A Novel Seminested Polymerase Chain Reaction Assay With High Sensitivity and Robust Specificity Enables Simultaneous Detection of Human and Animal Bocaviruses

Abstract

Background Bocaviruses (BoVs), belonging to the Parvoviridae family, pose significant challenges in detection due to their genetic diversity and cross-species transmission capabilities. Efficient and broad-spectrum detection methods are essential for understanding BoV epidemiology and addressing potential zoonotic risks. Methods We developed a seminested polymerase chain reaction (PCR) assay for simultaneous detection of diverse BoV species across human and animal hosts. Primers were designed by analyzing 765 BoV genome sequences, targeting conserved regions spanning the NP1 to VP2 genes. Sensitivity was determined through analytical tests, and specificity was evaluated against 39 non-BoV viruses. Validation was performed using spiked biological samples, and the method was applied to 552 clinical samples from 542 hosts, encompassing a broad range of mammalian species. Results The assay demonstrated high sensitivity, detecting BoVs at concentrations as low as 0.2 copies/µL. Specificity tests confirmed no cross-reactivity with other viral families. Validation using 37 strains representing 29 BoV species affirmed its broad efficacy. BoVs were identified across diverse hosts, including humans, bats, canines, porcines, rodents, and felines. Additionally, novel host associations were observed, such as Panthera uncia bocaparvovirus (PuBoV) in a tiger and serval cat, canine bocavirus 2 (CBoV-2) in raccoon dogs, and feline bocaviruses (FBoV) in murid rodents. Human bocaviruses were also detected in monkey samples, indicating potential pathogen spillover. Conclusions This seminested PCR method provides a sensitive and specific tool for BoV detection, enhancing surveillance in human and animal populations. It is instrumental in monitoring zoonotic risks and emerging infectious threats, offering critical insights into BoV epidemiology and cross-species transmission dynamics.