Diagnóstico molecular e filogenia de morbilivírus canino

Abstract

Canine distemper virus (CDV) is an important pathogen of domestic dogs with worldwide distribution. CDV presents high genetic variability, with several viral lineages being described to date, mainly based on phylogenetic analysis of the hemagglutinin (H) gene. The variability of CDV can also influence its molecular diagnosis, being essential the use of primers that allow amplification of different viral variants. In this context, the first study of this thesis aimed to investigate the suitability of H and other viral genes for phylogenetic classification of CDV. For this, the classification of 116 complete genomes (CGs) of CDV (GenBank) was compared with that obtained by the analysis of nucleotide (nt) and amino acid (aa) sequences of H. Also, the geodesic distance between CG and H trees was calculated. These analyzes were then repeated with the other viral genes, and a recombination analysis was performed with the CG sequences. The classifications based on H sequences showed disagreements in comparison to those from CG. In addition, strong recombination signals were identified in H. The H gene phylogeny presented the fifth furthest geodesic distance in relation to the CG. On the other hand, analyses of the phosphoprotein (P) and C genes were able to reproduce the classification of the CG, and presented the first and third shortest geodesic distances from CG, respectively. No strong recombination signals were identified in P and C. These findings alert to the classification of CDV based solely on H analysis and suggest the use of P and C to correctly identify viral lineages. In the second study, a pair of high coverage primers for CDV detection were designed, targeting an internal region of C gene, and that could be used in RT-PCR and RT-qPCR. To this, 194 complete/nearly complete CDV genome sequences (GenBank) were screened for the presence of conserved regions for primer design capable of generating an amplicon suitable for RT-PCR and RTqPCR. Reactions based on these primers were optimized and evaluated for analytical sensitivity and specificity, and diagnostic performance; the latter was analyzed with 70 clinical samples suspected of distemper. The coverage of these primers and their diagnostic performance were compared to the PP-I primer pair, largely used in CDV studies. Forward and reverse primers developed annealed (in silico) in 100 and 99% of the analyzed sequences, respectively, while the PP-I forward and reverse primers failed to anneal in 85% and 90% of the sequences, respectively. The RT-PCR and RT-qPCR assays with the primers from this study, as well as the reaction with PP-I, showed a detection limit of 0.01 TCID50/mL. Reactions with the primers developed here also showed high specificity and did not amplify other important viruses included in the differential diagnosis of CDV. Out of 70 clinical samples used to assess diagnostic performance, 38 were identified as positive in RT-PCR and RT-qPCR reactions. Out of 28 positive samples subjected to reaction with PP-I, only 19 amplified. These results demonstrate the sensitivity and efficiency of the reactions with the primers described here. In conclusion, the studies described in this thesis contribute to the epidemiology and molecular diagnosis of CDV.