Discussion
This study shows that CPV-2b has
emerged for the first time as the dominant antigenic CPV variant
circulating in dogs with parvoviral enteritis in Australia. This finding
is consistent with an increasing selective pressure for CPV-2b in
Australia that was identified by analysis of time-stamped CPV isolates
in a previous study in which samples were collected from all Australian
States and territories (Clark et al., 2017). Since our samples were
largely collected in three States (NSW, Victoria and Western Australia)
we compared strain prevalence in these States between the two studies
and identified that CPV-2b predominance was not the result of sampling
bias; between 2008 and 2016 CPV-2b comprised 44% of strains from NSW,
Victoria and Western Australia (Clark et al., 2017), whereas between
2015 and 2019 CPV-2b comprised 57%.
It is surprising that CPV-2c was not identified among the VP2 sequences
in this study. CPV-2c was identified in faecal samples from three dogs
hospitalised with acute gastroenteritis in 2015 in Australia (Woolford,
Crocker, Bobrowski, Baker, & Hemmatzadeh, 2017). However, testing of
faecal samples obtained 1-2 days earlier from the same dogs in the
laboratories of two of the authors did not confirm this and the viruses
were characterized as CPV-2a or CPV-2b (data not shown). A possible
explanation for this discrepancy is that the dogs were co-infected with
multiple CPV variants, with a change in strain predominance over time.
Alternatively, the PCR and sequencing methodologies used in the three
laboratories may have preferentially amplified different variants.
In the Asia Pacific region, the CPV-2c variant is not widespread
although it has become the dominant strain in Taiwan (Lin et al., 2017)
and Vietnam (Hoang et al., 2019). By contrast, in the most recent
molecular survey of 70 CPV isolates collected in New Zealand, one of
Australia’s closest neighbouring countries, CPV-2c was not identified
(Ohneiser, Hills, Cave, Passmore, & Dunowska, 2015).
Some of the CPV strains we identified had VP2 sequences that were
intermediate between CPV and FPV (Figure 3). Although CPV and FPV share
>99% nucleotide identity they have specific host ranges,
antigenic and haemagglutination properties, which are controlled by the
capsid VP2 gene (Chang, Sgro, & Parrish, 1992; Shackelton et al., 2005;
Truyen, Agbandje, & Parrish, 1994). Subsequent to adaptations of an
FPV-like ancestral virus, CPV emerged as a new virus in dogs in the late
1970s, although a specific evolutionary pathway has not been identified
(Allison et al., 2016). On phylogenetic analysis, most of the viruses in
Clade 2 in this study contained two FPV-defining residues, 564-Asn and
568-Ala (Figure 1 and Table 2). CPV differs from FPV at 7 VP2 residues
and substitutions at three of these (564 (Asn to Ser), 568 (Ala to Gly)
and 80 (Lys to Arg)) are associated with loss of replication ability of
CPV-2 in cats, as determined using viruses recombinant between FPV and
CPV-2 (U. Truyen et al., 1994). The 300 VP2 loop region (residues 299 to
301), structurally proximal to residues 80, 564 and 568, is a critical
determinant of host range due to its interaction with the transferrin
type I receptor (TfR) to mediate infection (Allison et al., 2016).
Mutations in residues in close proximity to the 300 VP2 loop region can
alter the efficiency of TfR binding and infection. One unique CPV-2b
strain in our study, in Clade 2, contained another FPV-defining residue,
323-Asp (Table 2). VP2 aa residues 93 and 323 are critical in
controlling CPV host range and a CPV-specific antigenic site on the
capsid (Chang et al., 1992; Hueffer et al., 2003).
We also identified CPV-2a-like viruses that contained substitutions
typical of CPV-2 in Clade 2, including four with 305-Asp and two with
87-Met (Figure 3). CPV-2a-like viruses isolated from dogs differ from
CPV-2, at VP2 residues 87, 101, 300, and 305. The CPV-2a-specific
residues 87-Leu and 101-Thr were likely acquired during evolution of the
virus in raccoons, while the substitutions at 300 (Gly) and 305 (Tyr)
were acquired when the virus transferred back to the canine host
(Allison et al., 2012). Importantly, residues 87, 300, and 305 all lie
within the binding footprint of the TfR, while residue 101 lies close to
residue 87, just below the capsid surface (Hafenstein et al., 2007). The
evolutionary trajectory between CPV-2 and CPV-2a-like viruses was likely
facilitated by passage through an alternative host (Stucker et al.,
2012). Another interesting finding of this study is that several
Australian CPV strains displayed signatures in the VP2 protein that are
typical of Asian CPVs, including 5-Gly, 267-Tyr, 324-Ile (Mira et al.,
2018; Vannamahaxay et al., 2017; J. Wang et al., 2016), suggesting the
introduction of CPV strains from Asia or, at least, for CPV circulation
between Asia and Australia.
Our finding of intermediates between FPV and CPV-2 is unusual since
intermediates have rarely been identified in molecular surveillance
studies in the field. One exception was the detection of 568-Ala in a
CPV-2a strain from Italy (Battilani et al., 2019), which was not
accompanied by 564-Asn as seen here. The lack of identification of
intermediates in other studies is likely due to the limited number of
epidemiological investigations carried out before the 1990s, which
restricts information on the possible geographical and temporal
variations of CPV worldwide. The reversion of some VP2 aa residues to
those of FPV, as identified here, could reflect alternate cycles of
replication in dogs and cats. While there are no endemic wildlifeFelidae in Australia, there are large populations of free-roaming
feral cats (Felis catus ). Alternatively, these changes could
represent residues retained from CPV-2 intermediates during adaption
from an FPV-like ancestor to CPV-2 rather than reverse mutations. We
consider this explanation less likely since Australia does not harbor
endemic wild Felidae or Canidae hosts (apart from a small
population of dingoes (Canis lupus dingo )), thought to be
required in viral evolution (Allison et al., 2012). Also, the strains
with these “reverse” mutations were characterised by 297-Ala and
555-Val, both recent acquisitions among CPV-2a-like viruses (Meers et
al., 2007). Finally, since recombination has been shown to play a role
in the evolution of CPV (Shackelton et al., 2005), these findings could
be the result of multiple evolutionary mechanisms, confounding the
actual evolutionary patterns. Site-directed mutagenesis of CPV-2 virus
to CPV-2b and vice versa has shown that some mutations in a given
background are not well tolerated and markedly decrease virus fitness
(Stucker et al., 2012). Accordingly, the discovery of intermediates with
a vast repertoire of combinations of key mutations between FPV, CPV-2
and CPV-2a-like variants could inform a better understanding of the
trajectory of evolution of CPV.
We identified two CPV-2 sequences, original type, from dogs without
available vaccination history. These are most likely vaccine strains
that were not the cause of enteritis in these dogs, since CPV-2 is no
longer circulating in the field but attenuated vaccines containing this
strain are used commonly in Australia. Reversion to virulence of the
vaccine strains is theoretically possible but in the absence of
corroborating evidence considered unlikely. Also, three dogs for which
vaccination history was available, were infected with a CPV-2b strain
closely related to a vaccine strain (SAH strain, accession no. FJ222822;
Figure 1, Clade 4). All three dogs were from different litters from a
breeding facility and had been vaccinated with the same CPV-2b SAH
vaccine strain. The VP2 sequences of the virus from these dogs also had
100% nucleotide identity with two clinical isolates collected from dogs
in the US in 2003 (AY742951) and 2009 (JN867605), for which accompanying
vaccination data were not available (Allison et al., 2012; Shackelton et
al., 2005). Viruses from these dogs differ from the SAH vaccine strain
at one residue, 570-Lys, which is 570-Glu in the vaccine strain.
Reversion to virulence of the vaccine strain in these cases is possible,
or alternatively the CPV-2b strain in these cases may have been a
vaccine mutant, but not the cause of disease. Another possible
explanation is that these were wild-type viruses with a similar VP2
sequence to the vaccine virus.
Primary immunization failures (3.8%) among adult dogs that had
completed a primary puppy vaccination course were rare, supporting the
results of a previous Australian study where the failure rate was 3.3%
(Ling, Norris, Kelman, & Ward, 2012). Although immunization failures
can occur for many reasons including vaccine factors (e.g. improper
storage or expiry) and various animal factors (e.g. immune-suppression
due to disease or poor nutrition) (Roth & Spickler, 2010), the most
likely cause of immunization failure of young adult dogs that have
completed their primary vaccination course and been re-vaccinated is a
genetic inability to synthesise antibodies against CPV, resulting in
“non-responders” (Day et al., 2016). Some reports suggest that dogs
infected with CPV-2c that have been vaccinated with a CPV-2 containing
product, may be at higher risk of immunization failure compared to those
receiving a CPV-2b vaccine (Decaro et al., 2008). If this is the case,
then the rate of CPV immunization failures identified in Australia may
not be broadly representative since only a single report exists of
CPV-2c in Australia (Woolford et al., 2017). Commercial vaccines
containing CPV-2c are not available. However, whether CPV-2 vaccines
confer less protection in vivo than those containing CPV-2b, is
controversial. It has been shown in vitro that dogs vaccinated
with CPV-2 develop significantly lower antibody titres against
CPV-2a-like viruses compared to antibody titres against CPV-2 (Cavalli
et al., 2008). Whether the duration of immunity to CPV-2c is shorter in
animals vaccinated with CPV-2 has not been investigated.
Almost half of the dogs with available vaccination histories were
incompletely vaccinated. These dogs were likely permissive to infection
due to interference of vaccine virus by maternally derived antibodies
(MDA), since disease onset occurred before the completion of the primary
vaccination series or in dogs completing the series before the age of 16
weeks. MDA in dogs generally persist until 13 to 15 weeks of age, and
interfering titres of MDA have long been recognised as the most common
cause of CPV immunisation failure (Decaro, Buonavoglia, & Barrs, 2020;
Pollock & Carmichael, 1982).
The large proportion of dogs with parvoviral disease that were
incompletely vaccinated in this study reflects the importance of final
vaccination of puppies at 16-weeks of age or older to reduce
immunization failure rate. However, almost half of the incompletely
vaccinated dogs developed disease before completion of the primary
vaccination series. Taken together, these findings indicate that
strategies to overcome titres of MDA capable of interfering with vaccine
protection in puppies are as important as increasing vaccination
coverage overall to reduce the incidence of canine parvovirosis. Such
strategies include measurement of MDA titres in puppies using
haemagglutination assays to determine the optimal time for vaccination.
Another tool, which has been shown to be at least partially effective in
an experimental setting is the use of intranasal or oral MLV CPV
vaccines (Cavalli et al., 2020; Martella et al., 2005).
In conclusion, passive surveillance between 2015 and 2019 shows that
CPV-2b has emerged, for the first time, as the dominant antigenic CPV
variant circulating in dogs with parvoviral enteritis in Australia.
Disease was diagnosed in unvaccinated or incompletely vaccinated dogs in
near equal proportions and immunization failures were uncommon. CPV-2c
strains were not identified in this study. However, analysis of
translated VP2 sequences revealed a vast repertoire of aa mutations.
Strains of CPV that were intermediate between CPV and FPV, displayed
reverse mutations, or, more likely, residual mutations retained from
CPV-2 during adaptation from an FPV-like viral ancestor. Similarly,
intermediates between CPV-2a-like viruses and CPV-2 were also
identified. These findings could be helpful to inform a better
understanding of the evolution of CPV in dogs.