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Host range relationships and the evolution of canine parvovirus
Veterinary Microbiology 69 (1999) 29±40
Host range relationships and the evolution of canine parvovirus Colin R. Parrish* James A. Baker Institute, College of Veterinary Medicine, Cornell University Ithaca, NY 14853, USA
Abstract Canine parvovirus (CPV) is an example of an unusual class of emerging virus±those that gain an altered host range through genetic variation and subsequently become widespread pathogens of their new and previously resistant host species. CPV was first detected in 1978 as the cause of new diseases in dogs throughout the world, when it rapidly spread throughout domestic populations, as well as becoming widespread in wild dogs. CPV was soon shown to be a variant of the long recognized feline panleukopenia virus (FPV), from which it differed in less than 1% at the nucleotide sequence level. Genetic analysis showed that virtually all of the biological differences between CPV and FPV, including the canine host range, were determined by three or four sequence differences in the viral capsid protein gene. Analysis of the atomic structures of the CPV and FPV capsids showed that the differences controlling host range were located within two different structural regions and were exposed on the capsid surface. The CPV which first emerged in 1978 appeared to be derived from a single ancestral sequence, which has allowed the ready analysis of the subsequent evolution of the virus in nature. Sequence analysis has also revealed that CPV strains have undergone a series of evolutionary selections in nature which have resulted in the global distribution of new virus variants. This was first seen in the global replacement between 1979 and 1981 of the original (1978) strain of the virus by a genetically and antigenically variant strain, and the subsequent widespread selection of other variants which have also become globally distributed. The genetic and antigenic variation in the virus strains was also correlated with changes in the host range of the virus, in particular in the ability to replicate in cats, and in canine host range differences seen in tissue culture cells. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Parvovirus; Evolution; Host range; Capsid structure
*
Tel.: +1-607-256-5649; fax: +1-607-256-5608 E-mail address: [email protected] (C.R. Parrish) 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 8 4 - X
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1. Introduction The emergence of new microbial pathogens can occur through a number of different mechanisms (Nathanson et al., 1993). In some cases this occurs through the identification of a long existing agent and its association with a disease of previously unknown etiology, most often through the use of new technologies such as PCR for identifying the agent. In other cases changes in environmental, vector, or the host leads to greater incidence or recognition of a pre-existing disease. Such changes can be of the insect vector density due to environmental change, changes in location of the hosts, or immune deficiency of the host. Another mechanism of emergence is through the selection of a host range variant of a pathogen in a host that was previously resistant to such a virus. That type of emergence is unusual, although for viruses it most likely has happened on many occasions to give rise to the current range of animal and plant viruses. However, when such a new agent with an altered host range emerges it has the potential to spread rapidly through the naõÈve and non-immune host population, and there may also be a period of rapid agent variation as the virus becomes adapted to its new host. Examples of viral host range variants of this type which have been documented in recent history include the pandemic influenza A viruses, human immunodeficiency virus (HIV) type-2 and possibly HIV type-1. 1.1. Emergence and initial characterization of CPV In early 1978 canine diseases were observed in Asia, Australia, New Zealand, the Americas, and in Europe which were characterized by vomiting and diarrhea in dogs older than about 6 weeks, and myocarditis in neonatal pups, and these diseases differed from those previously recognized in dogs. A small, round, non-enveloped virus was observed by electron microscopy in stool specimens and in tissues of the affected animals, and a parvovirus was soon isolated in canine and feline tissue cultured cells from both forms of the disease. The virus was named canine parvovirus (CPV), and has also been referred to as CPV type-2 to distinguish it from the previously described, although unrelated parvovirus minute virus of canines (MVC) (Carmichael and Binn, 1981; Carmichael et al., 1994). The enteric disease in dogs was pathologically similar to a disease of cats caused by the feline panleukopenia virus (FPV), and polyclonal antibody and in vivo cross-protection studies soon showed that CPV and FPV were closely related antigenically. Modified live virus vaccines were soon developed, and since 1979 these have allowed much of the disease to be controlled. However, the virus is still widely distributed in nature, and if pups are not vaccinated, and or when maternal antibodies interfere with vaccination in pups, they generally become naturally infected. Testing for antibodies in the sera collected from dogs or related canids showed that the first positive titers were present in dogs in Europe between 1974 and 1976, and the first positive sera in the USA, Japan, Australia, were reported in early 1978. Wild coyotes in the USA became widely infected during 1979, and wild wolves in a national park in Alaska were infected by 1980. Since 1978 the virus has been ubiquitous in dogs throughout the world.
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1.2. Properties of CPV and FPV 1.2.1. Genetic structure The parvoviruses are both small and genetically simple. The genome of CPV is a single stranded DNA of about 5,200 nucleotides in length, which has two promoters which result in the expression of two structural (VP1 and VP2) and two non-structural proteins (NS1 and NS2) through alternative splicing of the viral mRNAs (Reed et al., 1988, 1991). Only the -ve DNA strand is packaged into the capsid. A non-coding region near the right hand end of the genome that contains a variable number of direct repeat sequences, and there are palindromic hairpins at either end of the genome that are used in the replication of the viral DNA (Fig. 1). 1.2.2. Capsid structure As described below, virtually all the biological differences between CPV and FPV are determined by the viral capsid. The structures of the full (DNA-containing) CPV capsids or empty CPV capsids, or of empty FPV capsids have been determined by X-ray crystallography (Tsao et al., 1991; Agbandje et al., 1993). These structures showed that the capsids were assembled from a total of 60 copies of the VP1 and VP2, with about 5±6 copies of VP1 and 54±55 copies of VP2. The two proteins most likely acted in a structurally equivalent way, since empty capsids could be assembled from VP2 alone
Fig. 1. The genetic structure of the genome of CPV and FPV. The ssDNA genome is about 5150 nucleotides in length, and has terminal palindromes of about 150 bases at the 30 and 50 ends. Promoters at genomic map units 4 and 40 give rise to messages (R1±R3) for the non-structural proteins (NS) and capsid proteins (VP), respectively. NS1 and NS2, and VP1 and VP2 are formed by alternative splicing from the same mRNA. Three sequences in the 50 end of the genome may be present as single or multiple copies.
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Fig. 2. The surface of the 25 nm diameter non-enveloped capsid of CPV as determined from the atomic model. One asymmetric unit of the particle is shown by the large triangle. The three icosahedral axes of symmetry of the particle are indicated by the pentamer (five-fold axis), triangle (three-fold axis), and diamond (two-fold axis). Surface features indicated include a depressed canyon surrounding the five-fold axis, a dimple spanning the twofold axis, and a spike at the threefold axis of symmetry. The positions of two residues (VP2 residues 93 and 323) which affect canine host range of CPV are indicated.
expressed by mammalian plasmid or baculovirus vectors, or from VP1 and VP2 expressed from mammalian plasmid expression vectors (Saliki et al., 1992; Tresnan et al., 1995). The VP2 monomer comprised of an 8-stranded anti-parallel b-barrel with large loops inserted between the b-strands making up most of the exposed surface of the capsid. Surface features of the capsid included a raised area (spike) surrounding the threefold axis of symmetry, a depression (dimple) spanning the two-fold axis of symmetry, and a further depressed area (canyon) surrounding the five-fold axis of symmetry of the capsid (Fig. 2). Two primary antigenic sites on the three-fold spike of the virus were defined using monoclonal antibodies (mAb) were found on the three-fold spike (Strassheim et al., 1994; Wikoff et al., 1994), although epitopes have also been defined by peptide mapping in other regions of the capsid (Langeveld et al., 1993, 1994).
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1.2.3. Nature antigenic variation The antigenic type of the virus appears relatively stable, and viruses that appear antigenically identical have been isolated many decades apart. A relatively small amount of antigenic variation is observed between viruses isolated from an individual host, and there has also been variation identified between viruses from different hosts. Within host variation was clearly demonstrated for the mink enteritis virus isolates, where three different antigenic types were seen (Parrish et al., 1984). 1.2.4. Virus variation and evolution Studies of the DNA sequences of the viruses have revealed the phylogenetic relationships between CPV isolates from dogs and the viruses from cats, raccoons, mink, and arctic foxes (Fig. 3). All the CPV isolates clearly formed a single clade, and were most likely derived from a single common ancestral sequence. The viruses isolated from cats, mink, raccoons or foxes could not be clearly distinguished from each other, suggesting inter-species transmission of those latter viruses. During the analysis of canine viruses collected at different times, it became clear that there was genetic and antigenic variation between the viruses collected at different times after 1978. The early viruses (CPV type-2), collected between 1978 and 1980 were the same worldwide, but between 1979 and 1980 an antigenic variant was identified in many different countries of the world using mAb testing, and that strain was termed CPV type-2a (Parrish et al., 1985, 1991). Although those viruses only differed in 5±6 amino acids from the CPV type-2 isolates, they differed in two different neutralizing antigenic sites on the surface of the capsid (Fig. 4). In about 1984 a further antigenically variant virus strain was detected, which differed in only a single epitope, and that virus was designated CPV type-2b. Despite the small numbers of differences between those viruses, in each case the viruses became globally distributed, indicating that they must have been under strong selection (Fig. 5). As described below, those viruses also differ in host range as well as antigenicity. Initial studies examined the antigenicity of the viruses, and mAb analysis showed that the isolates from dogs differed from those from cats or other carnivores (mink, raccoons) in at least two virus strain specific epitopes one present only on CPV, and the other present on FPV isolates. Further analysis showed that CPV and FPV also differed in their hemagglutination (HA), which was a reflection of the ability to bind to the specific sialic acids. The first difference was that the FPV isolates would HA only in buffers at pHs below 6.6, while the CPV isolates HA at higher pHs up to at least pH 7.5 (Parrish et al., 1988, 1991). The HA specificity of the virus appears to be specific for the sialic acid N-glycolylneuraminic acid, which is present on feline erythrocytes of the A blood group (about 70% of almost all the breeds of domestic cat), but which is not present on the erythrocytes of most breeds of dogs (C. R. Parrish, unpublished results). 1.2.5. Host range The host range differences between the various viruses proved to be complex, and they differed in their ability to infect cells in vitro, or to infect animals. The overall relationships are summarized in Table 1. All of the viruses replicated in most feline cells in tissue culture, but only the isolates from dogs replicated in canine cells in culture, indicating that CPV isolates had a species-specific host range which enabled them to
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Fig. 3. Phylogeny of the DNA sequences of the capsid protein genes of various isolates of from dogs and Asiatic raccoon dogs (CPV and RD), cats (FPV), mink (MEV), raccoons (RPV), and Arctic (blue) foxes (BFPV). The numbers shown are the number of nucleotide differences which are present in each branch, the percentage of alternative trees which had that branching order (in italics), and the bootstrap support for that branching order (in parentheses). The tree was rooted using the PPV capsid protein gene sequence as an outgroup. From Truyen et al., 1995 with permission.
replicate in the new host, and that this was determined at the level of infection of individual cells (Truyen and Parrish, 1992). When the viruses were inoculated into animals different host ranges were observed (Truyen and Parrish, 1992). FPV isolates replicated efficiently in cats, as expected, but CPV isolates differed in their ability to infect and replicate in cats, depending on the antigenic type of the CPV tested. CPV type-2 isolates did not replicate detectably in cats, but the CPV type-2a and CPV type-2b isolates infected cats and replicated efficiently after experimental inoculation (Truyen et al., 1995). That this was a natural host range for the viruses was confirmed by the finding that CPV-2a and CPV-2b were isolated from
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Fig. 4. The coding sequences differences in the capsid protein gene which were found in the various isolates of CPV type-2, CPV type-2a and CPV type-2a and CPV type-2 (Parrish et al., 1991). Those sequences which differ from the reference sequence of CPV type-2 are shaded. The nucleotide position in the complete sequence of CPV, and the VP2 residue that changed are also indicated (Parrish, 1991).
about 10±20% of cats which had natural parvovirus disease in Japan, Germany and the USA (Truyen et al., 1995, 1996). The host range for dogs also differed from that seen in tissue culture. CPV isolates infected and replicated in dogs in a number of lymphoid tissues and the intestinal epithelium. FPV, which did not replicate to a detectable level in dog cells in tissue culture, replicated in the thymus and bone marrow of the dog, but not in the peripheral lymphoid tissue or the intestine (Truyen and Parrish, 1992). 1.2.6. Mapping host range and other virus-specific properties of CPV and FPV In order to understand the basis of the changes which gave rise to the new properties of CPV, infectious plasmid clones of CPV and FPV isolates were analyzed by recombination
Fig. 5. The distribution of CPV antigenic types-2, 2a and 2b in various parts of the world during various years between the year of the viruses first spread (1978) and 1994. Open boxes represent viruses from the USA, closed boxes are isolates from Europe, and shaded boxes are isolates from New Zealand and Australia. For the European isolates in 1980, 1981 and 1982 a large number of isolates were tested, and the number that were CPV2a antigenic type are indicated in parenthesis. Adapted from Parrish et al., 1991, with permission.
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Fig. 6. Genetic mapping of the sequence differences between CPV and FPV that affected canine cell infected. Recombination mapping indicated that the region of the VP2 gene that included the differences of VP2 residues 93, 103, and 323 was important in controlling host range. Site direct mutagenesis was used to alter the positions in the genome of CPV (solid bars) or FPV (hatched bars). The viable viruses generated were tested for their ability to infect either feline cells (solid bar), or canine cells (hatched bar). The reactivity of the virus capsids with monoclonal antibodies recognizing a CPV-specific epitope was also examined, and reactivity seen is indicated.
mapping and site-directed mutagenesis (Parrish et al., 1988; Chang et al., 1992; Horiuchi et al., 1994). Virtually all the defined specific differences between CPV and FPV canine host range, antigenic structure, and the pH dependence of HA were determined by two residues which differed between CPV and FPV in the capsid protein gene, as summarized in Fig. 6. Residue 93 was a Lys in FPV and an Asn in CPV, and residue 323 was an Asp in FPV and an Asn in CPV. Changing either of those two residues in the CPV sequence to the FPV sequence resulted in a virus that infected dog cells inefficiently, while if both those residues in FPV were changed to the CPV sequence (Asn) the virus gained the ability to infect dog cells and dogs. The CPV-specific antigenic epitope was determined by the sequence at residue 93, as the specific mAb bound when that was an Asn but not when it was an Asp. The difference in the pH dependence of HA of the two viruses was also controlled primarily by residue 323, and both CPV and FPV showed a CPV-like pH dependence of HA (HA at pHs up to pH 7.5) when residue 323 was an Asn, and an FPVlike HA (HA only below pH6.6) when it was an Asp. An Asp±Asn difference of VP2 residue 375 also affected the pH-dependence of HA, although the effect seen depended on the amino acid at position 323, which showed a dominant effect over the sequence at position 375 (Chang et al., 1992). 1.2.7. Other capsid sequences which determine canine host range Although VP2 residues 93 and 323 were the main determinants of the canine host range of CPV type-2, other sequences in the capsid also play important roles in determining the ability of the virus to infect canine cells. This was initially revealed by a CPV type-2 mutant which was selected during passage in feline cells (Parrish and
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Fig. 7. The structural variation of an additional region on the CPV capsid which affects canine host range (Llamas-Saiz et al., 1996). The mutation examined was the change of VP2 residue Ala(A)300 to Asp(D). The structure shows the amino acids which interact with the loop around the residue 300, and the adjacent loops derived from different VP2 molecules. For the region containing the mutation, both the wild type (dark) and mutant (shaded) structures are shown. The new bond which is likely formed between the mutant Asp(D))300 and Arg(R)81 is indicated.
Carmichael, 1986). That virus lost the ability to infect dogs or dog cells in culture, and also differed antigenically as it no longer reacted with several neutralizing monoclonal antibodies. Recombination analysis mapped the host range determining difference to a region of the genome which contained two differences in sequence from the wild type CPV type-2-VP2 residues 300 Ala-Asp, and 301 Ile-Val. In further analysis of that structural region, series of site-directed mutants was prepared at those positions, or in adjacent structural locations, and those showed that most mutations reduced the ability of the virus to infect dog cells, indicating that wild type structure of the region that was important for maintenance of canine host range (Parker and Parrish, 1997). The atomic structure of one mutant of CPV (with the change of VP2 residue 300 from La to Asp) was determined, and that showed that the new Asp side chain formed a new hydrogen bond with the Lys side chain of a three-fold-related VP2 molecule, and perhaps stabilized the structure of the capsid (Fig. 7) (Llamas-Saiz et al., 1996). Although that region was not directly associated with the canine host range differences between FPV and CPV type-2, in the later emerging CPV type-2a and CPV type-2b viruses there were a series of differences (87 Met-Leu; 300 Ala-Gly; 305 Asp-Tyr) in the vicinity of this region of the structure (Parrish et al., 1991), and those may have contributed additional host range effects that allowed the rapid selection of the newer strains of CPV in dogs throughout the world. 1.2.8. Feline host range Although CPV type-2 viruses did not replicate detectably in cats, CPV type-2a and CPV type-2b both replicated efficiently in that host. The viral sequences which restricted the feline host range of CPV type-2 proved difficult to define, in part because that could only be tested by infecting cats, but CPV/FPV recombinants only replicated efficiently in cats if sequences from the two ends of the capsid protein gene were included in the recombinant (Fig. 8) (Truyen et al., 1994). If only one or the other region were included
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Fig. 8. Genetic mapping of the feline host range difference between CPV type-2 and FPV. A series of recombinant viruses were prepared between the CPV (solid bars) and FPV genomes (crosshatched bars), and the viruses were tested for their ability to infect and replicate in cats. Tissue titers recovered in several tissues are shown. Adapted from (Truyen et al., 1994) with permission.
the virus would replicate to a higher titer in the cat than CPV type-2, but not to the level of FPV. 2. Conclusions The emergence of canine parvovirus provides a useful model for the process of emergence through host range variation and evolution of an animal virus. Although the type of change seen here (Fig. 9) is not common, such a genetic change only has to happen once under the right circumstances for the variant virus to become established in its new host. It is likely that viruses of humans and other animals have periodically emerged in this way during historical time periods, and current trends towards xenotransplantation and other new associations between humans and animals may favor the development and selection of new variants. It is also likely that this type of change happened on many occasions in prehistory to give rise to the present range of animal viruses. Once emerged the subsequent progress of such a new virus depends on it
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Fig. 9. Summary of the viruses examined in these studies and the probable relationships. We cannot clearly determine which of the viruses of cats, mink, raccoons, foxes, or other hosts gave rise to the original virus that was the single common ancestor of all the viruses of dogs (CPV), but that appears to have been present in Europe during the mid-1970s. Subsequently that ancestral virus gave rise separately to CPV type-2 and CPV type-2a, and CPV type-2b was derived from CPV type-2a or a very closely virus. CPV type-2 appears to be largely extinct in nature after about 11981.
properties, modes of persistence or transmission, and on the responses of the host. As seen for CPV, once the new virus becomes established in its new host it is likely to undergo a series of changes leading to further adaptation to become well adapted to the host, and also variation may be selected by the immune pressure of the host. In addition, it is worth noting that the types of change involved can occur DNA viruses such as parvoviruses, as well as the genetically more variable RNA viruses, making it important that all types of viruses be evaluated for these types of changes.
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Llamas-Saiz, A.L., Agbandje-McKenna, M., Parker, J.S.L., Wahid, A.T.M., Parrish, C.R., Rossmann, M.G., 1996. Structural analysis of a mutation in canine parvovirus which controls antigenicity and host range. Virology 225, 65±71. Nathanson, N., McGann, K.A., Wilesmith, J., Desrosiers, R.C., Brookmeyer, R., 1993. The evolution of virus diseases: their emergence, epidemicity, and control. Virus Res. 29, 3±20. Parker, J.S.L., Parrish, C.R., 1997. Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid. J. Virol. 71, 9214±9222. Parrish, C.R., 1991. Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones. Virology 183, 19±205. Parrish, C.R., Aquadro, C.F., Carmichael, L.E., 1988. Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink and raccoon parvoviruses. Virology 166, 293±307. Parrish, C.R., Aquadro, C., Strassheim, M.L., Evermann, J.F., Sgro, J.-Y., Mohammed, H., 1991. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J. Virol. 65, 6544±6552. Parrish, C.R., Carmichael, L.E., 1986. Characterization and recombination mapping of an antigenic and hostrange mutation of canine parvovirus. Virology 148, 121±132. Parrish, C.R., Gorham, J.R., Schwartz, T.M., Carmichael, L.E., 1984. Characterisation of antigenic variation among mink enteritis virus isolates. Am. J. Vet. Res. 45, 2591±2599. Parrish, C.R., O'Connell, P.H., Evermann, J.F., Carmichael, L.E., 1985. Natural variation of canine parvovirus. Science 230, 1046±1048. Reed, A.P., Jones, E.V., Miller, T.J., 1988. Nucleotide sequence and genome organization of canine parvovirus. J. Virol. 62, 266±276. Saliki, J.T., Mizak, B., Flore, H.P., Gettig, R.R., Burand, J.P., Carmichael, L.E., Wood, H.E., Parrish, C.R., 1992. Canine parvovirus empty capsids produced by expression in a baculovirus vector; use in analysis of viral properties and immunization of dogs. J. Gen. Virol. 73, 369±374. Strassheim, L.S., Gruenberg, A., Veijalainen, P., Sgro, J.-Y., Parrish, C.R., 1994. Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid. Virology 198, 175±184. Tresnan, D.B., Southard, L., Weichert, W., Sgro, J.-Y., Parrish, C.R., 1995. Analysis of the cell and erythrocyte binding activities of the dimple and canyon regions of the canine parvovirus capsid. Virology 211, 123±132. Truyen, U., Agbandje, M., Parrish, C.R., 1994. Characterization of the feline host range and a specific epitope of feline panleukopenia virus. Virology 200, 494±503. Truyen, U., Gruenberg, A., Chang, S.F., Obermaier, B., Veijalainen, P., Parrish, C.R., 1995. Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo. J. Virol. 69, 4702±4710. Truyen, U., Parrish, C.R., 1992. Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: Distinct host cell tropisms of each virus in vitro and in vivo. J. Virol. 66, 5399±5408. Truyen, U., Platzer, G., Parrish, C.R., 1996. Antigenic type distribution among canine parvoviruses in dogs and cats in Germany. Vet. Record 138, 365±366. Tsao, J., Chapman, M.S., Agbandje, M., Keller, W., Smith, K., Wu, H., Luo, M., Smith, T.J., Rossmann, M.G., Compans, R.W., Parrish, C.R., 1991. The three-dimensional structure of canine parvovirus and its functional implications. Science 251, 1456±1464. Wikoff, W.R., Wang, G., Parrish, C.R., Cheng, R.H., Strassheim, M.L., Baker, T.S., Rossmann, M.G., 1994. The structure of a neutralized virus: Canine parvovirus complexed with neutralizing antibody fragment. Structure 2, 595±607.
Host range relationships and the evolution of canine parvovirus Colin R. Parrish* James A. Baker Institute, College of Veterinary Medicine, Cornell University Ithaca, NY 14853, USA
Abstract Canine parvovirus (CPV) is an example of an unusual class of emerging virus±those that gain an altered host range through genetic variation and subsequently become widespread pathogens of their new and previously resistant host species. CPV was first detected in 1978 as the cause of new diseases in dogs throughout the world, when it rapidly spread throughout domestic populations, as well as becoming widespread in wild dogs. CPV was soon shown to be a variant of the long recognized feline panleukopenia virus (FPV), from which it differed in less than 1% at the nucleotide sequence level. Genetic analysis showed that virtually all of the biological differences between CPV and FPV, including the canine host range, were determined by three or four sequence differences in the viral capsid protein gene. Analysis of the atomic structures of the CPV and FPV capsids showed that the differences controlling host range were located within two different structural regions and were exposed on the capsid surface. The CPV which first emerged in 1978 appeared to be derived from a single ancestral sequence, which has allowed the ready analysis of the subsequent evolution of the virus in nature. Sequence analysis has also revealed that CPV strains have undergone a series of evolutionary selections in nature which have resulted in the global distribution of new virus variants. This was first seen in the global replacement between 1979 and 1981 of the original (1978) strain of the virus by a genetically and antigenically variant strain, and the subsequent widespread selection of other variants which have also become globally distributed. The genetic and antigenic variation in the virus strains was also correlated with changes in the host range of the virus, in particular in the ability to replicate in cats, and in canine host range differences seen in tissue culture cells. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Parvovirus; Evolution; Host range; Capsid structure
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Tel.: +1-607-256-5649; fax: +1-607-256-5608 E-mail address: [email protected] (C.R. Parrish) 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 8 4 - X
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1. Introduction The emergence of new microbial pathogens can occur through a number of different mechanisms (Nathanson et al., 1993). In some cases this occurs through the identification of a long existing agent and its association with a disease of previously unknown etiology, most often through the use of new technologies such as PCR for identifying the agent. In other cases changes in environmental, vector, or the host leads to greater incidence or recognition of a pre-existing disease. Such changes can be of the insect vector density due to environmental change, changes in location of the hosts, or immune deficiency of the host. Another mechanism of emergence is through the selection of a host range variant of a pathogen in a host that was previously resistant to such a virus. That type of emergence is unusual, although for viruses it most likely has happened on many occasions to give rise to the current range of animal and plant viruses. However, when such a new agent with an altered host range emerges it has the potential to spread rapidly through the naõÈve and non-immune host population, and there may also be a period of rapid agent variation as the virus becomes adapted to its new host. Examples of viral host range variants of this type which have been documented in recent history include the pandemic influenza A viruses, human immunodeficiency virus (HIV) type-2 and possibly HIV type-1. 1.1. Emergence and initial characterization of CPV In early 1978 canine diseases were observed in Asia, Australia, New Zealand, the Americas, and in Europe which were characterized by vomiting and diarrhea in dogs older than about 6 weeks, and myocarditis in neonatal pups, and these diseases differed from those previously recognized in dogs. A small, round, non-enveloped virus was observed by electron microscopy in stool specimens and in tissues of the affected animals, and a parvovirus was soon isolated in canine and feline tissue cultured cells from both forms of the disease. The virus was named canine parvovirus (CPV), and has also been referred to as CPV type-2 to distinguish it from the previously described, although unrelated parvovirus minute virus of canines (MVC) (Carmichael and Binn, 1981; Carmichael et al., 1994). The enteric disease in dogs was pathologically similar to a disease of cats caused by the feline panleukopenia virus (FPV), and polyclonal antibody and in vivo cross-protection studies soon showed that CPV and FPV were closely related antigenically. Modified live virus vaccines were soon developed, and since 1979 these have allowed much of the disease to be controlled. However, the virus is still widely distributed in nature, and if pups are not vaccinated, and or when maternal antibodies interfere with vaccination in pups, they generally become naturally infected. Testing for antibodies in the sera collected from dogs or related canids showed that the first positive titers were present in dogs in Europe between 1974 and 1976, and the first positive sera in the USA, Japan, Australia, were reported in early 1978. Wild coyotes in the USA became widely infected during 1979, and wild wolves in a national park in Alaska were infected by 1980. Since 1978 the virus has been ubiquitous in dogs throughout the world.
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1.2. Properties of CPV and FPV 1.2.1. Genetic structure The parvoviruses are both small and genetically simple. The genome of CPV is a single stranded DNA of about 5,200 nucleotides in length, which has two promoters which result in the expression of two structural (VP1 and VP2) and two non-structural proteins (NS1 and NS2) through alternative splicing of the viral mRNAs (Reed et al., 1988, 1991). Only the -ve DNA strand is packaged into the capsid. A non-coding region near the right hand end of the genome that contains a variable number of direct repeat sequences, and there are palindromic hairpins at either end of the genome that are used in the replication of the viral DNA (Fig. 1). 1.2.2. Capsid structure As described below, virtually all the biological differences between CPV and FPV are determined by the viral capsid. The structures of the full (DNA-containing) CPV capsids or empty CPV capsids, or of empty FPV capsids have been determined by X-ray crystallography (Tsao et al., 1991; Agbandje et al., 1993). These structures showed that the capsids were assembled from a total of 60 copies of the VP1 and VP2, with about 5±6 copies of VP1 and 54±55 copies of VP2. The two proteins most likely acted in a structurally equivalent way, since empty capsids could be assembled from VP2 alone
Fig. 1. The genetic structure of the genome of CPV and FPV. The ssDNA genome is about 5150 nucleotides in length, and has terminal palindromes of about 150 bases at the 30 and 50 ends. Promoters at genomic map units 4 and 40 give rise to messages (R1±R3) for the non-structural proteins (NS) and capsid proteins (VP), respectively. NS1 and NS2, and VP1 and VP2 are formed by alternative splicing from the same mRNA. Three sequences in the 50 end of the genome may be present as single or multiple copies.
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Fig. 2. The surface of the 25 nm diameter non-enveloped capsid of CPV as determined from the atomic model. One asymmetric unit of the particle is shown by the large triangle. The three icosahedral axes of symmetry of the particle are indicated by the pentamer (five-fold axis), triangle (three-fold axis), and diamond (two-fold axis). Surface features indicated include a depressed canyon surrounding the five-fold axis, a dimple spanning the twofold axis, and a spike at the threefold axis of symmetry. The positions of two residues (VP2 residues 93 and 323) which affect canine host range of CPV are indicated.
expressed by mammalian plasmid or baculovirus vectors, or from VP1 and VP2 expressed from mammalian plasmid expression vectors (Saliki et al., 1992; Tresnan et al., 1995). The VP2 monomer comprised of an 8-stranded anti-parallel b-barrel with large loops inserted between the b-strands making up most of the exposed surface of the capsid. Surface features of the capsid included a raised area (spike) surrounding the threefold axis of symmetry, a depression (dimple) spanning the two-fold axis of symmetry, and a further depressed area (canyon) surrounding the five-fold axis of symmetry of the capsid (Fig. 2). Two primary antigenic sites on the three-fold spike of the virus were defined using monoclonal antibodies (mAb) were found on the three-fold spike (Strassheim et al., 1994; Wikoff et al., 1994), although epitopes have also been defined by peptide mapping in other regions of the capsid (Langeveld et al., 1993, 1994).
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1.2.3. Nature antigenic variation The antigenic type of the virus appears relatively stable, and viruses that appear antigenically identical have been isolated many decades apart. A relatively small amount of antigenic variation is observed between viruses isolated from an individual host, and there has also been variation identified between viruses from different hosts. Within host variation was clearly demonstrated for the mink enteritis virus isolates, where three different antigenic types were seen (Parrish et al., 1984). 1.2.4. Virus variation and evolution Studies of the DNA sequences of the viruses have revealed the phylogenetic relationships between CPV isolates from dogs and the viruses from cats, raccoons, mink, and arctic foxes (Fig. 3). All the CPV isolates clearly formed a single clade, and were most likely derived from a single common ancestral sequence. The viruses isolated from cats, mink, raccoons or foxes could not be clearly distinguished from each other, suggesting inter-species transmission of those latter viruses. During the analysis of canine viruses collected at different times, it became clear that there was genetic and antigenic variation between the viruses collected at different times after 1978. The early viruses (CPV type-2), collected between 1978 and 1980 were the same worldwide, but between 1979 and 1980 an antigenic variant was identified in many different countries of the world using mAb testing, and that strain was termed CPV type-2a (Parrish et al., 1985, 1991). Although those viruses only differed in 5±6 amino acids from the CPV type-2 isolates, they differed in two different neutralizing antigenic sites on the surface of the capsid (Fig. 4). In about 1984 a further antigenically variant virus strain was detected, which differed in only a single epitope, and that virus was designated CPV type-2b. Despite the small numbers of differences between those viruses, in each case the viruses became globally distributed, indicating that they must have been under strong selection (Fig. 5). As described below, those viruses also differ in host range as well as antigenicity. Initial studies examined the antigenicity of the viruses, and mAb analysis showed that the isolates from dogs differed from those from cats or other carnivores (mink, raccoons) in at least two virus strain specific epitopes one present only on CPV, and the other present on FPV isolates. Further analysis showed that CPV and FPV also differed in their hemagglutination (HA), which was a reflection of the ability to bind to the specific sialic acids. The first difference was that the FPV isolates would HA only in buffers at pHs below 6.6, while the CPV isolates HA at higher pHs up to at least pH 7.5 (Parrish et al., 1988, 1991). The HA specificity of the virus appears to be specific for the sialic acid N-glycolylneuraminic acid, which is present on feline erythrocytes of the A blood group (about 70% of almost all the breeds of domestic cat), but which is not present on the erythrocytes of most breeds of dogs (C. R. Parrish, unpublished results). 1.2.5. Host range The host range differences between the various viruses proved to be complex, and they differed in their ability to infect cells in vitro, or to infect animals. The overall relationships are summarized in Table 1. All of the viruses replicated in most feline cells in tissue culture, but only the isolates from dogs replicated in canine cells in culture, indicating that CPV isolates had a species-specific host range which enabled them to
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Fig. 3. Phylogeny of the DNA sequences of the capsid protein genes of various isolates of from dogs and Asiatic raccoon dogs (CPV and RD), cats (FPV), mink (MEV), raccoons (RPV), and Arctic (blue) foxes (BFPV). The numbers shown are the number of nucleotide differences which are present in each branch, the percentage of alternative trees which had that branching order (in italics), and the bootstrap support for that branching order (in parentheses). The tree was rooted using the PPV capsid protein gene sequence as an outgroup. From Truyen et al., 1995 with permission.
replicate in the new host, and that this was determined at the level of infection of individual cells (Truyen and Parrish, 1992). When the viruses were inoculated into animals different host ranges were observed (Truyen and Parrish, 1992). FPV isolates replicated efficiently in cats, as expected, but CPV isolates differed in their ability to infect and replicate in cats, depending on the antigenic type of the CPV tested. CPV type-2 isolates did not replicate detectably in cats, but the CPV type-2a and CPV type-2b isolates infected cats and replicated efficiently after experimental inoculation (Truyen et al., 1995). That this was a natural host range for the viruses was confirmed by the finding that CPV-2a and CPV-2b were isolated from
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Fig. 4. The coding sequences differences in the capsid protein gene which were found in the various isolates of CPV type-2, CPV type-2a and CPV type-2a and CPV type-2 (Parrish et al., 1991). Those sequences which differ from the reference sequence of CPV type-2 are shaded. The nucleotide position in the complete sequence of CPV, and the VP2 residue that changed are also indicated (Parrish, 1991).
about 10±20% of cats which had natural parvovirus disease in Japan, Germany and the USA (Truyen et al., 1995, 1996). The host range for dogs also differed from that seen in tissue culture. CPV isolates infected and replicated in dogs in a number of lymphoid tissues and the intestinal epithelium. FPV, which did not replicate to a detectable level in dog cells in tissue culture, replicated in the thymus and bone marrow of the dog, but not in the peripheral lymphoid tissue or the intestine (Truyen and Parrish, 1992). 1.2.6. Mapping host range and other virus-specific properties of CPV and FPV In order to understand the basis of the changes which gave rise to the new properties of CPV, infectious plasmid clones of CPV and FPV isolates were analyzed by recombination
Fig. 5. The distribution of CPV antigenic types-2, 2a and 2b in various parts of the world during various years between the year of the viruses first spread (1978) and 1994. Open boxes represent viruses from the USA, closed boxes are isolates from Europe, and shaded boxes are isolates from New Zealand and Australia. For the European isolates in 1980, 1981 and 1982 a large number of isolates were tested, and the number that were CPV2a antigenic type are indicated in parenthesis. Adapted from Parrish et al., 1991, with permission.
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Fig. 6. Genetic mapping of the sequence differences between CPV and FPV that affected canine cell infected. Recombination mapping indicated that the region of the VP2 gene that included the differences of VP2 residues 93, 103, and 323 was important in controlling host range. Site direct mutagenesis was used to alter the positions in the genome of CPV (solid bars) or FPV (hatched bars). The viable viruses generated were tested for their ability to infect either feline cells (solid bar), or canine cells (hatched bar). The reactivity of the virus capsids with monoclonal antibodies recognizing a CPV-specific epitope was also examined, and reactivity seen is indicated.
mapping and site-directed mutagenesis (Parrish et al., 1988; Chang et al., 1992; Horiuchi et al., 1994). Virtually all the defined specific differences between CPV and FPV canine host range, antigenic structure, and the pH dependence of HA were determined by two residues which differed between CPV and FPV in the capsid protein gene, as summarized in Fig. 6. Residue 93 was a Lys in FPV and an Asn in CPV, and residue 323 was an Asp in FPV and an Asn in CPV. Changing either of those two residues in the CPV sequence to the FPV sequence resulted in a virus that infected dog cells inefficiently, while if both those residues in FPV were changed to the CPV sequence (Asn) the virus gained the ability to infect dog cells and dogs. The CPV-specific antigenic epitope was determined by the sequence at residue 93, as the specific mAb bound when that was an Asn but not when it was an Asp. The difference in the pH dependence of HA of the two viruses was also controlled primarily by residue 323, and both CPV and FPV showed a CPV-like pH dependence of HA (HA at pHs up to pH 7.5) when residue 323 was an Asn, and an FPVlike HA (HA only below pH6.6) when it was an Asp. An Asp±Asn difference of VP2 residue 375 also affected the pH-dependence of HA, although the effect seen depended on the amino acid at position 323, which showed a dominant effect over the sequence at position 375 (Chang et al., 1992). 1.2.7. Other capsid sequences which determine canine host range Although VP2 residues 93 and 323 were the main determinants of the canine host range of CPV type-2, other sequences in the capsid also play important roles in determining the ability of the virus to infect canine cells. This was initially revealed by a CPV type-2 mutant which was selected during passage in feline cells (Parrish and
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Fig. 7. The structural variation of an additional region on the CPV capsid which affects canine host range (Llamas-Saiz et al., 1996). The mutation examined was the change of VP2 residue Ala(A)300 to Asp(D). The structure shows the amino acids which interact with the loop around the residue 300, and the adjacent loops derived from different VP2 molecules. For the region containing the mutation, both the wild type (dark) and mutant (shaded) structures are shown. The new bond which is likely formed between the mutant Asp(D))300 and Arg(R)81 is indicated.
Carmichael, 1986). That virus lost the ability to infect dogs or dog cells in culture, and also differed antigenically as it no longer reacted with several neutralizing monoclonal antibodies. Recombination analysis mapped the host range determining difference to a region of the genome which contained two differences in sequence from the wild type CPV type-2-VP2 residues 300 Ala-Asp, and 301 Ile-Val. In further analysis of that structural region, series of site-directed mutants was prepared at those positions, or in adjacent structural locations, and those showed that most mutations reduced the ability of the virus to infect dog cells, indicating that wild type structure of the region that was important for maintenance of canine host range (Parker and Parrish, 1997). The atomic structure of one mutant of CPV (with the change of VP2 residue 300 from La to Asp) was determined, and that showed that the new Asp side chain formed a new hydrogen bond with the Lys side chain of a three-fold-related VP2 molecule, and perhaps stabilized the structure of the capsid (Fig. 7) (Llamas-Saiz et al., 1996). Although that region was not directly associated with the canine host range differences between FPV and CPV type-2, in the later emerging CPV type-2a and CPV type-2b viruses there were a series of differences (87 Met-Leu; 300 Ala-Gly; 305 Asp-Tyr) in the vicinity of this region of the structure (Parrish et al., 1991), and those may have contributed additional host range effects that allowed the rapid selection of the newer strains of CPV in dogs throughout the world. 1.2.8. Feline host range Although CPV type-2 viruses did not replicate detectably in cats, CPV type-2a and CPV type-2b both replicated efficiently in that host. The viral sequences which restricted the feline host range of CPV type-2 proved difficult to define, in part because that could only be tested by infecting cats, but CPV/FPV recombinants only replicated efficiently in cats if sequences from the two ends of the capsid protein gene were included in the recombinant (Fig. 8) (Truyen et al., 1994). If only one or the other region were included
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Fig. 8. Genetic mapping of the feline host range difference between CPV type-2 and FPV. A series of recombinant viruses were prepared between the CPV (solid bars) and FPV genomes (crosshatched bars), and the viruses were tested for their ability to infect and replicate in cats. Tissue titers recovered in several tissues are shown. Adapted from (Truyen et al., 1994) with permission.
the virus would replicate to a higher titer in the cat than CPV type-2, but not to the level of FPV. 2. Conclusions The emergence of canine parvovirus provides a useful model for the process of emergence through host range variation and evolution of an animal virus. Although the type of change seen here (Fig. 9) is not common, such a genetic change only has to happen once under the right circumstances for the variant virus to become established in its new host. It is likely that viruses of humans and other animals have periodically emerged in this way during historical time periods, and current trends towards xenotransplantation and other new associations between humans and animals may favor the development and selection of new variants. It is also likely that this type of change happened on many occasions in prehistory to give rise to the present range of animal viruses. Once emerged the subsequent progress of such a new virus depends on it
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Fig. 9. Summary of the viruses examined in these studies and the probable relationships. We cannot clearly determine which of the viruses of cats, mink, raccoons, foxes, or other hosts gave rise to the original virus that was the single common ancestor of all the viruses of dogs (CPV), but that appears to have been present in Europe during the mid-1970s. Subsequently that ancestral virus gave rise separately to CPV type-2 and CPV type-2a, and CPV type-2b was derived from CPV type-2a or a very closely virus. CPV type-2 appears to be largely extinct in nature after about 11981.
properties, modes of persistence or transmission, and on the responses of the host. As seen for CPV, once the new virus becomes established in its new host it is likely to undergo a series of changes leading to further adaptation to become well adapted to the host, and also variation may be selected by the immune pressure of the host. In addition, it is worth noting that the types of change involved can occur DNA viruses such as parvoviruses, as well as the genetically more variable RNA viruses, making it important that all types of viruses be evaluated for these types of changes.
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