Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

  • Animals (Basel). 2026 May 3;16(9):1402. doi: 10.3390/ani16091402.
Liangkai Liu  1 ,  Maohua Xia  2 ,  Chengyao Hou  1 ,  Danyu Chen  3 ,  Chengyao Li  3 ,  Xinggui Chen  1 ,  Qinyuan Chu  1 ,  Yue Sun  1 ,  Shujun Liu  1 ,  Yuqing Li  1 ,  Hanlin Wang  1 ,  Yan Zhu  3 ,  Mengfang Yang  3 ,  Hongning Wang  1 ,  Caiwu Li  3 ,  Xin Yang  1
Affiliations
  • 1. Animal Disease Prevention and Green Development Key Laboratory of Sichuan Province, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China.
  • 2. Beijing Zoo Administration Office, Beijing Key Laboratory of Captive Wildlife Technologies, Beijing 100044, China.
  • 3. China Conservation and Research Center for the Giant Panda, Key Laboratory of SFGA on the Giant Panda, Chengdu 610057, China.
PMID: 42121821 DOI: 10.3390/ani16091402
Abstract

Although canine parvovirus (CPV) vaccination has been widely implemented, CPV continues to circulate in dog populations and poses a potential cross-species transmission risk to wildlife, including giant pandas. Recent increases in CPV-2c detection in China highlight the need for molecular surveillance and standardized immunoreagents for diagnosis and epitope mapping. This study aimed to isolate a representative CPV-2c strain from China and develop VP2-targeted nanobody-based recognition molecules to support antigen monitoring and detection optimization. Canine and giant panda samples were collected in Sichuan Province, and CPV was isolated in F81 cells, followed by VP2 gene Sequencing and phylogenetic analysis. A secretion expression system in Bacillus subtilis was established to produce VP2-targeting Nanobodies, and a canine Fc-fused format of Nb10 (Nb10-Fc) was constructed. Immunoreactivity was evaluated via immunoassays, and structural modeling and molecular docking were performed to predict binding interfaces. The results showed that CPV-2c was the dominant genotype in Sichuan, with CPV L4 being a representative strain that exhibited 100% identity in VP2 with a giant panda-derived CPV-2c strain. Nb10 and Nb10-Fc demonstrated strong reactivity in Western blotting and immunofluorescence assays. The Fc-fusion improved detection sensitivity, offering potential in vivo application benefits. This study provides a standardized VP2-specific nanobody and molecular system for CPV-2c surveillance, antigenic studies, and diagnostic optimization.

Keywords
canine parvovirus; immunodetection; nanobody; virus isolation.