canine adenovirus type 2 (CAV-2) vaccine candidate using the recently isolated Korean CAV-2 strain; we termed the vaccine APQA1701-40P and evaluated its safety and immunogenicity in dogs.MATERIALS AND METHODS: To generate the anti-CAV-2 vaccine, APQA1701 was passaged 40 times in MDCK cells growing in medium containing 5 mM urea and the virus was inactivated using 0.05% (volume per volume) formaldehyde. Two vaccines were prepared by blending inactivated APQA1701-40P with two different adjuvants; both were intramuscularly injected (twice) into guinea pigs. The safety and immunogenicity of the Cabopol-adjuvanted vaccine were evaluated in seronegative dogs. The humoral responses elicited were measured using an indirect enzyme-linked immunosorbent assay (I-ELISA), and via a virus neutralization assay (VNA).RESULTS: The new, inactivated CAV-2 vaccine strain, APQA1701-40P, lacked six amino acids of the E1b-19K protein. In guinea pigs, the Cabopol-adjuvanted vaccine afforded a slightly higher VNA titer and I-ELISA absorbance than an IMS gel-adjuvanted vaccine 4 weeks post-vaccination (p>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.]]>
Adenoviruses, Canine ; Amino Acids ; Animals ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Formaldehyde ; Guinea Pigs ; Madin Darby Canine Kidney Cells ; Urea ; Vaccines

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.
Adenoviridae ; Adenoviruses, Canine ; Animals ; Base Sequence ; Canada ; Dogs ; Erythrocytes ; Fluorescence ; Fluorescent Antibody Technique ; Guinea Pigs ; Hemagglutination ; Hepatitis ; Madin Darby Canine Kidney Cells ; Microscopy, Electron ; Quality Control

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.
Adenoviridae Infections ; Adenoviruses, Canine* ; Animals* ; Antibodies ; Cats ; Cattle ; Dogs ; Horses ; Incidence* ; Korea ; Neutralization Tests ; Plants ; Quarantine ; Raccoon Dogs ; Vaccination

Wild raccoon dogs (Nyctereutes procyonoides koreensis) may play a role transmitting several pathogens to humans and pet animals. Information concerning the incidence of rabies, canine distemper virus (CDV), canine parvovirus (CPV), canine adenovirus type 2 (CAdV-2), canine parainfluenza virus type 5 (CPIV-5), and canine herpesvirus (CHV) is needed in wild raccoon dogs. In total, 62 brain samples of raccoon dogs were examined for rabies virus (RABV) and CDV, and 49 lung samples were screened for CDV, CAdV-2, CPIV-5, and CHV. No RABV, CAdV-2, CPIV-5, or CHV was identified, but nine CDV antigens (8.1%, 9/111) were detected. Moreover, 174 serum samples from wild raccoon dogs were screened for antibodies against the five major viral pathogens. The overall serosurveillance against CDV, CPV, CAdV-2, CPIV-5, and CHV in wild raccoon dogs was 60.3%, 52.9%, 59.8%, 23.6%, and 10.3%, respectively. Comparisons of the sero-surveillance of the five pathogens showed that raccoon dogs of Gyeonggi province have slightly higher sero-positive rates against CDV, CPV, and CHV than those of Gangwon province. These results indicate high incidences of CDV, CPV, and CAdV-2 in wild raccoon dogs of two Korean provinces and a latent risk of pathogen transmission to companion and domestic animals.
Adenoviruses, Canine ; Animals ; Animals, Domestic ; Antibodies ; Brain ; Disease Transmission, Infectious ; Distemper ; Distemper Virus, Canine ; Friends ; Gangwon-do ; Gyeonggi-do ; Humans ; Incidence ; Lung ; Paramyxoviridae Infections ; Parvovirus, Canine ; Rabies ; Rabies virus ; Raccoon Dogs* ; Raccoons*

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.
Adenoviruses, Canine ; Animals ; Base Sequence ; Canada ; Dogs ; Erythrocytes ; Fluorescent Antibody Technique ; Genome ; Guinea Pigs ; Gyeonggi-do ; Hemagglutination ; Kidney ; Korea ; Madin Darby Canine Kidney Cells ; Microscopy, Electron ; Polymerase Chain Reaction ; Vaccines ; Vero Cells ; Vitis

Canine adenovirus type 2 (CAV-2) has been proposed as a vector for recombinant vaccine. Alternatively, it may be an attractive tool for gene transfer due to lack of pre-existing immunity in humans. In this study, a transfer vector based on CAV-2, in which the 1381bp fragment of the E3 region was deleted, and a linker containing the Not I, Cla I, Fse I restriction enzyme sites were cloned into the deleted region. The recombinant CAV-2 genome was released from the plasmids enzyme digestion and transfected into MDCK cells by lipofectamine to obtain the recombinant virus. No significant difference in morphology, hemagglutination and replication between the recombinant and the wide type CAV-2 was found. These results indicated that this recombinant virus CAV-2-deltaE3 (NF) may be an efficient vector for gene transfer and the capacity of the vector for inserted foreign gene was up to 3.3kb.
Adenoviruses, Canine ; genetics ; ultrastructure ; Animals ; Binding Sites ; genetics ; Cell Line ; Cloning, Molecular ; DNA Restriction Enzymes ; metabolism ; DNA, Viral ; chemistry ; genetics ; Dogs ; virology ; Genetic Vectors ; genetics ; Humans ; Lipids ; chemistry ; Microscopy, Electron ; Transfection ; methods ; Virus Replication ; genetics