Molecular Analysis of Full-Length VP2 of Canine Parvovirus Reveals Antigenic Drift in CPV-2b and CPV-2c Variants in Central Chile

Abstract

Canine parvovirus (CPV) is a major pathogen in canines, with a high mortality rate in unvaccinated puppies. CPV is traditionally classified into three antigenic variants (CPV-2a, CPV-2b and CPV-2c) based on the amino acid sequence of the VP2 protein. Currently, various mutations are described in the receptor-binding area or in the regions of greatest antigenicity of the VP2 protein, giving rise to new viral variants that are capable of immunological escape, affecting the protective immunity of traditional vaccines. In the present study, a molecular characterization of the VP2 gene was performed, which included phylogenetic analysis, amino acid characterization and determination of selection pressures. Blood samples were initially collected from canine patients with clinical signs of gastrointestinal infection, of which 69 were positive for CPV as measured by means of PCR and 18 samples were selected for the amplification of the complete VP2 gene. The analysis revealed a higher rate of CPV-2c-positive patients compared to CPV-2b. Furthermore, the amino acid characterization of VP2 indicated mutations in the regions of highest antigenicity previously described in the literature (CPV-2b: 297 and 324; CPV-2c: 440), as well as others not previously documented (CPV-2b: 514; CPV-2c: 188, 322, 379, 427 and 463). Our analysis of selection pressure showed that the VP2 gene is under negative selection. However, positive selection point sites were identified, both in CPV-2c (324, 426 and 440) and CPV-2b (297 and 324), at sites that have been associated with evasion of the immune response via antigenic drift, which possibly has implications for the protective immunity generated by traditional vaccines.

Keywords: antigenic drift; canine parvovirus; full length VP2; immune escape; molecular characterization; selection pressures.

Figure 1

Phylogenetic tree using the maximum likelihood (ML) method. Phylogenetic inferences were carried out using complete nucleotide sequences of the VP2 gene of CPV-2a, CPV-2b and CPV-2c, including different countries in Europe (GER: Germany; ITA: Italy; SPA: Spain; FRA: France; BEL: Belgium; and POR: Portugal), Asia (CHINA; TW: Taiwan; and KS: South Korea), Africa (NIG: Nigeria), North America (US: United States) and South America (ARG: Argentina; UY: Uruguay; EC: Ecuador; BR: Brazil; including the viral variants from Chile: CL). Each sequence was identified according to GenBank accession number, country of origin and viral variant. The tree included as its root an FPV sequence. The blue branches include the variants associated with the reference gene for CVP-2 and commercial vaccines (CVP-2); the green, red and purple branches indicate the viral variants CVP-2a, CVP-2b and CVP-2c, respectively.

Figure 2

Modeling of the VP2 structure, indicating the substitutions on the triple spike. (A) Representation of more relevant substitutions in variant CPV-2b at sites A297N and Y324I; and (B) CPV-2c at sites T322P, D427N and T440A. For the modeling of amino acid substitutions, the Phire² platform (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 10 May 2021) [32] was used and visualized using Discovery Studio Visualizer Server Copyright © 2019.

Figure 2

Modeling of the VP2 structure, indicating the substitutions on the triple spike. (A) Representation of more relevant substitutions in variant CPV-2b at sites A297N and Y324I; and (B) CPV-2c at sites T322P, D427N and T440A. For the modeling of amino acid substitutions, the Phire² platform (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 10 May 2021) [32] was used and visualized using Discovery Studio Visualizer Server Copyright © 2019.