4 | Discussion
In this study, 44 rectal swabs or intestinal tissue samples were collected from several animal hospitals in Changchun City and Liaoyuan City, Jilin Province. A total of 44 dogs were confirmed to be infected with CPV-2 by PCR. Then, the VP2 genes of the 44 strains of CPV-2 identified in this study were sequenced and compared with the GenBank database. The subtypes were classified with reference to different amino acids at positions 297 and 426. The results showed that 1 strain of CPV-2, 8 strains of new CPV-2a, 4 strains of new CPV-2b and 31 strains of CPV-2c were found in this study. These results showed that the CPV-2c subtype was the main epidemic strain of CPV-2 in the Jilin area, accompanied by the coexistence of the new CPV-2a and new CPV-2b subtypes.
CPV-2 was discovered in the United States in the late 1970s. In China, CPV-2 was first reported to spread among dogs in 1983, and several variants (including CPV-2a, CPV-2b, new CPV-2a, new CPV-2b and CPV-2c) have cocirculating in recent years. In 2010, CPV-2c was detected for the first time in Jilin Province, China (Chiang, Wu, Chiou, Chang, & Lin, 2016). In 2014, the CPV-2c strain emerged in Shandong province (Zhao et al., 2017). From 2014 to 2015, 14 CPV-2c-positive samples (out of 95 samples) in Heilongjiang province and 18 CPV-2c-positive samples (out of 43 samples) in Beijing were found, and several strains were isolated (Geng et al., 2015; J. Wang et al., 2016) . It was reported in 2018 that CPV-2c had spread to Henan, Guangxi and Jiangsu provinces (Wu et al., 2018). Eleven CPV-2c samples (out of 24 samples) were reported in Sichuan in 2019 (Zhuang et al., 2019). The isolation rate of CPV-2c has increased year by year. It can be seen that in the past 10 years, CPV-2c has gradually replaced the old genotypes and become the new major epidemic genotype.
VP2 is the main antigenic determinant of canine parvovirus and is related to the host immune response. A small number of mutations in the VP2 gene may lead to a change in pathogenicity (Lin et al., 2014). To trace the evolution of antigenic variants, the mutations at 5, 267, 324 and 370 of VP2 were analyzed. Compared with the classical European original CPV-2c isolate G7/97 in 1997, all of the strains of CPV-2c identified in this study had aa substitutions of Ala5Gly, Phe267Tyr, Tyr324Ile, and Gln370Arg, while the earliest Chinese Jilin isolate 08/09 (GU380305) in 2009 did not have any amino acid mutations (Table 2). In contrast, the original Jilin isolate 06/09 (GU380303) showed a mutation of Tyr324Ile. Another early Jilin isolate 06/09 (GU380303) harbored the F267Y mutation. The Phe267Tyr mutation was also reported in 2013 in the Chinese CPV-2c isolates G15 (KF482471) and G1 (KF482468). The mutation of residue 267 may not affect the antigenicity of CPV because residue 267 is not exposed on the capsid surface (Decaro et al., 2006). However, the Tyr324Ile mutation, which affects the binding to the canine transferrin receptor, could lead to a change of the host range of canine parvovirus (Hueffer et al., 2003). In 2014, the amino acid substitution Gln370Arg appeared in Chinese isolates WANGQING-1, YANJI-1 and HRB-A6, and it might be involved in a required conformational change, or mediate an effect on receptor binding through the neighboring residues (Guo et al., 2013). In 2014 and 2015, a novel mutation at residue 5, substituting glycine for the highly conserved alanine present in all CPV-2c strains, was isolated in Beijing (J. Wang et al., 2016). Residue 5 is one of the surface and core residues of the antigen site. Therefore, the Ala5Gly mutation may change its antigenicity and immunogenicity (Li, Tang, Chen, Niu, & Liu, 2019). So far, these four amino acid mutations have simultaneously appeared in CPV-2c strains recently isolated from China and they are also present in the 31 CPV-2c strains identified in this study.
In the present study, we detected a mutation of Arg481Lys that has not been reported previously and its potential functional consequence remains to be determined. Residue 481 is located in the GH loop comprising aa 267-498 of VP2 protein (Agbandje & Rossmann, 1995). The residues inserted in the GH loop have the greatest variability, and the residues inserted in the GH loop have been proven to bind to neutralizing antibodies in CPV. Therefore, the substitution of amino acid 481 may result in changes in its antigenicity and immunogenicity. Further study is needed for confirmation.
According to the phylogenetic tree of VP2, the earliest Chinese CPV-2c strains were clustered in the same branch with the European original CPV-2c isolates, while the Chinese CPV-2c isolated in recent years forms a monophyletic cluster that is obviously different from the foreign strains in their evolutionary relationship. This consistent with the results of previous phylogenetic analysis (Zhao et al., 2017; Zhuang et al., 2019). The Chinese CPV-2c strains may be derived from foreign strains and subsequently evolved locally during distribution. It may be that in the development of the pet industry, the pet dog transportation trade has also broken the original regional barriers of CPV and led to the spread and prevalence of foreign CPV-2c strains in China. It has also been reported that CPV migration is likely to spread between geographically close countries through the movement of infected animals or mechanical vectors (Hoelzer et al., 2008).
An additional finding is that all of the CPV-2 strains collected here in this study were separated from the vaccine strains except the strain CPV-CC-33, which may be a vaccine-like strain. Furthermore, CPV-2c is becoming the most predominant strain in China. However, current commercial vaccines available in China are based on CPV-2. Therefore, special concerns regarding the efficacy of the current vaccines have been raised. Although previous studies have claimed that vaccination with CPV-2 type vaccine can cross-protect against challenge with virulent CPV-2a, CPV-2b, and CPV-2c (Siedek, Schmidt, Sture, & Raue, 2011; Spibey, Greenwood, Sutton, Chalmers, & Tarpey, 2008), some evidence suggests that dogs with a complete vaccination program still suffer from infection with CPV-2c or CPV-2c variants (Decaro et al., 2008; Faz et al., 2019). CPV-2c viruses have been isolated not only from unvaccinated but also from vaccinated dogs (J. Wang et al., 2016). Considering the inability of vaccine strains to provide adequate protection against field viruses, further and extensive epidemiological investigations are required in the future, and the CPV vaccines needed to be updated by replacing the original type 2 with the CPV variants currently circulating.