Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

Orf virus (ORFV) is the causative agent of a severe infectious skin disease (also known as contagious ecthyma) in goats, sheep and other small ruminants. Importantly, ORFV also infect humans which causes a public health concern in the context of changing environment and increase in human populations. The rapid detection is critical in effective control of the disease and urgently needed. A novel “point of care” molecular amplification assay for rapid visual detection of ORFV was developed based on isothermoal recombinase polymerase amplification (RPA) technology in combination with a simpler lateral flow immunoassay strip (ORFV RPA- LFD assay). The developed ORFV RPA- LFD assay was able to detect ORFV in less than 25 min. This assay was highly sensitive, with detection limit of as low as 80 copies/reaction, and highly specific, with no cross-reactions with capripox virus, foot-and-mouth disease virus and peste des petits ruminants virus. Furthermore, the ORFV RPA- LFD assay has good correlation with qPCR assay for detection of ORFV present in clinical samples. The developed ORFV RPA-LFD assay was a sensitive and specific method for rapid detection of ORFV, and has great potential as an onsite molecular diagnostic tool in control of Orf.

An important part of the innate immune response is the activation of NF-κB signaling pathway and NLPR3 inflammasome, which is induced upon exposure to pathogens. Peste des petits ruminants virus (PPRV) is a highly contagious virus causing fever, stomatitis, and pneumoenteritis in goats by inducing many proinflammatory cytokines.

Contagious ecthyma (CE) appears in the countries and regions containing goat and sheep farms, and it is considered a global epidemic. CE not only severely endangers the healthy development of the sheep and goat industries but also threatens human health. For viral infectious diseases, fast and effective isolation and culture of the pathogen is critical for CE diagnosis, and for disease prevention and control. Therefore, the sensitivity of bovine Sertoli cells to ORFV was estimate in this study. The sensitivities of bovine Sertoli cells, primary neonatal bovine testicular cells, and Madin-Darby bovine kidney (MDBK) cell line to ORFV were compared. Our results showed that the isolated bovine Sertoli cells were sensitive to inoculated ORFV, and viral titers were approximately 1 log higher than those in primary neonatal bovine testicular cells and in MDBK cell lines. Appropriately sensitive cells for the highly efficient isolation and culture of the ORFV were obtained. Culture of ORFV using the Sertoli cells showed good consistency and stability and also avoided the risk of other pathogens presenting during viral culture using a primary cell line. In addition, using these passaged bovine Sertoli cells to proliferate ORFV may simplify the CE diagnosis process, thereby reducing detection time and cost. Hence, this test has important practical significance for the diagnosis of CE and the research on the pathogenic mechanism of ORFV.