Review paper on the recent divergence and historical evolutionary patterns of CPV and CIV

1.
Kara Moloney May 3, 2016
BIOL.526 Evolutionary Biology
The Evolution of Viral Pathogens in Veterinary Medicine: Canine
Parvovirus and Canine Influenza
Abstract
Infectious diseases caused by viral pathogens are commonly encountered in the field
of veterinary medicine. Considering the rapid rate at which viruses mutate and evolve, one
can foresee the formidable challenge veterinary professionals around the world face in
treating their patients. Canine parvovirus (CPV) is an enteric virus that spreads rapidly
through canine populations. Despite the widespread use of the parvovirus vaccine, a high
frequency of outbreaks still occur worldwide. The original form of the virus has diverged
multiple times, with a number of unique variants now able to infect canine populations.
Another newly emerging canine virus, canine influenza virus H3N8, is an influenza A virus.
Canine H3N8 is believed to have been was transmitted from equines to canines at racing
tracks in the U.S. a decade ago. This virus has yet to cause as many violent outbreaks as
CPV, however the threat of an epidemic still looms. Both of these viruses have undergone a
multitude of evolutionary adaptations to increase their transmissibility and broaden their
host range. Herein I present a brief review of the current literature on this topic.
Introduction
Viral evolution often occurs at a rapid rate, as pathogen and host are constantly
locked in an evolutionary “arms race”. This so‐called Red Queen scenario leads to a high
rate of viral evolution as the pathogen attempts to overcome the host’s defences.
Mutations that are advantageous in overcoming the host’s immune system are naturally

2. Moloney 2
selected for, as they improve viral fitness. Other ways a virus mutates to increase its
transmission rate (and therefore its fitness) is by evolving to infect new host organisms. The
influenza A virus is a classical example of a pathogen that frequently crosses species to
infect multiple types of hosts. The unique segmented genome of the virus as well as
coinfection due to high instances of intraspecies contact give the virus multiple
opportunities to infect a wide variety of species such as avians, swines, humans, equines,
and canines. (Crawford et al. 2005, Song et al. 2008)
The field of veterinary medicine provides a unique set of challenges for doctors and
their canine patients. Infectious viruses such as canine parvovirus and, more recently,
canine influenza virus have emerged in regional and worldwide populations. Canine
parvovirus is believed to have evolved from feline form of the virus, feline panleukemia
(Hueffer et al. 2003). This often fatal enteric virus remains pandemic in canine populations
across the globe despite efforts to develop vaccines and improve hygiene protocols. The
frequent divergence of new strains of canine parvovirus as well as the ability for wildlife to
act as a reservoir for the pathogen also pose unique challenges in eradication.
The H3N8 strain of canine influenza virus emerged in 2004 and is believed to have
evolved from the equine version of the virus (Crawford et al. 2005). Like most influenza A
viruses, these cross‐species jumps are not uncommon and can lead to acute outbreaks.
However, unlike canine parvovirus, the canine version of influenza A has yet to cause
massive nationwide or global outbreaks, despite the fact that a small minority of dogs are
vaccinated against the pathogen. There are a variety of biological, geographical, as well as
sociological factors that account for the abnormal distribution of this virus.

3. Moloney 3
Herein, a sampling from the literature on the recent divergence and evolutionary
patterns of canine parvovirus and canine influenza subtype H3N8 is reviewed and analyzed.
Canine Parvovirus
Canine parvovirus (CPV) emerged fairly recently in the companion animal population.
CPV was first isolated in 1978 (Parrish 1990) and has since undergone a number of dramatic
mutations. The strain that became globally pandemic in canine populations at that time
was termed CPV type 2 (CPV‐2) (Hueffer et al. 2003). The virus was thought to have
evolved from its analogous feline form, feline panleukemia virus (FPV) (Truyen et al. 1995),
which shares up to 90% of its genome with CPV (Hueffer et al. 2003). CPV is a
single‐stranded non‐enveloped DNA virus with an icosahedral structure (Wang et al. 2016).
Its 5.2 kb genome encodes for VP1 and VP2, capsid proteins that have been thought to play
major roles in viral host specificity ( Hueffer et al. 2003, Perez et al. 2012). Infection with
the virus is characterized by hemorrhagic diarrhea as well as lethargy, vomiting, and the
presence of a fever and commonly presents in puppies (American Veterinary Medical
Association) and has shown a mild degree of breed specificity, though the underlying
mechanisms responsible for this breed preference are unclear. Vaccines for CPV are
available and are routinely administered by veterinarians as part of yearly wellness exams
for all companion animal canines. To help deter the spread of the virus, a CPV vaccination
is often a requirement of dogs intended to be boarded, participate in training classes, or to
attend “doggy daycare”. However, due to the rapidly evolving nature of the virus, vaccines
are not always entirely effective (Wang et al. 2016).

4. Moloney 4
Evolution of Novel Strains of CPV
There are currently three predominant antigenic variants of CPV‐2 found circulating
in the canine population: CPV‐2a, CPV‐2b, and CPV‐2c (Wang et al. 2016). The highest
variability between these strains lies in the viral capsid protein, VP2 (Calderón et al. 2011).
VP2, which makes up a large percentage (90%) of the total viral capsid (Xu et al. 2013), is
involved with host binding specificity and pathogenicity (Calderón et al. 2011). This protein
has been observed to be under high selection pressure and evolves rapidly (Xu et al. 2013).
Phylogenies of CPV are often constructed based on which variant of VP2 is present in a
population. In a 2013 study, Xu et al. identified a monophyletic group of CPV in a
population of Chinese dogs. In this experiment, fifty‐eight samples of rectal swabs were
collected from dogs who presented with classical CPV symptoms. Twenty‐seven of these
samples tested positive for CPV‐2. One sample was identified as the CPV‐2b strain, and the
remaining were typed as CPV‐2a strains. From the positive samples, the full‐length VP2
gene was then amplified using PCR and sequenced. The researchers analyzed the amino
acid substitutions in the VP2 gene and found three distinctive mutations in the differing
strains. With this data and reference strains of CPV‐2, ‐2a, ‐2b, and ‐2c, the researchers
constructed a phylogenetic tree based on maximum likelihood. The ratio of synonymous to
nonsynonymous amino acid substitutions in the VP2 genes evaluated in this study indicate
that the gene is evolving under strong positive selection. The amino acid substitutions
observed in VP2 affect the host range, transmission, and infection of CPV. The phylogenetic
tree data revealed that most of the Chinese strains occupied a large shared branch, with
differences existing between individual samples. Based on these findings, the authors

5. Moloney 5
conclude that CPV is rapidly evolving in China as the virus evolves to best suit its
environment.
In a 2011 study looking at an Argentinian canine population with CPV, authors
Calderon et al. aimed to discover why there was a high incidence of CPV‐vaccinated canines
in that area presenting with the virus. To shed some light on the problem, the researchers
collected 75 canine fecal samples positive for CPV. The full‐length VP2 gene from eleven
positive samples and a live attenuated vaccine was extracted and amplified by PCR. The
amplified DNA was then sequenced and identified by strain. CPV types‐2, ‐2a, ‐2b, and ‐2c
were all isolated from the samples. A phylogenetic tree using the neighbor‐joining method
was then constructed based on the sequenced VP2 data as well as data collected from
international strains of CPV. Relationships between global cases shown on the tree indicate
that Argentinean CPV‐2a and CPV‐2b strains had underwent direct contact and gene sharing
with analogous strains found abroad. A selection pressure analysis revealed that the VP2
gene of CPV is evolving by means of negative selection. The authors surmise that the rise in
CPV cases in vaccinated dogs could be attributed to the recent evolution and global spread
CPV type‐2c strain. They conclude by proposing that CPV vaccinations should be
refashioned with the addition of new adjuvants more effective to protect against this
strain. The authors also add that new vaccine schedules should be developed to best
control the spread of newly emerging CPV variants.
CPV and Host Specificity
Mutations in the VP2 gene of CPV can affect what type of host the virus is able to
infect (Calderón et al. 2011). The VP2 protein, located on the outside facing of the viral

6. Moloney 6
capsid (Hueffer et al. 2003), relies on interactions with the cellular receptor transferrin
receptor type‐1 (TfR) to successfully infect a host (Hueffer et al. 2003). In a 2003 study,
Hueffer et al. aimed to test the ability of canine and feline TfR to successfully cause
infection of CPV or feline panleukemia virus (FPV) in cells of canine origin. In this
experiment, researchers exposed canine Cf2Th cells that were engineered to express either
canine TfR or feline TfR to CPV‐2, CPV‐2b, FPV, or CPV type 2‐G299E strains. Their results
concluded that canine cells expressing the feline form of the transferrin receptor
successfully became infected with FPV as well as CPV type‐2 and CPV type 2‐G299E
mutant. Canine cells expressing the canine TfR became infected with CPV type 2 and 2b.
However, the presence of the canine TfR on the cell surface did not allow for the infection
of FPV. This implies that the canine TfR plays a major role in viral host specificity. The
finding that cells expressing feline TfR are susceptible to not only contracting FPV, but also
a variety of different strains of CPV implies that CPV could have diverged from an FPV‐like
virus at some point in its evolution. The authors conclude that while host‐switching by DNA
viruses such as CPV not a common event, understanding the mechanisms in which the virus
jumps species is key to preventing future outbreaks.
In a more recent study conducted on a wide variety of wildlife populations in the
United States, Allison et al. (2014) aimed to determine the mechanism in which
parvoviruses switch hosts both​in vitro​and in nature. The researchers passaged various CPV
and FPV strains through six different animal cell lines all from the order Carnivora. DNA
from the second, fifth, tenth, and twentieth passages was isolated and the VP2 capsid
protein gene was amplified by PCR. The resulting sequences were then analyzed to
determine the gene's mutational timeline. The authors also built a maximum‐likelihood

7. Moloney 7
phylogenetic tree based on full length VP2 sequences collected from the tissue samples of
68 wild carnivores as well as 275 complete VP2 sequences. The resulting data led to many
conclusions. Based on DNA data collected from 18 different species of animals from 30 U.S.
states, the authors were able to surmise that parvoviruses exist in a vast number and wide
variety of wild carnivores across the United States. The phylogenetic tree confirmed the
notion that the virus is present at a high frequency in wild carnivore populations, and often
exhibits host switching. CPV sequences found in wildlife were also found to be closely
related to domestic carnivores (companion animal cats and dogs). The authors also noted
that a certain region of the VP2 capsid gene (position 300) was continuously undergoing
mutations as it passed through different host species. The authors believe that the VP2
mutations observed at position 300 were undergoing parallel evolution. The capsid
mutations that allow these parvoviruses to adapt to new hosts seem to positively influence
viral fitness as well. It is then safe to assume that cross‐species transmission of parvoviruses
between members of the family Carnivora will continue to exist in wildlife and domestic
animal populations across the United States until strict control measures as well as more
effective vaccinations for companion animals are developed.
Canine Influenza Virus
Canine influenza virus (CIV) H3N8 emerged much more recently than Canine
Parvovirus and is currently enzootic in 41 US states, to date (Zhu et al. 2015, Merck Animal
Health). Much like the influenza strains that infect humans, CIV causes symptoms of
respiratory distress and fever in canines infected with the virus. CIV is classified as an
influenza A virus. It has a segmented, negative‐sense RNA genome that encodes for up to 14
proteins (Zhu et al. 2015). CIV is believed to have evolved from the Equine Influenza Virus

8. Moloney 8
(EIV) H3N8 sometime around 2004 (Crawford et al. 2005). Interactions between equines and
racing greyhounds at tracks are believed to be the probable cause of EIV's transmission to a
new host species. Vaccines have been developed against the H3N8 variant of CIV, but are
not strictly enforced by veterinary professionals. Another CIV that relaxed its host
specificity to infect canines, H3N2, was isolated in South Korea in 2007. This CIV is believed
to have evolved from an avian host (Song et al. 2008).
Evolution of CIV in Conjunction with EIV
By virtue of having a segmented RNA genome, CIV H3N8 carries a high mutation and
genetic reassortment rate (Zhu et al. 2015), as is the case with most Influenza A viruses. In
a 2010 study conducted using samples provided by the Animal Health Diagnostic Center at
Cornell, Rivailler et al. (2010) aimed to track the evolution of CIV H3N8. Viral RNA was
extracted from nasal swabs collected by the Animal Health Diagnostic Center at Cornell and
assayed for influenza virus using real time reverse transcriptase PCR. Isolated viral RNA was
extracted, amplified, and sequenced as DNA with reverse transcriptase PCR. The authors
then constructed individual phylogenetic trees for each of the virus' eight genes using the
neighbor‐joining method. Twenty‐nine canine and seven equine specimens collected from a
variety of distinct regions of the United States between the years 2005 to 2008 were
ultimately used to formulate the final results. Data from the constructed phylogeny implies
that, at the time of sampling, the virus occupied a few distinct geographical regions across
the United States. New York yielded the highest number of positive CIV samples, which was
thirteen. Four of the viral isolates were taken from racing dogs. Data from the eight gene
trees indicate that CIV and EIV evolved forming two separate clades in each of the viruses'

9. Moloney 9
respective species. This data also indicated that the seven EIV cases included in the data
had all recently diverged from from the previous year's strain. Comparative analysis based
on topology from all eight gene trees illustrate that the genes for EIV and CIV co‐evolved in
an independent manner, with no reassortment events. This implies that following the 2004
mutation that allowed EIV to infect canines, no cross‐species transmission of viral genetic
material has since taken place. The authors point out that this finding may be erroneous
due to a number of reasons. Lack of contact existed between the two species, as canines
were clustered in urban areas and equines were located in more rural areas. Another
reason to explain the apparent independent viral evolution is that while the virus may have
been present in any given canine or equine, the animal may have failed to display
respiratory symptoms severe enough to indicate that infection had taken place.
Evolution of CIV H3N8 Hemagglutinin Gene
The hemagglutinin (HA) protein of Influenza A viruses such as CIV is an envelope
glycoprotein located on the outside surface of the viral particle (Koday et al. 2016). This
protein plays an important role in viral entry into host cells (Ratnayake et al. 2016).
Temporal surveillance of the mutations the HA gene of a virus undergoes is often used to
track its evolution. In human influenza viruses carrying the HA subtype 3, antigenic drift is
thought to be responsible for the gene’s mutation (Smith et al. 2004). A 2014 study
published by Pecoraro et al. (2014) aimed to investigate the evolution of CIV H3N8 HA (H3)
genes. Nasal swabs were collected from shelter dogs located in major urban areas of the
United States. The geographical method of sample collection was intentional. Samples
came from three areas: 1) states where CIV has been described as endemic (CO, FL, NY), 2)

10. Moloney 10
states where CIV had yet to be detected in canine populations, and 3) states with a few
reported cases. Nineteen samples were chosen to be used. The H3 DNA of these nineteen
samples was amplified and isolated using real‐time reverse transcriptase PCR. A phylogeny
of 62 CIV H3 amino acid sequences was constructed using the maximum parsimony method.
Data from the tree suggested that H3N8 has mainly diverged with respect to geographical
distribution, with unique lineages forming in New York and Colorado. The authors also
suggest that based on amino acid substitutions observed specifically in antigenic regions of
H3, antigenic drift may also be acting as a possible mechanism of viral evolution.
Transmission and Distribution of CIV in the United States
Since emerging over a decade ago, CIV H3N8 has yet to become a nationwide
epidemic, despite the fact that there are many immunologically naive dogs in the United
States. In an in‐depth population dynamics analysis using data provided by shelters across
the United States, Dalzeil et al. (2014) investigated the mechanisms underlying the
“patchy” distribution of CIV H3N8 in the country. A phylogenetic tree using the maximum
likelihood method was constructed based on the HA1, NP, and M proteins of CIV H3N8.
Gene sequences were acquired from multiple sources, including GenBank and the Animal
Health Diagnostic Center at Cornell University. To create an accurate timeline of when
strains diverged, the authors included an uncorrelated lognormal relaxed molecular clock
model in their analysis. Data was additionally treated with phylogeny‐trait association
statistics to account for sampling errors that would skew the results regarding the
geographical distribution of the virus. The resulting phylogeny revealed that H3N8 CIV is
distributed regionally, with endemic areas located in New York and surrounding regions,

11. Moloney 11
Pennsylvania, and Colorado. The authors then simulated CIV H3N8 epidemics by
extrapolating upon population data from shelters. These hypothetical situations led to the
conclusion that, due to the poor transmission efficacy of the virus, it can only be sustained
in shelters with larger than average populations. The authors note that the average census
of a dog shelter in the U.S. is 43. Other factors that contribute to the erratic distribution of
CIV H3N8 include low rates of replication of the virus in its host species, which would lead
to reduced transmission rates among populations of canines. Another reason for “patchy”
virus distribution is due largely to shelter dynamics. These dogs stay in the shelter, on
average, between 10 to 15 days. Because of this, contact rates between susceptible hosts
are lower than necessary to sustain the virus in a population of that size. Therefore, the
authors conclude that CIV H3N8 exists in the canine population at a rate of near extinction,
but may continue to persist in certain geographic hotspots identified by the phylogenetic
tree.
Conclusion
Canine Influenza Virus and Canine Parvovirus are two pathogens that have
demonstrated the ability to evolve in order to successfully lower host specificity.
This ability to infect multiple hosts give them a major evolutionary advantage. In
doing so, CIV and CPV can be transmitted through a variety of intermediate or
reservoir hosts before ultimately infecting domestic canine populations. CPV
affects domestic dogs globally, and CIV has thus far been shown to emerge in
various geographic ‘hotspots’ across the United States. Both viruses are still
evolving towards better fitness and remain serious public health risks to canine
populations to this day.

12. Moloney 12
References
Allison, Andrew B., Dennis J. Kohler, Alicia Ortega, Elizabeth A. Hoover, Daniel M. Grove,
Edward C. Holmes, and Colin R. Parrish. (2014) Host­Specific Parvovirus Evolution in
Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species. PLoS
Pathog PLoS Pathogens, 10, no. 11.
American Veterinary Medical Association. " Canine Parvovirus." https://www.avma.org/.
Calderón, Marina Gallo, Maximiliano Wilda, Lorena Boado, Leticia Keller, Viviana Malirat,
Marcela Iglesias, Nora Mattion, and Jose La Torre. (2011) Study of Canine Parvovirus
Evolution: Comparative Analysis of Full­length VP2 Gene Sequences from Argentina and
International Field Strains. Virus Genes, 44, no. 1: 32­39.
Crawford, P.C., Dubovi, Edward J., Castleman, William L., Stephenson, Iain, Gibbs,
E.P.J., Chen, Limei, Smith, Catherine, Hill, Richard C., Ferro, Pamela, Pompey, Justine,
Bright, Rick A., Medina, Marie­Jo, Influenza Genomics Group, Johnson, Calvin M., Olsen,
Christopher W., Cox, Nancy J., Klimov, Alexander I., Katz, Jacqueline M., Donis, Ruben
O. (2005) Transmission of Equine Influenza Virus to Dogs. Science, 310, no. 5747:
482­85.
Dalziel, Benjamin D., Kai Huang, Jemma L. Geoghegan, Nimalan Arinaminpathy, Edward
J. Dubovi, Bryan T. Grenfell, Stephen P. Ellner, Edward C. Holmes, and Colin R. Parrish.
(2014) Contact Heterogeneity, Rather Than Transmission Efficiency, Limits the
Emergence and Spread of Canine Influenza Virus. PLoS Pathog PLoS Pathogens, 10, no.
10.
Hueffer, K., J. S. L. Parker, W. S. Weichert, R. E. Geisel, J.­Y. Sgro, and C. R. Parrish.
(2003) The Natural Host Range Shift and Subsequent Evolution of Canine Parvovirus
Resulted from Virus­Specific Binding to the Canine Transferrin Receptor. Journal of
Virology, 77, no. 3: 1718­726.
Koday, Merika Treants, Jorgen Nelson, Aaron Chevalier, Michael Koday, Hannah
Kalinoski, Lance Stewart, Lauren Carter, Travis Nieusma, Peter S. Lee, Andrew B. Ward,
Ian A. Wilson, Ashley Dagley, Donald F. Smee, David Baker, and Deborah Heydenburg
Fuller. (2016) A Computationally Designed Hemagglutinin Stem­Binding Protein Provides
In Vivo Protection from Influenza Independent of a Host Immune Response. PLoS Pathog
PLOS Pathogens 12, no. 2.
Merck Animal Health. “States with CIV.” http://www.doginfluenza.com/.
Parrish, Colin R. (1990) Emergence, Natural History, and Variation of Canine, Mink, and
Feline Parvoviruses. Advances in Virus Research Advances in Virus Research, 38: 403­50.
Pecoraro, Heidi L., Susi Bennett, Miranda E. Spindel, and Gabriele A. Landolt. (2014)
Evolution of the Hemagglutinin Gene of H3N8 Canine Influenza Virus in Dogs. Virus
Genes, 49, no. 3: 393­99.

13. Moloney 13
Pérez, Ruben, Pablo Bianchi, Lucía Calleros, Lourdes Francia, Martín Hernández, Leticia
Maya, Yanina Panzera, Katia Sosa, and Stephanie Zoller. (2012) Recent Spreading of a
Divergent Canine Parvovirus Type 2a (CPV­2a) Strain in a CPV­2c Homogenous
Population. Veterinary Microbiology, 155, no. 2­4: 214­19.
Ratnayake, Punsisi U., E.a. Prabodha Ekanayaka, Sweta S. Komanduru, and David P.
Weliky. (2016) Full­length Trimeric Influenza Virus Hemagglutinin II Membrane Fusion
Protein and Shorter Constructs Lacking the Fusion Peptide or Transmembrane Domain:
Hyperthermostability of the Full­length Protein and the Soluble Ectodomain and Fusion
Peptide Make Significant Contributions to Fusion of Membrane Vesicles. Protein
Expression and Purification, 117: 6­16.
Rivailler, Pierre, Ijeoma A. Perry, Yunho Jang, C. Todd Davis, Li­Mei Chen, Edward J.
Dubovi, and Ruben O. Donis. (2010) Evolution of Canine and Equine Influenza (H3N8)
Viruses Co­circulating between 2005 and 2008. Virology, 408, no. 1: 71­79.
Smith, D. J., Lapedes, Alan S., De Jong, Jan C.,Bestebroer, Theo M., Rimmelzwaan, Guus
F., Osterhaus, Albert D., Fouchier, Robert A.M.R. (2004) Mapping the Antigenic and
Genetic Evolution of Influenza Virus. Science, 305, no. 5682.
Song, Daesub, Bokyu Kang, Chulseung Lee, Kwonil Jung, Gunwoo Ha, Dongseok Kang,
Seongjun Park, Bongkyun Park, and Jinsik Oh. (2008) Transmission of Avian Influenza
Virus (H3N2) to Dogs. Emerg. Infect. Dis. Emerging Infectious Diseases, 14, no. 5:
741­46.
Truyen, U., Gruenberg, A., Chang, S.F., Obermaier, B., Veijalainen, P., Parrish, C.R.
(1995) Evolution of the feline­subgroup parvoviruses and the control of canine host
range in vivo. J. Virol., 69: 4702­4710.
Wang, Jianke, Peng Lin, Hang Zhao, Yuening Cheng, Zhong Jiang, Hongwei Zhu, Hua Wu,
and Shipeng Cheng. (2016) Continuing Evolution of Canine Parvovirus in China: Isolation
of Novel Variants with an Ala5Gly Mutation in the VP2 Protein. Infection, Genetics and
Evolution 38: 73­78.
Xu, J., H.­C. Guo, Y.­Q. Wei, L. Shu, J. Wang, J.­S. Li, S.­Z. Cao, and S.­Q. Sun. (2013)
Phylogenetic Analysis of Canine Parvovirus Isolates from Sichuan and Gansu Provinces of
China in 2011. Transboundary and Emerging Diseases, 62, no. 1: 91­95.
Zhu, Henan, Joseph Hughes, and Pablo R. Murcia. (2015) Origins and Evolutionary
Dynamics of H3N2 Canine Influenza Virus. J. Virol., 89, no. 10: 5406­5418.