Nanobodies against porcine CD163 as PRRSV broad inhibitor

PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is a major swine pathogen causing economic losses. To the date, effective broad PRRSV inhibitory strategies have not been available in practice yet. Targeting the key viral receptor CD163 to block PRRSV entry has emerged as an alternative approach beside vaccines for PRRSV inhibition. As an effective therapeutic tool, nanoantibodies (Nbs) have been widely used in antiviral research. In this study, a phage display VHH library was constructed for the selection of Nbs against porcine CD163 scavenger receptor cysteine-rich 5-9 domain (SRCR5-9). After five rounds of bio-panning and indirect ELISA, seven CD163-specific Nbs (Nb1-Nb7) were identified. All obtained Nbs displayed strong affinity to CD163 receptor and excellent antiviral activity. In particular, Nb2 exhibited significant broad inhibitory effects on variable PRRSV lineages and downregulated virus-related NF-κB signaling. Further studies suggested that Nbs mainly exerted antiviral functions by interfering with virus attachment stage, and also decreased the transcription of CD163. The conformational epitopes recognized by Nbs were localized in the SRCR5 domain of CD163, a crucial region in PRRSV infection. Overall, our findings provide a novel insight into the biofunction of CD163 in antiviral infection and the development of broad-spectrum strategies against PRRSV.