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Journal of General Virology (2001), 82, 3021–3025. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Evidence for evolution of canine parvovirus type 2 in Italy Canio Buonavoglia,1 Vito Martella,1 Annamaria Pratelli,1 Maria Tempesta,1 Alessandra Cavalli,1 Domenico Buonavoglia,2 Giancarlo Bozzo,1 Gabriella Elia,1 Nicola Decaro1 and Leland Carmichael3 1 Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, S.p. per Casamassima km 3, 70010 Valenzano, Bari, Italy 2 Department of Pathology and Infectious Diseases, University of Messina, Messina, Italy 3 James Baker Institute, Cornell University, Ithaca, NY, USA Two isolates of canine parvovirus (CPV) were obtained from dogs affected with severe haemorrhagic diarrhoea. Type 2b antigenic specificity was predicted by both antigenic analysis with monoclonal antibodies and PCR characterization with type-specific primers. Nevertheless, sequence analysis of the capsid protein-encoding gene revealed two amino acid changes. One of the changes affected position 426 (Asp to Glu), in a major antigenic site of the viral capsid, determining the replacement of a residue unique to CPV type 2b. The failure of established typing methods to distinguish this antigenic variant was overcome by the development of an RFLP assay. During the early 1970s, a new infectious disease of pups, characterized by either gastroenteritis or myocarditis, was observed worldwide. A small, round, non-enveloped virus was observed by electron microscopy in stool specimens and in tissues of affected animals. Subsequently, a novel parvovirus was isolated both in canine and feline cell cultures (Kelly, 1978 ; Appel et al., 1979 ; Burtonboy et al., 1979 ; Johnson & Spradbrow, 1979). The virus was named canine parvovirus type 2 (CPV-2), to distinguish it from the previously described parvovirus canine minute virus (CMV or CPV-1), which is antigenically unrelated to CPV-2 (Carmichael & Binn, 1981 ; Carmichael et al., 1994). CPV possesses a single-stranded DNA genome of negative polarity, about 5200 nt in length. The CPV capsid is a 26 nm diameter icosahedron made up of a combination of two proteins, VP1 and VP2, formed by alternative splicing from the same RNA (Reed et al., 1988). The mutation rate of the CPV genome has not been determined ; however, since parvovirus DNA is replicated by host cell DNA polymerases (Cotmore & Author for correspondence : Canio Buonavoglia. Fax j39 080 4679843. e-mail c.buonavoglia!veterinaria.uniba.it 0001-7880 # 2001 SGM Tattersall, 1987), which have low error rates, mutational events affecting the biological properties of parvoviruses presumably occur very rarely. Estimates suggest that the rate of variation is 1i10−% to 4i10−% changes\nt\year (Parrish, 1991 ; Truyen et al., 1995). In 1979 and 1980, an antigenic variant of CPV was identified in several different countries by using monoclonal antibodies (MAbs) and the variant was termed CPV type 2a (Parrish et al., 1985, 1991). In the mid 1980s, the virus underwent a further antigenic change, and the new variant was referred to as CPV type 2b (Parrish et al., 1991). Currently, the antigenic variants of CPV have completely replaced the original type 2 and are variously distributed in canine populations worldwide (Mochizuki et al., 1993 ; de Ybanez et al., 1995 ; Greenwood et al., 1996 ; Truyen et al., 1996, 2000 ; Steinel et al., 1998 ; Sagazio et al., 1998 ; Buonavoglia et al., 2000 ; Pereira et al., 2000). While the antigenic variants differ from the original type in five or six amino acids, the variant type 2b differs from type 2a in only two positions, Asn-426 to Asp and Ile-555 to Val (Parrish et al., 1991). Residue 426 is placed in a major antigenic site (epitope A) over the three-fold spike of the capsid and the mutation Asn-426 to Asp differentiates CPV-2b not only from CPV types 2 and 2a, but also from feline parvovirus (FPV) and mink enteritis virus (MEV). Conversely, residue 555 lies in a minor antigenic site and the mutation of Ile-555 to Val represents a reversion to or a retention of the sequence of the original type 2 (Table 2) (Parrish et al., 1991 ; Agbandje et al., 1995 ; Strassheim et al., 1994). Following the onset and rapid spread of the antigenic variants 2a and 2b, there has been little evidence of further significant antigenic evolution : the analysis of CPV strains isolated from either domestic or wild felines and canines has demonstrated, with no or a few exceptions (Ikeda et al., 2000 ; Steinel et al., 2000 ; Truyen et al., 2000), that, during the last 15 years, both CPV types 2a and 2b have retained the antigenic configuration defined originally by Parrish et al. (1985, 1991). This communication describes the isolation in Italy of two unusual CPV strains collected during 2000. Both isolates were Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 01:12:45 DACB C. Buonavoglia and others Table 1. Primers used for genotyping and sequence analysis Primer* P2sa P2asa Pabsb Pabasb Pbsb Pbasb Hforc Hrevc 555forc 555revc Sequence (5h 3h) GAAGAGTGGTTGTAAATAATA CCTATATCACCAAAGTTAGTAG GAAGAGTGGTTGTAAATAATT CCTATATAACCAAAGTTAGTAC CTTTAACCTTCCTGTAACAG CATAGTTAAATTGGTTATCTAC CAGGTGATGAATTTGCTACA CATTTGGATAAACTGGTGGT CAGGAAGATATCCAGAAGGA GGTGCTAGTTGATATGTAATAAACA Sense Position† j k j k j k j k j k 3025–3045 3706–3685 3025–3045 3706–3685 4043–4062 4470–4449 3556–3575 4185–4166 4003–4022 4585–4561 * Primers sequences were obtained from : a, Senda et al. (1995) ; b, Pereira et al. (2000) ; c, this study. † Primer locations are indicated as reported in the corresponding papers. The positions of the primers designed in this study are referred to strain CPV-b (accession no. M38245). characterized as type 2b viruses by means of antigenic and PCR analysis. However, analysis of the sequence of the capsid protein-encoding gene revealed an amino acid change in a strategic epitope of the capsid that differentiates both strains from classical type 2b CPVs. Two dogs, respectively 2 years (F56\00) and 2 months (F136\00) old, were presented for clinical examination during early 2000 with signs of severe haemorrhagic gastroenteritis. Suspensions (10 %, w\v) of faecal samples from the animals were positive for CPV by a haemagglutination (HA) test and two isolates were made on A-72 cells grown in DMEM with 10 % foetal bovine serum. Virus growth on infected cells was monitored by HA test and by an indirect immunofluorescence test. A panel of four MAbs (A4E3, B4A2, C1D1 and B4E1), kindly supplied by C. R. Parrish (Cornell University, Ithaca, NY, USA), was used for antigenic typing in a haemagglutination inhibition test. MAb analysis was performed both on suspensions of faecal samples and on viruses after one, three and five passages in cell culture. In order to predict the antigenic type of strains 56\00 and 136\00, we also used a genotyping approach, based on the PCR strategies reported by Pereira et al. (2000) and Senda et al. (1995). Primer pairs Pabs\Pabas and P2s\P2as (Table 1), recognizing the variants and the original type, respectively, were selected in overlapping positions (3025–3045 for Pabs and P2s, 3685–3706 for Pabas and P2as) ; the nucleotide differences between the primers are restricted to one base in the 3h end of each primer. Since the PCR products were of the same size, the primers were used in separate sets of reactions. Primer pair Pbs\Pbas (4043–4062 and 4449–4470 ; Table 1) recognizes CPV type 2b selectively. Differential amplification of CPV type 2b is ensured by single nucleotide variations in the very 3h end of each primer and these nucleotide variations (A G at 4062 and G A at 4449) correspond to the DACC strategic amino acid changes Asn-426 to Asp and Ile-555 to Val that determine the antigenic variation from type 2a to type 2b. Therefore, CPV type 2a is recognized by the primer pair Pabs\Pabas only, while CPV type 2b is recognized by both primer pairs Pabs\Pabas and Pbs\Pbas. PCR analysis was performed both on faecal samples and on viruses after one, three and five passages in cell cultures. The sequence was determined of a fragment of about 1100 nt, encoding a large region of the capsid protein of strains 56\00 and 136\00. Sequence analysis was performed on the virus present in faecal samples and in the first, third and fifth virus passage in A-72 cells. Also, the partial sequences were determined of an additional eight CPV strains (Table 2) isolated in Italy during 1998 and 1999. All these strains had been characterized as type 2b by MAb analysis and by PCR genotyping. For sequence analysis, two primer pairs, Hfor\Hrev and 555for\555rev (Table 1), were selected to amplify a large fragment of the capsid protein-encoding gene of CPV, encompassing at least six or seven informative amino acids responsible for important biological properties of the virus (Parrish et al., 1991 ; Parker & Parrish, 1997 ; Chang et al., 1992). The PCR products were purified and then sequenced directly by using an ABI PRISM 377 sequencer (Perkin Elmer). For sequence comparison, sequences of type 2, 2a and 2b CPVs were selected from the databases. The DNA sequences of strains 56\00 and 136\00 were searched for matches to the recognition sites of a large database of restriction enzymes by computer-aided analysis. A restriction enzyme, MboII, was found to recognize selectively the sequence GAGAA at nt 4062–4066, allowing differentiation of 56\00- and 136\00-like strains from CPV type 2, 2a and 2b strains. Primer pair 555for\555rev was used to amplify a 583 nt fragment that included the MboII restriction site. The PCR product obtained was digested with 5 U MboII. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 01:12:45 Table 2. Amino acid variation in the VP2 capsid protein of the Italian CPV isolates Amino acid residue 87 101 297 3674–3676 TCT (Ser) GCT (Ala) 300 305 … Nucleotide positions … Codons observed Strain 555 Origin (year) Accession no. USA (1967) M38246 Leu Ile Ser Ala Asp Asp Thr Ile Asn Val USA (1975) M24001 Leu Ile Ser Val Asp Asp Thr Ile Asn Val USA (1978) USA (1978) M38245 M19296 Met Met Ile Ile Ser Ser Ala Ala Asp Asp Asn Asp Thr Thr Ile Ile Asn Asn Val Val USA (1984) USA (1983) M24003 M24000 Met Met Thr Thr Ser Ser Gly Gly Tyr Tyr Asp Asp Thr Thr Ile Ile Asn Asn Ile Ile USA (1984) USA (1990) Italy (1998) Italy (1998) Italy (1998) Italy (1998) Italy (1998) Italy (1999) Italy (1999) M74849 M74852 – – – – – – – Met Met Thr Thr Ser Ser Ala Ala Ala Ala Ala Ala Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Asp Asp Asp Asn Asn Asp Asn Asn Asp Thr Thr Thr Asn Asn Thr Asn Asn Thr Ile Ile Ile Thr Thr Ile Thr Ile Ile Asp Asp Asp Asp Asp Asp Asp Asp Asp Val Val Val Val Val Val Val Val Val Italy (2000) Italy (2000) – – Ala Ala Gly Gly Tyr Tyr Asp Asp Thr Thr Ile Ile Glu Glu Val Val , Not determined. DACD Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 01:12:45 Natural mutants of canine parvovirus FPV FPV-b MEV MEV-b CPV type 2 CPV-b CPV-Norden CPV type 2a CPV-15 CPV-31 CPV type 2b CPV-39 CPV-133 242\98 244\98 224\98 212\98 197\98 111\99 01\99 CPV type 2b ? 136\00 56\00 375 389 418 426 3909–3911 3951–3953 4038–4040 4062–4064 GAT (Asp) ACC (Thr) ATT (Ile) AAT (Asn) AAT (Asn) AAC (Asn) ACT (Thr) GAT (Asp) GAA (Glu) C. Buonavoglia and others Strains 56\00 and 136\00 were identified as type 2b by both MAb analysis and PCR genotyping. Nevertheless, in sequence comparisons with well-established type 2, 2a and 2b strains, strains 56\00 and 136\00 were found to contain two unexpected amino acids variations, Ser-297 to Ala and Asp426 to Glu, due to only two nucleotide changes (T to G at nt 3674 and T to A at nt 4064). Extensive comparative analysis by BLAST and FASTA indicated that Ser-297 to Ala is shared by recently isolated CPVs from various parts of the word (Truyen et al., 2000). In contrast, Glu-426 is a substitution that is not present in other CPVs or in closely related parvoviruses. While the change Ser-297 to Ala affects an antigenic residue close to epitope B over the shoulder region of the capsid, the change Asp-426 to Glu determines the replacement of a residue unique to CPV type 2b that is situated in a major antigenic site, epitope A, over the three-fold spike of the capsid (Parrish et al., 1991 ; Parrish, 1994 ; Agbandje et al., 1995). After serial passages in cell culture, there was no apparent modification of either antigenicity or nucleotide composition. All of the isolates collected in 1998 and 1999 were clearly identified as type 2b (Table 2). Interestingly, all strains but one (strain 01\99) had the variation Ser-297 to Ala, suggesting that this change is common in recent CPVs circulating in Italy. There were other recurring mutations in some strains, such as Asp-375 to Asn, Thr-389 to Asn (strains 197\98, 224\98, 244\98 and 111\99) and Ile-418 to Thr (strains 224\98, 244\98 and 197\98). Both strains 56\00 and 136\00 were collected in 2000, but on separate occasions and from two distinct veterinary clinics. This makes it possible to hypothesize that CPVs with mutations similar to those of strains 56\00 and 136\00 have been spreading in Italy since the beginning of 2000. An intriguing question arising from the isolation of strains 56\00 and 136\00 is whether they might represent a newly emerging variant of CPV. Within a few years of the first recognition of CPV type 2, the antigenic variants type 2a and 2b replaced the original CPV type 2 completely. The variants now co-exist, though with different distributions in different countries. In Italy, CPV type 2a is prevalent and type 2b is isolated with low frequency (Sagazio et al., 1998 ; Buonavoglia et al., 2000). Presumably, these atypical type 2b (56\00-like) CPVs might currently co-exist in Italy with the other CPV antigenic types. However, the existence of a new variant should be defined by a wide distribution throughout the world and by significant changes in either the biological or antigenic properties of the virus. A major point to consider is that all the methods currently used to distinguish the CPV antigenic types, and routinely employed in our laboratory, failed to reveal the Glu-426 mutants. The discrimination by MAbs of CPV type 2a from the variant type 2b is based on only a single MAb (B4A2), which recognizes type 2a but not type 2b, because the Asn\Asp change at residue 426 alters the epitope recognized by the MAbs (Parrish et al., 1991). This means that variations at DACE Fig. 1. RFLP analysis of 555for/555rev amplicons. Lanes : M, GeneRuler 100 bp ladder (Fermentas) ; 1, strain 56/00 ; 2, strain 136/00 ; 3, CPV type 2b ; 4, CPV type 2a. residue 426, as in strains 56\00 and 136\00, may not be revealed by this panel of MAbs. The PCR typing approach was also unable to identify the variation that occurred in Glu-426 mutants. Type 2b-specific primers have been designed to take advantage of only two nucleotide variations, and the variation A G at nucleotide 4062, responsible for the change Asn-426 to Asp, is placed at the very 3h end of the sense primer (Pereira et al., 2000). Unfortunately, the Asp Glu change at residue 426 of strains 56\00 and 136\00 is due to a change (T A) in the third codon position, at nucleotide 4064, and these variants are erroneously recognized as type 2b by this PCR strategy. However, since the nucleotide variation at residue 4064 of strains 56\00 and 136\00 created an MboII restriction site (GAAGA) unique to these strains, it was possible to distinguish these mutants from the other antigenic types by simple digestion of the 555for\555rev amplicon with this enzyme, which generates two fragments of about 500 and 80 bp (Fig. 1). Although the isolation of only two anomalous strains does not allow the definition of a new antigenic type of CPV, the occurrence of these natural Glu-426 mutants might indicate a direction in which evolution is leading CPV. It can not be excluded that a more detailed examination of recent CPV strains presenting type 2b may provide evidence for a wider distribution of 56\00- and 136\00-like CPVs. We are grateful to Professor C. R. Parrish for his critical comments and suggestions on the manuscript. References Agbandje, M., Parrish, C. R. & Rossmann, M. G. (1995). 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Two dominant neutralizing antigen determinants of canine parvovirus are found on the threefold spike of the virus capsid. Virology 198, 175–184. 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. Journal of Virology 69, 4702–4710. Truyen, U., Platzer, G. & Parrish, C. R. (1996). Antigenic type distribution among canine parvoviruses in dogs and cats in Germany. Veterinary Record 138, 365–366. Truyen, U., Steinel, A., Bruckner, L., Lutz, H. & Mostl, K. (2000). Distribution of antigen types of canine parvovirus in Switzerland, Austria and Germany. Schweizer Archiv fuW r Tierheilkunde 142, 115–119 (in German). Received 23 May 2001 ; Accepted 15 August 2001 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 01:12:45 DACF