关键词: 犬细小病毒, 感染性克隆, 反向遗传学, 病毒拯救, 病毒基因组

Abstract:

【Background】Canine parvovirus type 2 (CPV-2), a member of the genus Parvovirus within the family Parvoviridae, is a non-enveloped, single-stranded DNA virus that mainly infects canines and poses a great threat to puppies in particular. The virus has become one of the most significant epidemic threats to the global canine breeding industry. Currently, vaccination is the main strategy for preventing CPV-2 infection; however, cases of disease have still been reported in vaccinated dogs. Therefore, the fundamental research on the pathogenic mechanisms and immunological characteristics of circulating CPV-2 strains is essential for optimizing prevention and control strategies.【Objective】This study aimed to establish a reverse genetics system for the currently prevalent CPV-2c subtype, providing an essential experimental tool for future investigations into viral pathogenicity, immune escape mechanisms, and host adaptive evolution.【Method】Based on the published genome sequence of the CPV-Y1 strain (GenBank ID: D26079.1), specific primers were designed to amplify the near full-length genome of the CPV-2c SX-LC strain, which was isolated in our laboratory using segmented PCR. The inverted terminal repeats (ITRs) of CPV-2 were synthesized and combined with the PCR products to clone the complete CPV-2 genome into the pBluescript SK (+) vector. An XhoI restriction site was introduced into the genome as a genetic marker, facilitating the construction of a stable and operable reverse genetics plasmid. The purified recombinant plasmid was transfected into canine-derived F81 cells to rescue the virus, followed by serial passaging. Successfully rescue of the virus was confirmed through the observation of cytopathic effects (CPE), detection of the VP2 major capsid protein using indirect immunofluorescence assay (IFA), and morphological identification of viral particles by negative staining under transmission electron microscopy (TEM). Additionally, the proliferation kinetics of the rescued virus were assessed via a one-step growth curve assay, and hemagglutination activity (HA) was evaluated too. These characteristics were compared to those of the parental wild-type strain to verify the stability and utility of the constructed reverse genetics system.【Result】Restriction enzyme digestion and sequencing confirmed the successful construction of the full-length CPV-2c SX-LC genome plasmid. After transfection of the recombinant plasmid into F81 cells and five serial passages, the significant cytopathic effects were observed. IFA results confirmed stable expression of the VP2 antigen in infected cells. TEM analysis revealed virus particles with typical CPV-2 morphological characteristics, further validating the successful rescue of the recombinant virus rCPV-2c SX-LC. Growth curve analysis and hemagglutination assays demonstrated that the rescued virus exhibited replication kinetics and hemagglutination activity comparable to those of the parental strain, indicating similar biological characteristics.【Conclusion】By optimizing the cloning of ITRs, a plasmid containing the complete CPV-2 ITR structure was successfully constructed, which improved rescue efficiency and ensured genome integrity compared to traditional construction strategies. The established reverse genetics system for the CPV-2c SX-LC strain provided a robust technical platform for further basic and applied research on canine parvovirus.

Key words: canine parvovirus, infectious clone, reverse genetics system, virus rescue, viral genome