Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia

Abstract

States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (Ͼ87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with Ͼ87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.cholangiocytes; natural killer cells; rotavirus; RRV; VP4 BILIARY ATRESIA (BA) is a neonatal disease characterized by obstructive cholangiopathy of extrahepatic bile ducts, leading to chronic cholestasis and biliary cirrhosis. The incidence is estimated at 1 in 8,000 -15,000 live births (27). Afflicted infants present with jaundice, acholic stool, and failure to thrive. Early diagnosis and surgical intervention with a Kasai portoenterostomy to establish biliary drainage are essential. Even after successful drainage, the progressive nature of this disease causes end-stage liver disease requiring liver transplantation for survival. BA remains the most common indication for liver transplantation in children (2). The importance of understanding the pathogenesis of BA is crucial such that alternative treatment options can be developed.The etiology of BA remains unknown but likely is multifactorial and may include viral infection of the biliary tree (6,7,16,20,28) with subsequent immune-mediated duct injury (4,15,26). Several viruses have been isolated from the livers of BA patients including group C rotaviruses (20). A well-established murine model of BA is employed to study disease pathogenesis (21). Intraperitoneal injection of neonatal BALB/c mice with Rhesus rotavirus (RRV) leads to symptoms (jaundice, acholic stools, bilirubinuria) and bile duct obstruction that mimic human disease (1,18). Previous studies demonstrated the unique tropism of RRV to the cholangiocyte (biliary epithelial cells) (12,23) and that development of the murine model is virus strain dependent (1). In a previous study, it was sho...

A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

“…Several rotavirus strains were used to induce the murine model of BA, as shown in Table 1. We previously showed that the VP4 gene of RRV is the major determinant in the murine model of BA (31,32). The SPPIDER program (34) was utilized to predict potential protein-protein interactions and to analyze the VP4 sequences of various rotavirus strains.…”

“…When several different rotavirus sequences were compared, 3 strains of rotavirus (RRV, SA-11, and GRV) had an arginine (R) at position 446. All these strains were capable of inducing the murine model of BA (31). Computer-generated analysis using published crystalline structures illustrated that the amino acid sequence SRL was exposed on the surface of the VP4 protein, allowing for binding (Fig.…”

“…We previously showed that the VP4 gene of RRV plays an integral role in the induction of the murine model of BA by infection of cholangiocytes (32) and immune cell activation (31). Subsequent studies demonstrated that the peptide TRTRVSRLY within the VP4 protein was able to block RRV's ability to bind to and infect cholangiocytes (33), and when this peptide was injected into mice in vivo, it could attenuate the disease phenotype.…”

“…Livers were harvested from pups 7 days after injection and pooled at three per sample in RPMI medium plus 2% FBS. Samples were processed as described before (31) to purify the mononuclear cells. After red blood cell lysis, the cells were washed two times and suspended in 1ϫ PBS plus 1% FBS.…”

“…Our previous studies showed that the VP4 gene of RRV is the major determinant in modulating disease pathogenesis (31,32). We recently found that the VP4 gene plays a role in primary binding to cells (cholangiocytes and MA104 cells) prior to entry and that treating cells with the VP4-derived peptide TRTRVSRLY (amino acids 440 to 449 of VP4) can inhibit virus binding and subsequent replication (33).…”