J Bacteriol Virol.  2019 Sep;49(3):124-132. 10.4167/jbv.2019.49.3.124.

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

• Yang DK
• Kim HH
• Lee EJ
• Yoo JY
• Yoon SS
• Park J
• Kim CH
• Kim HR

• KMID: 2468032
• DOI: http://doi.org/10.4167/jbv.2019.49.3.124

Abstract

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.

Keyword

Canine adenovirus type 1; Genetic characterization; Vaccine

MeSH Terms

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

Figure

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  Figure 1. Cytophatic effects in MDCK cells infected with CAV1V (magnification, 200×) (A). Normal MDCK cells derived from kidney of an adult female cocker spaniel (B). Indirect IF using a monoclonal antibody against CAV-1 (C, D). Intranuclear fluorescence was observed in MDCK cells infected with CAV1V (C) but not in normal MDCK cells (D). Scale bars, 100 μm (A-D).

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  Figure 2. Growth kinetics of CAV1V in MDCK cells according to time of harvest. At 4 days post inoculation, CAV1V strain was present at 107.5 TCID50/mL and 256 HA U/50 μL.

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  Figure 3. Electron microscopic images of virus particles of 70–90 nm diameter. Fiber proteins of CAV1V purified with cesicum chloride gradient are protruding from the virion surface (A) (magnification, 100,000×). CAV particles are shown in the nucleus of MDCK celle (magnification, 50,000×) (B). Scale bars, 100 nm (A and B).

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  Figure 4. Three primer sets targeting the fiber gene of CAV-1 were used for PCR. PCR products of the expected sizes confirmed that CAV1V was a CAV-1 strain. Lane M, 1 kb ladder; lanes 1-3, DNA amplified from CAV1V.

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  Figure 5. Phylogenetic tree based on the nucleotide sequences of the fiber genes of 16 adenoviral strains. CAV1V belonged to the CAV-1 clade and had the highest homology with the GLAXO strain, which was isolated in Canada. The phylogenetic tree was constructed based on alignments of nucleotide sequences obtained using the neighbor-joining method.

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  Figure 6. Alignment of the predicted amino acids at positions 200-450 of the CAV1V fiber protein with the corresponding region of the fiber proteins of three other CAV strains. Dots indicate identical amino acids. Compared with CAV-2, there is one deletion (∗) and one insertion (∗∗) in the fiber protein of CAV1V. The region (∗∗∗) conserved in adenoviruses of all animal species was well preserved. Six potential N-linked glycosylation sites (N-X-S/T) are indicated by dotted-line boxes.

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