key: cord-0026821-z7pplg6a
authors: Galvis, Cristian Camilo; Jimenez-Villegas, Tatiana; Reyes Romero, Diana Patricia; Velandia, Alejandro; Taniwaki, Sueli; Oliveira de Souza Silva, Sheila; Brandão, Paulo; Santana-Clavijo, Nelson Fernando
title: Molecular diversity of the VP2 of Carnivore protoparvovirus 1 (CPV-2) of fecal samples from Bogotá
date: 2021-10-15
journal: J Vet Sci
DOI: 10.4142/jvs.21181
sha: 44ff03f8a6981fba234f1a2890f048ef62c394cf
doc_id: 26821
cord_uid: z7pplg6a

BACKGROUND: Carnivore protoparvovirus 1, also known as canine parvovirus type 2 (CPV-2), is the main pathogen in hemorrhagic gastroenteritis in dogs, with a high mortality rate. Three subtypes (a, b, c) have been described based on VP2 residue 426, where 2a, 2b, and 2c have asparagine, aspartic acid, and glutamic acid, respectively. OBJECTIVES: This study examined the presence of CPV-2 variants in the fecal samples of dogs diagnosed with canine parvovirus in Bogotá. METHODS: Fecal samples were collected from 54 puppies and young dogs (< 1 year) that tested positive for the CPV through rapid antigen test detection between 2014–2018. Molecular screening was developed for VP1 because primers 555 for VP2 do not amplify, it was necessary to design a primer set for VP2 amplification of 982 nt. All samples that were amplified were sequenced by Sanger. Phylogenetics and structural analysis was carried out, focusing on residue 426. RESULTS: As a result 47 out of 54 samples tested positive for VP1 screening, and 34/47 samples tested positive for VP2 980 primers as subtype 2a (n = 30) or 2b (n = 4); subtype 2c was not detected. All VP2 sequences had the amino acid, T, at 440, and most Colombian sequences showed an S514A substitution, which in the structural modeling is located in an antigenic region, together with the 426 residue. CONCLUSIONS: The 2c variant was not detected, and these findings suggest that Colombian strains of CPV-2 might be under an antigenic drift.

Canine parvovirus type 2 (CPV-2), which is classified into 3 subtypes (CPV-2a, CPV-2b, and CPV-2c), is from the Carnivore protoparvovirus 1 (Parvoviridae, Parvovirinae, Protoparvovirus) species [1] . The virus is a small, non-enveloped virus with a spherical shape, icosahedral capsid, and single-stranded DNA genome of ≤ 5 kb [2] . This virus has four genes in 2 Open Reading Frames (ORFs): ORF1 encodes non-structural proteins NS1 and NS2, and ORF2 encodes capsid proteins VP1, VP2, and VP3 (cleaved from VP2 by host proteases), with a role in receptor binding and stimulation of neutralizing antibodies [3, 4] . CPV-2 shows 98% genome identity to Feline Panleukopenia Virus (FPLV) and Mink Enteritis Virus and probably evolved from the FPLV. Mutations at only 3 amino acids (K93N, V103A, and D323Y) on the VP2 increase infection range from felines to canines [5, 6] .

Monoclonal antibody development in 1980 allowed the recognition of 2 antigenic variants-CPV-2a and CPV-2b. In 2000, a new variant (CPV-2c) was described using molecular biology techniques in Italy [7] . While CPV-2a and CPV-2b have different N426D and I555V residues of VP2, CPV-2c differs only in the N/D426E residue [8] . On the other hand, the V555 residue has been challenged because this amino acid does not present changes in the actuality variants [9] .

In Colombia, CPV-2 was reported for the first time in 1980 [10] . Since then, canine parvovirus disease has been diagnosed frequently in Colombian veterinary clinics. Nevertheless, laboratory confirmation is rare [11] . Few epidemiological studies have been conducted in the country. For example, in Medellin and Bucaramanga, the CPV-2a and 2b variants were detected in 6-month-old puppies [12] . The CPV-2c variant has not been described in the country yet. This study examined the presence of CPV-2 variants in fecal samples of dogs diagnosed with canine parvovirus in Bogotá to increase the knowledge of CPV-2 in Colombia.

Fifty-four fecal samples from puppies and young dogs under one year of age with hemorrhagic enteritis were collected from 2014 to 2018 in a private veterinary practice in Bogotá, Colombia. The dogs received at least one dose of vaccine against CPV, and others with a complete vaccination schedule (Supplementary Table 1 ) underwent a screening test using an Anigen Rapid CPV Ag (BioNote, Korea) test for antigen detection in fecal samples ( Table 1) . After collection, the fecal samples were stored at −20°C until DNA extraction.

All fecal samples were prepared as a 20% (v/v) suspension in DEPC-treated water and clarified at 5,000 × g for 15 min at 4°C. The total DNA was isolated from fecal suspensions using a guanidine-isothiocyanate protocol [13] . The integrity of the extracted DNA was confirmed by polymerase chain reaction (PCR) targeting the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene [14] . All DNA samples were screened for CPV-VP1 with the primers, CPV/FPV diag F and CPV/FPV diag R [15] . Both PCRs were performed separately in a total volume of 25 µL, with 1 × Go Taq Green Master Mix (Promega, USA), 0.5 µM each of the forward and reverse primer, ultrapure water and 2.5 µL of DNA, and with the amplification conditions described by Furtado et al [16] . A field strain of CPV-2c isolated in the A72 cell line was used as a positive control and ultrapure water as the negative control [17] . The PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen, USA).

For amplification of the partial VP2 sequence, primers were manually designed using 83 complete genomes and 305 partial VP2 sequences retrieved from the Genbank, which were aligned using the Clustal W tool included in BioEdit 7.2.5 [18] . The CPV primers for VP2 980Fw (5′-ACCAGCTGAGGTTGGTTATAG-3′, position 3780-3801 based on NC_001539) and CPV VP2 980Rv (5′-CTACTAACTATGATCTAAATGTTC-3′, position 4762-4739) primers were analyzed using the OligoAnalizer 3.1 software (https://www.idtdna.com/calc/analyzer), and the specificity was checked on BLAST-N suite/GenBank (www.ncbi.nlm.nih.gov/BLAST).

PCR reaction was performed in a total volume of 25 µL that contained 1 × Go Taq Green Master Mix (Promega), 0.5 µM of each primer, ultrapure water, and 2.5 µL of DNA. The PCR conditions included initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 55.5°C for 20 seconds, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. Finally, the PCR products were visualized by electrophoresis on 1.5% agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen). Reactions with 982 bp fragments were considered positive. VP2 amplicons were purified with Illustra ExoProStar 1-Step (GE Healthcare, UK) and sequenced bidirectionally using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA), and ABI-3500 Genetic Analyzer (Applied Biosystems) following the manufacturer's instructions. Sequences with Phred quality scores ≥ 20 (http://asparagin. cenargen.embrapa.br/phph/) were assembled with the CAP contig assembly program and aligned with homologous sequences retrieved from GenBank using CLUSTAL W, both implemented in Bioedit 7.2.5 [17] . The amino acid alignment was used to build a maximum likelihood tree (Dayhoff and JTT models) with 1,000 bootstrap replicates using MEGA X [19], rooted with Aleutian Mink Disease Virus as an outgroup (Fig. 1) .

The identity of the nucleotide and amino acids sequences were calculated using the Bioedit Sequence Alignment Editor version 7.2.5 program [18]. Partial VP2 amino acid sequences were analyzed on residue 426 to determine the CPV variant (CPV-2a, 2b, or 2c) [7] . The VP2 monomer and pentamer structure of CPV-2a were constructed on the SWISS-MODEL system [20] using the Canine Parvovirus Empty Capsid (strain D) VP2 (GenBank access PDB 2CAS_2) as homology reference Chain A. The mutagenesis function in PyMOL was used to generate the homology model A514S mutant. Each loop and residue important for this study was highlighted in a 5-fold axis and 3-fold axis using the PyMOL software v.4.6.0 (Fig. 2) . All sequences obtained in this study were submitted to GenBank (MN562440 to MN562462 and MN561010-MN561020).

Approximately 87.0% (47/54) of fecal samples tested positive for VP1 screening (Supplementary Table 1) , 53.3% (26/47) of which were purebred dogs. The positive samples average age was 4.8 months, and the distribution between females and males was 40.4% and 59.6%, respectively. All samples were from puppies that were vaccinated at least once for CPV (34% [16/47] 

Of the positive VP1 samples, 72.3% (34/47) were positive for VP2 reaction and were sequenced. Approximately 88.2% (30/34) were CPV-2a and 11.8% (4/34) were CPV-2b, based on the 426-residue being either N or D, respectively ( Table 2) .

The sequences obtained were segregated in 2 clusters within reference sequences for subtypes 2a and 2b, close to the sequences from Medellin 2013 (KX891194 2b and KX891195 2a). The formation of a cluster for each subtype of CPV was observed. The first at the top of the tree was the cluster of subtypes 2c (yellow). The formation of a cluster of 2b, where four sequences (blue) are located, was observed. A 2a cluster was observed with sequences from different regions (red). The formation of another 2a cluster, where 32 (black) the sequences of Colombia (3 cities) are located, was noted. Finally, the cluster with the sequences of feline panleukopenia was found (Fig. 1) . to a transversion (G to T), all samples were found with T440 ( Table 2) . VP2 3-dimensional reconstruction showed that amino acid 514 is located close to the high antigenic zone at the 5-fold axis (Fig. 3) 

Vaccination is the primary strategy to protect against CPV. Any failures by the presence of maternal antibodies or circulation of different CPV variants could cause an inappropriate immune response, leading to the presentation of severe hemorrhagic gastroenteritis [17, 22] . In the present study, PCR screening showed that all dogs testing positive had been vaccinated with at least one dose of the CPV vaccine.

In Colombia, the vaccination protocol includes 2 or 3 doses (WSAVA Vaccination Guidelines [23]), and vaccines with CPV-2b were licensed by the Colombian Agricultural Institute (ICA). Interestingly, VP2 sequencing showed the circulation of the CPV-2a variant (88.23%), including dogs with 3 vaccine doses ( Table 1) . Therefore, immunization failure could be caused by the lack of cross-immunity by different antigenic variants. Moreover, the four puppies diagnosed with CPV-2b had an incomplete vaccination protocol and were less than 3 months of age (Supplementary Table 1 ).

The prevalence of CPV in puppies or young dogs can vary depending on age, breed, and sex [24, 25] , in addition to the different primers set used in each study. The higher positive rates than other reports from Colombia [26] is due to differences in sampling. The samples used in this study previously tested positive in a rapid Anigen Rapid CPV Ag (BioNote) test because the main purpose of this study was the molecular characterization.

These samples were not amplified with primers 555for/rev reported by [7] . Therefore, it was necessary to design novel primers set CPV VP2 920Fw/Rv. When performing alignment of the whole genome sequences, mismatching was observed in Asian sequences in the hybridization site of the 555rev primer, which is located after the stop codon of the VP2 coding sequence, was observed in Asian sequences. A conserved region in 3' UTR of all 83 complete CPV genome sequences was used to design the VP2 980Rv primer to have a better perspective of Colombian samples amplification. Sequencing showed that the 555rev primer region is different in all samples and could explain the failure in amplification using this set of primers.

The phylogenetic tree revealed several clusters, with the formation of a specific cluster for Colombian CPV-2a, including the sequences reported by Duque et al. [12] and Giraldo- The A514S mutation observed in this study was also reported in Colombian sequences in 2017 and 2020 [12, 26] . In those studies, they specified the possibility of a new variant present in Colombia. When mutation 514 was first reported, it was present in 16/22 samples of CPV-2a [12] ; In this study, all samples came from vaccinated puppies. On the other hand, the samples were collected from puppies with a positive immunochromatography test, and it was not possible to determine the efficacy of the vaccine. Nonetheless, 60% of puppies seroconverted after vaccination with ages between 6 to 8 weeks, and only 10% of puppies with 2 and 3 re-vaccinated had not seroconverted [29, 41] . The aim is not to evaluate the efficiency of the vaccine, but it is important to highlight the necessity of studies in this field.

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 Supplementary Table 1 Features of all 54 samples and test development in this study Click here to view