DOI QR코드

DOI QR Code

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

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.

TSSOBU_2018_v58n4_177_f0001.png 이미지

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).

TSSOBU_2018_v58n4_177_f0002.png 이미지

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.

TSSOBU_2018_v58n4_177_f0003.png 이미지

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).

TSSOBU_2018_v58n4_177_f0004.png 이미지

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.

TSSOBU_2018_v58n4_177_f0005.png 이미지

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

TSSOBU_2018_v58n4_177_t0001.png 이미지

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

TSSOBU_2018_v58n4_177_t0002.png 이미지

Acknowledgement

Supported by : Animal and Plant Quarantine Agency

References

  1. Appel M, Carmichael LE, Robson DS. Canine adenovirus type 2-induced immunity to two canine adenoviruses in pubs with maternal antibody. Am J Vet Res 1975, 36, 1199-1202.
  2. Balboni A, Dondi F, Prosperi S, Battilani M. Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2. J Virol Methods 2015, 222, 34-40. https://doi.org/10.1016/j.jviromet.2015.05.009
  3. Benetka V, Weissenbock H, Kudielka I, Pallan C, Rothmuller G, Mostl K. Canine adenovirus type 2 infection in four puppies with neurological signs. Vet Rec 2006, 158, 91-94. https://doi.org/10.1136/vr.158.3.91
  4. Choi JW, Lee HK, Kim SH, Kim YH, Lee KK, Lee MH, Oem JK. Canine adenovirus type 1 in a fennec fox (Vulpes zerda). J Zoo Wildl Med 2014, 45, 947-950. https://doi.org/10.1638/2013-0286.1
  5. Goldstein T, Colegrove KM, Hanson M, Gulland FMD. Isolation of a novel adenovirus from California sea lions Zalophus californianus. Dis Aquat Organ 2011, 94, 243-248. https://doi.org/10.3354/dao02321
  6. Gur S, Acar A. A retrospective investigation of canine adenovirus (CAV) infection in adult dogs in Turkey. J S Afr Vet Assoc 2009, 80, 84-86.
  7. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 2016, 33, 1870-1874. https://doi.org/10.1093/molbev/msw054
  8. Lavan R, Knesl O. Prevalence of canine infectious respiratory pathogens in asymptomatic dogs presented at US animal shelters. J Small Anim Pract 2015, 56, 572-576. https://doi.org/10.1111/jsap.12389
  9. Linne T. Differences in the E3 regions of the canine adenovirus type 1 and type 2. Virus Res 1992, 23, 119-133. https://doi.org/10.1016/0168-1702(92)90072-H
  10. Loustalot F, Kremer EJ, Salinas S. The intracellular domain of the coxsackievirus and adenovirus receptor differentially influences adenovirus entry. J Virol 2015, 89, 9417-9426. https://doi.org/10.1128/JVI.01488-15
  11. Millan J, Lopez-Bao JV, Garcia EJ, Oleaga A, Llaneza L, Palacios V, de la Torre A, Rodriguez A, Dubovi EJ, Esperon F. Patterns of exposure of Iberian wolves (Canis lupus) to canine viruses in human-dominated landscapes. Ecohealth 2016, 13, 123-134. https://doi.org/10.1007/s10393-015-1074-8
  12. Mochizuki M, Hashimoto M, Ishida T. Recent epidemiological status of canine viral enteric infections and Giardia infection in Japan. J Vet Med Sci 2001, 63, 573-575. https://doi.org/10.1292/jvms.63.573
  13. Park NY, Lee MC, Kurkure NV, Cho HS. Canine adenovirus type 1 infection of a Eurasian river otter (Lutra lutra). Vet Pathol 2007, 44, 536-539. https://doi.org/10.1354/vp.44-4-536
  14. Reed LJ, Muench H. Simple method of estimating fifty per cent endpoints. Am J Hyg 1938, 27, 493-497.
  15. Sanjuan R, Donimgo-Calap P. Mechanisms of viral mutation. Cell Mol Life Sci 2016, 73, 4433-4448. https://doi.org/10.1007/s00018-016-2299-6
  16. Szelechowski M, Fournier A, Richardson J, Eliot M, Klonjkowski B. Functional organization of the major late transcriptional unit of canine adenovirus type 2. J Gen Virol 2009, 90, 1215-1223. https://doi.org/10.1099/vir.0.007773-0
  17. Takamura K, Ajiki M, Hiramatsu K, Takemitsu S, Nakai M, Sasaki N. Isolation and properties of adenovirus from canine respiratory tract. Nihon Juigaku Zasshi 1982, 44, 355-357. https://doi.org/10.1292/jvms1939.44.355
  18. Tham KM, Horner GW, Hunter R. Isolation and identification of canine adenovirus type -2 from the upper respiratory tract of a dog. N Z Vet J 1998, 46, 102-105. https://doi.org/10.1080/00480169.1998.36068
  19. Walker D, Abbondati E, Cox AL, Mitchell GBB, Pizzi R, Sharp CP, Philbey AW. Infectious canine hepatitis in red foxes (Vulpes vulpes) in wildlife rescue centres in the UK. Vet Rec 2016, 178, 421. https://doi.org/10.1136/vr.103559
  20. Yoon SS, Byun JW, Park YI, Kim MJ, Bae YC, Song JY. Comparison of the diagnostic methods on the canine adenovirus type 2 infection. Basic Appl Pathol 2010, 3, 52-56. https://doi.org/10.1111/j.1755-9294.2010.01073.x