Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-α/β and IFN-γ receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-α/β receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-γ receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.     

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.     

Antibody-Dependent and -Independent Protection following Intranasal Immunization of Mice with Rotavirus Particles

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.     

Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.     

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.     

Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice.

The capacity of intramuscular (i.m.) inoculation of mice with homologous or heterologous host rotaviruses to induce protection from challenge was evaluated. i.m. inoculation with live, wild-type rotavirus (murine strain EDIM) induced complete protection from viral shedding after challenge for at least 6 weeks after inoculation; protection was correlated with production of virus-specific immunoglobulin A (IgA) by lamina propria (LP) lymphocytes. i.m. inoculation with inactivated EDIM, cell culture-adapted EDIM, or simian strain RRV was associated with partial protection, characterized by reduced viral shedding after challenge. Partial protection after challenge was not associated with production of virus-specific IgA by LP lymphocytes. The mechanisms by which i.m. inoculation induces virus-specific humoral immune responses in the small intestinal LP were examined.     

The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice

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.     

Primary murine small intestinal epithelial cells, maintained in long-term culture, are susceptible to rotavirus infection

We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-IC(cl2). Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-IC(cl2) cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology.     

Effect of Water-Based Microencapsulation on Protection against EDIM Rotavirus Challenge in Mice

We determined the capacity of microcapsules formed by the combination of sodium alginate, an aqueous anionic polymer, and spermine hydrochloride, an aqueous cationic amine, to enhance protection against rotavirus challenge in mice. Adult BALB/c mice were orally inoculated with either free or microencapsulated rotavirus (simian rotavirus strain RRV) and challenged 6 or 16 weeks later with murine rotavirus strain EDIM. Virus-specific humoral immune responses were determined at the time of challenge and 4 days after challenge by intestinal fragment culture. We found that spermine-alginate microcapsules enhanced protection against challenge 16 weeks after immunization but not 6 weeks after immunization. Quantities of virus-specific immunoglobulin A produced by small intestinal lamina propria lymphocytes were correlated with the degree of protection against challenge afforded by spermine-alginate microcapsules. Possible mechanisms by which microcapsules enhance protection against rotavirus challenge are discussed.     

B Cell Deficient Mice Are Protected from Biliary Obstruction in the Rotavirus-Induced Mouse Model of Biliary Atresia

A leading theory regarding the pathogenesis of biliary atresia (BA) is that bile duct injury is initiated by a virus infection, followed by an autoimmune response targeting bile ducts. In experimental models of autoimmune diseases, B cells have been shown to play an important role. The aim of this study was to determine the role of B cells in the development of biliary obstruction in the Rhesus rotavirus (RRV)-induced mouse model of BA. Wild-type (WT) and B cell-deficient (Ig-α^-/-) mice received RRV shortly after birth. Ig-α^-/- RRV-infected mice had significantly increased disease-free survival rate compared to WT RRV-infected BA mice (76.8% vs. 17.5%). In stark contrast to the RRV-infected BA mice, the RRV-infected Ig-α^-/- mice did not have hyperbilirubinemia or bile duct obstruction. The RRV-infected Ig-α^-/- mice had significantly less liver inflammation and Th1 cytokine production compared to RRV-infected WT mice. In addition, Ig-α^-/- mice had significantly increased numbers of regulatory T cells (Tregs) at baseline and after RRV infection compared to WT mice. However, depletion of Tregs in Ig-α^-/- mice did not induce biliary obstruction, indicating that the expanded Tregs in the Ig-α^-/- mice were not the sole reason for protection from disease. Conclusion: B cell deficient Ig-α^-/- mice are protected from biliary obstruction in the RRV-induced mouse model of BA, indicating a primary role of B cells in mediating disease pathology. The mechanism of protection may involve lack of B cell antigen presentation, which impairs T-cell activation and Th1 inflammation. Immune modulators that inhibit B cell function may be a new strategy for treatment of BA.     

Cholangiocyte expression of alpha2beta1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as alpha2beta1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express alpha2beta1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether alpha2beta1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the alpha2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the alpha2beta1-integrin. Newborn mice were pretreated with a monoclonal antibody against the alpha2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed alpha2beta1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-alpha2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the alpha2beta1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.     

Derivation of Neutralizing Monoclonal Antibodies Against Rotavirus

Monoclonal antibodies were derived against the SA11 simian, NIC bovine, and Wa human rotavirus strains and characterized by enzyme-linked immunosorbent assay, plaque neutralization, and hemagglutination inhibition. Several strain SA11-specific antibodies were found to have neutralizing and hemagglutination-inhibiting capacity.     

Relative Importance of Rotavirus-Specific Effector and Memory B Cells in Protection against Challenge

Adult BALB/c mice were orally inoculated with murine (strain EDIM), simian (strain RRV), or bovine (strain WC3) rotavirus. Six or 16 weeks after inoculation, mice were challenged with EDIM. At the time of challenge and in the days immediately following challenge, production of rotavirus-specific immunoglobulin A (IgA), IgG, and IgM by small intestinal lamina propria lymphocytes (LPL) was determined by fragment culture, and quantities of virus-specific antibodies at the intestinal mucosal surface were determined by intestinal lavage. Mice immunized with EDIM were completely protected against EDIM challenge both 6 and 16 weeks after immunization. Protection was associated with production of high levels of IgA by LPL and detection of virus-specific IgA at the intestinal mucosal surface. In addition, animals immunized and later challenged with EDIM did not develop a boost in antibody responses, suggesting that they were also not reinfected. We also found that in mice immunized with nonmurine rotaviruses, (i) quantities of virus-specific IgA generated following challenge were greater 16 weeks than 6 weeks after immunization, (ii) immunization enhanced the magnitude but did not hasten the onset of production of high quantities of virus-specific IgA by LPL after challenge, and (iii) immunization induced partial protection against challenge; however, protection was not associated with either production of virus-specific antibodies by LPL or detection of virus-specific antibodies at the intestinal mucosal surface.     

哺乳期BALB/c小鼠胆道梗阻模型的探索

目的探讨新生BALB/c小鼠胆道梗阻模型的建立,并与报告的新生BALB/c小鼠感染猕猴轮状病毒(RRV)模型小鼠生存曲线进行比较。方法将出生后5～7 d的BALB/c小鼠随机分为实验组和对照组,实验组进行胆总管结扎,然后关腹。对照组打开腹部后关腹不结扎胆总管。实验完成后每天观察小鼠的体重变化、无毛区皮肤颜色变化、小鼠存活天数以及在术后第5、10天时分别取小鼠肝脏做病理及免疫组化。结果小鼠在结扎后随着时间的延长,小鼠的体重及肝体比、无毛区皮肤颜色、存活天数、肝脏病理等都存在一定变化。小鼠体重增长逐渐缓慢,术后第2天就会出现无毛区的皮肤变黄,在尿道口会有淡黄色的液体并随后出现陶土样便。在术后第5天及第10天时取肝脏做肝体比有统计学差异(P≤0.05),小鼠在术后第10天左右会出现死亡高峰。结论新生BALB/c小鼠胆总管结扎模型是研究胆道梗阻的可靠动物实验,其生存曲线与报告的猕猴轮状病毒致胆道闭锁大体类似。     

Antibodies specific for the carboxy-terminal region of the major surface glycoprotein of simian rotavirus (SA11) and human rotavirus (Wa)

Antibodies specific for the major outer capsid protein (VP7) of the simian rotavirus SA11 were obtained by immunization of rabbits with a synthetic peptide, Ser-Ala-Ala-Phe-Tyr-Tyr-Arg-Val, corresponding to the eight carboxy-terminal amino acids of the viral protein predicted from the nucleotide sequence of the gene segment 9 of the SA11 genome. As the carboxy-terminal region of the VP7 of human rotavirus Wa has an identical sequence, cross-reactivity of the raised antibodies was observed with this strain.     

Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia

Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.     

Experimental Adaptation of Rotaviruses to Tumor Cell Lines

A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death.     

Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo.

To identify the rotavirus protein which mediates attachment to cells in culture, viral reassortants between the simian rotavirus strain RRV and the murine strains EHP and EW or between the simian strain SA-11 and the human strain DS-1 were isolated. These parental strains differ in the requirement for sialic acid to bind and infect cells in culture. Infectivity and binding assays with the parental and reassortant rotaviruses indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains. Using ligated intestinal segments of newborn mice and reassortants obtained between the murine strain EW and RRV, we developed an in vivo infectivity assay. In this system, the infectivity of EW was not affected by prior treatment of the enterocytes with neuraminidase, while neuraminidase treatment reduced the infectivity of a reassortant carrying gene 4 from RRV on an EW background more than 80% relative to the controls. Thus, VP4 appears to function as the cell attachment protein in vivo as well as in vitro.