The increasing prevalence of CPV-2c in domestic dogs in China

Introduction

Canine parvovirus type 2 (CPV-2), which belongs to the genus Protoparvovirus, family Parvoviridae, is an important pathogen that leads to high morbidity and mortality in several wild carnivore species and even non-carnivores (Appel, Scott & Carmichael, 1979; Burtonboy et al., 1979; Nelson et al., 1979; Pletcher et al., 1979; Wang et al., 2020). CPV-2 is a non-enveloped virus, with a single-stranded DNA genome encoding 2 large open reading frames (ORFs). VP2, the main capsid protein of the virus, is involved in determining viral tissue orientation and host range (Hueffer et al., 2003; Nelson et al., 2007). NS1, a versatile phosphorylated protein, facilitates DNA packaging and viral replication (Gupta et al., 2016; Saxena et al., 2013).

CPV-2 was considered to be a change in host range due to mutations in the feline panleukopenia virus (FPV) (Appel, Scott & Carmichael, 1979; Black et al., 1979; Johnson & Spradbrow, 1979; Osterhaus, Steenis & Kreek, 1980; Parrish, 1990), and it has become global since its discovery in the 1970s (Appel et al., 1979; Appel & Carmichael, 1980; Carmichael & Binn, 1981). Subsequently, CPV-2a and CPV-2b, two new variants, have emerged and replaced the initial CPV-2 (Parrish et al., 1991; Perez et al., 2012). CPV-2c, a newer variant, was later found in Italy (Buonavoglia et al., 2001) and spread rapidly worldwide (Decaro & Buonavoglia, 2012; Decaro et al., 2005; Decaro et al., 2006; Joao Vieira et al., 2008; Kapil et al., 2007; Nakamura et al., 2004; Nandi et al., 2010; Perez et al., 2007; Puentes et al., 2012; Streck et al., 2009; Sutton et al., 2013; Touihri et al., 2009). Notably, the original CPV-2 cannot infect cats (Truyen et al., 1996); however, the strains, CPV-2a, CPV-2b and CPV-2c, not only susceptible to dogs but also circulate in cats. Consequently, these variants have become an essential pathogen for feline panleukopenia disease (Ikeda et al., 2000; Truyen et al., 1996). Since the emergence of the different strains, research on their associated detailed amino acid mutations has been inconsistent. According to the latest report on the VP2 gene (Zhou et al., 2017), six aa mutations (K80R, K93N, V103A, D323N, N564S, A568G) are present in CPV compared with FPV, with three or four aa mutations (M87 L, A300G/D, D305Y and I101T not sure) in CPV-2a compared with CPV-2. However, compared with CPV-2a, only one mutation residue (N426D) was observed in CPV-2b, and D426E was found in CPV-2c compared with CPV-2b (Miranda & Thompson, 2016). To trace the prevalence of CPV-2 in domestic dogs in Guangdong Province, China, an aetiological survey was carried out from 2016 to 2019.

CPV-2a, CPV-2b and CPV-2c co-circulate in most regions on a global scale. As previously noted, CPV-2a exists in most areas in Asia (Zhou et al., 2017); it is worth noting that CPV-2c is also present in some Asian regions (Charoenkul et al., 2019; Vannamahaxay et al., 2017). Although CPV-2c is identified as the dominant strain in Europe and Latin America (Hong et al., 2007; Tucciarone et al., 2018; Zhou et al., 2017), it has gradually become the dominant variant in Laos, Vietnam, Thailand and other regions (Charoenkul et al., 2019; Lin et al., 2017; Vannamahaxay et al., 2017). CPV-2c was reported in China in 2010 (Zhang et al., 2010a; Zhang et al., 2010b), but two recent studies reported CPV-2a as the major strain in dogs in China (Zhao et al., 2017; Zhuang et al., 2019). Regardless, based on the evolution of parvovirus in other Asian regions, it is clear that this is only a temporary phenomenon and that CPV-2c will be isolated in an increasing number of locations (Li et al., 2019; Lin et al., 2017). Therefore, the prevalence of CPV-2 in China needs to be re-studied.

Since CPV-2 was reported, it has been circulating in the dog population, despite the development of related vaccines and the certain efficacy of the vaccine in preventing CPV disease. Indeed, CPV-2 mutates constantly, and new variants are continuously produced. Moreover, immune failure occurs on occasion, and the prevention and treatment of parvovirus disease is very serious (De et al., 2018; Wu et al., 2015). In particular, the evolution of VP2 deserves attention, as genetic surveillance of circulating viruses is essential for the prevention and control of CPV infection and the development of more effective vaccines.

Materials & Methods

Results

Phylogenetic analysis

Phylogenetic analysis is a prominent method for studying the evolution of viruses. Phylogenetic analysis has been performed in many previous studies to elucidate the classification of CPV-2 (Geng et al., 2015; Wang et al., 2016a; Wang et al., 2016b). However, phylogenetic branches between different studies have been inconsistent, resulting in unclear evolution. The reason for the inconsistency among the studies is that the bootstrap values of the phylogenetic branches in those studies were low. For the current analysis, all available sequences of CPV-2, FPV, MEV, RPV, and BFPV in GenBank were obtained, and a phylogenetic tree was constructed with the neighbour-joining method with 1,000 bootstraps. Although all sequences of CPV-2 clustered closely together, the bootstrap values of the branches in this CPV-2 cluster were low. In fact, only the bootstrap value between CPV-2 and other viruses was 100. Therefore, we selected virus sequences from the neighbour-joining trees and used the maximum likelihood method to reconstruct a tree in which the CPV-2 cluster was separated from other viruses with a bootstrap value of 100 (Fig. 1). In the CPV-2 branch, the same variants did not cluster with each other, and the bootstrap values between the subbranches of CPV-2 were very low (Fig. 1). RPV clustered with FPV in one branch and BFPV with MEV in another branch, but the bootstrap values of these two branches were also low (Fig. 1). Hence, different animal parvoviruses derived from FPV were separated into only a CPV-2 branch and FPV branch, which includes FPV, RPV, MEV and BFPV. Additionally, all CPV-2 isolates belong to the CPV-2 branch (Fig. 1).

Increasing prevalence of CPV-2c in China

All 1,076 Chinese CPV-2 isolates were classified and analysed according to collection date and variant type (Fig. 2). The percentages of different types of CPV-2 varied consistently over time. The original CPV-2 strain was sporadically present in China and generally regarded as a vaccine isolate (Zhang et al., 2010a; Zhang et al., 2010b). CPV-2a was the dominant strain until 2015; the prevalence of CPV-2a was highest in 2014, after which it showed a declining trend. CPV-2b first appeared in 2004 and had a low but stable prevalence until 2015, with a similar declining trend after 2014. It is worth noting that CPV-2c was not recorded until 2009; it has been increasing since. CPV-2c was on the rise before 2017 (Qi et al., 2020). In 2017–2018, CPV-2c predominated over CPV-2a and became the primary variant in China. CPV-2a and CPV-2b were replaced by CPV-2c in just a few short years.

Discussion

As a virus with a DNA genome, CPV-2 is susceptible to mutation (Shackelton et al., 2005). Recently, the new variants CPV-2a, CPV-2b, and CPV-2c have gradually replaced the original CPV-2 (Ogbu et al., 2019), which mainly developed according to an aa mutation at site 426 in VP2 (N: CPV-2a, D: CPV-2b, E: CPV-2c). During evolution, CPV-2a/2b/2c, which not only infect dogs but also cats, completely replaced the original CPV-2, which infected only dogs (Ikeda et al., 2000; Parrish, 1991; Parrish et al., 1991; Perez et al., 2012; Truyen et al., 1996). Additionally, it has been reported that the appearance of novel variants of CPV-2 that affect both vaccinated and unvaccinated dogs is occurring (Calderon et al., 2011). Therefore, it is essential to investigate the evolution of CPV-2 in dogs. From 2016 to 2019, an aetiological survey was carried out in Guangdong, China, including Guangzhou, Shenzhen, and Dongguan cities.

Using PCR detection and virus isolation, 55.7% (34/61) of samples were identified as positive, which is a high rate, indicating that CPV-2 is a major canine enteric pathogen among dogs. CPV-2c is prevalent and predominates over CPV-2a in this regional aetiological survey in Guangdong. According to recent research in Vietnam, CPV-2c is more prevalent than other variants and shares an evolutionary origin with the Asian strain (Hoang et al., 2019). Hence, the prevention and treatment of CPV-2 disease should be given more attention in dog veterinary care.

In addition to multiple variants circulating in dogs, coinfection with variants and coinfection with other canine enteric pathogens in several cases were identified in this study. Moreover, coinfection with other pathogens can aggravate the disease (Zheng et al., 2018). These results explain the complex infection situation in CPV-2 disease, indicating that it is urgent to consider these factors in the prevention and treatment of CPV-2 disease.

Alanine at residue 297 discovered in this study is consistent with a 2017 report verifying that 297A has been predominant in CPV-2 after 1990 (Zhou et al., 2017). Furthermore, position 267 and 324 in all CPV-2 isolates was Y and I, respectively. It is worth noting that A5G and Q370R, which were recently reported in China, were also detected in some CPV-2c isolates from Guangdong. One hundred percent (38/38) presented 324I, and 57.89% (22/38) presented 440A, which may indicate that the mutated strains are adapting to dogs. Moreover, several newly identified CPV-2c strains carried mutations at residue 13, and among other CPV-2c variants, more conservative P residues were common. The P13S residue was found in European cats (Decaro et al., 2008) and Japanese dogs (Nakamura et al., 2004), and in China, the same mutation occurred in CPV-2 isolates (accession numbers: GU392236, GU392244, GU392243, GU392242, GU392241, KM083037, KM083038, KJ170679) from raccoon dogs and foxes in 2009. A mutation in K582N was observed in this investigation. Whether these mutated amino acids have an effect on viral evolution deserves further study.

It is essential to note that the phylogenetic analysis was inconsistent with those in previous studies, resulting in unclear evolution. After performing the phylogenetic analysis of all sequences representing all viruses derived from FPV, we concluded that these viruses are separated into only CPV-2 and FPV branches, which includes FPV, RPV, MEV and BFPV. Clarification of the phylogeny would be useful for virus evolution research.

Although a previous study indicated that CPV-2a remains the major variant in China, accounting for 61.81% of CPV-2 isolates (Zhuang et al., 2019), with the rise of an increasing number of CPV-2c isolates, CPV-2c has to some extent become the dominant variant in China. A recent review showed that CPV-2c had been on the rise before 2017 (Qi et al., 2020). However, according to our research, CPV-2c may have become the main variant of CPV-2 in China. CPV-2c appears to have succeeded in replacing other variants as the major strain in only 10 years. This situation is similar to that in neighbouring Vietnam. The first CPV-2c strain in Vietnam was reported in 2004, after which it gradually evolved into the predominant variant (Hoang et al., 2019). The first Chinese CPV-2c isolates were collected in only 2009, and the evolution of CPV-2c in China lagged behind that in other regions. CPV-2c appears to be present in many countries worldwide, which will be a great challenge for disease control. Notably, the most recently used vaccines in China are mainly CPV-2 (Nobivac, Holland) (Wang et al., 2016b), and whether this type of vaccine still confers full protection against novel variants is unclear. Vaccine immunity protects most dogs from CPV infection, but vaccine protection can fail. Some vaccinated dogs may still be re-infected with canine parvovirus, and the vaccine may not provide complete protection against some endemic strains (De et al., 2018; Wu et al., 2015). Therefore, continued monitoring for CPV-2 prevalence and effective vaccines are required in the future.

Conclusions

A recent aetiological survey conducted in Guangdong, China, showed that 34/61 dogs with diarrhoea tested positive for parvovirus. Among them, 38 specific VP2 sequences were amplified. The function of several newly discovered variant sites deserves further study. It is worth noting that CPV-2c infection in domestic dogs in China is increasing. Based on this analysis, CPV-2c has replaced CPV-2a as the dominant variant. The results of this study provide fundamental knowledge regarding CPV-2 strains circulating in China.

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