title: An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication.
abstract: Porcine reproductive and respiratory syndrome (PRRS) is a widespread viral disease affecting the swine industry, with no cure or effective treatment. Current vaccines are inefficient mainly due to the high degree of genetic and antigenic variation within PRRS virus (PRRSV) strains. Thus, the development of novel anti-PRRSV strategies is an important area of research. The nonstructural protein 9 (Nsp9) of PRRSV is essential for viral replication, and its sequence is relatively conserved, making it a logical antiviral target for PRRSV. Camel single-domain antibodies (nanobodies) represent a promising antiviral approach because of their small size, high specificity, and solubility. However, no nanobodies against PRRSV have been reported to date. In this study, Nsp9-specific nanobodies were isolated from a phage display library of variable domains of Camellidaeheavy chain-only antibodies (VHH). One of the isolated nanobodies, Nb6, was chosen for further investigation. Co-immunoprecipitation experiments indicated that Nb6 can still maintain antigen binding capabilities when expressed in the cell cytoplasm. A MARC-145 cell line stably expressing Nb6 was established to investigate its potential antiviral activity. Our results showed that intracellularly expressed Nb6 could potently suppress PRRSV replication by inhibiting viral genome replication and transcription. More importantly, Nb6 could protect MARC-145 cells from virus-induced cytopathic effect (CPE) and fully block PRRSV replication at an MOI of 0.01 or lower. To our knowledge, this is the first report of a nanobody based antiviral strategy against PRRSV, and this finding has the potential to lead to future developments of novel antiviral treatments for PRRSV infection.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: liuhongliang55@126.com.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: wangyanfushi@163.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: duanhong910924@sina.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Duan
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: zhangangke1112@126.com.
authors.forename: Angke
authors.initials: A
authors.lastname: Zhang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: chaonvlc@163.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Liang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: jimingao2006@163.com.
authors.forename: Jiming
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: leofighting@163.com.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: hbch228@163.com.
authors.forename: Baicheng
authors.initials: B
authors.lastname: Huang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: jean_lqy@sina.com.
authors.forename: Qiongyi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: lina330077@163.com.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: xiaoshuqi@nwsuaf.edu.cn.
authors.forename: Shuqi
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China. Electronic address: zhouem@nwsuaf.edu.cn.
authors.forename: En-Min
authors.initials: EM
authors.lastname: Zhou
mesh_terms: D000818:Animals; D002162:Camelus; D002460:Cell Line; D047468:Immunoprecipitation; D008297:Male; D019318:Porcine Reproductive and Respiratory Syndrome / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D011485:Protein Binding; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D017361:Viral Nonstructural Proteins
keywords: Intrabody; Nanobody; Nsp9; PRRSV; VHH