Virus isolation and full-length genome sequencing of a representative canine distemper virus wild type strain of the South America 2 clade.

Canine Distemper Virus (CDV) is a highly contagious pathogen of dogs that causes severe respiratory, gastrointestinal and nervous signs. Although vaccines have been used to prevent infections, CDV has been reported worldwide, even in vaccinated animals. In the present study, a representative wild type CDV strain (Arg24) was isolated from a sick vaccinated dog and its genome was completely sequenced using Illumina technology. This strain produced a strong cytopathic effect in Vero SLAM (Signaling Lymphocyte Activation Molecule) cells with a higher titer of 1.1 × 105 Median Tissue Culture Infectious Dose (TCID50/mL) at 32 h post infection, in cell-associated virus. The Arg24 strain genome, showed values of 97.1, 90.3, 96.7, 90.6, 89.8 and 97.3 % of amino acid identity with respect to the Onderstepoort vaccine strain (Nucleoprotein, Phosphoprotein, Matrix, Fusion, Hemagglutinin and Large polymerase, respectively). Focusing on the Hemagglutinin gene, which is the target for genetic characterization, Arg24 showed four additional potential glycosylation sites, with respect to the Onderstepoort. The availability of Arg24 strain, which can be easily grown in Vero SLAM cells, is an important tool to perform immunological and antigenic comparative studies, between wild type and vaccine CDV strains.

Inter- and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America.

Canine parvovirus (CPV) is a fast-evolving single-stranded DNA virus that causes one of the most significant infectious diseases of dogs. Although the virus dispersed over long distances in the past, current populations are considered to be spatially confined and with only a few instances of migration between specific localities. It is unclear whether these dynamics occur in South America where global studies have not been performed. The aim of this study is to analyze the patterns of genetic variability in South American CPV populations and explore their evolutionary relationships with global strains. Genomic sequences of sixty-three strains from South America and Europe were generated and analyzed using a phylodynamic approach. All the obtained strains belong to the CPV-2a lineage and associate with global strains in four monophyletic groups or clades. European and South American strains from all the countries here analyzed are representative of a widely distributed clade (Eur-I) that emerged in Southern Europe during 1990-98 to later spread to South America in the early 2000s. The emergence and spread of the Eur-I clade were correlated with a significant rise in the CPV effective population size in Europe and South America. The Asia-I clade includes strains from Asia and Uruguay. This clade originated in Asia during the late 1980s and evolved locally before spreading to South America during 2009-10. The third clade (Eur-II) comprises strains from Italy, Brazil, and Ecuador. This clade appears in South America as a consequence of an early introduction from Italy to Ecuador in the middle 1980s and has experienced extensive local genetic differentiation. Some strains from Argentina, Uruguay, and Brazil constitute an exclusive South American clade (SA-I) that emerged in Argentina in the 1990s. These results indicate that the current epidemiological scenario is a consequence of inter- and intracontinental migrations of strains with different geographic and temporal origins that set the conditions for competition and local differentiation of CPV populations. The coexistence and interaction of highly divergent strains are the main responsible for the drastic epidemiological changes observed in South America in the last two decades. This highlights the threat of invasion from external sources and the importance of whole-genome resolution to robustly infer the origin and spread of new CPV variants. From a taxonomic standpoint, the findings herein show that the classification system that uses a single amino acid to identify variants (2a, 2b, and 2c) within the CPV-2a lineage does not reflect phylogenetic relationships and is not suitable to analyze CPV evolution. In this regard, the identification of clades or sublineages within circulating CPV strains is the first step towards a genetic and evolutionary classification of the virus.

RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks.

Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains.

The continuous emergence of new strains of canine parvovirus (CPV), poorly protected by current vaccination, is a concern among breeders, veterinarians, and dog owners around the world. Therefore, the understanding of the genetic variation in emerging CPV strains is crucial for the design of disease control strategies, including vaccines. In this paper, we obtained the sequences of the full-length gene encoding for the main capsid protein (VP2) of 11 canine parvovirus type 2 (CPV-2) Argentine representative field strains, selected from a total of 75 positive samples studied in our laboratory in the last 9 years. A comparative sequence analysis was performed on 9 CPV-2c, one CPV-2a, and one CPV-2b Argentine strains with respect to international strains reported in the GenBank database. In agreement with previous reports, a high degree of identity was found among CPV-2c Argentine strains (99.6-100% and 99.7-100% at nucleotide and amino acid levels, respectively). However, the appearance of a new substitution in the 440 position (T440A) in four CPV-2c Argentine strains obtained after the year 2009 gives support to the variability observed for this position located within the VP2, three-fold spike. This is the first report on the genetic characterization of the full-length VP2 gene of emerging CPV strains in South America and shows that all the Argentine CPV-2c isolates cluster together with European and North American CPV-2c strains.