Minute Virus of Canines (MVC) was first isolated from canine fecal samples in 1967 (Binn et al., 1967). Since its discovery, it has been reported in many countries. Neonatal puppies infected with MVC often display symptoms such as dyspnea and severe diarrheal enteritis. Moreover, MVC frequently coinfects with other viruses, which can aggravate and extend the disease's progression in dogs and, in severe instances, may lead to mortality (Piewbang et al., 2018). Bocaparvovirus has various hosts, including bovines (bovine parvovirus, BPV), MVC, humans (human bocavirus, HBoV) (Allander et al., 2005), as well as several new members such as pigs, rats, and minks (Li et al., 2011; Wang et al., 2017; You et al., 2022). However, only partial sequences of the virus have been reported for most of these species. In our prior research, we successfully developed an infectious cloning vector for MVC, designated as pIMVC, significantly advancing the study of the viral genome's sequence characteristics and cell tropism. (Li et al., 2014; Sun et al., 2009). Collectively, this research has provided valuable insights into the molecular mechanisms of MVC.

MVC denotes a linear single-stranded DNA virus characterized by a 5402-nucleotide genome comprising three open reading frames (ORFs). The left and middle reading frames encode the nonstructural proteins NS1 and NP1, respectively. NS1 is a multifunctional protein involved in regulating viral replication and expression. The non-structural protein NP1 (21 kDa) is believed to play a critical role in the accumulation of viral capsid mRNA and proteins. The wide ORF on the right encodes two overlapping capsid proteins, VP1 (81 kDa) and VP2 (67/63 kDa). VP3 (61 kDa) is presumably generated by the cleavage of VP2 and is present in intact viral capsids (Mochizuki et al., 2002). Research on MVC-VP2 remains limited. However, studies on parvovirus capsid protein VP2 suggest that it can induce both humoral and cellular immune responses in the host (Deng et al., 2014; Nan et al., 2018; Tu et al., 2015), rendering it a viable option for vaccine development. Currently, there are no commercially available antibodies targeting MVC-VP2. Therefore, the development of monoclonal antibodies targeting MVC-VP2 is vital for elucidating the pathogenic mechanisms of MVC infection.

In this study, we designed and optimized a 900 bp gene fragment from the N terminal region of the VP2 coding sequence (CDS), which was inserted into the expression vector pET-32a(+). The recombinant fusion protein was obtained following the induction of expression. After immunization of BALB/c mice, we obtained nine hybridoma clones, each secreting monoclonal antibodies that specifically recognized the recombinant fusion protein. Western blot, immunofluorescence and immunoprecipitation assays demonstrated that three highly specific mAbs against VP2 effectively recognized both the eukaryotically expressed VP2 in transfected COS-1 cells and the natural VP2 in MVC-infected WRD cells.