Abstract
By antigenic and genetic characterization of canine parvovirus type 2 (CPV-2) strains collected in 2001 and 2002 in Italy, it was possible to observe the spread of viruses with an unusual mutation, Glu-426, affecting a major antigenic epitope of CPV-2. Out of 67 strains analyzed, 49 (73.13%) were characterized as CPV-2a, 6 (8.95%) were characterized as CPV-2b, and 12 (17.91%) were characterized as the Glu-426 mutant.
In the early 1970s, a new infectious disease with a high rate of mortality was observed in pups throughout the world, and a novel parvovirus was isolated in both canine and feline cell cultures (2, 7, 14, 15). The virus was referred to as canine parvovirus type 2 (CPV-2) to distinguish it from the unrelated parvovirus minute virus of canines (MVC or CPV-1) (8, 9).
CPV-2 possesses a single-stranded DNA genome of about 5,200 nucleotides in length, enclosed in a 26-nm-diameter icosahedral capsid made up of a combination of two proteins, VP1 and VP2 (1, 25). By sequence analysis, CPV-2 has been shown to be closely related to feline panleukopenia virus, from which it presumably originated, and also to parvoviruses from raccoons, minks, and arctic foxes, all of which are included in the feline parvovirus subgroup (20, 33). A few years after the emergence of CPV-2, two new antigenic types, designated type 2a and type 2b and distinguishable by means of monoclonal antibodies (MAbs), arose consecutively (21, 23). Currently, the antigenic variants of CPV have completely replaced the original type 2 virus and are variously distributed in canine populations worldwide (2, 4, 11, 12, 15, 24, 26, 28, 29, 32, 34).
Following the onset and rapid spread of the antigenic variants CPV-2a and CPV-2b, there has been little evidence for a further significant evolution, even if some unusual mutations, such as 300-Asp or 265-Pro, have been described sporadically and another mutation, Ala-297, is now detectable in most of the recent CPV strains irrespective of their antigenic type and geographical origin (3, 13, 29, 32, 34).
Analysis of CPV strains detected in Italy revealed the onset in 2000 of an unusual CPV-2 mutant (5), with a change (Asp-426 to Glu) occurring in the strategic residue 426 (1, 21, 30) (Table 1). The mutation, although affecting an important antigenic epitope of CPV-2, is not identifiable by a panel of MAbs used commonly to characterize CPV strains antigenically, as the Glu-426 mutant displays the same monoclonal reactivity as CPV-2b (5), i.e., they are not recognized by MAb B4A2 (Table 2). However, since the nucleotide variation responsible for the substitution Asp-426 or Asn-426 to Glu at nucleotide 4064 created a new MboII restriction site (GAAGA) unique to these strains, it is possible to distinguish easily the Glu-426 mutants from other antigenic types by restriction enzyme analysis (5).
TABLE 1.
Amino acid changes in the VP2 capsid protein of reference parvoviruses of the feline subgroupa
| Virus type | Country of origin, year of isolation | Strain | Source animal | Amino acid changes at residue:
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 87 | 101 | 265 | 297 | 300 | 305 | 426 | 555 | ||||
| FPV | U.S., 1967 | FPV-b | Cat | Met | Ile | Thr | Ser | Ala | Asp | Asn | Val |
| MEV | U.S., 1975 | MEV-b | Mink | Met | Ile | Thr | Ser | Val | Asp | Asn | Val |
| CPV-2 | U.S., 1978 | CPV-b | Dog | Met | Ile | Thr | Ser | Ala | Asp | Asn | Val |
| U.S., 1978 | CPV-Norden | Dog | Met | Ile | Thr | Ser | Ala | Asp | Asn | Val | |
| CPV-2a | U.S., 1984 | CPV-15 | Dog | Leu | Thr | Thr | Ser | Gly | Tyr | Asn | Ile |
| U.S., 1983 | CPV-31 | Dog | Leu | Thr | Thr | Ser | Gly | Tyr | Asn | Ile | |
| CPV-2b | U.S., 1984 | CPV-39 | Dog | Leu | Thr | Thr | Ser | Gly | Tyr | Asp | Val |
| U.S., 1990 | CPV-133 | Dog | Leu | Thr | Thr | Ser | Gly | Tyr | Asp | Val | |
| Asp-300 CPV-2 | Vietnam, 2000 | LCPV-V203 | Leopard | Leu | Thr | Thr | Ala | Asp | Tyr | Asp | Val |
| Vietnam, 2000 | LCPV-V140 | Leopard | Leu | Thr | Thr | Ala | Asp | Tyr | Asn | Val | |
| Pro-265 CPV-2 | Italy, 2000 | CPV-616 | Dog | Leu | Thr | Pro | Ser | Gly | Tyr | Asp | Val |
| Italy, 2000 | W42 | Wolf | Leu | Thr | Pro | Ser | Gly | Tyr | Asp | Val | |
| Glu-426 CPV-2 | Italy, 2000 | 136/00 | Dog | Leu | Thr | Thr | Ala | Gly | Tyr | Glu | Val |
| Italy, 2000 | 56/00 | Dog | Leu | Thr | Thr | Ala | Gly | Tyr | Glu | Val | |
Recently identified patrovirus mutants are shown in bold.
TABLE 2.
Reactivities of MAbs against CPV-2, CPV-2a, CPV-2b, and the Glu-426 CPV-2 mutants by hemagglutination inhibition
| Isolate | Reactivities of MAbsa
|
Antigenic type, amino acid change | |||
|---|---|---|---|---|---|
| A4E3 | B4A2 | C1D1 | B4E1 | ||
| DP/83 | 160 | 160 | 0 | 1,280 | CPV-2, Asn-426 |
| 188-01 (B) | 640 | 80-160 | 1,280 | 40 | CPV-2a, Asn-426 |
| 108-02 (44) | 640 | 80-160 | 2,560 | 20-40 | CPV-2a, Asn-426 |
| 119-02 (A) | 640 | 80 | 1,280 | 20-40 | CPV-2a, Asn-426 |
| 42-01 | 2,560 | 0 | 2,560 | 20-40 | CPV-2b, Asp-426 |
| 198-02 (A) | 640 | 0 | 1,280 | 0 | CPV-2b, Asp-426 |
| 205-02 | 1,280 | 20 | 1,280 | 10 | CPV-2b, Asp-426 |
| 56-00 | 320 | 0 | 1,280 | 20 | CPV-2?, Glu-426 |
| 136-00 | 1,280-2,560 | 5-10 | 2,560 | 20 | CPV-2?, Glu-426 |
| 157-02 (5) | 320 | 0 | 640-1,280 | 20-40 | CPV-2?, Glu-426 |
| 202-02 (1) | 160 | 0 | 640 | 20 | CPV-2?, Glu-426 |
| 202-02 (1) | 160 | 0 | 640 | 20 | CPV-2?, Glu-426 |
Values indicate the reciprocal of the highest dilution of MAb inhibiting eight hemagglutination units of the virus. MAb specificities are as follows: A4E3 for CPV-2, CPV-2a, and CPV-2b; B4A2 for CPV-2 and CPV-2a; C1D1 for CPV-2a and CPV-2b; and B4E1 for CPV-2.
By combining antigenic and genetic analyses, searches for CPVs displaying the Glu-426 mutation were conducted in different parts of Italy (Apulia, Sicily, and Campania) between 2001 and 2002. A total of 67 CPV strains were detected by a hemagglutination assay in either specimens or rectal swabs of pups affected by gastroenteritis. Attempts to adapt the CPV strains to in vitro cultivation in A-72 cells were made. The viruses identified underwent antigenic characterization in a hemagglutination inhibition assay using a panel of four MAbs (A4E3, B4A2, C1D1, and B4E1), kindly supplied by C. R. Parrish (Cornell University, Ithaca, N.Y.). The differential reactivity of CPV-2, CPV-2a, and CPV-2b to MAbs is shown in Table 2. All the strains characterized as type 2b by MAb analysis were subjected to genetic screening by restriction enzyme analysis. The PCR product generated with the primer pair 555for-555rev, corresponding to the COOH terminus of CPV open reading frame 2, was digested with the restriction enzyme MboII (5). To verify the accuracy of MboII digestion, the amplicons 555for-555rev were directly sequenced by using a BigDye sequencing kit and an ABI-377 automatic DNA sequencer (Applied Biosystems, Foster City, Calif.).
In 2001 and 2002, type 2a was the most common antigenic type. Three type 2b CPVs were detected in 2001 and three were detected in 2002. Although in 2000 only two Glu-426 strains had been detected, three strains in 2001 and nine strains in 2002 were identified (Table 3). It is of note that the Glu-426 strains were detected in different areas of southern Italy (Apulia, Sicily, and Campania). Sequence analysis of the strains identified as the Glu-426 mutant by MboII digestion confirmed the presence of the mutation T→A at nucleotide 4064 (data not shown).
TABLE 3.
Antigenic and genetic characterization of CPV-2 strains in Italya
| Antigenic type | MboII site (nt 4062-4066) | Origin of the strain | Yr of isolation |
|---|---|---|---|
| CPV-2a | |||
| 4-01 | Abruzzo | 2001 | |
| 41-01 | Apulia | 2001 | |
| 52-01 | Apulia | 2001 | |
| 57-01 | Apulia | 2001 | |
| 62-01 | Apulia | 2001 | |
| 70-01 (A) | Apulia | 2001 | |
| 77-01 | Apulia | 2001 | |
| 85-01 | Apulia | 2001 | |
| 89-01 (B) | Apulia | 2001 | |
| 91-01 (D) | Apulia | 2001 | |
| 93-01 (A) | Apulia | 2001 | |
| 93-01 (B) | Apulia | 2001 | |
| 96-01 | Apulia | 2001 | |
| 97-01 (B) | Apulia | 2001 | |
| 98-01 (A) | Apulia | 2001 | |
| 99-01 | Apulia | 2001 | |
| 102-01 (C) | Apulia | 2001 | |
| 102-01 (A) | Apulia | 2001 | |
| 104-01 (A) | Apulia | 2001 | |
| 106-01 | Apulia | 2001 | |
| 111-01 (B) | Apulia | 2001 | |
| 113-01 | Apulia | 2001 | |
| 120-01 | Apulia | 2001 | |
| 122-01 | Apulia | 2001 | |
| 146-01 | Apulia | 2001 | |
| 169-01 | Apulia | 2001 | |
| 184-01 (A) | Apulia | 2001 | |
| 188-01 (B) | Apulia | 2001 | |
| 228-01 | Apulia | 2001 | |
| 233-01 | Apulia | 2001 | |
| 239-01 (A) | Apulia | 2001 | |
| 255-01 (A) | Apulia | 2001 | |
| 273-01 (I) | Apulia | 2001 | |
| 273-01 (2) | Apulia | 2001 | |
| 288-01 | Apulia | 2001 | |
| 297-01 (A) | Apulia | 2001 | |
| 307-01 (A) | Apulia | 2001 | |
| 307-01 (B) | Apulia | 2001 | |
| 2-02 | Apulia | 2002 | |
| 63-02 | Apulia | 2002 | |
| 69-02 | Apulia | 2002 | |
| 95-02 (A) | Apulia | 2002 | |
| 108-02 (8) | Abruzzo | 2002 | |
| 108-02 (32) | Abruzzo | 2002 | |
| 108-02 (44) | Abruzzo | 2002 | |
| 119-02 (A) | Apulia | 2002 | |
| 133-02 | Apulia | 2002 | |
| 146-02 | Apulia | 2002 | |
| 202-02 (3) | Apulia | 2002 | |
| CPV-2b | |||
| 42-01 | No | Apulia | 2001 |
| 281-01 (A) | No | Apulia | 2001 |
| 281-01 (B) | No | Apulia | 2001 |
| 61/02 | No | Apulia | 2001 |
| 198-02 (A) | No | Apulia | 2002 |
| 205-02 | No | Apulia | 2002 |
| 56-00 | Yes | Apulia | 2000 |
| 136-00 | Yes | Apulia | 2000 |
| 161-01 | Yes | Apulia | 2001 |
| 189-01 (3) | Yes | Sicily | 2001 |
| 189-01 (5) | Yes | Sicily | 2001 |
| 125-02 (debbi) | Yes | Campania | 2002 |
| 125-02 (leti) | Yes | Campania | 2002 |
| 157-02 (2) | Yes | Apulia | 2002 |
| 157-02 (5) | Yes | Apulia | 2002 |
| 157-02 (5-bis) | Yes | Apulia | 2002 |
| 202-02 (1) | Yes | Apulia | 2002 |
| 202-02 (2) | Yes | Apulia | 2002 |
| 231-02 (1) | Yes | Sicily | 2002 |
| 231-02 (2) | Yes | Sicily | 2002 |
The Glu-426 CPV-2 mutants are boldfaced. nt, nucleotides.
An intriguing question arising from the initial identification of strains 56/00 and 136/00 was whether they represented a newly emerging variant of CPV or a natural mutant detected occasionally. The antigenic variants type 2a and type 2b totally replaced the original CPV-2 and now coexist, even if with different distributions in different countries. In Italy, CPV-2a is prevalent and CPV-2b is isolated with a low frequency (4, 6, 26). The findings of the present study demonstrate that these atypical 56/00- and 136/00-like CPVs are currently cocirculating in Italy together with the other CPV antigenic types.
A peculiarity of parvoviruses of the feline subgroup is that single nucleotide substitutions may determine drastic phenotypic changes affecting antigenicity, host range in vivo and in vitro, and hemagglutination (10, 13, 17, 21, 22, 27, 31, 33). The trajectory of CPV evolution may be considered as a paradigm of how viruses evolve. During its multistep process of evolution, CPV changed its antigenic profile, gained the ability to infect dogs, and lost and regained the ability to infect cats by changing a few amino acid residues (10, 13, 16, 18, 19, 20, 21, 33, 34). From this perspective, the appearance of new variants of CPV-2 as a result of the acquisition of additional changes represents a constant threat to domestic dogs. In recent years, some other CPVs with natural mutations, such as Asp-300 (13) and Pro-265 (3), that significantly affect either the antigenicity or the capsid structure have been identified, but there is no evidence for a further spread of these mutants. In contrast, evidence has now been collected that the Glu-426 mutant of CPV-2 is broadly present in both southern and northern Italy (4). It is therefore possible to speculate that the mutation at residue 426, within an immunodominant epitope of CPV (10, 18, 21, 30), has provided the mutant with a certain benefit.
Continued epidemiological surveillance of the distribution of the CPV types will help elucidate whether this mutant has become permanently established in the dog population and whether it is also spreading in other parts of the world, thus providing insights into the mechanisms driving the evolution of CPV-2.
Acknowledgments
This work was supported by grants from CEGBA (Centro di Eccellenza di Genomica in campo Biomedico ed Agrario) and from MURST (project “Enteriti dei piccoli animali,” Ministero dell'Università per la Ricerca Scientifica e Tecnologica).
We thank Paola Fiorente for expert technical assistance and Athina Papa for editorial suggestions. We are extremely grateful to Leland Eugene Carmichael for constant encouragement throughout the study.
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