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.