Biological and Genetic Characterization of Canine Distemper Virus Vaccine Candidate Named as CD1901-100

Abstract

Canine distemper virus (CDV) infections cause high morbidity and mortality in dogs. Changes in the molecular biological characteristics of the Korean CDV strain over multiple cell passages have not been reported. We investigated the biological and genetic characteristics of CD1901-100 for use as an inactivated vaccine strain. Vero cells expressing the dog nectin-4 gene (Vero/dNectin-4 cells) were used to adapt CD1901, which was passaged 100 times in four types of cells. We assessed the cytopathic effects and used immunofluorescence assays to identify biological features of CD1901 and CD1901-100. Seven types of cells were used to explore the tropisms of the two CDV strains. The genetic analyses were based on whole-genome sequencing data. Vero cells expressing dog signaling lymphocyte activation molecule were infected with the two CDV strains and showed different cytopathic effects and fluorescence properties. CD1901-100 attained the highest viral titer of 10 6.5 TCID 50 /mL at 4 days post-inoculation; the overall highest virus titer of 10 7.0 TCID 50 /mL was that after growth in Vero/dNectin-4 cells. CD1901-100 exhibited 25 nucleotide mutations and 15 amino acid substitutions in six structural proteins compared to the CD1901 sequences. Of the six proteins, the F protein had the highest number of amino acid replacements (5/663, 0.75%). We constructed a Vero/dNectin-4 cell line and passaged CD1901 100 times in four types of cells. CD1901-100 propagated well in Vero/dNectin-4 cells. This will aid the development of an inactivated CDV vaccine. passaged 40 times in Vero/dSLAM cells without any treatment and then again (passages 41 to 60) after 1 min of ultraviolet (UV) light exposure about 60 centimeters away in a biosafety cabinet. Passages 61 to 76 employed DF-1 cells, and passages 77 to 84 used normal Vero cells. Passages 85-95 employed Vero/dSLAM cells and were performed in the presence of 4 mM 5' bromouracil. Passages 96-100 used Vero/dNectin-4 cells. Vero/dNectin-4, Vero, DF-1, A72 (ATCC, CRL-1542), and MDCK (ATCC, CRL34) cells and grown in 25-cm flasks. After incubation for 5 days, each flask was frozen and thawed three times. The clarified supernatants were subjected to viral titration to know the proliferative ability of CD1901-100 as described above.