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Molecular epidemiological and phylogenetic analyses of canine parvovirus in domestic dogs and cats in Beijing, 2010–2013

Fifty-five samples (15.62%) collected from dogs and cats were identified as canine parvovirus (CPV) infection in Beijing during 2010–2013. The nucleotide identities and aa similarities were 98.2–100% and 97.7–100%, respectively, when compared with the reference isolates. Also, several synonymous and non-synonymous mutations were also recorded for the first time. New CPV-2a was dominant, accounting for 90.90% of the samples. Two of the 16 samples collected from cats were identified as new CPV-2a (12.5%), showing nucleotide identities of 100% with those from dogs. Twelve samples (15.78%) collected from completely immunized dogs were found to be new CPV-2a, which means CPV-2 vaccines may not provide sufficient protection for the epidemic strains.

Canine parvovirus type 2 (CPV-2) is the etiological agent of an epizootic severe gastroenteritis of dogs [2,10,38]. It has genomic substitution rates similar to those of RNA viruses, with values of about 10 −4 substitutions per site per year, and host-immunity pressure may contribute to the progressive emergence of CPV-2 antigenic variants [34]. During 1979During -1981, an antigenic variant, CPV-2a, was found. In 1984, a second variant, CPV-2b, was identified [23,27,28]. These antigenic types differ from the original CPV-2 in the VP2 gene, with five distinct amino acid differences that are mostly in the VP2 domain interacting with the host-cell transferrin receptor (TfR) [4,8,29]. During the last few decades, CPV-2a/2b with the Ser297Ala mutation was designated as new CPV-2a/2b [11,23,26]. Currently, new CPV-2a and new CPV-2b appear to have replaced the prototype CPV-2a and CPV-2b and to have become the predominant types, and they appear to be co-circulating in many countries [13,16,24,26,40,41]. Another antigenic variant having an amino acid substitution (Asp426Glu), named as CPV-2c, was first reported in Italy in 2000 [6], and it is the most predominant variant in Italy, Germany, Uruguay and Argentina currently [10-12, 25, 39]. CPV-2 is considered the main pathogen responsible for acute gastroenteritis in dogs. However, CPV variants can also infect feline hosts [5,35]. In Germany, CPV was previously detected in only approximately 10% of feline samples [37], but in Vietnam and Taiwan, reports estimated that up to approximately 80% of diseased cats were infected with CPV [19]. Some reports previously found that new-type CPV-2a and CPV-2b have been the predominant types in some provinces of China since the 1990s [42], while the epidemic situation of CPV variants in the domestic cat in mainland of China is still unknown.
In the present study, to clarify the evolution of CPV-2, which has recently been considered to be epidemic in China, and compare the epidemic isolates with the vaccine strain, the VP2 gene sequences of CPV-2 detected in Beijing from 2010 to 2013 were analyzed and compared with strains from China, Korea and other areas throughout the world. A total of 352 samples (blood, feces and secretions) from domestic dogs and domestic cats with diarrhea or bloody diarrhea were collected during 2010-2013, and subjected PCR. The primers used for PCR are shown in Table 1. A 1752 bp fragment covering the full-length sequence of the VP2 gene was successfully amplified from 55 samples (15.63%); among them, bJ-E13-2012 and bJ-E53-2012 collected from cats were identified as new CPV-2a (12.5%) and showed nucleotide identities of 100% with CPV strains collected from domestic dogs, but their infectiousness and pathogenicity in cats and dogs still need a further study. The sequences were submitted to Genbank. The Genbank accession numbers are KF803589 to KF803643. Detailed information on the origin and accession numbers of the CPV-positive samples is shown in Table 2. The resulting sequences were aligned with CPV reference isolates retrieved from the Genbank database. Sequences comparisons showed no deletion or shift in any sequences of the detected samples, and the nucleotide identities and deduced aa sequences identities were 98.2-100% and 97.7-100%, respectively, when compared with the CPV reference isolates.
Mutations in the VP2 protein are associated with the CPV genotypes, so the nucleotide sequences were translated into aa sequences. All samples, except one original CPV2a (BJ-A45-2010), were identified as "new CPV-2a/2b" reported worldwide and were identified as "new CPV-2a/2b" [9,20,22]. Previous research has shown residue 297 is under strong positive selection [30,35]. Also, the 297Ala variant can not be distinguished serologically and does not change the viral antigenic type, but it potentially has a marked influence on Phe267Tyr change also has been described in Asiatic CPV-2b from Vietnam [25] and in CPV-2a from Thailand [32] and China [31,35]. Residue 267 is not exposed on the capsid surface [1], so substitutions in this position may not affect the antigenicity of the virus. Fifty four samples presented Ala300Gly mutation (98.18%). Tyr324Ile mutation was first detected in China and Korea in 2004 [20,35], and reported among the CPV 2a/2b isolates in China [17,35]. Previous studies have shown that residue 324 is subject to strong positive selection in all parvoviruses of carnivores [17].
Residue 324 is likely to have had an effect on the parvovirus host range [35]. It is adjacent to residue 323, and together with residue 93, affects TfR binding [18]. In this study, fifty one samples presented Tyr324Ile mutation (92.73%) indicated that strain with mutation Tyr324Ile is the major epidemic strain. Thr440Ala has been described in CPV-2a and CPV-2b strains from China, Korea, India, Italy, Brazil and Uruguayan [3,22,30,31] and in CPV-2c strains from the United States [21] and Argentina [7]. The 440 residue is important, because it is located at the top of the three-fold spike, the main antigenic site of the virus [36]. This residue is undergoing positive selection and has evolved in different populations independently, which explains its world-wide presence in unrelated CPV-2 populations [12]. In this study, seventeen samples presented Thr440Ala mutation, which accounted for 30.91%. And, all samples presented Tyr at 305 position and Val at 555 position. Also, Ser192Phe mutation in BJ-A2-2010 was just previously reported in SC02-2011. Asp375Asn mutation in BJ-A45-2010 was just previously reported in CPV-b, GZ0201 and JL0201. Previous report has indicated that residue 375 is associated with the ability of CPV to hemagglutinate or alter pH dependence of hemagglutination [15,36], so BJ-A45-2010 may have different coagulation feature with others. While the mutations, Pro202Thr, Ile219Vla, Ala347Thr, Gln386Lys, Pro187Gln, Ser188 Gln and Val308Ile, present in this study were interesting, because it has not been detected previously in any other strains. Pro202Thr mutation was firstly reported in BJ-A2-2010, and Ile219Vla, Ala347Thr and Gln386Lys mutations were firstly reported in BJ-A45-2010. Pro187Gln and Ser188 Gln mutations were firstly reported in BJ-A69-2010. Also, Val308Ile mutation was firstly reported in BJ-D6-2012 and bJ-D14-2012. While, Asp427His, The445Asn and Pro-512His which appeared in Nanjing strains of 2009-2012 [41] and Thr442Ala and Gln370Arg reported in the giant panda strain B11 [15] were not present in this study. The detail mutation sites, types and rates in VP2 genes were shown in Table 3. The affects of these mutations to virus itself, which appeared in this study, need our further research. Twelve strains were detected in dogs completely immunized with CPV-2 vaccine (Table 2). Although the effectiveness of CPV-2 vaccine against CPV-2a type has not been evaluated in China, our results showed that 12 out of 55 samples were detected as "new CPV-2a/2b" in dogs completely immunized with CPV-2 vaccine, which suggested that complete immunity may not be provided to dogs even if CPV-2 vaccines are used as previously reported [14,15,33,35].
To examine the phylogenetic relationships of the 55 samples with the reference isolates, a phylogenetic tree based on the nucleotide sequence from 1 to 1752 nt of the VP2 gene was built. As shown in Fig. 1  shows, most of the viruses isolated in China formed a large cluster and certain mutations detected in Chinese CPVs probably arose during the process of local adaptation, as indicated by previous surveys [15,30]. Gln, Ile219Vla, Ala347Thr, Gln386Lys and Val308Ile mutations were firstly reported in present study. Also, new CPV-2a was detected in cats with diarrhea, and CPV-2 vaccines may not provide complete immunity to dogs in china. Due to the continuing evolution of CPV, monitoring the prominent type of CPV, and detecting genetic mutation and antigenic changes, will be necessary to find new vaccines and control CPV infection in China.
AcKNOWLEDGMENT. We are grateful to the animal health workers who were involved in this study and collected animal blood samples. This work was supported by the Special Fund for Agro-Scientific Research in the Public Interest (no. 201303042), the National High Technology Research and Development Program of china (no. 2012AA101302), and a project supported by the National Science Foundation for Young Scientists of china (no. 31302130).