Research Article: Silencing of the Rotavirus NSP4 Protein Decreases the Incidence of Biliary Atresia in Murine Model

Date Published: August 18, 2011

Publisher: Public Library of Science

Author(s): Jiexiong Feng, Jixin Yang, Shuaiyu Zheng, Yinrong Qiu, Chengwei Chai, Jean-Pierre Vartanian.

Abstract: Biliary atresia is a common disease in neonates which causes obstructive jaundice and progressive hepatic fibrosis. Our previous studies indicate that rotavirus infection is an initiator in the pathogenesis of experimental biliary atresia (BA) through the induction of increased nuclear factor-kappaB and abnormal activation of the osteopontin inflammation pathway. In the setting of rotavirus infection, rotavirus nonstructural protein 4 (NSP4) serves as an important immunogen, viral protein 7 (VP7) is necessary in rotavirus maturity and viral protein 4 (VP4) is a virulence determiner. The purpose of the current study is to clarify the roles of NSP4, VP7 and VP4 in the pathogenesis of experimental BA. Primary cultured extrahepatic biliary epithelia were infected with Rotavirus (mmu18006). Small interfering RNA targeting NSP4, VP7 or VP4 was transfected before rotavirus infection both in vitro and in vivo. We analyzed the incidence of BA, morphological change, morphogenesis of viral particles and viral mRNA and protein expression. The in vitro experiments showed NSP4 silencing decreased the levels of VP7 and VP4, reduced viral particles and decreased cytopathic effect. NSP4-positive cells had strongly positive expression of integrin subunit α2. Silencing of VP7 or VP4 partially decreased epithelial injury. Animal experiments indicated after NSP4 silencing, mouse pups had lower incidence of BA than after VP7 or VP4 silencing. However, 33.3% of VP4-silenced pups (N = 6) suffered BA and 50% of pups (N = 6) suffered biliary injury after VP7 silencing. Hepatic injury was decreased after NSP4 or VP4 silencing. Neither VP4 nor VP7 were detected in the biliary ducts after NSP4. All together, NSP4 silencing down-regulates VP7 and VP4, resulting in decreased incidence of BA.

Partial Text: Biliary atresia (BA) is a common biliary disease in infants. It is characterized by progressive destruction of extrahepatic bile ducts, resulting in obstruction of bile flow during the first few months of children’s lives [1], [2]. Interdisciplinary initiatives [3] focusing on BA suggests it is a virus-induced autoimmune disease [4], [5]. As supported by our clinical evidence [6], [7], [8] and animal models [9], [10], reovirus, especially rotavirus, infection could cause biliary injury or obstruction of bile duct lumens. Among all candidates, rhesus rotavirus (RRV) is considered to be the most potent virus in inducing experimental BA [9]. Nonstructural protein 4 (NSP4), viral protein 7 (VP7) and viral protein 4 (VP4) have become hotspots for the mechanism of rotavirus infection as they are important in the process of rotavirus replication [11], [12], [13]. However, studies investigating the relationship between rotavirus proteins and BA are lacking.

It has been over 30 years since the report of a model of reovirus induced BA in rodents [15]. Since then, there have been several advances in this model including the study of rotavirus, an important human pathogen [5]. Our previous findings indicate that rotavirus-induced biliary atresia is mediated by nuclear factor-kappaB [10] and abnormal activation of osteopontin inflammatory pathway in the liver [7]. Our newly established method for primary culture of biliary epithelia gives us a powerful means to study the relationship between rotavirus infection and biliary injury [16]. Rotavirus can infect biliary duct epithelial cells [17]. Therefore, EHBE is the presumptive target of rotavirus.