viruses

Review
Evolution and Interspecies Transmission of Canine
Distemper Virus—An Outlook of the Diverse
Evolutionary Landscapes of a Multi-Host Virus
July Duque-Valencia 1 , Nicolás Sarute 2,3 , Ximena A. Olarte-Castillo 4
and Julián Ruíz-Sáenz 1, *
1 Grupo de Investigación en Ciencias Animales-GRICA, Facultad de Medicina Veterinaria y Zootecnia,
Universidad Cooperativa de Colombia, sede Medellín 050012, Colombia
2 Sección Genética Evolutiva, Facultad de Ciencias, Universidad de la Republica, Montevideo 11200, Uruguay
3 Department of Microbiology and Immunology, UIC College of Medicine, Chicago, IL 60612, USA
4 Facultad de Ciencias Exactas, Naturales y Agropecuarias. Universidad de Santander (UDES), sede
Bucaramanga 680002, Colombia
* Correspondence: julian.ruizs@campusucc.edu.co; Tel.: +57-7-685-45-00 (ext. 7072)




Received: 26 March 2019; Accepted: 18 May 2019; Published: 26 June 2019


Abstract: Canine distemper virus (CDV) is a worldwide distributed virus which belongs to the
genus Morbillivirus within the Paramyxoviridae family. CDV spreads through the lymphatic, epithelial,
and nervous systems of domestic dogs and wildlife, in at least six orders and over 20 families of
mammals. Due to the high morbidity and mortality rates and broad host range, understanding
the epidemiology of CDV is not only important for its control in domestic animals, but also for
the development of reliable wildlife conservation strategies. The present review aims to give an
outlook of the multiple evolutionary landscapes and factors involved in the transmission of CDV by
including epidemiological data from multiple species in urban, wild and peri-urban settings, not only
in domestic animal populations but at the wildlife interface. It is clear that different epidemiological
scenarios can lead to the presence of CDV in wildlife even in the absence of infection in domestic
populations, highlighting the role of CDV in different domestic or wild species without clinical signs
of disease mainly acting as reservoirs (peridomestic and mesocarnivores) that are often found in
peridomestic habits triggering CDV epidemics. Another scenario is driven by mutations, which
generate genetic variation on which random drift and natural selection can act, shaping the genetic
structure of CDV populations leading to some fitness compensations between hosts and driving
the evolution of specialist and generalist traits in CDV populations. In this scenario, the highly
variable protein hemagglutinin (H) determines the cellular and host tropism by binding to signaling
lymphocytic activation molecule (SLAM) and nectin-4 receptors of the host; however, the multiple
evolutionary events that may have facilitated CDV adaptation to different hosts must be evaluated by
complete genome sequencing. This review is focused on the study of CDV interspecies transmission
by examining molecular and epidemiological reports based on sequences of the hemagglutinin gene
and the growing body of studies of the complete genome; emphasizing the importance of long-term
multidisciplinary research that tracks CDV in the presence or absence of clinical signs in wild species,
and helping to implement strategies to mitigate the infection. Integrated research incorporating the
experience of wildlife managers, behavioral and conservation biologists, veterinarians, virologists,
and immunologists (among other scientific areas) and the inclusion of several wild and domestic
species is essential for understanding the intricate epidemiological dynamics of CDV in its multiple
host infections.

Keywords: genome evolution; canine distemper virus; hemagglutinin gene; genotype

Viruses 2019, 11, 582; doi:10.3390/v11070582 www.mdpi.com/journal/viruses

Viruses 2019, 11, 582 2 of 23

1. Introduction
Canine morbillivirus also known as canine distemper virus (CDV) belongs to the
genus Morbillivirus within the family Paramyxoviridae, which also includes measles virus
(measles morbillivirus—MeV), phocine distemper virus (phocine morbillivirus—PDV), peste des
petits ruminants virus (small ruminant morbillivirus—PPRV), and rinderpest virus (rinderpest
morbillivirus—RPV), which has already been eradicated [1,2]. CDV has the ability to infect a
wide range of species and poses a risk to the conservation of free and captive wildlife [3,4]. The present
review will analyze different viral and host characteristics to elucidate the conditions that have allowed
the CDV to have this wide distribution, evidencing different successful genetic and evolutionary
scenarios in both domestic and different wildlife environments.
The virus is pleomorphic (spherical and filamentous shapes), with a size between 150–300 nm in
diameter and contains a genome comprising single-stranded, non-segmented ribonucleic acid (RNA)
of negative coding sense. The genome comprises 15,690 nucleotides (15.6 kb), including six gene
regions organized in separate and non-overlapping transcriptional units coding for six structural
proteins: the nucleocapsid protein (N), phosphoprotein (P), viral polymerase (L), matrix (M) and the
hemagglutinin (H) and fusion protein (F) glycoproteins [1].
The transcriptional CDV units are separated by untranslated regions (UTRs) that are relatively
uniform (107–155 nucleotides), except for the UTR between the M and F genes, which has approximately
405 nucleotides. This UTR modulates virulence through the translational control of the protein F [5].
The phosphoprotein (P) gene (1524 nucleotides) is highly conserved and encodes a viral polymerase
cofactor protein (P) and two additional nonstructural proteins V and C, which are produced by RNA
editing and an alternative start of the translation, respectively [6]. The protein V suppresses the
innate immune response by inhibiting the induction of type I and II interferons and the activity of
the NF-kappa B complex in the host cell [7,8]. It has been established for MeV that the protein C
suppresses the IFN-γ signaling pathway by inhibiting dimerization of the phosphorylated STAT1
protein [9,10]. The large protein (L) gene (6555 nucleotides) is highly conserved and encodes for the
viral polymerase, that transcribes and replicates the virus RNA. The N gene (1572 nucleotides) encodes
for the N protein, which encapsulates the viral RNA [1]. The P, L, and N proteins together make up the
transcription/replication complex, known as ribonucleoprotein [6]
The hemagglutinin gene has 1824 nucleotides and encodes for the protein H, which shows the
highest variability in CDV when compared to other morbilliviruses [11]. This gene has the highest
divergence within CDV genome with substitution rates between 5.4 × 10−4 –1.8 × 10−3 nucleotide
substitutions per site, per year [11]. The protein H determines the viral tropism and initiates the infection
by binding to the signaling lymphocytic activation molecule (SLAM) receptor in immune cells [12,13],
and nectin-4 receptor in epithelial cells [14]. When dogs recover from natural infection/disease due to
CDV, they develop neutralizing antibodies against the H protein which confers lifelong immunity to
the diseases [15]. It has been shown that for this gene wild type and vaccine strains are highly divergent
(7–10% for nucleotides and 8–11% for amino acids) with a nucleotide identity between 93–90% and
amino acid identity between 92–89%. It is because of this high variability, that this gene is used in for
the phylogenetic classification of CDV in different lineages or genotypes [16]. The fusion (F) protein is
encoded by the F gene, composed by 1989 nucleotides. After the binding of the protein H to the host
cellular receptor, the F protein mediates the fusion between the viral envelope and the host plasma
membrane at physiological pH [17,18]. A short region of this gene (405 nucleotides) encodes for the F
signal peptide; the signal peptide region is highly variable and has also been used in phylogenetic
classification, representing a rapid method for characterizing circulating CDV strains [19]. The matrix
(M) protein, encoded by the M gene with 1008 nucleotides, constitutes the inner layer of the viral
envelope and interacts with the cytoplasmic domains of the H and F proteins [1]. When this protein is
synthesized in the cytoplasm during the replication cycle, it also associates with the ribonucleoprotein
complex, connecting it to the envelope, thus playing an essential role in viral shedding [18].
Viruses 2019, 11, 582 3 of 23

2. Disease in Domestic Dogs and Wildlife

Canine distemper is a worldwide distributed viral disease that is highly contagious and has high
morbidity and mortality. CDV infection may result in subclinical (asymptomatic) or clinical disease
manifestation. The main route of efficient infection is thought to be due to contact with fomites or via
the aerosol route by nasal secretions [20]; however transmission through nasal contact with bodily
excretions such as urine and feces is also likely [21].
For the development of the clinical disease, CDV infection has to go through two phases: first,
there is an acute infection of the lymphatic system [22]; and second, there is an invasion of epithelial
cells followed by viral shedding which allows transmission to other susceptible individuals [22]. CDV
can also invade the central nervous system probably mediated by a putative third receptor called
GliaR located in glial cells [23–25]. The clinical manifestations of CDV infection include depression,
anorexia, nasal and ocular mucopurulent secretions, gastroenteritis, hyperkeratosis of the plantar
pads and snout, and tooth enamel hypoplasia in puppies [26] In addition, seizures, myoclonia with
hyperesthesia, ataxia, paresis, and tremors indicate central nervous system involvement [24].
CDV infects multiple species within the order Carnivora, including domestic dogs (Canis lupus
familiaris, family Canidae), and various wild species within Canidae (hereafter referred to as canids) and
other families such as Felidae, Procyonidae, Mustelidae, Hyaenidae, Ursidae, and Viverridae (hereafter referred
to as non-canids). Some studies have also reported CDV infection in other mammalian families, such
as Cricetidae (rodents), Cercopithecidae (Old World monkeys), Suidae (pigs), and Elephantidae (elephants)
(for a comprehensive review, see [3]). Furthermore, recently CDV was reported as the causative agent
of the neurological signs observed on a captive southern tamandua (Tamandua tetradactyla, family
Myrmecophagidae, order Pilosa) in Brazil [27].
Viruses with a broad host range such as CDV could persist in populations with great sizes and
may potentially cause disastrous epidemics in endangered species. Thus, the understanding of CDV’s
epidemiology in multiple species is of great importance from a conservation point of view [28–30]. For
example, different studies have reported the link between CDV infection and the population decline
of various wildlife species [3,28], highlighting the possible role of CDV as a cause of extinction of
certain endangered wildlife species [31]. CDV outbreaks have been reported in near-extinct species,
such as the giant panda (Ailuropoda melanoleuca) in a wild animal rescue and conservation center in
China with a morbidity of 27% and mortality of 23% [28] and the Ethiopian wolf (Canis simensis) with a
reported mortality of up to 68% in a wild population in Ethiopia [29]. CDV has also been reported in
monkey breeding sites with an incidence of 20–60% and a mortality of 5–30% in Macaca fuscata and
Macaca mulata [32]. In the Serengeti National Park, in Tanzania, CDV has been detected in canid and
non-canid species with varying outcomes of the disease depending on the genetic strain involved [33],
with changes in the CDV genome of the non-canid viruses that were consistent with expansion of
selected variants in non-canid hosts [34–36]. Most notably, a strain adapted to non-canids but not with
the recent spillover of a dog strain caused a fatal epidemic in 1993–1994 in two non-canid species,
killing approximately one third of the African lion population (Panthera leo) and several juveniles
spotted hyena (Crocuta crocuta) [33].
In the United States, a mortality rate of 45% was reported in free-living raccoons (Procyon lotor)
found within, around, and outside a zoo in 2001 [37]. CDV has also been stated as one of the most
important causes of mortality in the gray fox (Urocyon cinereoargenteus) [38]. Likewise, a CDV outbreak
almost led to the extinction of the black-footed ferret (Mustela nigripes) [39].
Epidemiological data from Europe show varying rates of CDV seroprevalence in wildlife species,
particularly in red foxes (Vulpes vulpes) with a prevalence of 4% to 30.5% in countries such as Spain,
Portugal, Italy, and Germany [30,40–42]. In Central and South America, authors have reported the
presence of CDV in 2% of wild felids in Costa Rica [43], while in Brazil CDV seroprevalence has been
reported in multiple free-ranging wild carnivore species with a prevalence ranging between 10.6 to
23% [44–46].
Viruses 2019, 11, 582 4 of 23

The lethal epidemics of CDV in multiple species, have increased the understanding of the capacity
of this virus to cause high mortality in carnivores, impacting negatively wild populations [47]. Those
data allow us to propose that CDV, by infecting endangered wildlife species, decreases the populations
of these species, facilitating its extinction.
The role of the adaptive immunity and host immune response to the CDV, has been widely
discussed and studied in the domestic dog [48]. However due to the impossibility to experimentally
study this topic in wildlife, it is only possible to speculate how the evasion of the host immune system
is involve in CDV infection in different species and its role in the pathogenesis.

3. Reservoirs vs Viral Adaptation: Two Different Transmission Sceneries

3.1. The Role of Reservoirs in CDV Epidemiology

CDV transmission between domestic dogs and wildlife depends on the dynamics between their
populations [49], genetic characteristics of the virus [50], host receptors [51] and other factors that are
not fully understood.
Although the initial spread of CDV resulting from the interactions between domestic canids
and wildlife has been shown to trigger high mortality in wildlife [49], CDV spreading from wildlife
to domestic canids is also possible [52,53]. In 2015, authors analyzed CDV outbreaks in dogs and
lions in the Serengeti National Park, revealing that initially the outbreaks in lions were due to virus
transmission by domestic dogs; however, over the years the outbreaks in lions (1993–1994, 1999 and
2007) were asynchronous with those in domestic dogs, suggesting that CDV may persist in other wild
species interacting with this lion population [3,49] and possibly in other ecosystems. This hypothesis
is supported by the variety of species reported as susceptible for CDV infection [3].
Molecular phylogeography analysis of the origin and global evolution of CDV found that the
probability of CDV first infecting domestic canids and then wildlife is higher than that for CDV
infecting wildlife at first. Analyzing 208 sequences of the H gene the study propose that CDV strains
emerged in the United States during 1858–1913 and from there through migrations—perhaps pet
trade-it expands to Europe, Asia, Africa and America. Thus, domestic canids are probably the main
CDV reservoir worldwide [54].
Due to the lack of knowledge about the ecoepidemiology of the virus in wild-life ecosystems, it is
necessary to look for virus reservoirs, defined as one or more epidemiologically connected populations
or environments in which the CDV can be permanently maintained and from which infection is
transmitted to the defined target population. Populations in a reservoir may be the same or a different
species as the target population [55,56].
In the case of CDV in peridomestic settings, CDV seropositivity has been reported in clinically healthy
animals belonging to various orders and families. Although it is not known if those animals showed
clinical signs before the studies, these findings reveal that CDV virus circulation in wild ecosystems
occurs through certain species acting as a “meta-reservoir”. Those wild animals transmit the virus to
diverse interconnected populations, explaining the asynchronous outbreaks of CDV reported in wildlife
ecosystems in relation to domestic dog outbreaks [3]. This complex situation has been reported for CDV
in hyenas and cheetahs months after a CDV outbreak in lions of South Africa in 2015 [4].
A similar scenery was established in the United States when two strains with just 56 different
nucleotides in their genome, and belonging to the same CDV lineage, were found circulating in different
hosts of distant geographical areas [57]. However, in this case the raccoon (Procyon lotor) has already
been identified as a second CDV reservoir given that the target population is wildlife and the primary
reservoir is the domestic dog [53]. The raccoon populations are often found in peridomestic habits,
having contact with infected dogs, thus spreading the infection to other wildlife in wild ecosystems
and to healthy dogs in urban ecosystem [53,57].
Viruses 2019, 11, 582 5 of 23

3.2. Role of Asymptomatic Animals in CDV Transmission

Multiple epidemiological scenarios can lead to the presence of CDV in wildlife even in the absence
of infection in domestic populations [3]. Although CDV has been linked to the mass death of wild
species both free-living [58] and in captivity [59], CDV can also occur in different domestic or wild
species without clinical signs. For example, domestic dogs can be CDV-positive without developing
signs but can still transmit the virus since viral RNA is found on fecal swabs, thus these individuals
act as potential CDV reservoirs [60,61]. Recently, CDV shedding was observed up to several months
after the cessation of clinical signs in recovered domestic dogs [62]; therefore emphasizing the role of
asymptomatic domestic dog carriers in CDV epidemiology in this species in urban areas.
The report of an outbreak in giant panda (Ailuropoda melanoleuca) suggests that subclinical disease
is also possible in this species, as during the outbreak one individual did not show clinical signs
despite the detection of CDV genome from blood and nasal swabs samples. This animal had been
previously vaccinated suggesting that the protective immune responses elicited by vaccination against
CDV were not sufficient to prevent CDV infection acquired naturally, but may have attenuated disease,
as this was the only individual that survived the outbreak [28]. In other wild species such as raccoons
(Procyon lotor) and gray foxes (Urocyon cinereoargenteus) a high percentage of asymptomatic individuals
were positive for CDV (55%) [63]. These could be a source of the virus, especially in the coldest months,
when the virus can survive longer outside the host [63].
On the other hand, the experimental inoculation of cats (Felis silvestris catus, family Felidae) and
domestic pigs (Sus scrofa domesticus, family Suidae) with a CDV virulent strain for domestic dogs
resulted in infection and viral replication in lymphoid tissues and macrophages, but without clinical
signs or virus shedding [64]. Interestingly, the inoculation of two cats with a strain that was lethal in a
captive Chinese leopard (Panthera pardus orientalis, family Felidae) resulted in viremia in peripheral
blood mononuclear cells and mild transient leukopenia and lymphopenia, however no clinical signs of
disease were observed in the domestic cats [59].
‘Silent’ CDV epidemics have been observed in the Serengeti–Mara ecosystem, where serological
analyses have shown an increase in the exposure to CDV in spotted hyenas and lions without obvious
clinical signs, high mortality, or a reduction in population size [49,65–67], as observed during the lethal
epidemic of 1993–1994 in these species. Spotted hyenas from the Maasai Mara National Reserve were
more exposed to CDV than those near to human settlements, showing that in this case, domestic dogs
may not be the only animals that transmit CDV to these wild species [66].
These results show the importance of long-term research when studying CDV in free-ranging
wild species in which virus detection and the recognition of clinical signs of disease (or their absence) is
quite challenging [33,68,69]. For this reason integrated research incorporating the expertise of wildlife
managers, behavioral and conservation biologists, veterinarians, virologists, and immunologists
(among other scientific areas) and the inclusion of several wild and domestic species is needed for
understanding the intricate epidemiological dynamics of CDV in multiple hosts. By monitoring CDV
in target populations, regardless of whether clinical signs are observed or not, the complex scenarios of
CDV transmission may be explored; including the possibility of the circulation of different strains that
may vary in their virulence in different species [33], the epidemiological importance of asymptomatic
individuals in the spread of the disease [61], the existence of dead-end hosts—which are asymptomatic
and do not shed the virus—such as the cat and pig [70] or perhaps either lions or hyenas when infected
with CDV canid strains [33].
Multidisciplinary research is also needed to implement accurate strategies to mitigate CDV
infection, particularly at the interface between wildlife and domestic animals. For example, limiting
the contact between wild and domestic species [71] or administering vaccines to domestic animals to
reduce the impact of CDV on populations at risk of extinction [3] have been proposed for different
populations. Although the vaccination of wild carnivore species has been proposed based on successful
examples of vaccination against CDV in different species [28,39,72,73], there are also multiple cases
Viruses 2019, 11, 582 6 of 23

where after vaccination, typical CDV clinical signs have appeared with fatal consequences in different
wild carnivore species [38,74–76], including those at risk of extinction [28,71,77,78]
In these last cases it is not possible to differentiate between vaccine failure—a vaccine that does
not provide protection against wild CDV strains [77,79]—or possible vaccine escape—the ability of
the wild strains to re-infect or reactivate the infection in the presence of neutralizing antibodies that
cannot neutralize the virus, caused by mutations that change surface antigens [80,81]. However, as
the effectiveness of different vaccines (adapted modified live vaccine in canid cells or in eggs) or even
including the canary pox vectored vaccines can vary even between closely related species [38,74,82],
the efficacy, safety, and possible risks of administering vaccines to free-living wildlife should be
critically evaluated to avoid possible lethal consequences, especially in areas of high biodiversity.
Moreover, the handling of the animals to administer the vaccine [83,84] or the vaccine itself [85] may
result in negative immunological consequences for the animals, leaving them susceptible to CDV and
other pathogens.

3.3. Viral Adaptation: the Role of Specialist vs Generalist Strains

The study of the factors determining viral host range is critical for our understanding of the
diversity, evolution, and emergence of CDV and for the potential to predict virus changes in a given
host [86–89]. One of the dominant forces driving virus evolution is mutation, which generate genetic
variation on which random drift and natural selection can act, shaping the genetic structure of virus
populations [86,90].
Concepts such as fitness—defined as ”the capacity of a virus to produce infectious progeny in a
given environment” [90], quasispecies—“collections of closely related viral genomes subjected to a
continuous process of genetic variation, competition and selection” [91], coevolution—“evolutionary
change in a trait of the individuals in one population in response to a trait of the individuals of a
second population, followed by an evolutionary response by the second population to the change in
the first” [92], and antagonistic pleiotropy—defined as ”a beneficial mutation in one environment is
either harmful or neutral in another environment, or mutations that are neutral in the environment in
which they arose are deleterious in another” [93]—have been suggested to play a role on the adaptation
and evolution of CDV in its different hosts.
For example, CDV is thought to have specialized in two major host orders: Caniformia and Feliformia,
in which the virus quasispecies with better host fitness are considered specialized strains [33,93]. Those
have acquired the ability to interact with SLAM receptors of different species, and they have coevolved
with their hosts, thus fixing mutations allowing the virus to specialize in a particular host; thus, antagonistic
pleiotropy in CDV shows that substitutions at the hemagglutinin are related to species jump. However,
the capacity for evasion of the immune response should be studied in the other viral proteins in each
affected host [93,94].
Virus adaptability to different environments can be viewed in terms of an adaptive landscape in
which the peaks and valleys represent fitness estimates of the relative ability of the virus to enter and
successfully replicate in an environment, such as different host species [90]. Functional mutations allow
the virus to move through the fitness landscape, and selection helps fix adaptive mutations [95,96].
CDV cross-species transmission can be viewed within this adaptive landscape (Figure 1) in which
each peak represents the optimized viral fitness of a virus strain in a given host. These peaks are
separated by a fitness gradient because mutations optimizing the ability of a viral strain to successfully
infect a different host can reduce its fitness in the original host, a phenomenon known as antagonistic
pleiotropy [97]. In this evolutionary scenario, viral genetic variation is studied in a coevolutionary
framework to understand the degree to which hosts influence virus diversity.
Viruses 2019, 11, 582 7 of 23

A.
Donor Recipient
species species

Mutation Frecuency

B.

Figure 1. Evolutionary model for cross-species transmission of canine distemper virus (CDV). In the
model, each peak represents the optimized viral fitness in a given host species, and specific mutations
(colored circles) are essential to reach this optimum in the new host. (A) The two fitness peaks of CDV
are separated by a shallow fitness valley, thus few adaptive mutations are required. One mutation
allows the infection of other species but also decreases the fitness in the donor species (light blue circle,
generalist trait). Coevolution with the new species can select for further mutations that optimize the
fitness (dark blue circle, specialist trait). (B) CDV infection in canids and non-canids as an example of
the model presented in (A). A simplified phylogeny of the H gene showing the relationships between
CDV strains from canid and non-canid hosts, indicated at the tip of the tree as black and blue squares,
respectively. The distribution of the amino acid site 549, which has been shown to be involved in CDV
infection in non-canid hosts and is under positive selection, is mapped onto the branches of the tree.
Commensurate with the coloring in panel (A), the substitution of site 549 is shown in light blue because
it is considered a generalist trait that permits the infection of various non-canid species, but with lower
fitness than the specialist trait in canids, marked in black.

A comprehensive comparative genetic study using several complete CDV genomes showed that
all the residues found to be under positive selection in CDV are located within the H protein, which is
the viral protein interacting directly with the SLAM receptor in immune cells [50]. Within the receptor
binding region of the H protein, substitutions for one of these residues (site 549) were systematically
observed in non-canid hosts, pointing to their importance in host adaptation (Figure 1b) [50,94].
It is hypothesized that even when a few substitutions are needed for successful cross-host infection,
the likelihood of extinction of ‘partially-adapted’ viruses (i.e., those in the fitness valleys) should be
high because their fitness is not optimal in either host and therefore would be out-competed by fitter
strains (i.e., those at the fitness peaks) [87]. However, for CDV the results of fitness analyses to assess
the impact of substitutions at site 549 for cell entry and viral replication in canid and non-canid hosts
Viruses 2019, 11, 582 8 of 23

revealed an alternative scenario for the reconstruction of viral evolution when considering fitness
trade-offs across hosts: the evolution of specialist and generalist traits [93,94].
In a homogeneous environment with a single host species, strong coevolution promotes the
optimization of viral fitness in that host (i.e., fitness peaks), resulting in low fitness of the virus
in other species (i.e., fitness valleys). Therefore, specialists should be favored in homogeneous
environments [97]. By contrast, in heterogeneous environments where multiple hosts are available,
the evolution of generalists with the highest average fitness that allow the virus to infect all hosts
should be favored, which results in suboptimal fitness in any single host in comparison to single-species
specialists [98]. Therefore, specialists should have greater fitness in the host species to which they are
well adapted than generalists in that same host [99].
For the case of CDV, the large domestic dog world population could represent such a favorable,
homogeneous environment that permitted the optimization of CDV fitness in this particular host.
Accordingly, fitness assays showed that the CDV-H protein from domestic dog had a higher performance
in cell lines expressing domestic dog than non-canid (African lion and domestic cat) SLAM, as expected
in antagonistic pleiotropy. On the other hand, non-canid CDV-H proteins performed equally well
regardless of which host SLAM (canid or non-canid) was expressed, but the overall performance was
lower than that of domestic dog CDV-H protein in cells expressing domestic dog SLAM. Moreover,
the substitution of site 549 from a canid to non-canid residue in the domestic dog CDV-H protein
showed a reduction in fitness in domestic dog cells [93,94]. Together the results of the comparative
genetic analysis and the fitness assays of CDV in canid and non-canid species allow another view of
the model of virus cross-species transmission (Figure 1) that is consistent with the presence of specialist
(i.e., fitness peak in the domestic dog) and generalist (i.e., shallow fitness valley, possibility to infect
several species) traits (Figure 1) in the relation of the CDV-H protein with the SLAM receptor of canid
and non-canid hosts.
The diverse examples shown above show that the transmission and epidemiology of CDV may be
driven by three different mechanisms: (1) dissemination by means of animals with subclinical infection
which can transmit virus; (2) in urban and peridomestic settings where wild animals come into direct
contact with domestic animals which in this case would be considered reservoirs, and (3) strains that
have adapted to a specific host mainly driven by the existence of specialist vs. generalist strains.

4. CDV and Measles: Complementary Infection Models

Both CDV and MeV belong to the genus Morbillivirus and have the same genomic architecture.
The high nucleotide and amino acid similarities between both viruses results in high levels of functional
and structural conservation, this is why the CDV has been used as a model to understand the
pathogenesis of measles, and often has been used in genetic engineering to develop vaccines and
antiviral treatments [100,101].
Previous studies have shown that morbilliviruses including CDV, phocine distemper, rinderpest
and peste des petits ruminants virus circulate in nature [102] by using two primary receptors: SLAM,
expressed in cells of the innate immune response, and in activated T and B cells [51], and nectin-4,
expressed in polarized epithelial cells [103]. The SLAM receptor is considered the main morbillivirus
receptor because it allows its initial entry into cells of the immune system [104] Comparative genetic
analysis between SLAM sequences from different species—domestic dog (Canis lupus familiaris) and
domestic cat (Felis catus) [105]—revealed four important interaction sites with MeV and CDV including
amino acids at positions 123, 127, 129, and 131 [102,105,106].
The nectin-4 receptor is involved in cell-to-cell propagation of CDV and MeV, and also in cell-cell
fusion (syncytia formation) [103,107]. Four binding sites on the nectin-4 receptor have been identified
(F132, P133, A134, and G135) for the optimal binding to CDV H both in dogs and humans [107]. In both
species, nectin-4 is expressed in epithelial cells [103,108], however it has been found that in domestic
dogs nectin-4 is also found in neurons, ependymal cells, epithelia of choroid plexus, meningeal cells,
granular cells, and Purkinje’s cells, which could indicate its partial involvement in the neurovirulence
Viruses 2019, 11, 582 9 of 23

of CDV observed in dogs [109]. Besides the presence of nectin-4 in the white matter or astrocyte
cultures of dogs has not been confirmed, suggesting the existence of a third cellular receptor [109].
Indeed, it has been hypothesized the presence of a viral receptor, found mainly in astrocytes,
which allows the noncytolytic cell-to-cell transfer of CDV and its spread into the white matter. This
proposed third receptor was named glia R [23], which other authors have reported as the means of
colonization of other morbilliviruses in the brain of dolphins [110]. However, all authors conclude that
this new receptor should be fully identified and characterized in canines because it is very important
for the neurovirulence of CDV and other morbilliviruses [23,109,110].
The SLAM receptor has 39 residues that are involved in the interaction with CDV protein H [105];
when comparing the aminoacidic sequences of the SLAM receptor in different species -human, dog,
raccoon, mink, fox, cow, mouse- an identity of 57.2% is observed between the human SLAM and the
dog, fox and cow receptors. Likewise, there are an amino acid identity of 93.1% between nectin-4
receptor in humans and dogs [101]. The possible explanation for the inability of CDV to infect humans
and domestic cats could be the differences in certain residues that alter the SLAM interaction region
determining a low affinity interaction between the cellular receptor and CDV protein H [105].
Naturally acquired and experimental inoculation of CDV in non-human primates have shown high
neuropathogenicity potential in those animals with severe encephalitis [32,111–113]. Thus, the ability
of the virus to adapt to human cells and to infect humans is subjected to speculation [3,114]. It has been
established that CDV can gain the ability to infect human cells by acquiring certain specific mutations
in the protein H [106,115,116]. Vero cells expressing human SLAM were infected with CDV and in
the third passage syncytia formation was observed; when analyzing substitutions of the protein H a
single amino acid change was found (Table 1) which allowed the CDV-H to use the human SLAM
receptor [106]. On the other hand, the human cell line H358 (expressing Nectin-4 receptor) was infected
with CDV and authors find mutations in H and other proteins at the eighth passage [116] (Table 1).
In another study by Otsuki et al. [115], the authors showed that CDV was adapted to the two human
receptors in H358 and Vero-human-SLAM cells by acquiring amino acid substitutions in the V/C, F and
H proteins after 8 culture passages [115]. Taking together, these results allow to presume that, at least
at the receptor level, humans are susceptible to CDV infection, however further studies must be done
in order to establish if human cells could be also permissive to CDV replication [106,115].

Table 1. Amino acid substitutions in the CDV H protein reported in strains adapted to human cell lines.

CDV
Author
V Gene F Gene P Gene H Gene
Y267C C116Y M267V M548T Otsuki et al. [115]
D540G Bieringer et al. [106]

Given the identity (86%) between the human and non-human primates SLAM receptor [13] and
the similar pathogenesis between CDV and MeV, CDV infected animals have been used as study
models for MeV replication in vivo. However, this similar pathology opens the question if CDV can
cause the same pathogenesis in humans [117]. In the mid-1990s, an attempt was made to establish a
possible association between CDV with multiple sclerosis by finding CDV antibodies in patients with
multiple sclerosis [118].
It is well known that experimental MeV vaccination in pups usually leads to an immune response
with cross-reactive activity to CDV even in the presence of maternal antibodies [119–122]. The produced
antibodies recognize epitopes conserved between MeV and CDV thereby conferring a certain level of
protection [123,124]. Therefore, it is possible to hypothesize that immunity against MeV may have
protected humans against CDV infection or at least avoided the presentation of clinical signs [116].
In non-human primate models infected with CDV and vaccinated against MeV, the antibodies generated
in these animals partially cross-react against CDV protecting them from the clinical disease [102,125].
The possibility that MeV vaccination stops in the future in several geographical regions due to the
Viruses 2019, 11, 582 10 of 23

success of the eradication program, allows us to think that the CDV can possibly jump the species barrier,
infect humans and cause clinical disease in humans similar to that already reported in non-human
primates [106,115,125].

Genetic Variability of CDV

As mentioned earlier, CDV protein H determines the cellular tropism and host range when
interacting with the SLAM receptor in host lymphoid tissues [13] and with nectin-4 in epithelial
tissues [103]. Therefore, the H gene has the highest variability and thus it has been widely used in
genetic and phylogenetic analyses [126,127].
Studies using the H gene show a worldwide distribution of CDV genotypes, classifying the circulating
strains into 17 lineages (Figure 2): America-1 (vaccine strains), America-2, America-3, America-4,
America-5 Arctic-like, Rockborn-like, Asia-1, Asia-2, Asia-3, Asia-4, Africa-1, Africa-2, European Wildlife,
Europe/South
Viruses 2018, 10,America-1,
x South America-2 and South America-3 [33,57,59,80,81,128–133]. 10 of 22

Figure2.2. Phylogenetic
Figure Phylogeneticrelationship between
relationship 69 CDV
between 69 strains based onbased
CDV strains the Hon genethesequence.
H geneThe sequence.
phylogenetic
The phylogenetictree tree
was was
obtained by the by
obtained Maximum likelihood
the Maximum method with
likelihood a Bootstrap
method with a of Bootstrap
1000 based of 1000
on the
based on evolution model
the evolution T92 +T92
model G, +69G,
sequences were analyzed
69 sequences with a with
were analyzed total aoftotal
1824ofnucleotides.
1824 nucleotides.
Evolutionary analyses were conducted in MEGA7.
Evolutionary analyses were conducted in MEGA7.
These lineages are defined according to the amino acid divergence of the H protein between the
These lineages are defined according to the amino acid divergence of the H protein between the
different strains. Thus, it is considered that two given strains belong to a certain lineage when the
different strains. Thus, it is considered that two given strains belong to a certain lineage when the
protein H amino acid divergence is less than 3.5% [134]. On the other hand, following the guidelines
protein
basedH onamino acid divergence
MeV studies, sub-lineageisstrains
less than 3.5%
present [134]. On“bootstrap”
a minimum the other hand,
value following
of 70% andthe guidelines
at least
based
98%on MeV studies,
of aminoacidic sub-lineage
identity [61]. strains present a minimum “bootstrap” value of 70% and at least
98% of Evolutionary
aminoacidic comparative
identity [61].analyses of both virus receptor-binding proteins and host receptor
Evolutionary
proteins may help comparative analyses of
elucidate virus–host both virus receptor-binding
co-evolutionary proteins comparative
dynamics. Additionally, and host receptor
genomics in a phylogenetic framework permits the assessment of evolutionary forces
proteins may help elucidate virus–host co-evolutionary dynamics. Additionally, comparative that influence
genomics
genetic variation in those interacting regions reflecting virus–host arms races [89,96].
in a phylogenetic framework permits the assessment of evolutionary forces that influence genetic Comparative
genetic in
variation studies
thoseincluding
interactingseveral complete
regions genomevirus–host
reflecting sequences of CDV
arms showed
races that certain
[89,96]. residuesgenetic
Comparative
are under positive selection, almost all located in the receptor binding region of the H protein [50,93].
studies including several complete genome sequences of CDV showed that certain residues are under
In particular specific substitutions in sites 530 and 549 were systematically observed in non-dog
positive selection, almost all located in the receptor binding region of the H protein [50,93]. In particular
species pointing their importance in host adaptation [50].
Domestic dogs have eight possible substitutions at position 530 (G/D/N/E/R/S/A/K) and two
substitutions at 549 (H/Y) present in at least ten lineages; in non-canid wild animals there are seven
substitutions at position 530 (G/D/N/E/R/C/V) and two substitutions at position 549 (H/Y) present in
seven lineages. On the other hand, wild canids have three substitutions at position 530 (G/N/R) and
two substitutions at position 549 (H/Y) that are present in five lineages. (Table 2) [135].
Viruses 2019, 11, 582 11 of 23

specific substitutions in sites 530 and 549 were systematically observed in non-dog species pointing
their importance in host adaptation [50].
Domestic dogs have eight possible substitutions at position 530 (G/D/N/E/R/S/A/K) and two
substitutions at 549 (H/Y) present in at least ten lineages; in non-canid wild animals there are seven
substitutions at position 530 (G/D/N/E/R/C/V) and two substitutions at position 549 (H/Y) present in
seven lineages. On the other hand, wild canids have three substitutions at position 530 (G/N/R) and
two substitutions at position 549 (H/Y) that are present in five lineages. (Table 2) [135].
Recent analyses of the CDV strain responsible for the outbreak in lions of Serengeti National Park
in the 1990s demonstrated that all the species affected (including dogs and wild canids) presented the
530D substitution [33], agreeing with Liao et al. [135] who reported the 530 site is not directly related
to host adaptation because it is conserved in different species. In addition, although an association
has not been statistically established, it has been shown that dogs have a greater tendency to present
549Y, while substitution 549H occurs most commonly in wildlife, which shows a possible association
between this substitution and the affected species [94].
Likewise, studies conducted in China characterizing different CDV isolates in mink breeding sites
(non-canid wild species), raccoon dogs and foxes (wild canid species) showed the trend 530G and
549H [137]. In monkeys (non-canid species), there was a trend of 530G and 549Y with an additional
substitution at another site of the protein H (542F) that may play an important role in CDV adaptation
to primates (Table 2) [137].
However, in an outbreak in urban raccoons (non-canid species) in Europe, researchers found the
trend 530G and 549Y, and concluded that the Y549H substitution apparently is not essential to facilitate
infection in wildlife other than canids. The explanation for this outbreak was that urban raccoons are
in contact with foxes (wild canids), which are widespread species in urban areas thus having great
contact with domestic dogs. Therefore, foxes act as a wild reservoir of domestic dog strains in which
the substitution 549Y is predominant [136].
Contributing to this debate, there was an 11.5% presence of the Y549H substitution in dogs from
Brazil. In addition, all the dogs presented 530G and 580R, the latter representing 89% of the circulating
strains (Table 2). The authors suggest that this trend may be associated with the condition of the “stray”
dogs that were sampled, who are more likely to have contact with wildlife acting like reservoirs in
these ecosystems [126].
Regarding to wildlife, more sequences of the H gene are needed to determine the trend of the presence
of substitutions at positions 530 and 549 because the 549H substitution has not been reported in the
lineages of Asia, South America, and Africa due to the lack of studies in wild species at these areas [94].
With respect to CDV jump to humans, the literature reports that substitutions D540G and M548T in
the protein H, besides Y267C in the protein V, C116Y in the protein F, and M267V in the phosphoprotein
have allowed cell invasion in H358 cells (Table 2). However, different authors suggest there must be
mutations in other genes allowing the intracellular adaptation of CDV [106,115]. We point out that
CDV complete genome sequences must be evaluated to identify other substitutions that can define its
ability to jump between hosts, because most studies aimed to explain an adaptation process from a
single component (protein H) ignoring the genetic changes in other regions that may contribute to the
inter-species jump and adaptation.
Viruses 2019, 11, 582 12 of 23

Table 2. Summary of amino acids present at different positions of the H protein interacting with the SLAM receptor.

H Gene
530 549 Animal Lineage Author
G Y Dogs Europe, Asia 1, America 2, Arctic-like, Asia 2 Nikolin et al. [94], Liao et al. [135]
G Y Wild canids Europe, Asia 1, Nikolin et al. [94], Liao et al. [135]
G Y Wild species (other than canids) Europe, Asia 1, Nikolin et al. [93], Rentería-Solís et al. [136], Liao et al. [135]
G H Wild canids Europe, Asia 1, Nikolin et al. [93], Panzera et al. [54], Liao et al. [135]
G H Wild species (other than canids) Europe Asia 1, America 2 Nikolin et al. [93], Panzera et al. [54], Liao et al. [135]
G H Dogs America 2, Europe, South America 1 Nikolin et al. [93], Liao et al. [135], Fischer et al. [126]
D Y Dogs European Wildlife, South America 2 Nikolin et al. [93], Liao et al. [135]
D Y Wild species (other than canids) European Wildlife, Africa 2 Nikolin et al. [33]
D Y Rockborn-like, Candur, China, Sweden Vaccine Nikolin et al. [93], Liao et al. [135]
N H Wild canids European Wildlife Nikolin et al. [93]
N H Dogs European Wildlife, America 1 Liao et al. [135]
N H Wild species (other than canids) America 1 Rentería-Solís et al. [136], Liao et al. [135]
D H Wild species (other than canids) European Wildlife, Africa 2 Nikolin et al. [93], Rentería-Solís et al. [136], Liao et al. [135]
C H Wild species (other than canids) European Wildlife Nikolin et al. [93],
N Y Dogs Arctic-like, Africa 1, America 1 Nikolin et al. [93], Liao et al. [135]
N Y Wild species (other than canids) America 1 Nikolin et al. [93], Liao et al. [135]
N Y Wild canids Arctic-like Liao et al. [135]
E Y Dogs Asia 2 Nikolin et al. [93], Liao et al. [135]
E H Wild species (other than canids) Asia 2 Rentería-Solís et al. [136]
R Y Wild canids Asia 2 Nikolin et al. [93]
R Y Dogs Asia 2 Liao et al. [135]
R H Wild species (other than canids) America 2 Nikolin et al. [93], Liao et al. [135]
R H Wild species (other than canids) America 2 Rentería-Solís et al. [136],
S H Vaccines Nikolin et al. [93], Liao et al. [135]
S L Vaccines Liao et al. [135]
S Y Dogs Arctic-like Liao et al. [135]
A Y Dogs Asia 1 Liao et al. [135]
V H Wild species (other than canids) European Wildlife Nikolin et al. [93], Liao et al. [135]
K Y Dogs Asia 2 Liao et al. [135]
Viruses 2019, 11, 582 13 of 23

Table 2. Cont.

Other Positions Reported in the H Gene

542 549 Animal Lineage Author
F Y Wild Species (Other Than Canids) Asia 1 Zhao et al. [137]
Animal Lineage Author
530 549 580
G H Q Dogs South America 1 Fischer et al. [126]
276 392 542
V R F Wild species (other than canids) Asia 1 Feng et al. [138]
530 519 549
N R Y Dogs South America 3 Espinal et al. [129]
Other Positions Reported in the H and V Genes
GENE
GENE V Animal Lineage Author
H
519 149 134
I H S Wild species (other than canids) Africa 2
R Y G Wild canids Africa 2
R H G Wild canids Africa 2 Nikolin et al. [33]
R Y G Dogs Africa 2
I H Wild species (other than canids) America 2
Viruses 2019, 11, 582 14 of 23

5. Evolutionary Rates and Molecular Clock of CDV

Different studies have tried to elucidate the time of the most recent common ancestor (tMRCA) for
CDV [11,54,126,139]. However, the number of sequences available in the GenBank throughout years
has led to diverse results, making the data from different studies not comparable. A first approach,
published in 2008, estimated the tMRCA for CDV to happened 58 years ago using a sequence from 2001
as reference, suggesting that the ancestor existed in 1943, with an interval from 1894 to 1974 and 95%
higher probability density (HPD) [11]. Next, a tMRCA was estimated at 125 years by analyzing a strain
from 2011, corresponding to the year 1886 with an interval from 1858 to 1913 and a 95% HPD [54].
In Taiwan, the tMRCA was established in 1945, with an interval from 1918 to 1966 and a 95% HPD [139].
Likewise, in Brazil, another tMRCA was found to be 92 years from 1919, with an interval from 1899 to
1944 and a 95% HPD [126].
The differences found in the tMRCA studies for CDV are ascribed both to the difference in the
number of sequences evaluated in the different studies (from 35 to 208 sequences of the H gene) and
to the geographical and temporal diversity of those sequences. In this sense, it is important to note
that the intervals intersect and that more reliable results will be obtained by including sequences with
greater temporal and spatial divergence in the analysis. A time-measured Bayesian maximum clade
credibility (MCC) tree for the CDV H gene is presented in the Figure 3 by using 69 CDV H sequences
retrieved from Genbank.

Figure 3. Time-measured Bayesian maximum clade credibility (MCC) tree for the CDV H gene using
BEAST [140]. The time of the most recent common ancestor (tMRCA) of CDV is shown using 68
available sequences of the H gene for the lineages reported to date. Posterior probability limit: 0.7 are
showed on nodes.

CDV evolutionary rates (substitution rates per site per year [subs/site/yr]) for the H gene were
estimated in 4.8E-4 subs/site/yr, with 95% HPD and a range of 3.9 × 10−4 to 5.9 × 10−4 subs/site/yr,
Viruses 2019, 11, 582 15 of 23

representing a high evolutionary capacity [54]. Other substitution rates have been reported, including
11.65 × 10−4 and 7.41 × 10−4 subs/site/yr [11,139]. This shows that substitution rates of CDV are slightly
higher compared to other morbilliviruses such as MeV (6.5 × 10−4 subs/site/yr) [11]. The high genetic
variability of CDV may have facilitated the inter-species jump, and its adaptation and specialization to
other hosts [93]. It is important to remember that tMRCA studies for CDV and for other viruses are
based on available sequences in the Genbank, generating a bias that must be taken into account in the
analysis of the results, since it can not necessarily coincide with the historical records present in the
literature as has been recently discussed by others [141].

6. Pathogenicity Predictions
Genetic approaches are one of the multiple tools used to analyze viral spread and pathogenesis.
Association predictions between morbidity and lethality related to the presence of specific substitutions
at certain genomic positions have been presented by several authors [33,126,139]. It is important
to note that these predictions are hypotheses that arise from the analysis of the sequences obtained
by the authors and that due to the lack large epidemiological studies no statistical association has
been established.
In the non-canid CDV strains from the 1993–1994 Serengeti outbreak a new substitution in the
H protein (R519I) was associated with the presence of clinical signs in these wild species (African
lion and spotted hyena). In this study, the authors evaluated the presence of various combinations
of substitutions at positions 519 and 549 of the protein H in strains from domestic dogs, wild canids,
and non-canids wild species. They found that a rare combination of substitutions in these positions
(519I and 549H) was only observed in non-canid strains and was related to a fatal outcome only in
non-canids (lion and hyena). The other combinations found (519R/549Y and 519R/549H) caused death
only in domestic and wild canids (Table 2) [33].
Similarly, in a Brazilian study all the strains isolated from dogs harbored the substitution 549H
causing the death of every animal, with a statistically significant association (p < 0.05). In addition,
the authors concluded that this substitution is associated with the creole breeds (p < 0.05) [126]. In dogs
from Medellin, Colombia, our research group reported the presence of CDV of the South America−3
lineage and the combination of 549Y, 519R, and 530N; one dog presented 549H (Table 2). All of these
cases showed evidence of nervous symptomatology and respiratory disease with fatal outcomes [129].
The mutations association studies of the H gene are the first performed for CDV, and should be
extended to the study of the complete genome to explain the pathogenesis in the various susceptible
species. It has been reported that mutations in intergenic regions, and in the 50 and 30 UTRs can affect
the virulence and pathogenicity of field strains [142]. Specifically, mutations in CDV M–F intergenic
region were associated with a different viral phenotype [5], highlighting the importance of conducting
association studies with complete genomes.

7. H Gene vs. Complete Genome

There is controversy about which position substitutions in the H gene are associated with CDV host
adaptation and pathogenicity in the various affected species, which has led the scientific community to
conclude that more sequences of the H gene in wildlife should be obtained to clarify this point [94].
As was mentioned, it is necessary to broaden the search for molecular markers to explain relevant
aspects such as the species barrier jump and the pathogenicity of field strains. Studies on CDV infection
in human cells have characterized the complete genome of these adapted strains, and in addition
to the H gene sites, substitutions associated with host adaptation and immune system evasion were
found in other genes [115]. Additionally, other authors have characterized the complete genome of
wildlife strains and have reported important substitutions (at CDV-H 519/549) that could explain the
adaptation, immune system evasion, and the presence/absence of clinical signs in these animals [33].
Also, studies of CDV complete genome have reported homologous recombination between the P,
H, and L genes in vaccine and field strains from different hosts [143]. In addition, phylogenetic studies
Viruses 2019, 11, 582 16 of 23

from the complete genome reflect a more accurate strain classification [144]. Likewise, all glycosylation
sites that may mask antigenic epitopes—which could explain vaccine failures—can be established as
other sites of positive, neutral, or negative selection in CDV membrane proteins [57,143,145].

8. Conclusions
Viruses with a broad host range such as CDV are highly contagious and have high morbidity and
mortality in wild and domestic animal populations. Epidemiological data from around the world
show that CDV has become an important threat for conservation of multiple species even outside of
the Carnivora order. The lack of ecoepidemiological information of CDV transmission beyond the
infection between dogs, has led to investigate the importance of the infection in a multihost scenario
that is not totally yet understood. The capability of the virus to adapt to multiple species and evolve in
those, makes CDV not only a well-known pathogen of domestic dogs, but one of the most important
pathogen of mammals with the ability to jump the species barrier and perhaps even infect humans.
The different transmission scenarios discussed in this paper are an attempt to elucidate the transmission
of CDV in different environments, and encourage uniting multiple areas of research for understanding
the intricate epidemiological dynamics of CDV in multiple hosts.
The H gene is the basis for the phylogenetic classification of CDV due to the high evolutionary
pressure to which its protein subjected. The H gene study has also allowed researchers to infer key
amino acid substitutions involved in the inter-species jump. The developing of new studies of the CDV
complete genome would enable us to explain in detail its evolution and to establish substitution rates,
glycosylations, and homologous recombination points that would explain the pathogenicity, species
jump ability and vaccine failure of this virus.

Author Contributions: J.R.-S. conceived the study; J.D.-V., X.A.O.-C., N.S. and J.R.-S. were involved in all other
aspects of the study: data collection, data analysis, drafting and editing the paper. All authors read and approved
the final manuscript.
Funding: This work was financially supported by the Departamento Administrativo de Ciencia, Tecnología e
Innovación–COLCIENCIAS Grant No. 123171249669 (Grant to J.R.-S.).
Conflicts of Interest: The authors declare no conflict of interest.

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