Residues in the apical domain of the feline and canine transferrin receptors control host-specific binding and cell infection of canine and feline parvoviruses

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses.     

Combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses

Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2 residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells. Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR binding but that binding is not always sufficient to mediate infection.     

The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor

Canine parvovirus (CPV) is a host range variant of a feline virus that acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37 degrees C only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type 2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs the later variant strain of CPV gained the ability to more efficiently use the canine TfR for infection and also showed reduced binding to feline and canine cells compared to CPV type 2. Differences on the top and the side of the threefold spike of the capsid surface controlled specific TfR binding and the efficiency of binding to feline and canine cells, and these differences also determined the cell infection properties of the viruses.     

Role of multiple hosts in the cross-species transmission and emergence of a pandemic parvovirus

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.     

Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 A resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.     

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts.     

Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells

Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919-1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.     

Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization, and capsid-binding domains

The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.     

Early Steps in Cell Infection by Parvoviruses: Host-Specific Differences in Cell Receptor Binding but Similar Endosomal Trafficking

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are closely related parvoviruses that differ in their host ranges for cats and dogs. Both viruses bind their host transferrin receptor (TfR), enter cells by clathrin-mediated endocytosis, and traffic with that receptor through endosomal pathways. Infection by these viruses appears to be inefficient and slow, with low numbers of virions infecting the cell after a number of hours. Species-specific binding to TfR controls viral host range, and in this study FPV and strains of CPV differed in the levels of cell attachment, uptake, and infection in canine and feline cells. During infection, CPV particles initially bound and trafficked passively on the filopodia of canine cells while they bound to the cell body of feline cells. That binding was associated with the TfR as it was disrupted by anti-TfR antibodies. Capsids were taken up from the cell surface with different kinetics in canine and feline cells but, unlike transferrin, most did not recycle. Capsids labeled with fluorescent markers were seen in Rab5-, Rab7-, or Rab11-positive endosomal compartments within minutes of uptake, but reached the nucleus. Constitutively active or dominant negative Rab mutants changed the intracellular distribution of capsids and affected the infectivity of virus in cells.Cell infection by animal viruses involves a specific sequence of steps that deliver the virus and its genome from the cell surface to the compartment where replication can occur. For nonenveloped viruses, infection initiates with binding to a specific cell receptor and uptake into the cell by receptor-mediated endocytosis. Various factors can control the process of viral uptake, including the characteristics of the receptor(s) bound by the virus and its signaling and endocytic properties, the affinity of the virus for the receptor, and the structural features of the interaction in different environments (36, 61). Receptors may be located on the cell body or may also be displayed on the extended lamellipodia or filopodia with greater surface areas. Viruses binding to filopodia can be either passively delivered to the cell body for endocytosis by dynamic movement of the entire structure or actively trafficked by retrograde actin transport as well as the action of myosin-2 motors on the actin (32, 57). Cross-linking and clustering of receptors by viral particles can influence the rate and pathways of uptake from the cell surface (23), and many viral receptors activate signaling pathways that alter the structure of the underlying cytoskeleton to enhance uptake (see, e.g., references 12, 30, and 51). Receptor-bound viruses then enter one or more endosomal pathways that results in the capsid being enclosed in vesicles and trafficked within the endosomal pathways of the cell, where clustered virus and receptors (23) may undergo structural alterations upon exposure to conditions such as low pH or proteases (36, 61). The specific receptor-mediated binding and entry pathways often provide signals for viruses that allow endosomal escape and establish infection. A variety of markers of the endosomal compartments have been used in studies of viral entry. Rab proteins are monomeric small GTPases which regulate endosomal membrane trafficking, and specific Rab proteins are associated with different endosomal compartments. Among the many Rab proteins in the cells, Rab5 is primarily associated with the early endosome and regulates trafficking through that compartment, Rab7 is associated with the late endosome, and Rab11 is associated with the recycling endosome (14, 58). Tracking viral particles within the endosomal pathways during cell entry has been used to define the steps in the entry and infection processes of a variety of different viruses and has revealed many of the common features and variant processes that are used (7-9, 33, 71).Here, we examine the uptake and infection of cells by parvovirus capsids and compare some of the steps followed by capsids that differ in their receptor binding properties and host ranges. Feline panleukopenia virus (FPV) infects cats (50, 66), binds the transferrin receptor-1 (TfR) on feline cells, and uses that receptor for uptake and infection (27, 44). FPV does not bind the canine TfR or infect dogs or cultured canine cells. Canine parvovirus (CPV) is a natural variant of FPV which emerged in 1978 after acquiring a small number of mutations that allow its capsid to bind the canine TfR (27). The original strain of CPV (designated CPV type 2 [CPV-2]) spread worldwide in dogs during 1978, but some of the same mutations that gave it the canine host range rendered it unable to infect cats (66, 67). CPV-2 was replaced worldwide during 1979 and 1980 by a natural variant, CPV type 2a (CPV-2a), which contained an additional four to five changes in its coat protein gene (48, 49). Subsequently, the canine viruses have continued to evolve, and additional single mutations have been selected that alter antigenic epitopes. Strains altered at VP2 residue 426 are designated CPV-2b (Asn426Asp) and CPV-2c (Asp426Glu) (13, 48). CPV-2a and its variants are able to infect both dogs and cats but show reduced binding to the feline TfR on cells and in vitro (27, 42). In addition, the affinity of binding to the canine TfR is much lower than that seen for the feline TfR (42).The TfR is a type II membrane protein expressed in nonlipid raft regions of the plasma membrane, and it binds iron-loaded (holo) transferrin (Tf) at neutral pH (2). TfR expression is tightly regulated, and it is more highly expressed on dividing cells with high iron needs, which would favor binding of these viruses. The TfRs of mice and humans are used as receptors for cell infection by the mouse mammary tumor virus and the New World hemorrhagic fever arenaviruses (52, 56).The TfR is assembled as a homodimer, and each monomer of the ectodomain is composed of protease-like, apical and helical domains, as well as a 30-Å membrane-proximal stalk (5, 20, 31). The transmembrane domain mediates membrane insertion and influences some aspects of trafficking within the cell, while the cytoplasmic domain contains a tyrosine-threonine-arginine-phenylalanine (YTRF) sequence that engages the clathrin-mediated endocytic machinery through AP-2 (adaptor protein-2) (53, 55). The TfR sequence also includes one or two cysteines adjacent to the inner leaflet of the membrane that may be palmitoylated to influence the rate of receptor recycling, and it also contains sequences that control basolateral localization in polarized cells (41). In the normal pathway of TfR-mediated entry, the TfR-holo-Tf complex is transported into the endosomal system, where low pH results in conformational changes and iron release. The TfR-Tf complex enters the early endosome, from which some of the complex is rapidly recycled to the cell surface while most passes to the perinuclear recycling endosome. From there it recycles to the cell surface where the iron-free apo-Tf is released at neutral pH (21, 22, 24, 37, 38, 69, 70). The rate of uptake and the efficiency of TfR recycling depend on the form of the ligand, and more than 97% of monomeric Tf recycles to the cell surface within 10 to 30 min. However, cross-linking TfRs with oligomeric Tf or antibodies causes the complexes to be retained within endosomes for longer times, and a higher proportion is trafficked to late endosomes and lysosomes for degradation (35).Holo-Tf binds the membrane-proximal side of the feline and canine TfR ectodomain (11), while virus binding involves the apical domain of the receptor as mutations in that structure affect the ability to bind FPV and CPV capsids (43) The feline and canine TfRs differ in ∼10% of their sequences, but a major difference controlling the CPV-specific binding is a unique glycosylation site in the apical domain of the canine TfR (43). Alteration of the glycosylated Asn to Lys (the feline TfR residue) allowed the canine TfR to bind FPV and also greatly increased the affinity of binding to CPV-2 and CPV-2a-related capsids (42).CPV and FPV have small (25 nm) nonenveloped capsids that package a single-stranded DNA genome of ∼5,120 bases (68). The particles are made up of two overlapping proteins, VP1 and VP2, with 90% of the capsid protein being VP2. VP1 contains a 143-residue amino (N)-terminal sequence that encodes a phospholipase A₂ enzymatic activity, as well as basic amino acid motifs that play a role in nuclear localization (72). The VP1 unique region becomes exposed during cell entry without capsid disintegration, and the phospholipase A₂ modifies the endosomal membrane to enhance endosomal escape (19, 75).Previous studies of cell entry by CPV, minute virus of mice, and various adeno-associated viruses (AAVs) show that viral uptake primarily occurs through clathrin-mediated endocytosis. However, when the AP-2-interacting sequences in the cytoplasmic tail of the feline TfR were mutated or deleted, the altered receptor still allowed CPV infection at a similar efficiency to that of wild-type TfR (26). The intracellular pathways of viral entry and trafficking have been examined by using cells fixed at various times after uptake and then antibody stained for virus and cellular markers or by expressing green fluorescent protein (GFP)-labeled markers. Time courses examined were between 1 and 6 h, and sequential steps of trafficking were suggested, with the virus passed from the early endosomes to the recycling endosome, followed by localization in late endosomes and lysosomes after uptake (65). By fluorescent antibody staining, VP1 release occurred only hours after uptake, possibly in a low-pH degradative compartment (64, 72). In addition, CPV capsids appear to remain associated with the receptor for 1 to 2 h after virus uptake as antibodies against the TfR cytoplasmic tail microinjected into feline CRFK cells block infection in this time period (44). Infection is also blocked by neutralizing the low pH of the endosomal system with ammonium chloride or bafilomycin A1, although it is not clear whether this is due to direct effects on the capsid or to indirect alterations in endosomal trafficking. When the X-ray crystal structures of capsids of CPV and FPV were determined at low pH or in the presence of EDTA or when capsids were examined for changes in protease susceptibility, only small changes in surface loops of the viral structure were present (40, 60).Here, we used microscopy to examine dynamic steps in the binding, uptake, and early trafficking of parvovirus capsids in live canine and feline cells. Labeled capsids were seen to undergo rapid movement into multiple endosomal compartments shortly after entry. Initial binding of CPV to canine cells involved filopodia while in feline cells the virus bound primarily to receptors on the cell body. In cells expressing GFP-conjugated Rab proteins, particles rapidly localized to multiple endosomal compartments in the cytoplasm after uptake, which gradually accumulated near the microtubule-organizing center. The distribution of intracellular viruses and the viral infectivity in feline cells were altered by expression of either constitutively active (CA) or dominant negative (DN) mutants of the Rab proteins.     

The role of evolutionary intermediates in the host adaptation of canine parvovirus

The adaptation of viruses to new hosts is a poorly understood process likely involving a variety of viral structures and functions that allow efficient replication and spread. Canine parvovirus (CPV) emerged in the late 1970s as a host-range variant of a virus related to feline panleukopenia virus (FPV). Within a few years of its emergence in dogs, there was a worldwide replacement of the initial virus strain (CPV type 2) by a variant (CPV type 2a) characterized by four amino acid differences in the capsid protein. However, the evolutionary processes that underlie the acquisition of these four mutations, as well as their effects on viral fitness, both singly and in combination, are still uncertain. Using a comprehensive experimental analysis of multiple intermediate mutational combinations, we show that these four capsid mutations act in concert to alter antigenicity, cell receptor binding, and relative in vitro growth in feline cells. Hence, host adaptation involved complex interactions among both surface-exposed and buried capsid mutations that together altered cell infection and immune escape properties of the viruses. Notably, most intermediate viral genotypes containing different combinations of the four key amino acids possessed markedly lower fitness than the wild-type viruses.     

Host range and variability of calcium binding by surface loops in the capsids of canine and feline parvoviruses

Canine parvovirus (CPV) emerged in 1978 as a host range variant of feline panleukopenia virus (FPV). This change of host was mediated by the mutation of five residues on the surface of the capsid. CPV and FPV enter cells by endocytosis and can be taken up by many non-permissive cell lines, showing that their host range and tissue specificity are largely determined by events occurring after cell entry.We have determined the structures of a variety of strains of CPV and FPV at various pH values and in the presence or absence of Ca(2+). The largest structural difference was found to occur in a flexible surface loop, consisting of residues 359 to 375 of the capsid protein. This loop binds a divalent calcium ion in FPV and is adjacent to a double Ca(2+)-binding site, both in CPV and FPV. Residues within the loop and those associated with the double Ca(2+)-binding site were found to be essential for virus infectivity. The residues involved in the double Ca(2+)-binding site are conserved only in FPV and CPV.Our results show that the loop conformation and the associated Ca(2+)-binding are influenced by the Ca(2+) concentration, as well as pH. These changes are correlated with the ability of the virus to hemagglutinate erythrocytes. The co-localization of hemagglutinating activity and host range determinants on the virus surface implies that these properties may be functionally linked. We speculate that the flexible loop and surrounding regions are involved in binding an as yet unidentified host molecule and that this interaction influences host range.     

The emergence and evolution of canine parvovirus—an example of recent host range mutation

Canine parvovirus (CPV) emerged in 1978 an a new pathogen of dogs, which spread around the world and now appears endemic in the domesticated and wild dog populations in all countries. CPV is over 98% identical in DNA sequence to viruses which had been known for many years in cats, mink and raccoons, and genetic analysis has revealed that the differences in canine host range are determined by a small number of changes in the capsid protein gene. Comparison of the atomic structures of the CPV and FPV capsids shows that the changes affecting host range and virus-specific antigenic sites are exposed on the capsid surface in three different positions within a raised region at the threefold axis of symmetry, which is also the site of major antigenic determinants on the capsid. Three types of CPV have been defined by antigenic analysis with monoclonal antibodies. The original CPV strain (called CPV type-2) was only present in nature for a few years, and by 1981 it had been largely replaced in nature by a variant of CPV (CPV type 2a), which in turn replaced between 1984 and 1990 by a further variant (CPV type-2b). Those viruses differed by less than 0.2% of their genome sequences, but in each case the replacement apparently occurred on a global scale. The true ancestry of CPV is not clear, but the apparent emergence of the new types of CPV and its subsequent evolution suggest that this is a useful model for the emergence of new viruses with extended host ranges and their continuing adaptation.     

Limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structures

Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here we used fluorescence microscopy to directly visualize the association of single canine parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that most capsids associated with fewer than five TfRs and that ～25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissociated from the cell surface with a half-life of ～30 s. Together, our results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.     

Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are over 98% similar in DNA sequence but have specific host range, antigenic, and hemagglutination (HA) properties which were located within the capsid protein gene. In vitro mutagenesis and recombination were used to prepare 16 different recombinant genomic clones, and viruses derived from those clones were analyzed for their in vitro host range, antigenic, and HA properties. The region of CPV from 59 to 91 map units determined the ability to replicate in canine cells. A complex series of interactions was observed among the individual sequence differences between 59 and 73 map units. The canine host range required that VP2 amino acids (aa) 93 and 323 both be the CPV sequence, and those two CPV sequences introduced alone into FPV greatly increased viral replication in canine cells. Changing any one of aa 93, 103, or 323 of CPV to the FPV sequence either greatly decreased replication in canine cells or resulted in an inviable plasmid. The Asn-Lys difference of aa 93 alone was responsible for the CPV-specific epitope recognized by monoclonal antibodies. An FPV-specific epitope was affected by aa 323. Amino acids 323 and 375 together determined the pH dependence of HA. Amino acids involved in the various specific properties were all around the threefold spikes of the viral particle.     

Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid.

We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range.     

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.     

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

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.     

Parvovirus host range,cell tropism and evolution

The past few years have seen major advances in our understanding of the controls of evolution, host range and cell tropism of parvoviruses. Notable findings have included the identification of the transferrin receptor TfR as the cell surface receptor for canine parvovirus and feline panleukopenia virus, and also the finding that specific binding to the canine TfR led to the emergence of canine parvovirus as a new pathogen in dogs. The structures of the adeno-associated virus-2 and porcine parvovirus capsids, along with those of the minute virus of mice, have also advanced our understanding of parvovirus biology. Structure-function studies have shown that in several different parvoviruses the threefold spikes or peaks of the capsid control several aspects of cell tropism and host range, and that those are subject to selective pressures leading to viral evolution. The cell and tissue tropisms of different adeno-associated virus serotypes were demonstrated to be due, in part, to specific receptor binding.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

Antibodies to canine and feline viruses in spotted hyenas (Crocuta crocuta) in the Masai Mara National Reserve

Spotted hyenas (Crocuta crocuta) are abundant predators in the Serengeti ecosystem and interact with other species of wild carnivores and domestic animals in ways that could encourage disease transmission. Hyenas also have a unique hierarchical social system that might affect the flow of pathogens. Antibodies to canine distemper virus (CDV), feline immunodeficiency virus (FIV), feline panleukopenia virus/canine parvovirus (FPLV/CPV), feline coronavirus/ feline infectious peritonitis virus (FECV/IPV), feline calicivirus (FCV), and feline herpesvirus 1 (FHV1) have been detected in other Serengeti predators, indicating that these viruses are present in the ecosystem. The purpose of this study was to determine whether spotted hyenas also had been infected with these viruses and to assess risk factors for infection. Serum samples were collected between 1993 and 2001 from 119 animals in a single clan for which behavioral data on social structure were available and from 121 hyenas ill several other clans. All animals resided in the Masai Mara National Reserve. Antibodies to CDV, FIV, FPLV/CPV, FECV/FIPV, FCV, and FHV1 were present in 47%, 3.5%, 81%, 36%, 72%, and 0.5% of study hyenas, respectively. Antibody prevalence was greater in adults for FIV and FECV/FIPV, and being a female of high social rank was a risk factor for FIV. Hyenas near human habitation appeared to be at lower risk to have CDV, FIV, and FECV/FIPV antibodies, whereas being near human habitation increased the risk for FPLV/CPV antibodies. Canine (distemper virus and FECV/FIPV antibody prevalence varied considerably over time, whereas FIV, FPLV/CPV, and FCV had a stable, apparently endemic temporal pattern. These results indicate that hyenas might play a role in the ecology of these viruses in the Serengeti ecosystem. The effect of these viruses on hyena health should be further investigated. The lower prevalence of CDV antibody-positive hyenas near human habitation suggests that reservoirs for CDV other than domestic dogs are present in the Serengeti ecosystem.