Abstract
Canine parvovirus (CPV) is a contagious and highly pathogenic virus of dogs. After its first report in 1978, the CPV original type (CPV-2) was rapidly and totally replaced by three antigenic variants named CPV-2a, CPV-2b and CPV-2c that circulate in various countries at different frequencies and recently reported in Nigeria. This study describes the molecular characterization of 28 CPV strains in dogs presenting with gastroenteritis in veterinary clinics at Lagos and Ibadan, Nigeria. The results show the predominance (92.8%) of CPV-2a, while CPV-2c was found only in two samples. Phylogenetic analyses revealed that the CPV Nigerian strains were closely related to Asian strains and 26 CPV-2a out of 28 CPV sequences fell into 2 different subclades consistent with predicted amino acid mutations at position 267, 321, 324 and 440. Lys321Asn was evident in all the Nigerian strains whilst Phe267Tyr and Tyr324Ile were observed in 96.4% of the sequences, respectively. Thr440Ala occurred in 89.3% of sequences from this study. The new CPV-2a was predominant and appears to have replaced other CPV-2a strains in South-western Nigeria whilst the CPV-2c strain which is identical to the isolate recently reported in Northern Nigeria, may have been introduced in this country at the time of this study. Monitoring virus epidemiology is important to better understand the dynamics of CPV evolution and the eventual need to change or improve existing vaccination strategies.
Keywords: Protoparvovirus, Canine parvovirus, Mutations, Molecular characterization, Predicted amino acid changes, Nigeria
Canine parvovirus (CPV) is a highly contagious virus of dogs with mortality rates up to 70% in puppies [11]. It was reported in 1978, when it was associated with canine enteric disease [15, 47]. CPV belong to the new species Carnivore protoparvovirus 1, under the genus Protoparvovirus in the family Parvoviridae along with feline panleukopenia virus (FPV) and other canine parvoviruses [7, 8]. CPV is one of the smallest animal DNA viruses, with a diameter ranging from 18 to 26 nm [46]. The virus has an icosahedral symmetry with a linear, single-stranded DNA genome (5.2 kb), which has two major open reading frames, one encoding the non-structural proteins (NS1 and NS2) and the other encoding the capsid proteins (VP1 and VP2) [42, 45]. VP2 constitutes most of the capsid protein, including the most important antigenic epitopes and is the main target for neutralizing antibodies [47]. CPV and FPV, as well as FPV-like parvoviruses from wild carnivores are closely related genetically and antigenically [34, 39].
CPV has spread worldwide since its emergence in 1978 [35]. In the 1980s, two antigenic variants of CPV-2 emerged, namely CPV-2a and CPV-2b [39] with both variants bearing 5 and 1 amino acid (aa) substitutions respectively, in the VP2 protein [53]. A new antigenic variant, CPV-2c, was reported in 2000 and soon partially replaced the earlier strains in Italy [4]. Other mutants reported since then include the-new CPV-2a and -new CPV-2b with the Ser297Ala mutation [38]. CPV-2c mutants were also recently reported in China, suggesting continuing evolution of the virus [57].
These three strains (CPV-2a, CPV-2b and CPV-2c) are circulating around the world at various frequencies [42]. For example, CPV-2a has been reported to be prevalent in Argentina [5, 6], while CPV-2b was found to be prevalent in India [34] and CPV-2c is prevalent in Mexico [40]. The tendency of the virus to change has been attributed to the peculiarity of the CPV genome [10], which possesses the ability to continuously mutate thereby producing new virus strains with evolutive advantages over the parental viruses. This may explain the subsequent emergence of the antigenic variants and their total replacement of the original strain CPV-2 in the field [11, 55]. With respect to the old type CPV-2, its antigenic variants have been found to cause a more severe disease and are able to infect cats, showing an extended host range [11].
In Nigeria, CPV infection was first reported in locally acquired indigenous breeds of dogs kept in communal cages [24]. Although the disease is enzootic in the country [49], CPV 2a was the only variant reported [12], until recently when all three variants were found to be widespread in Ibadan, Nigeria [13]. However, the most recent CPV study which was focused on the northern part of the country reported only two strains in this region [37].
This study was designed to determine the CPV strains in dogs presenting with diarrhea in veterinary clinics at Lagos and Ibadan, Nigeria, and provide more information regarding the molecular evolution of the virus in this country.
A total of 75 rectal swab samples were collected from dogs presenting with the clinical signs of gastroenteritis (diarrhea, vomiting or both), regardless of age, breed or sex. The samples were obtained between 2014 and 2016 from different veterinary hospitals located in Lagos and Ibadan, Nigeria.
Viral DNA was extracted from the samples using the “fast boiling preparation” method [48]. Commercial DHLPP vaccine (PRO-VAC® by Komipharm international Co. limited) and distilled water were used as positive control and blank, respectively. The lyophilized vaccine was re-suspended in 1 ml PBS and processed using the same extraction protocol. The 12.5 µl reaction contained 2.5 µl of Jenna Bioscience® red load PCR mix, 0.5 µl of each primer (corresponding to 10 pmol/l), 7.5 µl of PCR grade water, and 1.5 µl of template. This PCR protocol amplified a 1042-bp fragment of the VP2 gene (from position 3385 to 4426 of the genome). The following set of primers was used: Forward, 5′-GGAAACCAACCATACCAACTCC-3′ and Reverse, 5′-GGATTCCAAGTATGAGAGGC-3′ [42]. An Applied Biosystems® PCR Thermal Cycler was used for amplification. The cycling conditions included an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation for 30 s at 94 °C, annealing for 1 min at 50 °C, and extension for 1 min at 72 °C and final extension at 72 °C for 10 min. PCR products were run on 1.5% agarose gel with Red Load Taq Master (Jenna bioscience ®) at 120 V for 35 min and visualized under fluorescent light on the Gel Doc EZ imaging system (GelDocTMEZ System with Image Lab software, Bio- Rad Laboratories).The amplified DNA was purified using Promega® PCR Preps DNA purification kit (Promega®, Wisconsin, USA) and quantified by fluorometry using PicoGreen (Invitrogen®). DNA concentration of 10 to 40 ng, required for sequencing 1000 to 2000 bp, ascertained before submission to Inqaba Biotec Lab TM (Pretoria, South Africa) for direct sanger sequencing using the same primers as those used for PCR. Forward and reverse sequences derived from each sample were assembled to generate consensus sequences by CLC Main Workbench 5.5 program. The assembled nucleotide sequences (945–946 nt) were submitted to the NCBI nucleotide blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to search related sequences in public domain databases [25]. Partial VP2-econding gene sequences were aligned with reference sequences retrieved from NCBI database using the Clustal W algorithm, translated into amino-acid (aa) sequences (315 aa) and analyzed using MEGA 7 software program [26]. CPV variants typing was based on the analysis of VP2 amino-acid (aa) residues differentiating CPV2a/b/c [28]. Translated aa sequences were compared with the aa sequence of the Fortdodge® vaccine CPV-2b strain (GenBank accession number FJ222822). Sequence data were submitted to the GenBank database under accession numbers: MH128383, MH137937-MH137938, MH168679- MH168701, MH179302 and MK248596 (Table 1).
Table 1.
VP2 aa changes of the CPV strains described in this study compared with the aa residues of the CPV-2b reference sequence of the Fortdodge® vaccine strain (GenBank accession number FJ222822)
| STRAIN | GenBank Access. nr | VP2 amino acids (Nucleotides) | ||||||
|---|---|---|---|---|---|---|---|---|
| 251 (751–753) | 267 (799–801) | 321 (961–963) | 324 (970–972) | 370 (1108–1110) | 426(1276–1278) | 440(1318–1320) | ||
| Vaccine | FJ222822 | Pro (CCA) | Phe (TTT) | Lys (AAA) | Tyr (TAT) | Gln (CAA) | Asp (GAT) | Thr (ACA) |
| NIG2010 | HQ602995 | Pro (CCA) | Tyr (TTT) | Asn (AAT) | Tyr (TAT) | Gln (CAA) | Asn (AAT) | Thr (ACA) |
| NIG2018 | MK895488 | Pro (CCA) | Tyr (TAT) | Asn (AAC) | Ile (ATT) | Arg (CGA) | Glu (GAA) | Thr (ACA) |
| 1PVCAN | MHI28383 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 4PVCAN | MH168679 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 5PVCAN | MH168680 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 6PVCAN | MH168681 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 7PVCAN | MH168682 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 8PVCAN | MH168683 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 9PVCAN | MH168684 | Ser (TCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 10PVCAN | MH168685 | Ser (TCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 11PVCAN | MH168686 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 12PVCAN | MH168687 | Ser (TCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 13PVCAN | MH168688 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 14PVCAN | MH163689 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 15PVCAN | MH163690 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 24PVCAN | MH137937 | Ser (TCA) | Tyr (TAT) | Asn (AAC) | Ile (ATT) | Arg (CGA) | Glu (GAA) | Ala (GCA) |
| 25PVCAN | MH137938 | Pro (CCA) | Tyr (TAT) | Asn (AAC) | Ile (ATT) | Arg (CGA) | Glu (GAA) | Thr (ACA) |
| 29PVCAN | MH168691 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Thr (ACA) |
| 30PVCAN | MH168692 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 35PVCAN | MK248596 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 36PVCAN | MH168693 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 38PVCAN | MH168694 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 42PVCAN | MH168695 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 46PVCAN | MH168696 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 48PVCAN | MH168697 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 50PVCAN | MH168698 | Pro (CCA) | Tyr (TTT) | Asn (AAT) | Leu (CTT) | Gln (CAA) | Asn (AAT) | Thr (ACA) |
| 54PVCAN | MH168699 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 56PVCAN | MH168700 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 62PVCAN | MH168701 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
| 66PVCAN | MH179302 | Pro (CCA) | Tyr (TAT) | Asn (AAT) | Ile (ATT) | Gln (CAA) | Asn (AAT) | Ala (GCA) |
aAmino acid and nucleotide (in brackets) positions refer to the Fortdodge® vaccine strain (GenBank Accession nr. FJ222822)
In the PCR-based screening 39/75 (52%) specimens collected from dogs were positive for CPV along with commercial DHLPP vaccine used as positive control. No product was obtained for the sterile water sample used as blank.
Out of 39 samples amplified by PCR, 28 amplicons yielded sequence reads with good quality that were assembled to generate consensus sequences. Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) comparison analysis showed a 99% similarity with most CPV sequences. By sequence analysis of aa residues, 26 out of 28 (92.8%) sequences were characterized as CPV-2a (n = 26) and 2 were identified as CPV-2c. Deduced aa sequences of the Nigerian strains were also compared to the reference sequence of the Fortdodge® vaccine CPV-2b strain and two other Nigerian strains obtained eight years apart. The residue Ala at position 297 was conserved in the Nigerian and reference strains. Mutations in a total of 7 aa positions were evidenced when comparing the Nigerian and the reference strains (Table 1). The most common mutation was Lys321Asn, observed in 100% of the sequences identified in this study. Moreover, Phe267Tyr, Tyr324Ile and Thr440Ala mutations were observed in 96.4% (27/28), 96.4% (27/28) and 89.3% (25/28), respectively, of the Nigerian strains.
Phylogenetic tree based on a 945-bp fragment of VP2-encoding gene revealed that CPV-2a and CPV-2c sequences identified in this study clustered with CPV-2a/2c reference sequences retrieved from the GenBank database, respectively (Fig. 1). In general, Nigerian CPV sequences clustered with sequences from Asia and Italy. Two Nigerian strains (24PVCAN-MH137937 and 25PVCAN-MH137938) clustered with CPV-2c strains identified from Vietnam, Italy, and China. These strains revealed aa substitutions at position 267 (Tyr), 324 (Ile), 370 (Arg) and 440 (Ala) compared to the reference strain. Furthermore, three different CPV-2a sub-clades were depicted in the phylogenetic tree (Fig. 1). The sequence of 25 Nigerian CPV-2a strains clustered in the major sub-clade. These sequences revealed predicted aa changes at positions 267 (Tyr), 297 (Ala), 324 (Ile) and 440 (Ala) compared to the reference strain. A second sub-clade contained Chinese and Canadian strains with aa changes evidenced in two residues 267 (Phe) and 449 (Thr) compared to strains of the first sub-clade. A unique sequence (strain 50PVCAN- MH168698) formed a separate sub-clade together with an Italian isolate (Italy 2016-MG434738). The strain showed aa mutations at position 267 (Phe), 324 (Leu) and 440 (Thr) compared with the sequences of the major sub-clade. Interestingly, it was 100% identical to the Italian strain (GenBank accession number MG434738), both sharing the mutation Tyr324Leu [29].
Fig. 1.
Maximum-likelihood tree based on 945-bp fragment of the VP2-econding gene of CPV. The strains identified in this study (evidenced by black dots and triangles) and reference strains retrieved from NCBI are labelled as CPV type, country of origin, year of isolation, isolate name and GenBank accession number. Bootstrap values > 70% (considered to be significant) are indicated at the nodes of the branches. On the right-side aa mutations are evidenced. The scale bar indicates the estimated numbers of nucleotide substitutions per site
Despite the availability of safe and effective vaccines, CPV remains an important cause of mortality in dogs globally [9, 36]. It is regarded as the most important enteric virus affecting canids worldwide [10, 31, 36]. This study aimed to provide more information about the CPV type distribution in southern Nigeria.
A total of 39 out of 75 (52%) faecal samples collected from dogs presented at Nigerian veterinary clinics with gastro-enteritis tested positive by for CPV PCR.
Until recently, the only variant reported in Nigeria was CPV-2a [1, 12]. Notably, only two strains identified in this study were characterized as CPV-2c. Whether this different variant had been infecting dogs before in southern Nigeria or it was introduced within the period of sample collection for this study could not be clearly assessed. The restricted geographic area of Northern Nigeria which was formerly sampled [12], does not rule out the previous circulation of other strains in different regions of the same country. Circulation of all CPV variants, including CPV-2c, in Ibadan was later reported [13]. Recently, in Northern Nigeria, only CPV types 2a and 2c were detected with CPV-2c being predominant [37] and nearly identical to the CPV-2c from this study. This relatedness suggests that this variant may have been newly introduced in western Nigeria early in 2016. Furthermore, only one CPV-2a strain clustered with older isolates of the virus [12] whilst all others were related to the CPV-2a strain from the recent study [37]. This shows a remarkable change in the epidemiological situation of the virus in this country. International accessibility of the two regions (north and south) by their respective airports, one seaport located in the west, in addition to access by different land borders may explain the differences in epidemiology of CPV in the north versus the southern parts of Nigeria. Accordingly, the real circulation of CPV- 2a, -2b and -2c across the country needs to be assessed by larger simultaneous epidemiological studies. Monitoring virus epidemiology is also important to better understand the dynamics of CPV evolution and the eventual need to change or improve existing vaccination strategies, considering that differences between vaccine and field strains may lead to vaccination failures [44]. Phylogenetic analysis showed that CPV-2a strains clustered with Chinese reference strains, which supports earlier theories of Asian origin of these viruses, as suggested in other continents [30]. Although, at the moment, there is no established exotic dog trade with this country, Nigeria has fostered strong business relations with them in recent years [2]. Therefore, virus introduction from Asian countries cannot be ruled out. The presence of some predicted aa residues detected in this study, have been previously reported. Ala-297 was observed in all of the Nigerian sequences and described elsewhere [3, 21, 27, 35, 52, 54]. Mutant viruses presenting Ala-297 have been designated as “new CPV-2a” [14, 58]. The emergence and spread of this mutant strain may have been influenced by the process of host adaptation [14, 41]. The residue 297 has been shown to be under strong positive selection pressure [41]. This site has been suggested to affect an antigenic residue close to epitope B over the shoulder region of the capsid [4]. In the study by Dogonyaro et al. [12], South African isolates showed Asn at position 297, while the Nigerian strains contained Ser at the same position. This mutation was also present in the most recent CPV-2a isolates [37], suggesting that the”new CPV- 2a” may have replaced the older variants of the CPV-2a circulating in Nigeria now. Further research is needed to better understand the role of this aa change in the virus adaptation to the host.
Phe267Tyr mutation, observed in all Nigerian strains except MH168698, had been identified previously in Asia [14, 58] and Uruguay [42]. However, this mutation has been predicted not to have effect on antigenicity as it is unexposed on the capsid of the virus [42]. Another interesting mutation observed in 96.4% (27/28) of the Nigerian strains was Tyr324Ile. This aa change has been previously reported in CPV-2a isolates from Taiwan [27] (Lin et al., 2014), China [14, 56, 57], Korea [22], Thailand [43], Uruguay [42], Japan [51], India [33], Nigeria and South Africa [12, 37]. Tyr324Ile mutation was found in 96.4% (27/28) sequences, either CPV-2a or CPV-2c strains from this study. This residue is adjacent to residue 323, which affects binding of canine transferrin receptor (TfR), resulting in altered host range of CPV [20] and a stronger receptor binding [14]. Residue 324 has been shown to be under strong positive selection in all carnivore parvoviruses [19]. Gln370Arg substitution was observed in the CPV-2c strains from this study and this change has been previously reported in China [14, 56]. This residue is adjacent to residues 379 and 384, which affect the binding to canine transferrin receptor [23], and is therefore suggested to be involved in receptor binding through the neighboring residues [14]. The aa residue 370 is also close to residues 375 and 377, which affect the ability of the virus to haemagglutinate [16]. A total of 25 sequences (89.2%) analyzed in this study contained the Thr440Ala mutation, which has been described in CPV-2a/2b from China [14, 18, 56, 59, 60], and Uruguay [42]. This residue is located at the top of the threefold spike, which is the major antigenic site of the virus and has been suggested to be undergoing positive selection [42]. One isolate from Nigeria as well as 4 from South Africa have previously been described with the same mutation [12].
To the best of our knowledge, aa change Pro251Ser, evidenced in this study, was not previously observed. This aa residue does not elicit antibody as it is unexposed on the capsid surface [17]. Further studies will elucidate the significance of this mutation. However, mutation Lys321Asn, observed in 100% of the strains in this study, was previously reported in recombinant cell culture isolates [32], and the change Tyr324Leu was described in Italy [29, 30], and Brazil [50]. Genotypic surveillance of CPV-2 variants circulating in Nigeria will enable early identification of new genotypic variants with distinct pathogenic or antigenic potentials. The biological implications of these mutations must be assessed by functional assays in order to better understand their role in the severity of clinical signs, as well in the virus infection at the cellular level.
Acknowledgements
We thank the staff of the Department of Virology, Faculty of Basic Medical Sciences, University of Ibadan for providing an enabling environment for the laboratory aspect of this research work, Dr Abdulrazaq Lawal of Mokola Veterinary Clinic, staff of Tessy’s Veterinary Clinic, Idishin, and Dr O. Bankole for their help in the collection of samples for this study.
Conflict of interest
The authors declare that there is no conflict of interests regarding the publication of this article.
Footnotes
Publisher's Note
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