title: Short hairpin RNA-mediated silencing of bovine rotavirus NSP4 gene prevents diarrhoea in suckling mice.
abstract: While RNA interference (RNAi) has been widely used to study rotavirus gene function in vitro, the potential therapeutic role for RNAi in vivo has not been explored. To this end, we constructed two recombinant lentiviral vectors containing short hairpin RNA (shRNA) against non-structural protein-4 (NSP4) of bovine rotavirus (BRV), RNAi-351 and RNAi-492. RNAi-351 and RNAi-492 strongly suppressed the transient expression of a FLAG-tagged NSP4 fusion protein in 293T cells. In BRV-susceptible MA104 cells, RNAi-492 more potently silenced NSP4 mRNA than RNAi-351 and combination of the two shRNAs almost completely silenced viral NSP4 gene expression. While 100% of suckling mice exposed to BRV and control shRNA developed severe diarrhoea, no suckling mice exposed to BRV in the presence of RNAi-492 or a combination of RNAi-492/RNAi-351 developed severe diarrhoea, and only 20 and 3.3% developed mild diarrhoea, respectively. In addition, RNAi-492 and RNAi-351 markedly abrogated rotaviral replication in MA104 cells and significantly inhibited BRV replication in mouse pups. These results indicated that shRNAs silencing NSP4 gene had substantial antiviral properties and inhibited replication of BRV in a sequence-specific manner that may have clinical application.
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, PR China.
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Chen
authors.forename: Hongmei
authors.initials: H
authors.lastname: Wang
authors.forename: Hongbin
authors.initials: H
authors.lastname: He
authors.forename: Lingling
authors.initials: L
authors.lastname: Song
authors.forename: Jianming
authors.initials: J
authors.lastname: Wu
authors.forename: Yundong
authors.initials: Y
authors.lastname: Gao
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.forename: Chengqiang
authors.initials: C
authors.lastname: He
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yang
authors.forename: Lili
authors.initials: L
authors.lastname: Chen
authors.forename: Liqun
authors.initials: L
authors.lastname: Wang
authors.forename: Guangpeng
authors.initials: G
authors.lastname: Li
authors.forename: Yonghai
authors.initials: Y
authors.lastname: Li
authors.forename: David E
authors.initials: DE
authors.lastname: Kaplan
authors.forename: Jifeng
authors.initials: J
authors.lastname: Zhong
mesh_terms: D000818:Animals; D000833:Animals, Suckling; D003967:Diarrhea / Q000517:prevention & control* / Q000821:virology; D020868:Gene Silencing; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012401:Rotavirus / Q000235:genetics / Q000472:pathogenicity*; D014118:Toxins, Biological / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D037521:Virulence Factors / Q000037:antagonists & inhibitors* / Q000096:biosynthesis
chemical_list: D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D034741:RNA, Small Interfering; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D037521:Virulence Factors