Strain SA11 Research Articles

Potential of avian and mammalian species A rotaviruses to reassort as explored by plasmid only-based reverse genetics

Species A rotavirus (RVA) is an important gastrointestinal pathogen that is widely distributed in humans, mammalian animals and birds. The RVA genome consists of eleven double-stranded RNA segments, enabling the generation of novel strains with new pathogenic or antigenic features by genetic reassortment. While reassortants between human and mammalian animal RVAs have been repeatedly described, data on the reassortment potential of avian RVA strains are rare. To investigate genome segment exchanges between avian and mammalian RVA strains, a plasmid-based reverse genetics strategy originally developed for the simian RVA strain SA11 was used here. All eleven genome segments of the chicken RVA strain 02V0002G3 were cloned into similar plasmids as in the SA11 system. However, in contrast to SA11, no infectious virus could be generated by transfection of the eleven 02V0002G3 plasmids into cell culture under the same conditions. In another series of experiments, each of the genome segments of 02V0002G3 was transfected together with the remaining ten genome segments of SA11. Viable mono-reassortants were only retrieved for the avian VP3 and VP4 genes. The reassortant viruses were structurally indistinguishable from their parental viruses, but grew to slightly lower titers in cell culture. The results indicate that the VP3 and VP4 genes, but not the other genes of avian RVA, can functionally substitute their mammalian homologs and create viable reassortants. Further research should focus on the reasons behind the reassortment incompatibility and on the optimization of the system for the generation of viable avian RVA rescued entirely from cloned avian RVA genome segments.

A novel amino acid site closely associated with the neurovirulence of live, attenuated Japanese encephalitis vaccine (SA14-14-2 strain)

Japanese encephalitis (JE) poses a serious threat to the world’s public health yet without a cure, the only way to prevent Japanese encephalitis virus (JEV) infection is vaccination. Live attenuated vaccine (SA14-14-2 strain) is the most widely used JE vaccine, and clinical data have confirmed its safety and effectiveness. Eight sitesassociated with virulence in the Envelope (E) protein are often the focus of quality control of JE vaccine. However, sequences retrieved from NCBI, as well as our previous results showed that the wild strain SA14 may harbor two different amino acids at amino acid residue 244 of the E glycoprotein (E244), and it may be related to virulence. In this study, we introduced a single mutation at nt1708 (G → A) in the full-length cDNA clone of SA14-14-2, replacing a Gly with Glu at amino acid residue 244 of the E glycoprotein, and successfully constructed the mutant virus (JEV E244). JEV E244 exhibited a similar plaque morphology and growth characteristics to JEV SA14-14-2 in cell culture. However, it had lethal neurovirulence in mice and could enter the brain following intraperitoneal inoculation. Moreover, the virulence of JEV E244 in the context of vaccine in mice is significantly different from that of the JEV E244 alone. These results suggested that E244 site should be included in the assessment of the genetic stability of the attenuated JE vaccine. The detection of minor mutations in vaccine population and influence on the safety of vaccine is discussed.

Generation of simian rotavirus reassortants with diverse VP4 genes using reverse genetics.

Species A rotaviruses (RVAs) are a major cause of gastroenteritis in animals and humans. Their genome consists of 11 segments of dsRNA, and reassortment events between animal and human strains can contribute to the high genetic diversity of RVAs. We used a plasmid-based reverse genetics system to investigate the reassortment potential of the genome segment encoding the viral outer capsid protein VP4, which is a major antigenic determinant, mediates viral entry and plays an important role in host cell tropism. We rescued reassortant viruses containing VP4 from porcine, bovine, bat, pheasant or chicken RVA strains in the backbone of simian strain SA11. The VP4 reassortants could be stably passaged in MA-104 cells and induced cytopathic effects. However, analysis of growth kinetics revealed marked differences in replication efficiency. Our results show that the VP4-encoding genome segment has a high reassortment potential, even between virus strains from highly divergent species. This can result in replication-competent reassortants with new genomic, growth and antigenic features.

The antibacterial activities of bacteriocin Pediococcus acidilactici of breast milk isolate to against methicillin-resistant Staphylococcus aureus

Resistance has occurred if an antibiotic cannot kill pathogenic bacteria therefore an antibiotic therapeutic effect cannot be achieved. Broad and free use of antibiotics effected in health problems, one of the health problems is bacteria are experienced the resistance such as Methicillin-Resistant Staphylococcus aureus (MRSA). Therefore an antibiotic alternative is needed from natural ingredients have the potential as anti-MRSA such as bacteriocin. This research was conducted to determine the antibacterial activity of bacteriocin from Pediococcus acidilactici of breast milk isolate (ASI) against MRSA. Bacteriocin isolation was carried out by growing of P.acidilactici on the media of Rogosa Sharpe (MRS) broth. Antibacterial activity test was carried out by diffusion method are to see the inhibition zone and dilution to determine the value of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The test results shown that bacteriocin P.acidilactici strains A11 and C12 strains had antibacterial activity against MRSA. The A11 strain inhibition zone is 15 mm, MIC and MBC values are 25%. While the C12 strain inhibition zone produced 18 mm, the value of MIC and MBC was 12.5%.

Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication

Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conventional human intestinal Caco2 cell line as well as in human primary intestinal organoids. The antiviral effect is conserved in both laboratory strain SA11 and rotavirus strain 2011K isolated from clinical sample. Mechanistic study indicated that BQR and LFM exerted their anti-rotavirus effect through targeting DHODH to deplete pyrimidine nucleotide pool. Therefore, targeting pyrimidine biosynthesis represents a potential approach for developing antiviral strategies against rotavirus.

GC-MS analysis and in-vitro hypocholesterolemic, anti-rotavirus, anti-human colon carcinoma activities of the crude extract of a Japanese Ganoderma spp

Background and objective Medicinal mushrooms are mines of various biologically active compounds. Therefore, chemical analysis and in-vitro evaluation of some biological activities of the Japanese originated mushroom Ganoderma spp. were conducted. Materials and methods Extraction of the fruiting bodies of Ganoderma spp. was accomplished using 80% methanol. This extract was investigated for its in-vitro cholesterol-lowering activity, anti-rotavirus effect, and anti-human colon cancer influence. Moreover, a gas chromatography–mass spectrometry analysis for this extract was performed. Results and conclusion The gas chromatography–mass spectrometry analysis resulted in the detection of 39 compounds, which were generally saturated and unsaturated fatty acids, and alkenes. The crude extract exhibited a promising in-vitro cholesterol-lowering activity (100±0%) after 96 h of incubation at room temperature. The same crude extract showed a moderate anti-rotavirus SA-11 strain effect with a therapeutic index of 9.3. Moreover, Ganoderma spp. extract displayed a strong activity toward HCT116 human colon carcinoma cell line, resulting in a cytotoxicity of 84.03±0.93% on HCT116 cell line monolayers. Ganoderma spp. crude extract represents a promising source of biologically active compounds that could by further investigations represent support and/or alternative to the currently used drugs.

Immune and cytokine/chemokine responses of PBMCs in rotavirus-infected rhesus infants and their significance in viral pathogenesis.

The rotavirus (RV) is the most important causative agent of severe gastroenteritis in infants and children aged less than 5 years worldwide. However, the response and the roles of peripheral blood mononuclear cell (PBMC) in RV clearance have yet to be fully elucidated. In this study, we established the neonatal rhesus monkey model of RV infection with histopathological changes in the small intestine. Then, we investigated gene expression changes in PBMCs from the monkey model of RV infection. Similar pathways regulatedin rhesus monkeys that received intragastric administration of the RV monkey SA11 strain (G3P[2]) and the human wild-type strain ZTR-68 (G1P[8]). Gene profiling showed differences in functional genes mainly associated with chemokine signaling pathways and cytokine-cytokine receptor interactions post RV infection. Transferrin and C-C motif chemokine ligand 23 (CCL23) gene expression were upregulated in PBMCs of monkeys when stimulated by simian and human RV strains. Monkeys infected with RV had an enhanced and prolonged inflammatory response that was associated with increased levels of CCL20, CCL23, and C-X-C motif chemokine ligand 1; while inhibition of major histocompatibility complex class I expression may be important for immune evasion by RV. The RV infection was also characterized by pathological changes in the small intestine with a cytokine and chemokine storm. This study identified the chemokine signaling pathway and immune response genes involved in RV infection in infant rhesus monkeys. The SA11 RV strain is more suitable for establishing a monkey diarrhea model than the ZTR-68 RV strain.

Chimeric Japanese Encephalitis Virus SA14/SA14-14-2 Was Virulence Attenuated and Protected the Challenge of Wild-Type Strain SA14.

The attenuated Japanese encephalitis virus (JEV) live vaccine SA14-14-2 prepared from wild-type (WT) strain SA14 was licensed to prevent Japanese encephalitis (JE) in 1989 in China. Many studies showed that the premembrane (prM) and envelope (E) protein were the crucial determinant of virulence and immunogenicity of JEV. So we are interested in whether the substitution of prM/E of JEV WT SA14 with those of vaccine strain SA14-14-2 could decrease neurovirulence and prevent the challenge of JEV WT SA14. Molecular clone technique was used to replace the prM/E gene of JEV WT strain SA14 with those of vaccine strain SA14-14-2 to construct the infectious clone of chimeric virus (designated JEV SA14/SA14-14-2), the chimeric virus recovered from BHK21 cells upon electrotransfection of RNA into BHK21 cells. The results showed that the recovered chimeric virus was highly attenuated in mice, and a single immunization elicited strong protective immunity in a dose-dependent manner. This study increases our understanding of the molecular mechanisms of neurovirulence attenuation and immunogenicity of JEV.

Neonatal rhesus monkeys as an animal model for rotavirus infection.

AIMTo establish a rotavirus (RV)-induced diarrhea model using RV SA11 in neonatal rhesus monkeys for the study of the pathogenic and immune mechanisms of RV infection and evaluation of candidate vaccines.METHODSNeonatal rhesus monkeys with an average age of 15-20 d and an average weight of 500 g ± 150 g received intragastric administration of varying doses of SA11 RV ( 107 PFUs/mL, 106 PFUs/mL, or 105 PFUs/mL, 10 mL/animal) to determine whether the SA11 strain can effectively infect these animals by observing their clinical symptoms, fecal shedding of virus antigen by ELISA, distribution of RV antigen in the organs by immunofluorescence, variations of viral RNA load in the organs by qRT-PCR, histopathological changes in the small intestine by HE staining, and apoptosis of small intestinal epithelial cells by TUNEL assay.RESULTSThe RV monkey model showed typical clinical diarrhea symptoms in the 108 PFUs SA11 group, where we observed diarrhea 1-4 d post infection (dpi) and viral antigen shed in the feces from 1-7 dpi. RV was found in jejunal epithelial cells. We observed a viral load of approximately 5.85 × 103 copies per 100 mg in the jejunum at 2 dpi, which was increased to 1.09 × 105 copies per 100 mg at 3 dpi. A relatively high viral load was also seen in mesenteric lymph nodes at 2 dpi and 3 dpi. The following histopathological changes were observed in the small intestine following intragastric administration of SA11 RV: vacuolization, edema, and atrophy. Apoptosis in the jejunal villus epithelium was also detectable at 3 dpi.CONCLUSIONOur results indicate that we have successfully established a RV SA11 strain diarrhea model in neonatal rhesus monkeys. Future studies will elucidate the mechanisms underlying the pathogenesis of RV infection, and we will use the model to evaluate the protective effect of candidate vaccines.

New tetrameric forms of the rotavirus NSP4 with antiparallel helices.

Rotavirus nonstructural protein 4, the first viral enterotoxin to be identified, is a multidomain, multifunctional glycoprotein. Earlier, we reported a Ca2+-bound coiled-coil tetrameric structure of the diarrhea-inducing region of NSP4 from the rotavirus strains SA11 and I321 and a Ca2+-free pentameric structure from the rotavirus strain ST3, all with a parallel arrangement of α-helices. pH was found to determine the oligomeric state: a basic pH favoured a tetramer, whereas an acidic pH favoured a pentamer. Here, we report two novel forms of the coiled-coil region of NSP4 from the bovine rotavirus strains MF66 and NCDV. These crystallized at acidic pH, forming antiparallel coiled-coil tetrameric structures without any bound Ca2+ ion. Structural and mutational studies of the coiled-coil regions of NSP4 revealed that the nature of the residue at position 131 (Tyr/His) plays an important role in the observed structural diversity.

Ginsenoside-Rb2 and 20(S)-Ginsenoside-Rg3 from Korean Red Ginseng Prevent Rotavirus Infection in Newborn Mice.

It is well known that Korean red ginseng has various biological activities. However, there is little knowledge about the antiviral activity of Korean red ginseng and its ginsenosides. In this study, we addressed whether oral administration of ginsenoside-Rb2 and -Rg3 is able to protect against rotavirus (RV) infection. The protective effect of ginsenosides against RV infection was examined using an in vivo experiment model in which newborn mice (10-day-old) were inoculated perorally (p.o.) with 1.5 × 106 plaque-forming units/mouse of RV strain SA11. When various dosages of ginsenoside-Rb2 (25-250 mg/kg) were administered 3days, 2 days, or 1 day before virus challenge, treatment with this ginsenoside at the dosage of 75 mg/kg 3days before virus infection most effectively reduced RV-induced diarrhea. In addition, consecutive administration of ginsenoside-Rb2 (75 mg/kg) at 3 days, 2 days, and 1 day before virus infection was more effective than single administration on day -3. The consecutive administration of ginsenoside-Rb2 also reduced virus titers in the bowels of RV-infected mice. In an experiment to compare the protective activity between ginsenoside-Rb2 and its two hydrolytic products (20(S)- and 20(R)-ginsenoside-Rg3), 20(S)-ginsenoside-Rg3, but not 20(R)-ginsenoside-Rg3, prevented RV infection. These results suggest that ginsenoside-Rb2 and its hydrolytic product, 20(S)-ginsenoside-Rg3, are promising candidates as an antiviral agent to protect against RV infection.

Enhanced cytopathic effect of Japanese encephalitis virus strain SA14-14-2: Probable association of mutation in amino acid of its envelope protein

Japanese encephalitis (JE) is a mosquito-borne viral disease. It is a global public health concern since it causes an acute encephalitis syndrome (AES). A large number of JE/AES cases are reported to occur in areas with established or developing JE vaccination program. Partial vaccine coverage and emergence of new variants of Japanese encephalitis virus (JEV) might be playing an important role. The envelope protein (E) of JEV is a major antigenic determinant and responsible for immunogenic responses as well as membrane fusion and virion assembly. In the present study, we have characterized the JEV live attenuated vaccine strain SA14-14-2 in baby hamster kidney cells (BHK-21). The vaccine strain showed enhanced replication following its passage in BHK-21 cells. Nucleotide sequence analysis of the E protein gene of the cell-culture adapted vaccine strain showed an important point mutation. The mutation in the E protein gene was identical to its wild-type parent strain SA14. This study suggests the possibility of reversion mutation and exaltation of vaccine strains following adaptation in the host cells.

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.

Mechanism of Anti-rotavirus Synergistic Activity by Epigallocatechin Gallate and a Proanthocyanidin-Containing Nutraceutical.

Epigallocatechin gallate (EGCG) of green tea and the nutraceutical CystiCran®-40 (containing 40% proanthocyanidins) of the cranberry plant have been associated with antiviral activity. The purpose of this work was to determine the mechanism of antiviral synergy between each compound. Coliphage T4II (phage T4) and the rotavirus strain SA-11(RTV) were used as model virus systems. Individual and combined flavonoids structural and molecular weight analyses were performed by NMR and HPCL/MS, respectively. A suboptimal concentration of EGCG or C-40 alone or in combination reduced phage infectivity by ≤10%. Similarly, EGCG (30µg/ml) and C-40 (25µg/ml), respectively, reduced RTV titers by 3 and 13%. However, RTV titers were reduced by 32% (p<.05) with both flavonoids used in combination. RTV was not recognized in host cells by electron microscopy 24-h post-inoculation. NMR and HPLC/MS findings revealed significant structural and potential changes in molecular weight of the flavonoids in complex.

A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity.

Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.

An Inhibitory Motif on the 5'UTR of Several Rotavirus Genome Segments Affects Protein Expression and Reverse Genetics Strategies.

Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5’UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5’UTRs of eight genome segments (gs3, gs5-6, gs7-11) of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3’UTR sequences. Analysis of several mutants of the 21-nucleotide long 5’UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5’ terminal 6-nucleotide long pyrimidine-rich tract 5’-GGY(U/A)UY-3’. The 5’ terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5’UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies.

Comparison of the live-attenuated Japanese encephalitis vaccine SA14 -14-2 strain with its pre-attenuated virulent parent SA14 strain: similarities and differences in vitro and in vivo

Japanese encephalitis virus (JEV) is the main cause of acute viral encephalitis, primarily affecting children and young adults in the Asia-Pacific region. JEV is a vaccine-preventable pathogen, with four types of JE vaccine licensed in different regions of the world. To date, the most common JEV strain used in vaccine development and production is SA14-14-2, an attenuated strain derived from its wild-type parental strain SA14. In this study, we directly compared the phenotypic and genotypic characteristics of SA14 and SA14-14-2 to determine the biological and genetic properties associated with their differential virulence. In susceptible BHK-21 cells, SA14-14-2 grew slightly more slowly and formed smaller plaques than SA14, but unlike SA14, it showed almost no expression of the viral protein NS1', the product of a conserved predicted RNA pseudoknot-mediated ribosomal frameshift. In weanling ICR mice, SA14-14-2 was highly attenuated in terms of both neuroinvasiveness and neurovirulence, with its median lethal doses invariably over five logs higher than those of SA14 when inoculated intramuscularly and intracerebrally. Interestingly, the neurovirulence of SA14-14-2 was dependent on mouse age, with the 1- to 7-day-old mice being highly susceptible and the 14- to 21-day-old mice becoming resistant to intracerebral inoculation. At the genome level, SA14-14-2 differed from SA14 by 57 nucleotides, including one silent G-to-A substitution at position 3599 within the predicted RNA pseudoknot for NS1' synthesis; of the 57 differences, 25 resulted in amino acid substitutions. Our data pave the way for the development of new genetically modified JE vaccines.

Generation of an Avian-Mammalian Rotavirus Reassortant by Using a Helper Virus-Dependent Reverse Genetics System

The genetic diversity of rotavirus A (RVA) strains is facilitated in part by genetic reassortment. Although this process of genome segment exchange has been reported frequently among mammalian RVAs, it remained unknown if mammalian RVAs also could package genome segments from avian RVA strains. We generated a simian RVA strain SA11 reassortant containing the VP4 gene of chicken RVA strain 02V0002G3. To achieve this, we transfected BSR5/T7 cells with a T7 polymerase-driven VP4-encoding plasmid, infected the cells with a temperature-sensitive SA11 VP4 mutant, and selected the recombinant virus by increasing the temperature. The reassortant virus could be stably passaged and exhibited cytopathic effects in MA-104 cells, but it replicated less efficiently than both parental viruses. Our results show that avian and mammalian rotaviruses can exchange genome segments, resulting in replication-competent reassortants with new genomic and antigenic features. This study shows that rotaviruses of mammals can package genome segments from rotaviruses of birds. The genetic diversity of rotaviruses could be broadened by this process, which might be important for their antigenic variability. The reverse genetics system applied in the study could be useful for targeted generation and subsequent characterization of distinct rotavirus reassortant strains.

Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

Isolation and characterization of Saccharomyces species for bioethanol production from sugarcane molasses: Studies of scale up in bioreactor

Decreases in oil reserves and gas fields around all over the world justify the deepening of studies to render viable the larger-scale use of new energy sources. Therefore, the use of microorganisms to convert sugars into ethanol is a feasible process to be performed in a short period of time and at low costs. In this context, this study aimed to select ethanol-producing yeasts, after isolating samples in molasses obtained from companies in the Province of Tucumán (Argentina) and grapes obtained from farms located in Cafayate (Salta, Argentina). Among the twenty-nine samples studied A2, A10 and A11 isolates showed higher ethanol productions of 12.87; 13.64 and 13.46% respectively. A2 showed a homogeneous growth meanwhile the growth of strains A10 and A11 was flocculent. Molecular taxonomic characterization of these isolates showed a percentage of similarity of 100% with the strain Saccharomyces cerevisiae. The behavior of the non-flocculent A2 strain at laboratory scale was faster using a sugarcane molasses based medium, reaching 11.36% ethanol without adding nutrients and other growth factors, probably because its disperse form facilitates the transfer of nutrients and products. These values were improved to 12.02% when the process was scaled up to a 10L bioreactor. All these studies allowed concluding that S. cerevisiae A2 strain is a promising microorganism for the production of bioethanol with potential environmental, energy and economic benefits to be projected into industrial scale.