Korean Journal of Veterinary Research (대한수의학회지)

The Korean Society of Veterinary Science (대한수의학회)
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Isolation and identification of canine adenovirus type 2 from a naturally infected dog in Korea

  • Yang, Dong-Kun (Viral Disease Research Division, Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs) ;
  • Kim, Ha-Hyun (Viral Disease Research Division, Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs) ;
  • Yoon, Soon-Seek (Viral Disease Research Division, Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs) ;
  • Lee, Hyunkyoung (Viral Disease Research Division, Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs) ;
  • Cho, In-Soo (Viral Disease Research Division, Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs)
  • Received : 2018.08.13
  • Accepted : 2018.12.05
  • Published : 2018.12.31
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Abstract

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.

Keywords

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Fig. 1. Cytopathic effects (CPEs) of CAV type 2 (CAV-2) isolates in infected Vero cells (A) and normal Vero cells (B). Indirect immunofluorescence assay using monoclonal antibodies against CAV-1 (C and D) and CAV-2 (E and F). Vero cells infected with the APQA1601 isolate showed specific CPEs, and intranuclear fluorescence (E) was observed in Vero cells stained with the CAV-2 antibody. 200× (A-F).

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Fig. 2. Growth curves of the APQA1601 strain at passage 10 according to the time of harvest from Vero and Madin-Darby canine kidney (MDCK) cells. The APQA1601 strain showed higher proliferation in MDCK cells than in Vero cells.

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Fig. 3. Viral particles from the APQA1601 strain propagated in Vero cells. CAV-2 particles of 60–80 nm in diameter are visible in the nucleus and cytoplasm. Scale bars = 500 nm (A), 100 nm (B).

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Fig. 5. Comparison of the full nucleotide sequences among four CAVs (A). A phylogenetic analysis based on the nucleotide sequences of the fiber genes from 11 adenoviral strains (B). The APQA1601 strain had the highest homology with the Toronto A26/61 strain isolated in Canada. The phylogenetic tree was constructed based on alignments of nucleotide sequences obtained using the neighborjoining method.

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Fig. 4. Three primer sets targeting the F gene of the APQA1601 isolate were used for polymerase chain reaction (PCR). PCR products of the expected sizes confirmed the identification of the isolate as CAV-2.

Table 1. List of primers used for polymerase chain reaction analysis of canine adenovirus (CAV) type 2

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Table 2. Hemagglutination activity of CAV isolates using red blood cells from several species

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