Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range. Canine parvovirus (CPV) is an important example of a viral pathogen that evolved by cross-species transmission and mutation to initiate a disease pandemic. Carnivore parvoviruses infect many species, and their passage in different hosts may select mutations that facilitate host jumping; for example, natural passage of CPV in raccoons may have facilitated its adaptation to dogs. Conversely, some raccoon-adapted viruses are non-infectious to dogs, illustrating that host range barriers exist among different carnivores. Here we demonstrate that these barriers can be overcome by only a few mutations in the virus that likely alter host receptor binding, and that host adaptation can differ dramatically among very similar viruses. Importantly, we also show that passage of viruses in cell cultures of different hosts results in mutations at the same sites that vary in nature and confer fitness increases, strongly suggesting that they are adaptively important. These findings demonstrate that parvoviruses may cross species barriers to infect less susceptible hosts through single or only a few mutations, and that differences in the genetic background, host range, and/or evolutionary history of the viruses influence their propensity to jump hosts. Overall, these discoveries help reveal the mechanisms that control host switching and viral emergence.

Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus (“FPV-like”) or canine parvovirus (“CPV-like”). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host. Parvoviruses in cats and dogs have been studied as a model system to understand how viruses gain the ability to infect new host species. By studying the evolution of the transferrin receptor, which the virus uses to enter a cell, we discovered that the ancestors of dogs were likely infected by a parvovirus millions of years ago until they evolved and became resistant; this was caused by their transferrin receptor changing so it no longer bound the virus. When a variant virus that infects dogs emerged in the 1970s, it had adapted to overcome this block. This story suggests that diseases which were once eliminated from a species can evolve and regain the infectivity for that host, therefore having high potential to be emerging diseases. We identified features of the receptor that were important to the evolution of this host-virus interaction and confirmed their role in regulating virus binding in cell culture.

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Sylvatic carnivores, such as raccoons, have recently been recognized as important hosts in the evolution of canine parvovirus (CPV), a pandemic pathogen of domestic dogs. Although viruses from raccoons do not efficiently bind the dog transferrin receptor (TfR) or infect dog cells, a single mutation changing an aspartic acid to a glycine at capsid (VP2) position 300 in the prototype raccoon CPV allows dog cell infection. Because VP2 position 300 exhibits extensive amino acid variation among the carnivore parvoviruses, we further investigated its role in determining host range by analyzing its diversity and evolution in nature and by creating a comprehensive set of VP2 position 300 mutants in infectious clones. Notably, some position 300 residues rendered CPV noninfectious for dog, but not cat or fox, cells. Changes of adjacent residues (residues 299 and 301) were also observed often after cell culture passage in different hosts, and some of the mutations mimicked changes seen in viruses recovered from natural infections of alternative hosts, suggesting that compensatory mutations were selected to accommodate the new residue at position 300. Analysis of the TfRs of carnivore hosts used in the experimental evolution studies demonstrated that their glycosylation patterns varied, including a glycan present only on the domestic dog TfR that dictates susceptibility to parvoviruses. Overall, there were significant differences in the abilities of viruses with alternative position 300 residues to bind TfRs and infect different carnivore hosts, demonstrating that the process of infection is highly host dependent and that VP2 position 300 is a key determinant of host range. IMPORTANCE Although the emergence and pandemic spread of canine parvovirus (CPV) are well documented, the carnivore hosts and evolutionary pathways involved in its emergence remain enigmatic. We recently demonstrated that a region in the capsid structure of CPV, centered around VP2 position 300, varies after transfer to alternative carnivore hosts and may allow infection of previously nonsusceptible hosts in vitro . Here we show that VP2 position 300 is the most variable residue in the parvovirus capsid in nature, suggesting that it is a critical determinant in the cross-species transfer of viruses between different carnivores due to its interactions with the transferrin receptor to mediate infection. To this end, we demonstrated that there are substantial differences in receptor binding and infectivity of various VP2 position 300 mutants for different carnivore species and that single mutations in this region can influence whether a host is susceptible or refractory to virus infection.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).