Simian Rotavirus SA11 Research Articles

Comparative Removal of Poliovirus, Rotavirus SA11 and MS2 Coliphage by Point-of-Use Devices used to Treat Drinking Water and Water Disinfectants: A Review.

Test protocols have been developed to test water treatment devices/systems for use for treating drinking water that are used at the individual and home level to ensure the removal of waterborne viruses. Current test procedures call for the use of poliovirus type 1 and/or rotavirus SA11. Recently we suggested that selected coliphages could be used as surrogates for poliovirus for testing of point-of-use (POU) water treatment devices, however, rotavirus was not used in those studies. The purpose of this review was to compare studies of POU devices which were tested with poliovirus type 1, simian rotavirus SA11 and coliphage MS2 to determine if the behavior of rotavirus SA11 was significantly different. In addition, an attempt was made to compare the relative resistance of these viruses by various disinfectants used to treat drinking water. In all cases SA11 was removed to an equal or greater degree than poliovirus. SA11 was found to be less resistant to halogens, although one study found it to be more resistance to chloramines than poliovirus and MS2. Based on this review, use of coliphages for testing POU devices appear justified. Additionally, data on chloramines for these viruses would be useful to determine if rotavirus is more resistant than poliovirus and MS2.

Genetic determinants restricting the reassortment of heterologous NSP2 genes into the simian rotavirus SA11 genome

Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784–820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784–820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.

Study of the Ability of Bifidobacteria of Human Origin to Prevent and Treat Rotavirus Infection Using Colonic Cell and Mouse Models.

Rotavirus is the leading cause of severe acute gastroenteritis among children worldwide. Despite effective vaccines, inexpensive alternatives such as probiotics are needed. The aim of this study was to assess the ability of probiotic candidate Bifidobacterium thermophilum RBL67 to inhibit rotavirus infection. Bacterial adhesion to intestinal cells and interference with viral attachment were evaluated in vitro. B. thermophilum RBL67 displayed adhesion indexes of 625 ± 84 and 1958 ± 318 on Caco-2 and HT-29 cells respectively and was comparable or superior to four other bifidobacteria, including B. longum ATCC 15707 and B. pseudolongum ATCC 25526 strains. Incubation of B. thermophilum RBL67 for 30 min before (exclusion) and simultaneously (competition) with human rotavirus strain Wa decreased virus attachment by 2.0 ± 0.1 and 1.5 ± 0.1 log10 (by 99.0% and 96.8% respectively). Displacement of virus already present was negligible. In CD-1 suckling mice fed B. thermophilum RBL67 challenged with simian rotavirus SA-11, pre-infection feeding with RBL 67 was more effective than post-infection feeding, reducing the duration of diarrhea, limiting epithelial lesions, reducing viral replication in the intestine, accelerating recovery, and stimulating the humoral specific IgG and IgM response, without inducing any adverse effect. B. thermophilum RBL67 had little effect on intestinal IgA titer. These results suggest that humoral immunoglobulin might provide protection against the virus and that B. thermophilum RBL67 has potential as a probiotic able to inhibit rotavirus infection and ultimately reduce its spread.

Antiviral activity of Myracrodruon urundeuva against rotavirus

Myracrodruon urundeuva Allemão, Anacardiaceae, is a medicinal plant widely found in Brazil, especially in the northern region. In our previous study, the ethanolic extract from leaves of M. urundeuva showed antiviral activity against simian rotavirus SA-11. Here, the crude extract was subjected to fractionations in order to subsequently work with more concentrated and pure bioactive compounds, which were analyzed by TLC and HPLC methods to support a better understanding of their virucidal effect. The antiviral activity was evaluated using a rotavirus infection model in MA-104 cells treated with the maximum non-cytotoxic concentration of the crude extract and its fractions. Data were expressed as the percentage inhibition of viral replication calculated by the inhibition of cytopathic effect in the treated cells compared to untreated controls after 48h of incubation. First, we conducted a fractionation, generating five fractions (F1–F5) which were submitted to antiviral assay. Then, the fraction that showed the highest virucidal effect (F3, PI=75%) was subjected to a larger partition, yielding eighteen subfractions, which were submitted to new antiviral assays. Terpenes, flavonoids and tannins were the major secondary metabolites detected by TLC analysis in F3. SF1, a flavonoid-enriched fraction, showed the strongest in vitro activity against rotavirus (PI=92%), preventing cytopathic effect. Chromatographic profiles were obtained by HPLC for the crude extract and SF1, the most potent subfraction. Overall, our data point to the potential anti-rotavirus activity of flavonoid-enriched fraction (SF1) of M. urundeuva leaves, corroborating the traditional use of this species to treat diarrhea and broadening our perspectives on in vivo assays in mice with SF1 isolated or associated with other fractions.

Prevention of Rotavirus‐induced Diarrhea by Preferential Secretion of IgA in Breast Milk via Maternal Administration of Lactobacillus gasseri SBT2055

Rotavirus (RV)-induced diarrhea poses a major health problem, particularly to infants. An effective measure to prevent RV infection is to consume breast milk with higher levels of protective IgA. We therefore examined whether Lactobacillus gasseri SBT2055 (LG2055) could augment immunoglobulin (Ig) A levels and reduce the incidence of diarrhea in a mouse model of RV infection. Female BALB/c mouse dams were fed a diet containing 0.1% heat-treated LG2055 or a control, beginning 4 weeks before mating with male mice and continuing until the experiment ended. One week after mating, female dams were immunized orally with simian RV SA-11. Five days after birth, mouse pups were infected orally with RV and the incidence of diarrhea was determined 4 days later. RV-specific and total IgA were quantified by an enzyme-linked immunosorbent assay. LG2055-fed dams immunized with RV (LG2055/RV) secreted breast milk that significantly lowered the incidence of RV-induced diarrhea in their pups as compared with dams immunized with RV alone (C/RV). The LG2055/RV dams also produced a significantly greater amount of RV-specific IgA in breast milk obtained from the pups' stomach, but not in feces or Peyer's patch cell cultures. In addition, LG2055 stimulated total IgA production in splenocyte cultures from Toll-like receptor (TLR)-4-knockout mice, but not those from TLR-2-knockouts. LG2055-fed dams reduced RV infection in their pups and elevated RV-specific IgA levels in breast milk of stomach origin, the possible mechanism of which may be TLR-2 stimulation by LG2055.

Screening of Brazilian medicinal plants for antiviral activity against rotavirus

Ethnopharmacological relevanceBrazilian medicinal plants traditionally used for the treatment of diarrhoea were investigated for their in vitro antiviral activity against the simian rotavirus SA11. Materials and methodsThe ethanolic crude extracts of plants collected in the cerrado of Minas Gerais, Brazil were submitted to phytochemical screening. The cytotoxicity of the extracts was inferred by cellular morphologic alterations. Antiviral activity was assessed by the ability of the extracts to inhibit the cytopathic effect (CPE) of rotavirus on the treated cells. RT-PCR was performed to confirm and/or confront antiviral assay data. ResultsThe maximum non-toxic concentration ranged from 50 to 500μg/mL. All extracts were toxic at a concentration of 5000μg/mL but no extract showed cytotoxicity at 50μg/mL. The species Byrsonima verbascifolia, Myracrodruon urundeuva, Eugenia dysenterica and Hymenaea courbaril exhibited the strongest in vitro activity against rotavirus. Their extracts prevented the formation of CPE, and RT-PCR analysis detected no amplification of genetic material from rotavirus. Tannins, flavonoids, saponins, coumarins and terpenes were the major classes of natural products found in the leaf extracts that showed antiviral activity. ConclusionAmong the species studied, Byrsonima verbascifolia, Eugenia dysenterica, Hymenaea courbaril and Myracrodruon urundeuva showed potential activity against rotavirus and are worthy of further study. The present study corroborates ethnopharmacological data as a valuable source in the selection of plants with antiviral activity and to some extent validates their traditional uses.

ROTAVIRUS DISTRIBUTION IN OYSTERS CRASSOSTREA GIGAS TISSUE

Shellfish are commonly associated with viral gastroenteritis outbreaks in human consumers. However, several attempts to depurate contaminated bivalve mollusks have been halted by increased persistence of viruses in mollusks’ tissues, mainly in digestive tract. RT-PCR and immunohistochemistry were applied to evaluate the localization of simian rotavirus SA11 strain in experimentally contaminated Crassostrea gigas oysters. Highest concentrations of SA11 persisted in digestive tract along 12-h experiment, whereas gills, mantle, labial palps and hemolymph presented fewer SA11. SA11 was found in close association with mucus filling oyster stomach. It can account for increased virus accumulation in digestive tract. DOI: http://dx.doi.org/10.17525/vrr.v15i2.45

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Supplementing Suckling Rats with Whey Protein Concentrate Modulates the Immune Response and Ameliorates Rat Rotavirus-Induced Diarrhea

Group A rotaviruses (RV) are the most common causative agents of acute gastroenteritis in children <2 y. The present study was designed to establish the effect of a bovine whey protein concentrate (WPC) in a RV infection model in suckling rats. From d 3 of life, suckling Lewis rats received daily supplements of WPC, WPC plus lactoferrin (LF), standard infant formula (SIF), or water (RV-infected group and an untreated, uninfected reference group). On d 8 of life, heterologous simian RV SA-11 was inoculated orally in the WPC-RV, WPC+LF-RV, SIF-RV, and RV groups. WPC and WPC+LF reduced diarrhea incidence from ∼90% in RV group to ∼60% in WPC-RV and WPC+LF-RV groups (P < 0.05), whereas the area under the curve (AUC) of severity along time diminished from ∼10 AUC in the RV group to ∼6 AUC in both supplemented groups (P < 0.05). Serum levels of anti-RV antibodies, splenocyte proliferation, and interferon-γ secretion after specific stimulation were significantly lower in the WPC-RV and WPC+LF-RV groups than in the SIF-RV and RV groups. In the intraepithelial intestinal compartment, RV infection increased the proportion of typical mucosal T cells (IE-T CD8 αα+); however, this modification was controlled by WPC and WPC+LF supplementation. In general, for most of the parameters studied, the SIF-RV and RV groups did not differ. In summary, daily supplementation with WPC or WPC+LF in early life considerably reduces the severity of RV-induced acute gastroenteritis and modulates the immune response against the pathogen.

Characterization of Clinical and Immune Response in a Rotavirus Diarrhea Model in Suckling Lewis Rats

Group A rotaviruses (RVs) are the leading pathogens causing diarrhea in children and animals. The present study was designed to establish an experimental model of RV infection and immune response in suckling rats. Wistar (W) and Lewis (L) suckling rats were inoculated orally with two different doses of a simian RV SA-11 strain. RV infection was evaluated by growth rate and clinical indexes. Virus-shedding and serum anti-RV antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Mucosal interferon-gamma (IFN gamma), specific splenocyte proliferation, and spleen and intestinal intraepithelial lymphocyte (IEL) phenotype were analyzed. No diarrhea was observed in any inoculated Ws. All Ls developed acute moderate diarrhea, and a high score and incidence of diarrhea were found in rats infected with higher titers of RV. Specific humoral and cell systemic immune response was confirmed by splenocyte proliferation and by the presence of serum anti-RV antibodies. Moreover, RV infection induced changes in IEL composition, which showed an increase in the proportion of innate immune cells with respect to cells involved in acquired immunity. This acute moderate diarrhea process constitutes a good experimental model that also provides some immune biomarkers that may allow establishing modulation by drugs or diet components.

Systemic immune response after rotavirus inoculation of neonatal mice depends on source and level of purification of the virus: implications for the use of heterologous vaccine candidates

Rotavirus is an important cause of morbidity and mortality worldwide and vaccines are currently under development, with clinical trails conducted in humans worldwide. The immune responses in infant BALB/c mice were examined following oral inoculation with murine rotavirus EDIM (2 x 10(4) focus-forming units) and with three CsCl gradient-purified fractions of heterologous simian rotavirus SA11 (standardized at 2 x 10(6) CCID(50)) that differed in antigen composition: fraction 1 was enriched for double-layered rotavirus particles, fraction 2 for triple-layered particles and fraction 3 consisted mainly of cell components. Diarrhoea and high IgG responses, but marginal IgA responses, were observed after inoculation with all three SA11 fractions. Virus shedding was observed in all EDIM-inoculated mice, but in none of the SA11-inoculated mice. Rotavirus-specific IgG1 : 2a ratios were similar in mice inoculated with EDIM and SA11 fraction 1, but higher for SA11 fraction 3- and lower for SA11 fraction 2-inoculated mice. A higher IgG1 : 2a ratio indicates a more Th2-like immune response. This undesirable response is apparently mostly induced by inoculation with heterologous rotavirus in the presence of abundant cell-associated and soluble rotavirus proteins, compared with infection with a more purified preparation or with homologous virus. These data show that, following inoculation with a standardized amount of infectious virus, the composition of the fraction influences the outcome of the immune responses significantly.

Antiviral effects on bacteriophages and rotavirus by cranberry juice

Studies were undertaken to investigate the antiviral effects of comestible juices, especially cranberry juice, on non-related viral species. After exposure of bacteriophage T2 to a commercially available cranberry (Vaccinium macrocarpon) juice cocktail (CJ), virus infectivity titer was no longer detectible. After a 60-min exposure to orange (OJ) and grapefruit juices (GJ), phage infectivity was reduced to 25–35% of control, respectively. Similar data were observed for the bacteriophage T4. CJ inactivation of phage T4 was rapid, dose-dependent, and occurred at either 4 or 23 °C. Neither pH nor differences in sugar/carbohydrate levels among the juices may be ascribed to the recognized antiviral effects. Further studies were performed to identify the occurrence of antiviral activity by CJ to a mammalian enteric virus. The treatment of the simian rotavirus SA-11 with a 20% CJ suspension was sufficient to inhibit hemagglutination. Under scanning and transmission electron microscopy, CJ was observed to inhibit the adsorption of phage T4 to its bacterial host cells and prevented the replication of rotavirus in its monkey kidney (MA-104) host cells, respectively. The data suggest, for the first time, a non-specific antiviral effect towards unrelated viral species (viz., bacteriophages T2 and T4 and the simian rotavirus SA-11) by a commercially available cranberry fruit juice drink.

Analysis of genetic factors related to preferential selection of the NSP1 gene segment observed in mixed infection and multiple passage of rotaviruses

Reassortment is one of the major evolutionary mechanisms of the rotavirus genome. Preferential selection (assortment) of the NSP1 gene segment from either of the parental viruses after coinfection of these viruses has been reported as a notable finding in reassortment. To analyze genetic factors which are associated with preferential selection of the rotavirus NSP1 gene segment into progeny viruses, mixed infection and multiple passages were performed using two panels of rotaviruses, i.e., bovine rotavirus A5 clones, and simian rotavirus SA11 and five strains of SA11-based single NSP1 gene-substitution reassortants. In the first experiment, three A5 clones (A5-10, A5-13, and A5-16) that had genetically distinct NSP1 genes in the same genetic background were used. In coinfection of these A5 clones, it was noted that the A5-10 NSP1 gene, which encodes an incomplete protein product due to presence of a nonsense codon at an unusual position, was selected more preferentially than the A5-13 NSP1 gene with intact length and structure. The A5-16 NSP1 gene, with a deletion of 500 bp, was least efficiently selected. In the second experiment, we prepared two reassortants, SOF and SRF, which have NSP1 genes from rotavirus strains OSU and RRV, respectively, in the genetic background of SA11, which were used together with previously prepared reassortants SKF, SDF, and SNF, which had NSP1 genes from strains KU, DS1, and K9, respectively. Among the 6 NSP1 genes analyzed, the NSP1 gene from SKF was most preferentially selected, followed by SNF, SOF, SDF, SA11, and SRF, in that order. Although SOF exhibited less growth efficacy than SA11, the growth rates of other reassortants were similar to that of SA11. These findings suggest that for the occurrence of preferential selection of the NSP1 gene, production of the intact NSP1 protein may not be involved, but the presence of intact length of the NSP1 gene may be required. Furthermore, it was also found that genetic similarity based on primary structure of this gene is not related to the selectivity of the NSP1 gene.

Isolation and characterization of a new simian rotavirus, YK-1

BackgroundTo effectively analyze the requirements for protection to rotavirus infection, a reliable animal model that reasonably mimics infection and disease in humans is needed. A requirement for an effective animal model is the availability of appropriate rotavirus stocks for challenge.ResultsA new simian rotavirus, designated YK-1, was isolated from a 2-year-old immunodeficient pigtailed macaque with chronic diarrhea. YK-1 was distinguishable by electropherotype from the other simian rotavirus strains, SA11 and RRV. One variant of YK-1, clone 311, which was isolated after adaptation and plaque purification in cell cultures, displayed an unusual RNA electropherotype with an abnormally migrating gene 11 segment. Sequence analysis demonstrated a genetic rearrangement that involved a partial duplication of the gene 11 ORF encoding NSP5. YK-1 was identified as a Group A rotavirus belonging to subgroup 1. To further characterize the YK-1 strain, the genes encoding VP4, VP7, and NSP4 were sequenced. Analysis of VP4 and VP7 gene fragments suggests that this strain is a G3P[3] rotavirus and is closely related to the simian rotavirus strain RRV. Serotype analysis also identified YK-1 as a G3 rotavirus. The NSP4 genotype of YK-1 is C, the same genotype as RRV.ConclusionThis newly isolated rotavirus, YK-1, is being used to establish a nonhuman primate model for studying the infectivity, immunity, and pathogenesis of rotavirus and for evaluating candidate rotavirus vaccines.

Ricin Toxin B Subunit Enhancement of Rotavirus NSP4 Immunogenicity in Mice

A 90-amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized in Escherichia coli. Recombinant RTB and the NSP4(90)::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4(90) peptide proteins or heat denatured NSP4(90)::RTB fusion protein generated higher titers of serum anti-NSP4(90) IgG antibodies than mice immunized with NSP4(90) peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB. Serum anti-NSP4(90) IgG titers were highest in mice immunized with native recombinant NSP4(90)::RTB fusion protein, confirming the immunostimulatory function of RTB. Results of experiments described here demonstrate the feasibility of using RTB mediated adjuvant functions for stimulation of the antigenicity of a rotavirus nonstructural protein. The ability of recombinant NSP4(90)::RTB fusion protein synthesized in E. coli to bind glycoprotein receptor molecules effectively indicates that protein linkage to the RTB N-terminus and synthesis of the recombinant NSP4(90)::RTB fusion protein in bacteria do not interfere with the immunostimulatory properties of the RTB subunit.

Synthesis of a Ricin Toxin B Subunit-Rotavirus VP7 Fusion Protein in Potato

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fusion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.

Mucosal immunization with a ricin toxin B subunit–rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response

The castor-oil plant Ricinus communis A–B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4 90 and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4 90–RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4 90–RTB ligand–antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4 90–RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4 90 alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4 90–RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.

Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

: Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized (sVP6). The VP6 and sVp6 genes were transformed into tobacco (Nicotiana tabacum L.) plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8–34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein (TSP). Then, BALB/c female mice that had been gavaged weekly with 10 mg TSP containing 34 μg VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea. (Managing editor: Li-Hui ZHAO)

Oral immunization with pBsVP6-transgenic alfalfa protects mice against rotavirus infection

A critical factor in edible plant-derived vaccine development is adequate expression of the exogenous antigens in transgenic plants. We synthesized a codon-optimized gene (sVP6) encoding the VP6 protein of human group A rotavirus and inserted it into the alfalfa genome using agrobacterium-mediated transformation. As much as 0.28% of the total soluble protein of the pBsVP6-transgenic alfalfa was sVP6. Female BALB/c mice were gavaged weekly with 10 mg of transgenic alfalfa extract containing 24 μg of sVP6 protein and 10 μg of CpG-rich oligodeoxynucleotides as mucosal adjuvant. Immunized mice developed high titers of anti-VP6 serum IgG and mucosal IgA. Offspring of immunized dams developed less severe diarrhea after challenge with simian rotavirus SA-11, indicating that antibodies generated in the dams provided passive heterotypic protection to the pups. These results suggest that oral immunization with pBsVP6-transgenic alfalfa provides a potential means of protecting children and young animals from severe acute rotavirus-induced diarrhea.

Synthesis and Assembly of a Cholera Toxin B Subunit-Rotavirus VP7 Fusion Protein in Transgenic Potato

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.