Rotavirus SA11 Research Articles

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.

Survival of SA11 Rotavirus in Fresh Fruit Juices of Pineapple, Papaya, and Honeydew Melon

Survival of rotavirus in fresh fruit juices of papaya (Caraca papaya L.), honeydew melon (Cucumis melo L.), and pineapple (Ananas comosus [L.] Merr.) was studied. Clarified juices were prepared from pulps of ripe fruits and sterilized by ultrafiltration. One milliliter of juice from each fruit was inoculated with 20 μlof 1 × 106 PFU of SA11 rotavirus and sampled immediately (0-h exposure) and 1 and 3 h later at 28°C. Mean viral titers in juices of papaya (pH 5.1) and honeydew melon (pH 6.3) at 1 and 3 h were not significantly different from titers at 0-h exposure. Mean viral titers in juices from pineapples with ripening color indices of 3 (pH 3.6) and 6 (pH 3.7) at 1-h exposure (color index 3: 4.0±1.7 × 104; color index 6: 2.3±0.3 × 105) and 3-h exposure (color index 3: 1.1±0.4 × 104; color index 6: 1.3±0.6 × 105) were significantly lower than titers at 0-h exposure (color index 3: 5.7±2.9 × 105; color index 6: 7.4±1.3 × 105). Virus titers in pineapple juices of color index 3 were significantly lower than titers of the virus in juices of index 6. In cell culture medium (pH 7.4), SA11 titer remained stable over 3 h at 28°C. However, at pH 3.6, the virus titer was reduced to a level not significantly different from that of the virus in pineapple juice of color index 6 (pH 3.7). In conclusion, papaya and honeydew melon juices, in contrast to pineapple juice, have the potential to transmit rotavirus. Inactivation of SA11 virus in pineapple juice can be possibly attributed to low pH and constituent(s) in the juice.

Virus Removal from Water by a Portable Water Treatment Device

Few portable point-of-use (POU) devices are available for treatment of water by recreational enthusiasts who may obtain water from untreated sources (rivers, lakes, etc.). This study evaluated a POU device containing a structure matrix capable of removing viruses from water without the use of a disinfectant. The unit was evaluated for the removal of poliovirus type 1, rotavirus SA-11, human norovirus, and a wide range of different coliphages exhibiting different isoelectric points, sizes, and shapes. The removal of all virus types tested exceeded 99.99%. The tested unit complied with the criteria guidelines for virus removal under the US Environmental Protection Agency's "Guide Standard and Protocol for Testing of Microbiological Water Purifiers."

Epitope mapping and use of epitope-specific antisera to characterize the VP5 ⁎ binding site in rotavirus SA11 NSP4

Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural protein 4 (NSP4), the first characterized viral enterotoxin, is a 28-kDa glycoprotein that has pleiotropic functions in RV infection and pathogenesis. NSP4 has multiple forms enabling it to perform its different functions. Dissecting such functions could be facilitated by use of epitope-specific antibodies. This work mapped the epitopes for the monoclonal antibody B4-2/55 and three polyclonal antisera generated against synthetic SA11 NSP4 peptides corresponding to residues 114–135, 120–147, and 150–175. The epitope for B4-2/55 mapped to residues 100–118, wherein residues E105, R108 and E111 are critical for antibody binding. Antiserum generated to two peptides (aa114–135 and aa120–147) with enterotoxin activity each recognize a single but distinct epitope. The epitope for the peptide antiserum to aa114–135 was mapped to residues 114–125 with highly conserved residues T117/T118, E120, and E122 being critical for antibody binding. The peptide antiserum to aa120–147 binds to NSP4 at residues 130–140 and residues Q137–T138 are critical for this epitope. Finally, the epitope for the antiserum to peptide aa150–175 mapped to residues 155–170, wherein residues E160 and E170 are critical for antibody binding. Knowledge of the binding sites of domain-specific antibodies can aid in further characterizing different functions of NSP4. To demonstrate this, we characterized the interaction between NSP4 and VP5 ⁎ [ K D = 0.47 μM] and show that binding of NSP4 to VP5 ⁎ is blocked by antibody to NSP4 aa114–135 and aa120–147, but not aa150–175. The use of single epitope-specific antibodies to differentially block functions of NSP4 is a feasible approach to determine the functional domain structure of this important RV virulence factor.

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.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

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.

Genome heterogeneity of SA11 rotavirus due to reassortment with “O” agent

Derivatives of the rotavirus SA11-H96 strain, isolated in 1958 from an overtly healthy vervet monkey, have been used extensively to probe the viral life cycle. To gain insight into the phenotypic and genotypic differences among SA11 isolates, we sequenced the segmented double-stranded RNA genomes of SA11-H96 (P5B[2]:G3), two SA11-4F-like viruses (P6[1]:G3), two SA11-4F-like viruses with gene 5 rearrangements, and relevant segments of SA11 temperature-sensitive mutants and the “O” (Offal) agent (P6[1]:G8), a rotavirus isolated in 1965 from abattoir waste. This analysis indicates that the only complete genomic sequence previously reported for SA11 (Both) is instead that of a reassortant, originating like the SA11-4F-like viruses, from the introduction of an “O” agent gene into the SA11 genetic background. These results, combined with identification of mutations that correlate with altered growth properties and ts phenotype, emphasize the importance of considering segment origin and sequence variation in interpreting experimental outcomes with SA11 strains.

UV disinfection of wastewater effluents for unrestricted irrigation

Wastewater reuse in arid regions is important for the production of a water resource to be utilised for non-potable purposes and to prevent the environmental transmission of disease-causing agents. This study was conducted to evaluate the effect of water quality on the comparative disinfection efficiency of viruses, bacteria and spores by UV irradiation. Furthermore, the microbial quality of effluent produced by coagulation, high rate filtration (HRF) and either UV irradiation or chlorination was determined. Using low pressure collimated beam, a UV dose of 80 mWs/cm2 was needed to achieve a 3-log10 inactivation of either rotavirus SA-11 or coliphage MS2, whereas over 5-log10 inactivation of E. coli was reached with a dose of only 20 mWs/cm2. B. subtilis inactivation was found to be linear up to a dose of 40 mWs/cm2 and then a tailing up to a UV dose of 120 mWs/cm2 was observed. It is worth noting that effluent turbidity of < 5 NTU did not influence the inactivation efficiency of UV irradiation. Operation of a pilot plant to treat secondary effluent by coagulation, HRF and UV disinfection at a UV dose of 80 mWs/cm2 resulted in the production of high quality effluent in compliance with the Israel standards for unrestricted irrigation (< 10 CFU/100 mL faecal coliform and turbidity of < 5 NTU). Sulphite reducing clostridia (SRC) were found to be more resistant than coliphages and F coliform for UV irradiation. The results of this study indicated that UV disinfection is suitable for the production of effluents for unrestricted irrigation of food crops.

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.

Rotaviruses interact with α4β7 and α4β1 integrins by binding the same integrin domains as natural ligands

Group A rotaviruses are major intestinal pathogens that express potential alpha4beta1 and alpha4beta7 integrin ligand sequences Leu-Asp-Val and Leu-Asp-Ile in their outer capsid protein VP7, and Ile-Asp-Ala in their spike protein VP4. Monkey rotavirus SA11 can use recombinant alpha4beta1 as a cellular receptor. In this study a new potential alpha4beta1, alpha4beta7 and alpha9beta1 integrin ligand sequence, Tyr-Gly-Leu, was identified in VP4. It was shown that several human and monkey rotaviruses bound alpha4beta1 and alpha4beta7, but not alpha9beta1. Binding to alpha4beta1 mediated the infectivity and growth of monkey rotaviruses, and binding to alpha4beta7 mediated their infectivity. A porcine rotavirus interacted with alpha4 integrins at a post-binding stage to facilitate infection. Activation of alpha4beta1 increased rotavirus infectivity. Cellular treatment with peptides containing the alpha4 integrin ligand sequences Tyr-Gly-Leu and Ile-Asp-Ala eliminated virus binding to alpha4 integrins and infectivity. In contrast, rotavirus recognition of alpha4 integrins was unaffected by a peptide containing the sequence Leu-Asp-Val or by a mutation in the VP7 Leu-Asp-Val sequence. VP4 involvement in rotavirus recognition of alpha4beta1 was demonstrated with rotavirus reassortants. Swapping and point mutagenesis of alpha4 surface loops showed that rotaviruses required the same alpha4 residues and domains for binding as the natural alpha4 integrin ligands: mucosal addressin cell adhesion molecule-1, fibronectin and vascular cell adhesion molecule-1. Several rotaviruses are able to use alpha4beta7 and alpha4beta1 for cell binding or entry, through the recognition of the same alpha4-subunit domains as natural alpha4 ligands.

A rotavirus strain isolated from pig-tailed macaque ( Macaca nemestrina) with diarrhea bears a P6[1]:G8 specificity

A distinct rotavirus strain (PTRV) was isolated in cell cultures from a stool sample obtained from a diarrheic 3-year-old female pig-tailed macaque ( Macaca nemestrina) that was born at the breeding colony of the University of Washington in Seattle. Unlike other known simian rotavirus strains including vervet monkey rotavirus SA11 which bears P5B[2]:G3 or P6[1]:G3 specificity, rhesus monkey rotavirus MMU18006 with P5B[3]:G3 specificity, pig-tailed macaque rotavirus YK-1 with P[3]:G3 specificity and rhesus monkey rotavirus TUCH with P[24]:G3 specificity, the cell-culture-grown PTRV strain was shown to bear P6[1]:G8 specificity as determined by VP4 (P)- and VP7 (G)-specific neutralization assays as well as gene sequence analyses. The virus in the original diarrhea stool was also shown to bear genotypes P[1] and G8. In addition, the PTRV strain exhibited a “long” electropherotype, subgroup I specificity and NSP4 genotype A specificity. The PTRV probe formed (i) 8–9 hybrid bands with genomic RNAs of various bovine rotavirus strains and (ii) only 2–3 hybrid bands with simian rotavirus RNAs as demonstrated by RNA–RNA hybridization, suggesting a possible bovine origin of the virus. Serologic analysis of serum samples obtained from selected pig-tailed macaques in the colony suggested that a rotavirus bearing P[1]:G8 specificity was endemic among macaques for at least 8 years (1987–1994). This is the first report describing an isolation of a simian rotavirus bearing a non-G3 VP7 and possibly a P6[1] specificities. Because of its unique simian serotype, this virus may prove to be valuable in challenge studies in a non-human primate model in studies of rotavirus 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 a shiga toxin B subunit::rotavirus NSP4 90 fusion protein protects mice against gastroenteritis

A fusion protein containing the shiga toxin-1 B subunit (STB) linked to a 90 amino acid peptide (aa residues 86–175) from simian rotavirus (SA-11) nonstructural protein NSP4 was synthesized in Escherichia coli. Mice orally inoculated with 60 μg of STB::NSP4 90 fusion protein per dose generated higher humoral and intestinal antibody titers than mice inoculated with 30 μg of NSP4 alone. Serum anti-NSP4 IgG2a isotype titers were substantially greater than IgG1 titers, suggesting a dominant Th1 immune response. ELISA measurement of cytokines secreted from splenocytes isolated from immunized mice confirmed the STB::NSP4 90 fusion protein stimulation of a strong Th1 cell mediated immune response. Diarrhea in SA-11 rotavirus challenged neonates suckling from STB::NSP4 immunized dams was significantly reduced in severity and duration in comparison with virus challenged neonates from unimmunized mice. Together, our experiments demonstrate for the first time that the shiga toxin B subunit provides ligand mediated delivery of virus antigens to the gut-associated lymphoid tissues for enhanced stimulation of humoral and cellular responses against rotavirus gastroenteritis.

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.