Abstract To trace the evolution process of CPV-2, all of the VP2 gene sequences of CPV-2 and FPV (from 1978 to 2015) from GenBank were analyzed in this study. Then, several new ideas regarding CPV-2 evolution were presented. First, the VP2 amino acid 555 and 375 positions of CPV-2 were first ruled out as a universal mutation site in CPV-2a and amino acid 101 position of FPV feature I or T instead of only I in existing rule. Second, the recently confusing nomenclature of CPV-2 variants was substituted with a optional nomenclature that would serve future CPV-2 research. Third, After check the global distribution of variants, CPV-2a is the predominant variant in Asia and CPV-2c is the predominant variant in Europe and Latin America. Fourth, a series of CPV-2-like strains were identified and deduced to evolve from modified live vaccine strains. Finally, three single VP2 mutation (F267Y, Y324I, and T440A) strains were caught concern. Furthermore, these three new VP2 mutation strains may be responsible for vaccine failure, and the strains with VP2 440A may become the novel CPV sub-variant. In conclusion, a summary of all VP2 sequences provides a new perspective regarding CPV-2 evolution and the correlative biological studies needs to be further performed.

Citation: Zhou P, Zeng W, Zhang X, Li S (2017) The genetic evolution of canine parvovirus – A new perspective. PLoS ONE 12(3): e0175035. https://doi.org/10.1371/journal.pone.0175035 Editor: Yongchang Cao, Sun Yat-Sen University, CHINA Received: December 28, 2016; Accepted: March 20, 2017; Published: March 31, 2017 Copyright: © 2017 Zhou 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: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Promote Scientific and Technological Development Program (2013B040200032), the Science and Technology Planning Project of Guangdong Province(2013B020202001), the Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases (2013A061401013). Pei Zhou received financial support from the China Scholarship Council (CSC). 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.

Introduction Canine parvovirus (CPV) belongs to the genus Protoparvovirus and the family Parvoviridae and causes a highly contagious and fatal disease in dogs [1]. CPV is a non-enveloped DNA virus with an approximately 5000-nucleotide, single-stranded DNA genome containing two open reading frames (ORFs). The first ORF encodes two non-structural proteins, NS1 and NS2. The second ORF encodes two structural proteins, VP1 and VP2 [2]. VP1 and VP2 each encode parts of the viral capsid, which is assembled from 54 copies of VP2 and 6 copies of VP1 [3]. VP2, the major capsid protein, is also the major antigenic protein and determines viral tissue tropism and host range [4, 5]. NS1, a pleiotropic nuclear phosphoprotein, plays an essential role in viral replication and is responsible for inducing cell apoptosis [6, 7]. Although CPV is a DNA virus, its genomic substitution rate is approximately 10−4 per site per year, which is similar to RNA viruses [8]. In 1978, CPV-2 was first identified from outbreaks in canines in the United States and Australia [9, 10]; it was then reported in many countries during 1978 and 1979 [11]. CPV-2 is closely related to feline parvovirus (FPV); therefore, it is assumed to be a host variant of FPV [12, 13]. CPV-2 was named to distinguish and differentiate it from an unrelated canine minute virus (CPV-1) [14]. In the 1980s, the original CPV-2 type was completely replaced by two new antigenic variants in canines, which were termed CPV types 2a (CPV-2a) and 2b (CPV-2b) [15, 16]. In 2000, CPV-2c with Asp426Glu substitution was reported in Italy[17]. With its antigenicity constantly drifting, an increasing number of further mutations of VP2 have been described, and various viral mutants have been named [17–20]. However, the nomenclature of these variants is inconsistent and confusing. To distinguish the nomenclature and to further understand CPV-2 evolution, the sequence characteristics of the VP2 gene of CPV-2 and FPV were updated in this study.

Materials and methods Sequence data By 17th June 2016, 2170 VP2 sequences of CPV-2 and 245 sequences of FPVwere downloaded from the NCBI (from 1978 to 2015). Among the 2170 sequences of CPV-2, 1679 have collection date and 491 not have collection date. Among the 245 sequences of FPV, 170 have collection date and 75 not have collection date. 1679 sequences of CPV-2 were ordered according to the collection date from 1978 to 2015 by using Microsoft Excel. Sequence analysis The sequences that have collection date were used for mutation analysis. The nucleotide alignments were performed using BioEdit and the nucleotide substitution were manually edited by checking 50 sequences every time. Then, the amino acid (aa) mutation was summarized according to the nucleotide substitution. Graph The aa mutational figs were created using the Prism 5.0 software (GraphPad Software). The VP2 structure was downloaded from SWISS-MODEL (https://swissmodel.expasy.org/), and the aa residues were depicted in PyMOL (Version 1.5.0.4).

Discussion The VP2 V555I mutation was formerly defined as one CPV-2 mutation site that was important for CPV-2a evolution. The VP2 aa 375 of CPV-2 feature N in most original CPV-2 isolates and feature D in FPV and others CPV-2 variants. Here, after analyzing all VP2 sequence of CPV-2 and FPV(from 1978 to 2015), the VP2 amino acid 555 and 375 positions of CPV-2 were first ruled out as a universal mutation site in CPV-2a and amino acid 101 position of FPV feature I or T instead of only I in existing rule. the VP2 V555I was first ruled out from the universal CPV-2a mutation sites. The previous definition may be due to the limited number of sequences available from that period. The nomenclature of CPV-2 variants is inconsistent and confusing. Here, the original CPV-2 and CPV-2a\2b\2c were defined to be the four unique variants that is a optional nomenclature would serve CPV-2 research. CPV-2a is the predominant variant in Asia and CPV-2c is the predominant variant in Europe and Latin America, which remind different regions may be need pay more attention to the predominant variant. There are some sporadic identifications of the original CPV-2 and CPV-2-like strains. The original CPV-2 were regarded as vaccine strains and CPV-2-like strains have been identified from sick and vaccinated dogs [31, 34]. Therefore, CPV-2-like strains may be the result of the reversion of vaccine strains to virulence. With antigenic drift, three new mutations at VP2 (F267Y, Y324I, and T440A) have occurred. Recently, inactivated vaccines and modified live virus vaccines have become widely used [24]. However, there are some concerns regarding the complete efficacy of existing vaccines against the antigenic variants [40–43]. Because viruses always evolve to escape the immune system via antigenic drift, the new mutations (F267Y, Y324I, and T440A) may be caused by vaccine immune pressure. Meanwhile, these same new mutations (F267Y, Y324I, and T440A) may be responsible for vaccine failure. The aa 440 residue located in the top protrusion of loop 4 of the VP2 protein. The aa 426 residue that is located in one top protrusion of loop 4 has been defined as the major mutation site for CPV-2 evolution. Mutation of the aa 440 residue that is located in the other top protrusion of loop 4 may cause viral antigenic drift. Of note, the T440A mutation has been reported in previous studies [44, 45]. Thus, strains with VP2 440A may become a novel CPV sub-variant. Certainly, the functional or other testing of the mutants needs to be further performed. Finally, this sequence summary reveals a new perspective regarding CPV-2 evolution and represents a significant way to discover new characteristics and to overcome the problems arising from periods with limited available sequencing data.

Supporting information S1 Table. The number of different amino acid of 375, 555 and 101375 and 555 sites in different CPV-2 variants and FPV. https://doi.org/10.1371/journal.pone.0175035.s001 (DOCX)

Author Contributions Formal analysis: PZ. Funding acquisition: SL. Investigation: WZ XZ. Methodology: PZ. Project administration: SL. Software: PZ. Supervision: SL. Validation: PZ. Visualization: PZ. Writing – original draft: PZ. Writing – review & editing: PZ SL.