Phylogenetic and Genome-Wide Deep-Sequencing Analyses of Canine Parvovirus Reveal Co-Infection with Field Variants and Emergence of a Recent Recombinant Strain Ruben Pe ´ rez 1 *, Lucı´a Calleros 1 , Ana Marandino 1 , Nicola ´ s Sarute 1 , Gregorio Iraola 1 , Sofia Grecco 1 , Herve ´ Blanc 2 , Marco Vignuzzi 2 , Ofer Isakov 3 , Noam Shomron 3 , Lucı´a Carrau 1 , Martı´n Herna ´ ndez 1 , Lourdes Francia 1 , Katia Sosa 1 , Gonzalo Toma ´s 1 , Yanina Panzera 1 1 Seccio ´ n Gene ´ tica Evolutiva, Instituto de Biologı ´a, Facultad de Ciencias, Universidad de la Repu ´ blica, Montevideo, Uruguay, 2 Institut Pasteur, Viral Populations and Pathogenesis Unit, Centre National de la Recherche Scientifique, Paris, France, 3 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Abstract Canine parvovirus (CPV), a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c) with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population) and a major recombinant strain (86.7%). The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity. Citation: Pe ´rez R, Calleros L, Marandino A, Sarute N, Iraola G, et al. (2014) Phylogenetic and Genome-Wide Deep-Sequencing Analyses of Canine Parvovirus Reveal Co-Infection with Field Variants and Emergence of a Recent Recombinant Strain. PLoS ONE 9(11): e111779. doi:10.1371/journal.pone.0111779 Editor: Eduard Ayuso, University of Nantes, France Received May 2, 2014; Accepted September 30, 2014; Published November 3, 2014 Copyright: ß 2014 Pe ´rez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All sequence files are available from the GenBank database (accession numbers KM457102-KM457143). Funding: Grant number: 2206, Comisio ´ n Sectorial de Investigacio ´ n Cientı ´fica (CSIC) (http://www.csic.edu.uy/) to KS. Grant number ANR-10-LABX-62-IBEID, French Investissement d’Avenir program, Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases (http://www.agence-nationale-recherche.fr/ investissementsdavenir/) to MV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: rperez@fcien.edu.uy Introduction Members of the Parvoviridae family are small (18–22 nm), non- enveloped icosahedral viruses that cause a wide range of diseases in animals [1–3] and humans [1,4]. Canine parvovirus (CPV) is an extremely relevant member of the Parvoviridae family because it is the causative agent of one of the most dangerous infectious disease in young dogs and is responsible for large numbers of animal deaths worldwide [5]. CPV has a linear single-stranded DNA (ssDNA) genome (5.2 kb) with two major open reading frames (ORFs) [6]. The left ORF encodes nonstructural proteins NS1 and NS2, which are essential for replication and DNA packaging [7]. The N-terminal regions of NS1 and NS2 are identical in sequence, whereas the C- terminal region of NS2 is derived from differential splicing of the mRNA and is translated from a different reading frame than NS1. The right ORF encodes the viral capsid proteins 1 and 2 (VP1 and VP2), which are the main antigens that induce protective antibodies [8–10]. VP1 and VP2 are splice variants and are identical in sequence, except for a 143-amino-acid (aa) N-terminal region that is unique to VP1. At both ends of the CPV genome, there are non-translated regions with hairpin structures that are necessary for priming replication [6]. CPV is a host-specific variant of the feline panleukopenia virus (FPV) that emerged in the 1970s because of an interspecies jump from other carnivores to dogs [11]. The newly emerged CPV, named CPV-2, rapidly spread worldwide in 1978 causing an acute enteritis disease in dogs [5]. The successful cross-species viral transfer and adaptation to the new canine host involved a small number of point mutations in the viral capsid proteins [12]. In 1979, CPV-2 was replaced globally by CPV-2a, a genetic variant that differs at five residues in VP2 and regained the ability to infect cats and other carnivores [12,13]. CPV-2a became the new dominant lineage and underwent further evolution, retaining several point mutations. Some of these mutations change the antigenic properties of the capsid and have reached high frequencies in viral populations. In addition to the original CPV-2a antigenic type, there are two known antigenic variants, PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e111779