibodies were measured in sera collected from 2,367 dogs and 894 cats between 2017 and 2021. A serum sample with a value of 0.5 IU/mL or higher was considered a pass. The overall pass rates for rabies virus were 96.4% in dogs and 98.4% in cats. The mean rabies virus neutralization assay titers were higher in cats than in dogs and in female than in male animals. According to age, 6-year-old dogs and 9-year-old cats had the highest virus neutralization assay titers. Of the failure cases, 53.0% (53/100) were dogs or cats less than 1 year old. Although the average failure rates in dogs and cats were low at 3.5% and 1.6%, respectively, the factors influencing failure were age and vaccine manufacturer. Therefore, it is necessary to observe the vaccination interval and timing of blood collection after boosting.

Immunogenicity of an inactivated rabies vaccine for animals derived from the recombinant ERAGS strain

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.

Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay

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.

Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay

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.

Isolation and Molecular Characterization of Feline Herpesvirus 1 from Naturally Infected Korean Cats

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.

Genotype-specific neutralizing antibody titers against Japanese encephalitis virus genotypes 1 and 3 in horses immunized with a genotype 3 vaccine

Purpose@#Japanese encephalitis is one of the most important mosquito-borne and zoonotic diseases in Asia and the Pacific region. Although the dominant Japanese encephalitis virus (JEV) genotype has shifted from G3 to G1 in Korea since 1990, a G3 strain (Anyang 300) has been used in vaccines for horses for almost 40 years. This study aimed to investigate the seroconversion rates and geometric mean titers (GMTs) of virus-neutralizing antibodies (VNAs) against JEV G1 and G3 in horses immunized with the G3 vaccine. @*Materials and Methods@#Serum samples of 1,231 horses immunized with the Anyang 300 vaccine were collected in 2018. VNA titers against JEV KV1899 (G1) and Anyang 300 (G3) were measured in all serum samples using the virus neutralization test. Titers were analyzed according to blood sampling time (prior to and following annual revaccination), age, and region. @*Results@#Rates of VNA titer >10 were 45.1% and 77.8% for G1, and 49.1% and 82.9% for G3 in samples taken before and after revaccination, respectively. GMTs of genotype-specific VNAs against JEV G1 and G3 were 8.3 and 11.6 before revaccination and rose to 27.2 and 65.4 following revaccination. Overall sero-positivity did not significantly differ between genotypes, but GMTs significantly differed among genotypes and sampling times. No significant difference was found in GMTs among age groups or regions. @*Conclusion@#Genotype-specific neutralizing antibody titers against JEV G1 and G3 differed significantly in horses immunized with the G3 vaccine. Antigenic differences between genotypes could reduce the vaccine’s efficacy, requiring the development of a new vaccine.

Biological and molecular characterization of feline caliciviruses isolated from cats in South Korea

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.

Isolation and Molecular Characterization of Feline Herpesvirus 1 from Naturally Infected Korean Cats

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.

Genotype-specific neutralizing antibody titers against Japanese encephalitis virus genotypes 1 and 3 in horses immunized with a genotype 3 vaccine

Purpose@#Japanese encephalitis is one of the most important mosquito-borne and zoonotic diseases in Asia and the Pacific region. Although the dominant Japanese encephalitis virus (JEV) genotype has shifted from G3 to G1 in Korea since 1990, a G3 strain (Anyang 300) has been used in vaccines for horses for almost 40 years. This study aimed to investigate the seroconversion rates and geometric mean titers (GMTs) of virus-neutralizing antibodies (VNAs) against JEV G1 and G3 in horses immunized with the G3 vaccine. @*Materials and Methods@#Serum samples of 1,231 horses immunized with the Anyang 300 vaccine were collected in 2018. VNA titers against JEV KV1899 (G1) and Anyang 300 (G3) were measured in all serum samples using the virus neutralization test. Titers were analyzed according to blood sampling time (prior to and following annual revaccination), age, and region. @*Results@#Rates of VNA titer >10 were 45.1% and 77.8% for G1, and 49.1% and 82.9% for G3 in samples taken before and after revaccination, respectively. GMTs of genotype-specific VNAs against JEV G1 and G3 were 8.3 and 11.6 before revaccination and rose to 27.2 and 65.4 following revaccination. Overall sero-positivity did not significantly differ between genotypes, but GMTs significantly differed among genotypes and sampling times. No significant difference was found in GMTs among age groups or regions. @*Conclusion@#Genotype-specific neutralizing antibody titers against JEV G1 and G3 differed significantly in horses immunized with the G3 vaccine. Antigenic differences between genotypes could reduce the vaccine’s efficacy, requiring the development of a new vaccine.

Immunogenicity of a new, inactivated canine adenovirus type 2 vaccine for dogs

0.05). Dogs inoculated with the former vaccine developed a significantly higher immune titer than non-vaccinated dogs.CONCLUSION: The Cabopol-adjuvanted, inactivated CAV-2 vaccine was safe and induced a high VNA titer in dogs.

Isolation and molecular characterizations of canine distemper virus from a naturally infected Korean dog using Vero cells expressing dog signaling lymphocyte activation molecule

Background@#Canine distemper virus (CDV) infection results in high morbidity and mortality in dogs. There has been no report about Isolation of Korean CDV since 1980 in Korea. @*Objectives@#To investigate the biological properties and the genetic characterization of Korean CDV. @*Methods@#Vero cells expressing dog signaling lymphocyte activation molecule (dSLAM) gene named as Vero/dSLAM were used to isolate CDV using 17 samples. Diagnostic methods such as cytopathic effects, immunofluorescence assay, peroxidase linked assay, electron microscopy, rapid immunodiagnostic assay, and reverse transcription polymerase chain reaction were used to confirm the Korean CDV isolate as a CDV. The genetic analysis was performed through cloning and sequencing of hemagglutinin gene of CDV isolate. @*Results@#A virus propagated in Vero/dSLAM cell was confirmed as CDV (CD1901 strain) based on the above methods. The CD1901 strain showed the highest viral titer (10 5.5 50% tissue culture infectious dose [TCID 50 ]/mL) in the Vero/dSLAM cells at 4 days post inoculation, but did not form a fork on chorioallantoic membrane of 7-day-old egg. Ribavirin, a nucleotide analogue anti-viral agent, inhibits moderately the Korean CDV propagation in the Vero/dSLAM cells. The nucleotide and amino acid sequences of the H gene of CD1901 strain were compared with those of other CDV strains. The CD1901 strain belonged to Asia 1 group and had the highest similarity (99.9%) with the BA134 strain, which was isolated in China in 2008. @*Conclusions@#We constructed successfully Vero/dSLAM and isolated one Korean CDV isolate (CD1901 strain) from a naturally infected dog. The CD1901 strain belonged to Asia 1 genotype.

Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID 50 /reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No crossreactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

Development of indirect ELISA for the detection of canine adenovirus type 2 antibodies in dog sera

Background@#Canine adenovirus type 2 (CAV-2) induces infectious laryngotracheitis in members of the family Canidae, including dogs. To date, no ELISA kits specific for CAV-2 antibody have been commercialized for dogs in Korea. @*Objectives@#We aimed to develop new indirect enzyme-linked immunosorbent assay (I-ELISA) to perform rapid, accurate serological surveys of CAV-2 in dog serum samples. @*Methods@#In total, 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces between 2016 and 2018. The Korean CAV-2, named the APQA1701-40P strain, was propagated in Madin–Darby canine kidney cells and purified in an anion-exchange chromatography column for use as an antigen for I-ELISA. The virus-neutralizing antibody titers of CAV-2 in the dog sera were measured by virus neutralization (VN) test. @*Results@#We compared the results obtained between the VN and new I-ELISA tests. The sensitivity, specificity, and accuracy of new I-ELISA were 98.6%, 86.4% and 97.0% compared with VN test, respectively. New I-ELISA was significantly correlated with VN (r = 0.91). @*Conclusions@#These results indicate that new I-ELISA is useful for sero-surveillance of CAV-2 in dog serum.

Evaluation of Commercial Immunochromatographic Test Kits for the Detection of Canine Distemper Virus

The rapid diagnosis of canine distemper virus (CDV) helps to determine the treatment of dogs in veterinary clinics. We evaluated the performance of seven commercial rapid immunochromatographic test (RICT) kits for the detection of CDV. Six core dog viral pathogens (canine adenovirus type 1 and 2, canine coronavirus, canine parainfluenza virus, canine parvovirus, and rabies virus), five CDV strains (CD1901, Lederle, Rockborn, Onderstepoort, and Synder Hill), and three bacteria (Bordetella bronchiseptica, Leptospira canicola, and Staphylococus aureus) were used to determine the cross-reactivity and detection limits of the kits. The seven commercial RICT kits did not yield positive results with the six dog viruses or the three bacteria. All the RICT kits for CDV detected the Korean CDV isolate. The detection limits of the RICT kits for the Korean CDV isolate, CD1901, belonging to Asia 1 genotype ranged from 103.0 to 104.0 TCID50/mL. There was an average difference of 1.1 in scores judged by eye between four CDV vaccine strains and CD1901 strain. Therefore, the RICT kits enable the detection of CDV vaccine strains, but need to be improved to detect CDV circulating in dog populations in Korea.

Development of an Indirect ELISA Featuring Plates Coated with Column Chromatographically Purified Canine Adenovirus Type-1 Antigen

Canine adenovirus type 1 (CAV-1) causes infectious hepatitis in members of the family Canidae, including dogs. An indirect enzyme-linked immunosorbent assay (I-ELISA) that detects CAV-1 antibodies is required for large-throughput tests of dog sera. We collected 165 serum samples from dogs of Chungbuk and Gyeongbuk provinces between February 2016 and October 2018. The Korean CAV-1 vaccine strain CAV1V was propagated in Madin-Darby canine kidney (MDCK) cells and purified via Nuvia cPrime anion-exchange chromatography; the virus served as an I-ELISA antigen. Virus-neutralizing anti-CAV-1 titers in dog sera were measured using the virus neutralization (VN) method. The I-ELISA was optimized using purified CAV-1 antigen and serum samples. This kit was used to evaluate dog sera. The VN and I-ELISA data were compared. The sensitivity, specificity, and accuracy of the I-ELISA were 97.0%, 74.2%, and 92.7% compared to the VN assay, respectively. The I-ELISA data significantly correlated with those of VN (r = 0.88). These results suggest that the I-ELISA is useful for serosurveillance of CAV-1 in dog sera.

Biological and molecular characterization of feline caliciviruses isolated from cats in South Korea

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.

Recharacterization of the Canine Adenovirus Type 1 Vaccine Strain based on the Biological and Molecular Properties

Canine adenovirus type 1 (CAV-1) infection results in hepatitis in dogs. In this study, we investigated the biologic and genetic characteristics of the CAV-1 vaccine strain (CAV1V) to improve quality control about CAV vaccine. The identity of CAV1V as CAV-1 was confirmed based on its cytopathic effects and the results of hemagglutination (HA) and immunofluorescence assays, and electron microscopy. The CAV1V strain reached 10(7.5) TCID(50)/mL in MDCK cells at 4 days post-inoculation and exhibited hemmagglutination activity of 256 U using guinea pig erythrocytes. Intranuclear fluorescence in the infected cells was observed and typical adenoviruses were observed in electon microscope. CAV1V strain was identified as a CAV-1 strain by nucleotide sequence analysis. In a comparison of the nucleotide sequences of the fiber genes of several CAV strains, CAV1V showed the highest similarity (99.8%) with the GLAXO strain, which was isolated in Canada. Our biological characterization of CAV1V will facilitate quality control of the canine hepatitis vaccine.

Rabies immune status of raccoon dogs residing in areas where rabies bait vaccine has been distributed

Since 2000, large amounts of rabies bait vaccine have been distributed in two provinces where raccoon dog-mediated rabies has occurred. A total of 146 raccoon dogs were caught in Gangwon and Gyeonggi Provinces from January 2017 to June 2018, and raccoon dog blood samples were collected. Of the 146 raccoon dogs, 13.7% (20/146) had rabies antibodies. In Gyeonggi and Gangwon provinces, the rate of rabies antibody was 8.5% (5/59) and 17.2% (15/87), respectively. Considering these results, it would be desirable to improve the distribution method or use a new bait vaccine to prevent animal rabies in South Korea.

Incidence and Sero-survey of Canine Adenovirus Type 2 in Various Animal Species

Canine adenovirus type 2 (CAV-2) is the cause of a major respiratory illness in dogs. In this study, we analyzed adenovirus infections in dogs using 2000–2017 data from the Animal and Plant Quarantine Agency (APQA) and conducted a serological survey of CAV-2 infection in six animal species in Korea. In total, 38 of the 3,179 dog samples were confirmed as canine adenovirus infections. In serological survey, 1,028 dog sera, 160 raccoon dog sera, 100 cattle sera, 257 sow sera, 206 horse sera, and 106 cat sera, collected from January 2016 to July 2018, were screened for the presence of anti-CAV-2 antibodies by virus neutralization test. The seropositivity rates for dogs, raccoon dogs, cattle, sows, horses, and cats were 88.5% (910/1,028), 51.3% (82/160), 85.0% (85/100), 48.6% (125/257), 35.0% (72/206), and 2.8% (3/106), respectively. Among dogs and raccoon dogs, 1.9% (20/1,028) and 8.8% (14/160), respectively, had a virus-neutralizing antibody (VNA) titer of over 1:256. A high CAV-2 VNA titer indicates a repeated vaccination or natural infection in Korean dogs and circulation of CAV-2 in raccoon dog populations.

Isolation and identification of canine adenovirus type 2 from a naturally infected dog in Korea

Canine adenovirus type 2 (CAV-2) infection results in significant respiratory illness in dogs. Isolating and culturing CAV-2 allows for investigations into its pathogenesis and the development of vaccines and diagnostic assays. In this study, we successfully isolated a virus from a naturally infected dog in Gyeonggi-do, Korea. The virus was propagated in Madin-Darby canine kidney (MDCK) and Vero cells and showed a specific cytopathic morphology that appeared similar to a bunch of grapes. The virus was first confirmed as CAV-2 based on these cytopathic effects, an immunofluorescence assay, hemagglutination assay, and electron microscopy. The viral titer of the isolate designated APQA1601 reached 10(6.5) 50% tissue culture infections dose per mL in MDCK cells and exhibited no hemagglutination units with erythrocytes from guinea pig. The virus was also confirmed by polymerase chain reaction and next-generation sequencing. The APQA1601 strain had the highest similarity (~99.9%) with the Toronto A26/61 strain, which was isolated in Canada in 1976 when the nucleotide sequences of the full genome of the APQA1601 strain were compared with those of other CAV strains. Isolating CAV-2 will help elucidate the biological properties of CAV-2 circulating in Korean dogs.