Discussion
According to the reports before 2015, CPV-2c was scarcely detected in Chinese strains(Han et al., 2011; Wang et al., 2016a; Zhang et al., 2010a; Zhang et al., 2010b; Zhao et al., 2016). After 2015, although its number increased, most reports indicated that New CPV-2a has the highest proportion in China(Geng et al., 2015; Wang et al., 2016b; Wu et al., 2018; Zhao et al., 2015; Zhuang et al., 2019). This study provided a different result compared to previous reports. By collecting available Chinese CPV-2 strains from Genbank and related publications, we further proved our finding, that CPV-2c has a dramatic increase in China and finally replaced the New CPV-2a variant as the predominant strain in most cities of China. However, since many cities were not investigated, the result was not conclusive. But anyway, this tendency should be caught concern since CPV-2c variants have been frequently associated with CPV-2 outbreak in vaccinated dogs(Cavalli et al., 2008; Decaro and Buonavoglia, 2012; Decaro et al., 2008a). So far, there is no consensus about the effectiveness of prototype CPV-2-based vaccines against heterologous CPV-2 variants(Decaro et al., 2020). However, the continuous and dynamic evolution of CPV-2c may pose a new challenge to the vaccines used currently.
In the past, CPV-2c has been found mainly in South American and European countries, and it was rarely detected in Asia, where more CPV-2a/2b variants prevailed(Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016). However, in recent years, the amount of CPV-2c increased in Asia(Moon et al., 2020). The result in this study was consistent with recent reports of Asian countries, such as Taiwan(Chiang et al., 2016; Lin et al., 2017), Laos(Vannamahaxay et al., 2017), Vietnam(Hoang et al., 2019; Nguyen Manh et al.), Thailand(Charoenkul et al., 2019; Inthong et al., 2020), and Korea (Moon et al., 2020). These reports either revealed the higher detection rate of CPV-2c or indicated that CPV-2c has become the predominant variant in recent years. It’s noteworthy that these reports have pointed out that the strains isolated shared similar features between Asian strains, which seemed to demonstrate the mutual transmission of CPV-2 between neighboring Asian countries. We could see the synchronous changes of CPV-2c in some Asian countries since 2015, thus it’s possible that CPV-2c variants have been continuing its evolution in Asia and gaining a stronger epidemiological advantage over other mutants.
In this study, some newly reported amino acid mutations in VP2 protein were found. Mutation Ala5Gly existed in all the CPV-2c strains, including one New CPV-2a strain. Mutation Ala5Gly first reported in Chinese strains in 2015, then detected in Asian countries like Taiwan(2017)(Lin et al., 2017), Thailand(2017)(Mira et al., 2018), and Vietnam(2018)(Hoang et al., 2019). In these studies, Ala5Gly was only observed in CPV-2c strains, while it also existed in one New CPV-2a strains. Formerly, this mutation was only observed in Asian strains. However, recently, it has also been found in CPV-2a/b variants in Australia(Kwan et al., 2020) and Italy(Mira et al., 2019), suggesting the possible introduction from Asian countries. Or they were not of unitary origin and evolved independently due to the increasingly selective pressure in this site since this mutation was not presented in the same variant. Currently, the potential biological function of this mutation is unclear.
Another specific amino acid mutation of CPV-2c was Gln370Arg, except for two CPV-2c strains. In 2012, this mutation was first observed in a New CPV-2a strain isolated from a giant panda(Guo et al., 2013). Then in 2015, it was reported again in Chinese CPV-2c strains from dogs(Geng et al., 2015; Zhao et al., 2015). After that, most Chinese CPV-2c strains harboured the same mutation(Wang et al., 2016a; Wang et al., 2016b; Wu et al., 2018; Zhuang et al., 2019). Interestingly, it was first observed in a New CPV-2a strain, but only presented in CPV-2c strains when it prevailed in China. It’s possible that this mutation occurred during the further adaptation to giant pandas and has spread back to dogs(Wang et al., 2016b). Currently, this mutation is not only prevailing in China, but also occurring in neighboring countries like Taiwan, Thailand, Korea, Laos, and African countries like Nigeria(Ukwueze et al., 2020) and Zambia(Kapiya et al., 2019). Probably, this mutation was exported from China. It was reported that amino acid site 370 is adjacent to residue 375, which affects the pH dependence of hemagglutination(Chang et al., 1993). Also, residue 370 is located in a flexible surface loop consisting of residues 359 to 375 of the capsid protein, and residues within this loop were considered essential for virus infectivity(Simpson et al., 2000). Then, the mutation in residue 370 may also affect the loop’s conformation then influence the host range.
In this study, mutation Thr440Ala was specific to New CPV-2a strains. According to previous reports, it also occurred in New CPV-2b strains(Geng et al., 2015; Zhang et al., 2010a; Zhuang et al., 2019). It was first detected in 1993 and became prevalent after 2005(Zhou et al., 2017). In China, Korea(Jeoung et al., 2008), Thailand(Inthong et al., 2020), India(Mittal et al., 2014), Colombia(Giraldo-Ramirez et al., 2020), Italy(Decaro et al., 2009), Brazil(de Oliveira et al., 2019), it presented in New CPV-2a/2b variants. However, in the United States(Kapil et al., 2008), Argentina(Calderón et al., 2011), and Mexico(Faz et al., 2019), it was unique to CPV-2c strains. Amino acid residue 440 is located in the GH loop, a region with the greatest variability due to its exposure on the capsid surface(Decaro et al., 2009). Also, it was found in the threefold axis, a site containing two major epitopes – epitope A and epitope B(Strassheim et al., 1994). Thus, mutations in this site could have antigenic significance and affect the host immune response. This mutation was estimated to have multiple evolutionary origins(Voorhees et al., 2019), which may explain why it presented in different CPV-2 variants in different areas.
Another substitution found was Pro13Ser, which presented both in New CPV-2a(n=1) and CPV-2c(n=2) strains. This mutation was identified in Uruguayan, Vietnamese, and Japanese dogs, European cats(Maya et al., 2013), and Chinese raccoon dogs (Lu et al., 2020). In Italy, it was observed in CPV-2b strains but was Ala rather than Ser(Decaro et al., 2009). It was estimated that this mutation may not be antigenically significant since residue 13 was not exposed to the surface(Lu et al., 2020).
Mutation Phe267Tyr and Tyr324Ile were identified in all CPV-2 strains, and it’s thought to be Asian strains characteristic(Mira et al., 2018). Phe267Tyr first appeared in 2002 and has become predominant since 2014, Tyr324Ile first appeared in 2006 and has consistently occurred, then becoming predominant in 2014(Zhou et al., 2017). As mentioned above, residue 267 and 324 are located in the GH loop, and they are subjected to positive selection(Giraldo-Ramirez et al., 2020; Zhao et al., 2015). Then they were thought to play an important role in the evolution of new variants (Nandi et al., 2019). Also, residue 324 is adjacent to 323, a key residue determining the host range. Then, it may also affect the host range. Recently, it was also reported in Australian(Kwan et al., 2020), Uruguayan(Maya et al., 2013), Colombian(Giraldo-Ramirez et al., 2020), and Italian(Mira et al., 2019) strains. The phylogenetic analysis compellingly indicated the intercontinental dissemination, possibly originated from Asian countries.
The last mutation was observed in residue 570, from Lys to Arg. However, in two Australian strains and one vaccine strain, it was Lys to Glu(Kwan et al., 2020). And it seemed to be the first case in China. Residue 570 is lying on the capsid surface and it closely approaches to the residue 300, a residue that has the greatest variability determining the cross-species transfer of viruses between different carnivores(Allison et al., 2016). Thus, its alteration may also affect the host TfR binding ability.
Based on the phylogenetic tree, all the CPV-2c strains in this study, along with other newly sequenced Chinese CPV-2c strains, were embraced in the same clade, indicating the intimate relationship between Chinese CPV-2c strains, which suggested the mutual transmission between different cities of China. In recent years, pets can be sold by online trading. Thus, the transportation of pets from cities to cities has increased, which could greatly contribute to the transmission of CPV-2. Other Asian strains were included in the same clade, revealing the possible introduction from neighboring Asian countries. Noteworthily, one Italian strain was also included. As the report concluded, this strain was of Asian origin(Mira et al., 2019). In recent years, several essays demonstrated the detection of CPV-2 strains originated from Asia in these Oceanian, African, American, and European countries(Giraldo-Ramirez et al., 2020; Kapiya et al., 2019; Kwan et al., 2020; Maya et al., 2013; Mira et al., 2019; Zaher et al., 2020). Therefore, stricter border controls may be needed as a preventive measure. All the strains located in this clade carried four important substitutions: Ala5Gly, Phe267Tyr, Tyr324Ile, and Gln370Arg. While clade CPV-2c Ⅱ contained CPV-2c strains retaining initial amino acids in these sites and circulated in American and European countries, suggesting the geographical isolation of CPV-2c variants with Asian countries.
Compared to CPV-2c variants, the molecular evolution of New CPV-2a strains was more complicated due to its genetic diversity. New CPV-2a variants were marked by residue 297Ala. However, in Colombia, Brazil, Uruguay and Argentina(Giraldo-Ramirez et al., 2020), residue 297 can be substituted with Asn. Thus, such categorization can be confusing. All the New CPV-2a strains isolated in this study were located in the same clade, characterized by mutations Phe267Tyr, Tyr324Ile and Thr440Ala. In contrast, clade New CPV-2a Ⅱ contained New CPV-2a strains with only one mutation Thr440Ala, and clade New CPV-2a Ⅲ included New CPV-2a variants with original residues in the sites mentioned above. The Phe267Tyr and Tyr324Ile changes together resulted in a distant phylogenetic relationship between clade New CPV-2a Ⅰ and clade New CPV-2a Ⅱ/Ⅲ, which highlighted the importance of Phe267Tyr and Tyr324Ile. All the FPLV strains were located in the FPLV clade. Compared with CPV-2, FPLV strains were highly conserved and exhibited a closer relationship with Asian strains.
Remarkably, two FPLV strains were detected in dog samples and both harboured a mutation Ile101Thr, which is CPV-2 mutants specific. Most FPLV detected recently owned the same mutation compared to original FPLV strains(Decaro et al., 2008b; Li et al., 2018). It is strange since FPLV was unable to replicate in the small intestine or mesenteric lymph nodes of dogs without being shed in the feces(Truyen and Parrish, 1992). Similar findings have been reported in Pakistani and Thai strains. This phenomenon may suggest the dynamic evolution of carnivore parvovirus, or the existence of the recombinant virus of CPV and FPLV(Ahmed et al., 2018; Inthong et al., 2020). Meanwhile, it may be caused due to the mistake made by a veterinary assistant, mislabeling cat samples with dog samples. However, similar reports in recent years may disprove this postulation. Also, as Nisar Ahmed suggested, it was possibly due to the coprophagous behavior of dogs(Ahmed et al., 2018). Anyway, further investigations are needed to explain this phenomenon.
In conclusion, 6 New CPV-2a, 19 CPV-2c, and 2 FPLV strains were isolated from 33 canine samples, and CPV-2c emerged as the dominant genotype, which was further proved by the analysis of 683 reported Chinese strains collected from 2014 to 2019. Currently, no ascertainable facts prove that CPV-2c could result in immunization failure. However, the obvious epidemic superiority of CPV-2c strains with mutation Ala5Gly and Gln370Arg mutation in Asian areas may pose a new challenge to the vaccines used at present. Further research should be undertaken to investigate the relationship between immunization failure with current CPV-2c strains. Phylogenetic tree revealed the national and international spread of CPV-2 variants, thus boundary administration should be stricter. One unanticipated finding was the identification of two FPLV strains in canine samples, which is an important issue for future research.