Search

Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction. NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.

Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRV VP4-R446G ) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRV VP4-R446G ) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro , the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo , it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia., (Copyright © 2017 American Society for Microbiology.)

Biliary atresia (BA) is a neonatal obstructive cholangiopathy that progresses to end-stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that the RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Using rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains that cause obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4-derived peptide (TRTRVSRLY) significantly reduced the ability of RRV to bind and infect cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains that do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by small interfering RNAs reduced RRV's ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice., Conclusion: The tripeptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70, defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response to this interaction to understand how it contributes to the pathogenesis of BA. (Hepatology 2017;65:1278-1292)., (© 2016 by the American Association for the Study of Liver Diseases.)

The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United 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 (>87%) 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 >87% 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., (Copyright © 2015 the American Physiological Society.)

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.

The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-microg doses of each of the chimeras and 10 microg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.

The ability to elicit protective immune responses after intranasal immunization with rotavirus particles, either with or without the attenuated Escherichia coli heat-labile enterotoxin LT(R192G) as an adjuvant, was examined in the adult mouse model. BALB/c mice were administered one or two inoculations of psoralen/UV-inactivated, triple-layered (tl) or double-layered (dl) purified rotavirus particles. Four weeks after immunization, mice were challenged with the murine rotavirus strain EDIM, and the shedding of rotavirus antigen was quantified. Rotaviruses used for immunization included EDIM and heterotypic simian (RRV), bovine (WC3), and human (89-12) strains. tl EDIM stimulated both systemic and intestinal rotavirus antibody responses and complete protection with as little as one 1-microgram dose. Inclusion of LT(R192G) (10 micrograms) significantly increased rotavirus antibody responses and reduced antigen concentrations needed for full protection. Both dl EDIM and heterotypic dl and tl particles stimulated protection, but they did so less than tl EDIM at comparable concentrations, either with or without LT(R192G). When B-cell-deficient microMt mice were immunized with tl EDIM particles, protection was reduced to levels similar to those induced with dl EDIM and heterotypic particles in BALB/c mice. However, dl EDIM particles induced similar levels of protection in both mouse strains. The protection stimulated by tl or dl EDIM particles was not diminished by CD8 cell depletion prior to immunization in either strain of mice. These results indicate that tl EDIM induced immunity at least partially through responses to its outer capsid proteins, presumably by stimulation of serotype-specific neutralizing antibody. In contrast, the other particles stimulated protection primarily by an antibody-independent mechanism. Finally, depletion of CD8 cells had no effect on protection by either mechanism.

We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge., (Copyright 1998 Academic Press.)

The rotavirus inner capsid protein VP6 contains conserved epitopes that are potential targets for eliciting protective immunity against different serotypes within the same group of rotavirus. In order to determine whether VP6 alone can induce protective immunity, an expression vector pcDNA1/EDIM6 containing gene 6 of rotavirus EDIM strain was constructed and used as a vaccine in an adult mouse model. Cloned gene 6 was determined to be 1356 nucleotides long and contained a 5' noncoding region of 23 nucleotides, a 3' noncoding region of 139 nucleotides, and a coding frame of 1194 nucleotides for a polypeptide of 397 amino acid residues. Recombinant VP6 was expressed in rabbit reticulocyte lysate and the heat-denatured recombinant VP6 migrated in SDS-gels with an apparent molecular weight of approximately 43 kDa. Five additional polypeptide bands corresponding to oligomers of recombinant VP6 were observed when the expressed product was not heat denatured. To determine the immunogenicity of recombinant VP6, female BALB/c mice were injected intramuscularly or intradermally with pcDNA1/EDIM6, or were inoculated epidermally with plasmid-coated gold beads using the Geniva Accell particle delivery device. Only intradermal injection and particle delivery elicited measurable serum anti-rotavirus IgG responses, but responses developed following particle delivery were significantly (P < 0.001) greater. However, none of the delivery methods induced serum or stool anti-rotavirus IgA responses and, when challenged with EDIM no protection against infection was observed in the immunized mice. Therefore, parenteral immunization with VP6 alone elicited large anti-rotavirus IgG responses but did not elicit protection against murine rotavirus infection in this model.

Neutralizing monoclonal antibodies 2F1 and 1C10, which are specific for VP7 of serotype 2 rotaviruses (G2), were used to select neutralization escape variants of the human serotype 2 rotavirus, DS-1. Neutralization survival patterns generated by monoclonal antibodies 2F1, 1C10, and RV5:3 indicated that 2F1 and 1C10 did not recognize identical epitopes. Direct sequencing of PCR products of gene 8, encoding VP7, revealed that each escape variant possessed only a single nucleotide mutation which resulted in a single amino acid substitution. In one variant, a second nucleotide change occurred, but did not result in an amino acid change. Four independently selected 2F1 mutants showed mutations in four separate sites in antigenic regions A, C, and D. Two independently selected 1C10 variants had mutations in either the A region or an unreported site at amino acid 190. Two of the mutations resulted in the creation of new glycosylation sites which were utilized, but did not appear to greatly affect antigenicity. Of note, three of the mutants also demonstrated alterations in the migration patterns of gene 8 on PAGE. Such electrophoretic mobility shifts caused by single base neutralization escape mutations have not previously been reported for rotavirus. Since multiple mutations were selected with the same monoclonal antibody it appears that the antigenic regions of VP7, although widely separated in the linear sequence, are parts of a single, large and complex neutralization domain which includes amino acid 190 as well as the other previously reported epitopes.

Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.