Feline panleukopenia virus and canine parvovirus: development of two qPCR assays with High-Resolution Melting analysis and molecular epidemiology in dogs and cats from Northen Italy in 2017-2023

Andrea Balboni^*Veronica FacileLorenza UrbaniMartina MaglioccaLaura GallinaMaddalena VigatoMara Battilani

• Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy

The Protoparvovirus carnivoran 1 viral species includes relevant pathogens as feline panleukopenia virus (FPV) and canine parvovirus type 2 (CPV-2), which are mainly responsible for immunosuppression and gastroenteritis in domestic and wild carnivores. Differently to FPV, CPV is frequently subjected to mutations. To date, the original antigenic type CPV-2 is mainly used in vaccine production, while field strains have been progressively replaced by the CPV antigenic variants 2a, 2b and 2c. In recent years, additional distinctive mutations have been identified in different CPV antigenic variants classified as "Asian-like". The variability of these viruses can impact on the reliability of molecular diagnostic tests potentially leading to false-negative results or delays in diagnosis. To improve diagnostic accuracy and efficiency, innovative molecular techniques such as High-Resolution Melting (HRM) analysis have been developed. These methods reduce execution time, facilitate diagnosis, and enable the differentiation of species or variants without the need for sequencing. In this study, two real-time PCR (qPCR) assays with HRM analysis were developed to complement existing tools for the detection and genetic differentiation of circulating FPV and CPV. Specifically, a test capable of differentiating FPV, original CPV-2 and CPV-2 antigenic variants, and a test for the identification of Asian-like CPV strains were validated. Furthermore, the FPV and CPV-2 identified in 33 dogs and cats diagnosed with parvoviral infection in a veterinary teaching hospital in Northern Italy between 2017 and 2023 were genetically characterized by sequencing. Based on specific VP2 amino acid residues, 33.3% viruses were FPV, 6.1% were original CPV-2, 6.1% were CPV-2a, 21.2% were CPV-2b and 33.3% were CPV-2c. FPV were detected only in cats and showed high amino acid similarity, confirming its evolutionary stasis. The 45.4% of CPV identified in this study, carried amino acid residues resembling those of Asian-like viruses, suggesting an origin linked to an initial importation and subsequent local diffusion. In contrast, the other CPV-2a, 2b and 2c viruses exhibited greater genetic heterogeneity and their autochthonous origin was supposed. The two qPCR-HRM assays successfully detected and classified all the FPV and CPV tested, highlighting their reliability and usefulness for both diagnostic and epidemiological purposes.