Establishment of a rotavirus-infected zebrafish model and its application in drug screening.

Rotavirus (RV) is one of the main pathogens that induce infantile diarrhea and by now no effective drugs are available for RV-induced infantile diarrhea. Thus the development of novel models is of vital importance for the pathological research of RV-induced infantile diarrhea, as well as the progress of the associated treatment strategy. Here we introduced for the first time that RV-Wa strain and RV-SA-11 strain could infect 5 dpf(day post fertilization) and 28 dpf larvae, to induce infantile diarrhea model that was highly consistent with the clinical infection of infants. RV infection significantly changed the signs, survival rate and inflammation of larvae. Some important indicators, including the levels of RV antigen VP4 and VP6, the in vivo RV tracking, and the RV particles were also analyzed, which collectively demonstrated that the model was successfully established. More importantly, we also determined the potentials of the proposed RV-infected zebrafish model for anti-viral drug assessment. In conclusion, we established a RV-infected zebrafish model with formulated relevant indicators both larvae and adult fish, which might be served as a high throughput platform for antiviral drug screening.

Study on the effectiveness and safety of Xingpi Yanger granule combined with Saccharomyces boulardii for rotavirus enteritis in children: A protocol for systematic review and meta-analysis.

BACKGROUND: To systematically evaluate the effectiveness and safety of traditional Chinese medicine preparation XPYEG combined with SBI and SBI alone in the treatment of REC, and to provide the reference in drugs for the clinical treatment of children with rotavirus enteritis. METHODS: Retrieving the English databases: PubMed, Cochrane Library and Embase; Chinese databases: CNKI, CBM and WANFANG Data. Retrieving a randomized controlled trial of XPYEG and SBI in the treatment of REC. The retrieval time is from the above database until September 2020. The retrieval strategy of combining free words and subject words is adopted, and the references included in the literature are searched manually in accordance with the literature studied in this paper and not included in the above database. Two researchers screen the literature according to the literature inclusion and exclusion criteria, extract valid data and evaluate the quality of the literature, and cross-check it. Using the RevMan 5.3 software to conduct the meta-analysis on the main outcome and secondary outcome indicators of the included literature, while assessing the evidence quality of included study. RESULTS: The effectiveness and safety of XPYEG and SBI in the treatment of REC are presented through the main and secondary outcome indicators. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/3QSZG. CONCLUSION: This study will conclude whether the combination of XPYEG and SBI is more effective than SBI alone in the treatment of REC, and whether the medication increases the risk of adverse reactions compared with single medication. ETHICS AND DISSEMINATION: This study does not involve the specific patients, and all research data comes from publicly available professional literature, so an ethics committee is not required to conduct an ethical review and approval of the study.

Influence of individual or group housing of newborn calves on rotavirus and coronavirus infection during the first 2 months of life.

Bovine rotavirus A (RVA) and bovine coronavirus (CoV) are the two main viral enteropathogens associated with neonatal calf diarrhea. The aim of the present work was to study the impact of group and individual housing systems in the epidemiology of RVA and CoV infection. Eleven calves reared in individual housing (FA) and nine calves in group housing (FB) were monitored during the first 7 weeks of life. Stool and serum samples were screened for RVA and CoV antigens by ELISA. IgG1 antibodies (Ab) to both antigens were also measured. From the 160 fecal samples collected, the proportion of positive samples to RVA and CoV was significantly higher in FB (23.6%) than in FA (9%) (p = 0.03). The geometric mean of colostral IgG1 Ab titers to CoV and RVA in FA (IgG1 anti-CoV 1024 and anti-RVA 1782.9) was lower than in FB (IgG1 anti-CoV 10,321.2 and anti-RVA 4096) at birth. Calves less than 2 weeks of life from FB had a higher risk of being infected by RVA (OR = 4.9; p = 0.01) and CoV (OR = 17.15; p = 0.01) than calves from FA. The obtained results showed that there was higher RVA and CoV shedding in group-housed calves than in individual-housed animals.

Valeriana jatamansi Jones Inhibits Rotavirus-Induced Diarrhea via Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway.

Rotavirus (RV), as the main cause of diarrhea in children under 5 years, contributes to various childhood diseases. Valeriana jatamansi Jones is a traditional Chinese herb and possesses antiviral effects. In this study we investigated the potential mechanisms of V. jatamansi Jones in RV-induced diarrhea. MTT assay was performed to evaluate cell proliferation and the diarrhea mice model was constructed using SA11 infection. Mice were administered V. jatamansi Jones and ribavirin. Diarrhea score was used to evaluate the treatment effect. The enzyme-linked immunosorbent assay was performed to detect the level of cytokines. Western blot and quantitative reverse transcription-PCR were used to determine protein and mRNA levels, respectively. Hematoxylin-eosin staining was applied to detect the pathological change of the small intestine. TdT-mediated dUTP nick-end labeling was conducted to determine the apoptosis rate. The results showed V. jatamansi Jones promoted MA104 proliferation. V. jatamansi Jones downregulated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in protein level, which was consistent with the immunohistochemistry results. Moreover, V. jatamansi Jones combined with ribavirin regulated interleukin-1ß (IL-1ß), interferon γ, IL-6, tumor necrosis factor α, and IL-10, and suppressed secretory immunoglobulin A secretion to remove viruses and inhibit dehydration. V. jatamansi Jones + ribavirin facilitated the apoptosis of small intestine cells. In conclusion, V. jatamansi Jones may inhibit RV-induced diarrhea through PI3K/AKT signaling pathway, and could therefore be a potential therapy for diarrhea.

Generation of recombinant rotaviruses encoding a split NanoLuc peptide tag.

Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.

Drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway.

Although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. Since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. We identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. We confirmed this effect in 2D cell cultures and 3D cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. Supplementation of UTP or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. Our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients.

A Systematic Review and Meta-Analysis: The Effectiveness of Probiotics for Viral Gastroenteritis.

BACKGROUND: Probiotics can be used for the treatment of viral gastroenteritis. OBJECTIVE: This systematic review is to evaluate the evidence regarding the effect of probiotics on human cases of viral gastroenteritis. METHODS: The objective of this review is to evaluate the effectiveness of probiotics against placebo or standard treatment for viral gastroenteritis. A comprehensive search of Cochrane Library, EMBASE, MEDLINE via PubMed and Ovid databases, and unpublished studies (till 27 January 2018) was conducted followed by a process of study selection and critical appraisal by two independent reviewers. Randomized controlled trials assessing probiotic administration in human subjects infected with any species of gastroenteritis viruses were considered for inclusion. Only studies with a confirmed viral cause of infection were included. This study was developed using the JBI methodology for systematic reviews, which is in accordance with the PRISMA guideline. Meta-analysis was conducted where feasible. Data were pooled using the inverse variance method with random effects models and expressed as Mean Differences (MDs) with 95% Confidence Intervals (CIs). Heterogeneity was assessed by Cochran Q statistic and quantified by the I2 statistic. We included 17 RCTs, containing 3,082 patients. RESULTS: Probiotics can improve symptoms of viral gastroenteritis, including the duration of diarrhea (mean difference 0.7 days, 95% CI 0.31 to 1.09 days, n = 740, ten trials) and duration of hospitalization (mean difference 0.76 days, 95% CI 0.61 to 0.92 days, n = 329, four trials). CONCLUSION: The results of this review show that the administration of probiotics in patients with viral gastroenteritis should be considered.

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed-batch fermentation.

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein-based rotavirus vaccine candidate derived from non-replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small-scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full-length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full-length material (60-75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).

Lentinan administration relieves gut barrier dysfunction induced by rotavirus in a weaned piglet model.

Rotavirus (RV) is a pathogen that induces severe diarrhea in infants and young animals. Shiitake mushroom is a traditional food, which can improve physiological function, including gut health. Lentinan (LNT) is the main functional component of Shiitake mushroom. This study aimed to verify whether LNT administration could improve intestinal barrier function, thereby decreasing RV-induced diarrhea in a porcine model. According to initial weight and origin, a total of 28 weaned piglets were randomly fed 2 diets containing 0 or 84 mg kg-1 LNT for 19 d (n = 14). On day 15, RV was orally infused to half of the pigs in each group. RV-induced diarrhea (P < 0.05), the positive rate of RV non-structural protein 4 (NSP4), impaired intestinal morphology, antioxidant capacity and microbiota (P < 0.05), and increased apoptosis of jejunal epithelial cells (P < 0.05) were assessed in the piglets. Dietary LNT supplementation was found to improve intestinal morphology, permeability, antioxidant capacity and microbiota (P < 0.05). Supplementation also further alleviated the effects of RV infection on diarrhea, intestinal morphology, permeability, antioxidant capacity, microbiota and apoptosis of jejunal epithelial cells in piglets (P < 0.05). Thus, these results suggest that LNT administration relieved RV-induced diarrhea in piglets, which could be due to the increase in antioxidant capacity, reduction in apoptosis and improvement of the microbiota-increased gut barrier.

Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing S gene from porcine epidemic diarrhea virus and VP7 gene from porcine rotavirus.

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Rotavirus Double Infection Model to Study Preventive Dietary Interventions.

Rotaviruses are the main cause of acute diarrhea among young children worldwide with an increased frequency of reinfection. Several life style factors, such as dietary components, may influence such processes by affecting the outcome of the first rotavirus infection and therefore having a beneficial impact on the anti-rotavirus immune responses during any subsequent reinfections. The aim of this research was to develop a double-infection model in rat that mimics real-life clinical scenarios and would be useful in testing whether nutritional compounds can modulate the rotavirus-associated disease and immune response. Three experimental designs and a preventive dietary-like intervention were conducted in order to achieve a differential response in the double-infected animals compared to the single-infected ones and to study the potential action of a modulatory agent in early life. Diarrhea was only observed after the first infection, with a reduction of fecal pH and fever. After the second infection an increase in body temperature was also found. The immune response against the second infection was regulated by the preventive effect of the dietary-like intervention during the first infection in terms of specific antibodies and DTH. A rotavirus-double-infection rat model has been developed and is suitable for use in future preventive dietary intervention studies.

Rotavirus Genotypes Circulating in Brazil Before and After the National Rotavirus Vaccine Program: A Review.

BACKGROUND: Rotavirus vaccines created the opportunity to control diarrhea in children. We describe the rotavirus genotypes before and after the rotavirus vaccine introduction in Brazil. METHODS: We reviewed the distribution of rotavirus genotypes in Brazil before and after vaccine introduction by searching publication. RESULTS: Eighty-six studies reported 6884 (15.2%) rotavirus episodes among 45,305 children. Rotavirus caused 22.4% and 11.6% of cases before and after vaccine introduction. G1P[8], G9P[8] and G2P[4] heterotypic strains were most common before and after vaccine introduction. CONCLUSIONS: The vaccines may have selected heterotypic strains in this highly vaccinated population.

A fermented milk concentrate and a combination of short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides/pectin-derived acidic oligosaccharides protect suckling rats from rotavirus gastroenteritis.

Human milk contains bioactive compounds that confer a protective role against gastrointestinal infections. In order to find supplements for an infant formula able to mimic these benefits of breast-feeding, two different concepts were tested. The products consisted of the following: (1) a Bifidobacterium breve- and Streptococcus thermophilus-fermented formula and (2) a combination of short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides with pectin-derived acidic oligosaccharides. A rotavirus infection suckling rat model was used to evaluate improvements in the infectious process and in the immune response of supplemented animals. Both nutritional concepts caused amelioration of the clinical symptoms, even though this was sometimes hidden by softer stool consistency in the supplemented groups. Both products also showed certain modulation of immune response, which seemed to be enhanced earlier and was accompanied by a faster resolution of the process. The viral shedding and the in vitro blocking assay suggest that these products are able to bind the viral particles, which can result in a milder infection. In conclusion, both concepts evaluated in this study showed interesting protective properties against rotavirus infection, which deserve to be investigated further.

Rotavirus specific maternal antibodies and immune response to RV3-BB neonatal rotavirus vaccine in New Zealand.

BACKGROUND: Maternal antibodies, acquired passively via placenta and/or breast milk, may contribute to the reduced efficacy of oral rotavirus vaccines observed in children in developing countries. This study aimed to investigate the effect of rotavirus specific maternal antibodies on the serum IgA response or stool excretion of vaccine virus after any dose of an oral rotavirus vaccine, RV3-BB, in parallel to a Phase IIa clinical trial conducted at Dunedin Hospital, New Zealand. At the time of the study rotavirus vaccines had not been introduced in New Zealand and the burden of rotavirus disease was evident. METHODS: Rotavirus specific IgG and serum neutralizing antibody (SNA) levels in cord blood and IgA levels in colostrum and breast milk samples collected ∼4 weeks, ∼20 weeks and ∼28 weeks after birth were measured. Infants were randomized to receive the first dose of vaccine at 0-5 d (neonatal schedule) or 8 weeks (infant schedule). Breast feeding was with-held for 30 minutes before and after vaccine administration. The relationship between rotavirus specific IgG and SNA levels in cord blood and IgA in colostrum and breast milk at the time of first active dose of RV3-BB vaccine and level of IgA response and stool excretion after 3 doses of vaccine was assessed using linear and logistic regression. RESULTS: Forty infants received 3 doses of RV3-BB rotavirus vaccine and were included in the analysis of the neonatal and infant groups. Rotavirus specific IgA in colostrum (neonatal schedule group) and breast milk at 4 weeks (infant schedule group) was identified in 14/21 (67%) and 14/17 (82%) of infants respectively. There was little evidence of an association between IgA in colostrum or breast milk IgA at 4 weeks, or between cord IgG or SNA level, and IgA response or stool excretion after 3 doses of RV3-BB, or after one dose (neonatal schedule) (all p>0.05). CONCLUSIONS: The level of IgA in colostrum or breast milk and level of placental IgG and SNA did not impact on the serum IgA response or stool excretion following 3 doses of RV3-BB Rotavirus Vaccine administered using either a neonatal or infant schedule in New Zealand infants.

Medicinal plants and natural molecules with in vitro and in vivo activity against rotavirus: A systematic review.

BACKGROUND: Rotaviruses can cause life-threatening health disorders, such as severe dehydrating gastroenteritis and diarrhea in children. Vaccination is the main preventive strategy to reduce rotavirus diarrhea and the severity of episodes, but vaccines are not fully effective and new episodes may occur, even in vaccinated children. The WHO recommends oral rehydration therapy and zinc supplementation for rotavirus-induced diarrhea management. There is little preclinical evidence to support the use of phytotherapeutics in the management of rotaviral infections. PURPOSE: We aim to review the use of medicinal plants and natural molecules in the management of rotavirus infections in experimental studies. METHODS: Articles, published in the English language between 1991 and 2016, were retrieved from PubMed, Scopus and Web of Science using relevant keywords. The scientific literature mainly focusing on plant natural products with therapeutic efficacies against experimental models of rotavirus, were identified and tabulated. In addition, an assessment of the reliability of animal experiments was determined under ``Risk of Bias'' criteria. CHAPTERS: After an initial search and a revision of the inclusion criteria, 41 reports satisfied the objectives of the study. 36 articles were found concerning the anti-rotaviral potential in rotavirus infected cell lines. Among the active secondary metabolites screened for rotavirus inhibition, the polyphenols of flavonoid structure had acquired the highest number of studies in our survey, compared to phenolic acids, stilbenoids, tannins, pectins, terpenoids and flavonoid glycosides. Also, many phytochemicals reduced the efficacy of viral capsid proteins foremost to their elimination and improved the tendency of host-cell inhibiting virus absorption or by prevention of viral replication. Furthermore, five in vivo studies reported that herbs, as well its components, reduced the duration and severity of diarrhea in mice and piglets. The anti-rotavirus efficacy were highlighted based on improvements in reduction on liquid stool, fecal virus shedding, small intestinal histology, levels of inflammation related cytokines and signaling receptors. However, the quality of the experiments in animal studies contained certain types of bias in terms of how they were conducted and reported. CONCLUSION: We identified and summarized studies on medicinal plants and natural molecules having anti-rotavirus activity in order to further future developments of cures for rotavirus gastroenteritis.

Anti-VP6 VHH: An Experimental Treatment for Rotavirus A-Associated Disease.

Species A Rotaviruses (RVA) remain a leading cause of mortality in children under 5 years of age. Current treatment options are limited. We assessed the efficacy of two VP6-specific llama-derived heavy chain antibody fragments (VHH) -2KD1 and 3B2- as an oral prophylactic and therapeutic treatment against RVA-induced diarrhea in a neonatal mouse model inoculated with virulent murine RVA (ECw, G16P[16]I7). Joint therapeutic administration of 2KD1+3B2 (200 µg/dose) successfully reduced diarrhea duration, RVA infection severity and virus shedding in feces. While the same dose of 2KD1 or 3B2 (200 µg) significantly reduced duration of RVA-induced diarrhea, 2KD1 was more effective in diminishing the severity of intestinal infection and RVA shedding in feces, perhaps because 2KD1 presented higher binding affinity for RVA particles than 3B2. Neither prophylactic nor therapeutic administration of the VHH interfered with the host's humoral immune response against RVA. When 2KD1 (200 µg) was administered after diarrhea development, it also significantly reduced RVA intestinal infection and fecal shedding. Host antibody responses against the oral VHH treatment were not detected, nor did viral escape mutants. Our findings show that oral administration of anti-VP6 VHH constitute, not only an effective prophylactic treatment against RVA-associated diarrhea, but also a safe therapeutic tool against RVA infection, even once diarrhea is present. Anti-VP6 VHH could be used complementary to ongoing vaccination, especially in populations that have shown lower immunization efficacy. These VHH could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Vitamin D3 supplementation alleviates rotavirus infection in pigs and IPEC-J2 cells via regulating the autophagy signaling pathway.

Vitamin D had an anti-infection effect and benefited to the intestinal health. Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the severe diarrheal disease in young children and young animals. Although evidence suggested that vitamin D3 attenuates the negative effects of RV infection via the retinoic acid-inducible gene I signaling pathway, little is known of its antiviral effect whether through the regulation of autophagy. The present study was performed to investigate whether vitamin D3 alleviates RV infection in pig and porcine small intestinal epithelial cell line (IPEC-J2) models via regulating the autophagy signaling pathway. RV administration increased the Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA expression under the condition of RV challenged. The viability of IPEC-J2 was significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3 significantly decreased the concentrations of RV antigen and non-structural protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3 treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles) treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA expression in the RV-infected cells. Taken together, these results indicated that vitamin D3 attenuates RV infection through regulating autophagic maturation and porcine cathelicidin genes expression.

Is there a relationship between low vitamin D and rotaviral diarrhea?

BACKGROUND: For children under 5 years of age, 1700 000 000 episodes of diarrhea are seen worldwide, and death occurs in 700 000 of these cases due to diarrhea. Rotavirus is an important cause of diarrhea in this age group, and many studies have shown that vitamin D plays a pivotal role in the immune system, as well as in antimicrobial peptide gene expression. In addition, lower vitamin D has been correlated with higher rates of infectious diseases such as respiratory tract infection, tuberculosis, and viral infection. METHODS: Seventy patients with rotaviral diarrhea and 67 healthy patients were enrolled in this study. Serum 25-hydroxy vitamin D(3) (25(OH)D(3)), parathormone, calcium, phosphate, alkaline phosphatase, complete blood count parameters, and C-reactive protein were compared between pre-school children hospitalized due to rotaviral diarrhea and healthy children. Additionally, birthweight, feeding habits in the first 6 months of life, vitamin D and multivitamin supplements, and rotaviral vaccinations were also evaluated in each group. RESULTS: There were no differences between the groups with regard to gender and age, but 25(OH)D(3) was significantly different: 14.6 ± 8.7 ng/mL in the rotaviral diarrhea patients versus 29.06 ± 6.51 ng/mL in the health controls (P < 0.001), and serum 25(OH)D(3) <20 ng/mL (OR, 6.3; 95%CI: 3.638-10.909; P < 0.001) was associated with rotaviral diarrhea. CONCLUSIONS: Low vitamin D is associated with rotaviral diarrhea. This is the first study in the literature to show this, and this result needs to be repeated in larger controlled clinical studies.

Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

BACKGROUND: Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. RESULTS: We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. CONCLUSION: Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.