Continuing evolution of canine parvovirus in China: Isolation of novel variants with an Ala5Gly mutation in the VP2 protein

Highlights

• This is the first report on isolation of novel CPV-2c from a dog in China.

• This study reveals that new CPV mutants are spreading in China.

• CPV vaccines failed to generate protection in vaccinated dogs In China.

Abstract

Canine parvovirus (CPV) type 2c is a new antigenic variant of CPV-2. Since the year 2000 it has spread to several countries, causing severe hemorrhagic enteritis in dogs. In 2014 and 2015, 58 fecal samples were collected from dogs in Beijing with suspected CPV infection. Regardless of the vaccination status of the dogs, 43 samples were found positive for CPV according to PCR results; i.e., 18, 7, and 18 respectively belonged to antigenic types new CPV-2a, new CPV-2b, and CPV-2c. A phylogenetic tree based on their VP2 gene sequences indicated that the Chinese CPV-2c strains form a separate cluster. In addition to synonymous mutations, the CPV-2c strains also contain a unique coding mutation in VP2 that leads to glycine at residue 5, instead of the highly conserved alanine at this position in all other CPV-2c strains sequenced to date. Using F81 cells, several novel isolates of CPV-2c, each with the Ala5Gly mutation, were obtained. One of these was used to infect experimentally beagle dogs, which subsequently developed the typical clinical symptoms of CPV infection. Hence, it appears that CPV-2c is still evolving in China, a finding that warrants continuous surveying and the eventual adaptation of current vaccines.

Introduction

Canine parvovirus (CPV) is an important pathogen in domestic dogs and several wild carnivore species (Kaelber et al., 2012). It belongs to the genus Protoparvovirus within the family Parvoviridae (the 2014 ICTV taxonomy, website: http://ictvonline.org/virusTaxonomy.asp). The parvoviral genome consists of a ~ 5000-nucleotide DNA molecule containing 2 large open reading frames (ORFs). One of these ORFs encodes 2 structural proteins, VP1 and VP2, by alternative splicing of the same mRNAs. The other encodes 2 non-structural proteins, NS1 and NS2 (Reed et al., 1988). The virus capsid is a 25-nm-diameter icosahedron that is assembled from 6 copies of VP1 and 54 copies of VP2 (Vihinen-Ranta et al., 2002).

CPV-2 was the original virus strain in dogs that spread worldwide in the 1970s (Appel et al., 1979) and soon gave rise to two distinct antigenic variants. One of them, CPV-2a, emerged in 1979 and contained 5 amino acid substitutions in VP2 (Met87Leu, Ile101Thr, Ala300Gly, Asp305Tyr, and Asn375Asp); the other, CPV-2b, appeared in 1984 and had a single additional substitution (Asn426Asp) in VP2. Both progressively replaced the original type (Stucker et al., 2012).

Subsequently, new variants appeared in various parts of the world, including new CPV-2a and new CPV-2b, each with a variety of amino acid substitutions (Ohshima et al., 2008). In the year 2000, yet another novel antigenic variant, characterized by an Asp426Glu substitution and called CPV-2c, was detected in Italy (Buonavoglia et al., 2001) and rapidly spread worldwide (Decaro and Buonavoglia, 2012, Decaro et al., 2006, Nakamura et al., 2004, Touihri et al., 2009). Relative to the original CPV-2, the antigenic variants of CPV-2a, CPV-2b, and CPV-2c are more highly pathogenic in dogs and have an extended host range that includes cats (Ikeda et al., 2000, Martella et al., 2005, Mochizuki et al., 1996). Epidemiological surveys indicate that CPV-2c is prevalent in different geographic regions and is often associated with severe disease in adult dogs, regardless of the dogs' vaccination status (Decaro and Buonavoglia, 2012).

In China, hemorrhagic enteritis in dogs caused by CPV-2 was first reported in 1983, and new CPV-2a and new CPV-2b strains have been in circulation (Wang et al., 2015). Sequencing first confirmed the existence of CPV-2c in China in 2010 (Zhang et al., 2010b), but it was not until 2014 that a CPV-2c virus was isolated and identified in China (the present study). In fact, in 2014 and 2015, CPV sequences were identified in 43 samples, and viruses were isolated from 8 of these. The nucleotide sequences of their VP2 capsid protein genes and their phylogenetic relationship to each other and to reference strains were determined.

It appears that CPV-2c, new CPV-2a, and new CPV-2b circulate simultaneously in China. Interestingly, the newly identified CPV-2c strains all harbor a novel mutation at residue 5, substituting glycine for the highly conserved alanine present in all other CPV-2c strains. This finding suggests that CPV continues to evolve in China and that current vaccination strategies might have to be re-evaluated.