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.

Structural insight into African horsesickness virus infection.

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T=13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines.

Whole-genome consensus sequence analysis of a South African rotavirus SA11 sample reveals a mixed infection with two close derivatives of the SA11-H96 strain.

Whole-genome, sequence-independent amplification and 454(®) pyrosequencing of a rotavirus SA11 cell culture sample with an unknown passage history yielded consensus sequences of twelve complete genome segments. Two distinct sequences for genome segment 8 (encoding NSP2) were present, indicating a mixed infection with two rotavirus SA11 strains. The genotypes of the viruses were G3-P[2]-I2-R2-C5-M5-A5-Nx-T5-E2-H5, where x was either 5 or 2. The strains were named RVA/Simian-tc/ZAF/SA11-N5/1958/G3P[2] and RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2]. The genotype (N2) and sequence of genome segment 8 of RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2] were identical to that of the bovine rotavirus O agent. Five novel amino acids were detected in minor population variants of three genome segments. Genome segment 1 (VP1) has a high nucleotide substitution rate, but the substitutions are synonymous. Distance matrices and Bayesian molecular clock phylogenetics showed that SA11-N2 is a reassortant containing genome segment 8 from the O agent, whereas SA11-N5 is a very close derivative of the prototype SA11-H96.

Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase.

Glycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. 2.3.1.13). GLYAT detoxifies various xenobiotics, such as benzoic acid, and endogenous organic acids, such as isovaleric acid, which makes GLYAT important in the management of organic acidemias in humans. We cloned the open reading frame encoding the bovine ortholog of GLYAT from bovine liver mRNA into the bacterial expression vector pColdIII. The recombinant enzyme was expressed, partially purified, and enzymatically characterized. Protein modeling was used to predict Glu²²6 of bovine GLYAT to be catalytically important. This was assessed by constructing an E226Q mutant and comparing its enzyme kinetics to that of the wild-type recombinant bovine GLYAT. The Michaelis constants for benzoyl-CoA and glycine were determined and were similar for wild-type recombinant GLYAT, E226Q recombinant GLYAT, and GLYAT present in bovine liver. At pH 8.0, the E226Q mutant GLYAT had decreased activity, which could be compensated for by increasing the reaction pH. This suggested a catalytic mechanism in which Glu²²6 functions to deprotonate glycine, facilitating nucleophilic attack on the acyl-CoA. The recombinant bovine GLYAT enzyme, combined with this new understanding of its active site and reaction mechanism, could be a powerful tool to investigate the functional significance of GLYAT sequence variations. Eventually, this should facilitate investigations into the impact of known and novel sequence variations in the human GLYAT gene.

Whole genome analyses of African G2, G8, G9, and G12 rotavirus strains using sequence-independent amplification and 454® pyrosequencing.

High mortality rates caused by rotaviruses are associated with several strains such as G2, G8, G9, and G12 rotaviruses. Rotaviruses with G9 and G12 genotypes emerged worldwide in the past two decades. G2 and G8 rotaviruses are however also characterized frequently across Africa. To understand the genetic constellation of African G2, G8, G9, and G12 rotavirus strains and their possible origin, sequence-independent cDNA synthesis, amplification, and 454(®) pyrosequencing of the whole genomes of five human African rotavirus strains were performed. RotaC and phylogenetic analysis were used to assign and confirm the genotypes of the strains. Strains RVA/Human-wt/MWI/1473/2001/G8P[4], RVA/Human-wt/ZAF/3203WC/2009/G2P[4], RVA/Human-wt/ZAF/3133WC/2009/G12P[4], RVA/Human-wt/ZAF/3176WC/2009/G12P[6], and RVA/Human-wt/ZAF/GR10924/1999/G9P[6] were assigned G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, G12-P[4]-I1-R1-C1-M1-A1-N1-T1-E1-H1, G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1, and G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotypes, respectively. The detection of both Wa- and DS-1-like genotypes in strain RVA/Human-wt/ZAF/3133WC/2009/G12P[4] and Wa-like, DS-1-like and P[6] genotypes in strain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] implies that these two strains were generated through intergenogroup genome reassortment. The close similarity of the genome segments of strain RVA/Human-wt/MWI/1473/2001/G8P[4] to artiodactyl-like, human-bovine reassortant strains and human rotavirus strains suggests that it originated from or shares a common origin with bovine strains. It is therefore possible that this strain might have emerged through interspecies genome reassortment between human and artiodactyl rotaviruses. This study illustrates the swift characterization of all the 11 rotavirus genome segments by using a single set of universal primers for cDNA synthesis followed by 454(®) pyrosequencing and RotaC analysis.

Insights into the pre-initiation events of bacteriophage phi 6 RNA-dependent RNA polymerase: towards the assembly of a productive binary complex.

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage phi 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.

Rescue of Infectious Rotavirus Reassortants by a Reverse Genetics System Is Restricted by the Receptor-Binding Region of VP4.

The rotavirus species A (RVA) capsid contains the spike protein VP4, which interacts with VP6 and VP7 and is involved in cellular receptor binding. The capsid encloses the genome consisting of eleven dsRNA segments. Reassortment events can result in novel strains with changed properties. Using a plasmid-based reverse genetics system based on simian RVA strain SA11, we previously showed that the rescue of viable reassortants containing a heterologous VP4-encoding genome segment was strain-dependent. In order to unravel the reasons for the reassortment restrictions, we designed here a series of plasmids encoding chimeric VP4s. Exchange of the VP4 domains interacting with VP6 and VP7 was not sufficient for rescue of viable viruses. In contrast, the exchange of fragments encoding the receptor-binding region of VP4 resulted in virus rescue. All parent strains and the rescued reassortants replicated efficiently in MA-104 cells used for virus propagation. In contrast, replication in BSR T7/5 cells used for plasmid transfection was only efficient for the SA11 strain, whereas the rescued reassortants replicated slowly, and the parent strains failing to produce reassortants did not replicate. While future research in this area is necessary, replication in BSR T7/5 cells may be one factor that affects the rescue of RVAs.

Accurate prediction of protein structures and interactions using a three-track neural network.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

Preparation and evaluation of a recombinant Rift Valley fever virus N protein for the detection of IgG and IgM antibodies in humans and animals by indirect ELISA.

This paper describes the cloning, sequencing and bacterial expression of the N protein of the Rift Valley fever virus (RVFV) ZIM688/78 isolate and its evaluation in indirect ELISAs (I-ELISA) for the detection of IgM and IgG antibodies in human and sheep sera. Sera used for the evaluation were from 106 laboratory workers immunised with an inactivated RVF vaccine, 16 RVF patients, 168 serial bleeds from 8 sheep experimentally infected with wild type RVFV and 210 serial bleeds from 10 sheep vaccinated with the live attenuated Smithburn RVFV strain. All human and animal sera that tested positive in the virus neutralisation test were also positive in the IgG I-ELISA. There was a high correlation (R2=0.8571) between virus neutralising titres and IgG I-ELISA readings in human vaccinees. In experimentally infected sheep IgG antibodies were detected from day 4 to 5 post-infection onwards and IgM antibodies from day 3 to 4. The IgG I-ELISA was more sensitive than virus neutralisation and haemagglutination-inhibition tests in detecting the early immune response in experimentally infected sheep. The I-ELISAs demonstrated that the IgG and IgM response to the Smithburn vaccine strain was slower and the levels of antibodies induced markedly lower than to wild type RVFV infection.

Consensus Sequence of 27 African Horse Sickness Virus Genomes from Viruses Collected over a 76-Year Period (1933 to 2009).

We announce the complete consensus genome sequence of 27 African horse sickness viruses, representing all nine African horse sickness virus (AHSV) serotypes from historical and recent isolates collected over a 76-year period (1933 to 2009). The data set includes the sequence of the virulent Office International des Epizooties AHSV reference strains which are not adapted to cell culture.

Conservation of the coding regions of the glycine N-acyltransferase gene further suggests that glycine conjugation is an essential detoxification pathway.

Thorough investigation of the glycine conjugation pathway has been neglected. No defect of the glycine conjugation pathway has been reported and this could reflect the essential role of glycine conjugation in hepatic metabolism. Therefore, we hypothesised that genetic variation in the open reading frame (ORF) of the GLYAT gene should be low and that deleterious alleles would be found at low frequencies. This hypothesis was investigated by analysing the genetic variation of the human GLYAT ORF using data available in public databases. We also sequenced the GLYAT ORF of a small cohort of South African Afrikaner Caucasian individuals. In total, data from 1537 individuals was analysed. The two most prominent GLYAT haplotypes in all populations analysed, were S156 (70%) and T17S156 (20%). The S156C199 and S156H131 haplotypes, which have a negative effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. In the Afrikaner Caucasian cohort a novel Q61L SNP occurring at a high frequency (12%) was detected. The results of this study indicated that the GLYAT ORF is highly conserved and supported the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. These findings emphasise the importance of future investigations to determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics.

Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.

Consensus sequence determination and elucidation of the evolutionary history of a rotavirus Wa variant reveal a close relationship to various Wa variants derived from the original Wa strain.

The consensus nucleotide sequence of a human rotavirus Wa strain, with only a partially known passage history, was determined with sequence-independent amplification and next generation 454® pyrosequencing. This rotavirus Wa strain had the expected genome constellation of G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and was designated RVA/Human-tc/USA/WaCS/1974/G1P[8]. Phylogenetic analyses revealed a close relationship to four human rotavirus Wa variants (Wag5re, Wag7/8re, ParWa and VirWa) derived from the original 1974 human isolate. There were rearrangements in the Wag5re- and Wag7/8re variants in genome segments 5 (Wag5re) and 7 and 8 (Wag7/8re), which were not present in WaCS. Pairwise comparisons and a combined molecular clock for the Wa rotavirus genome indicated a close relationship between WaCS and ParWa and VirWa. These results suggest that WaCS is most probably an early cell culture adapted variant from the initial gnotobiotic pig passaged Wa isolate. Evolutionary pressure analysis identified a possible negative selected amino acid site in VP1 (genome segment 1) and a likely positive selected site in VP4 (genome segment 4). The WaCS may be more appropriate as a rotavirus Wa reference sequence than the current composite Wa reference genome.

Sequence analysis of the whole genomes of five African human G9 rotavirus strains.

The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diarrhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and characterised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequence of the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and one G9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe (southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8] from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped as G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317 was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1) genome segments. All the genome segments of the five strains grouped strictly according to their genotype Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9 study strains from southern Africa clustered most closely with rotaviruses from the same geographical origin and with those with the same G and P types. The highest nucleotide identity of genome segments of the study strains from eastern and central Africa regions on a Wa-like backbone was not limited to rotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] and G11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirus genotypes. Rotavirus strain MRC-DPRU9317 is the first G9P[6] to be characterised on a DS-1-like genetic backbone with a reassortant segment 8 (NSP2) and fourth G9P[6] to be fully sequenced globally.

Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase.

Human glycine N-acyltransferase (human GLYAT) detoxifies a wide range of endogenous and xenobiotic metabolites, including benzoate and salicylate. Significant inter-individual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. To investigate the influence of single nucleotide polymorphisms (SNPs) in the GLYAT coding sequence on enzyme activity, we expressed and characterised a recombinant human GLYAT. Site-directed mutagenesis was used to generate six non-synonymous SNP variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. We also generated an E227Q mutant, which lacks the catalytic residue proposed by Badenhorst et al. (2012). This mutant was inactive compared to the wild-type recombinant human GLYAT. A molecular model of human GLYAT containing coenzyme A (CoA) was generated which revealed that the inactivity of the R199C variant could be due to the substitution of the highly conserved Arg(199) and destabilisation of an α-loop-α motif which is important for substrate binding in the GNAT superfamily. The finding that SNP variations in the human GLYAT gene influence the kinetic properties of the enzyme may explain some of the inter-individual variation in glycine conjugation capacity, which is relevant to the metabolism of xenobiotics such as aspirin and the industrial solvent xylene, and to the treatment of some metabolic disorders.

Whole genome sequence analyses of three African bovine rotaviruses reveal that they emerged through multiple reassortment events between rotaviruses from different mammalian species.

Animal-to-human interspecies transmission is one of the evolutionary mechanisms driving rotavirus strain diversity in humans. Although quite a few studies emanating from Africa revealed evidence of bovine-to-human rotavirus interspecies transmission, whole genome data of African bovine rotavirus strains are not yet available. To gain insight into the complete genome constellation of African bovine rotaviruses, the full genomes of three bovine rotavirus strains were extracted from stool samples collected from calves, amplified using a sequence-independent procedure, followed by 454(®) pyrosequencing. Strains RVA/Cow-wt/ZAF/1603/2007/G6P[5] and RVA/Cow-wt/ZAF/1605/2007/G6P[5] were both genotyped as G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and were probably two variants of the same rotavirus due to their close nucleotide sequence similarity. The genotype constellation of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] was G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The genetic relationships and phylogenetic analyses suggested that these three bovine rotavirus strains may have emerged through multiple reassortment events between bovine, giraffe and antelope rotaviruses. Due to the close relatedness of genome segments 1 (encoding VP1), 7 (NSP2), 9 (VP7) and 10 (NSP4) of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] to those of the corresponding segments of human rotaviruses, RVA strain 1604 may represent bovine strains that were transmitted to humans and possibly reassorted with human rotaviruses previously. The complete nucleotide sequences of the bovine rotavirus strains reported in this study represent the first whole genome data of bovine rotaviruses from Africa.

Determination of the whole-genome consensus sequence of the prototype DS-1 rotavirus using sequence-independent genome amplification and 454® pyrosequencing.

The prototype DS-1 rotavirus strain, is characterised by a short electropherotype and G2P[4] serotype specificity. Following sequence-independent genome amplification and 454(®) pyrosequencing of genomic cDNA, differences between the newly determined consensus sequence and GenBank sequences were observed in 10 of the 11 genome segments. Only the consensus sequence of genome segment 1 was identical to sequences deposited in GenBank. A novel isoleucine at position 397 in a hydrophobic region of VP4 is described. An additional 7 N-terminal amino acids was found in NSP1. For genome segment 10 the first 34 and last 30 nucleotides of the 5' and 3'-terminal ends, respectively, were identified. Genome segment 11 was found to be 821 bp long, which is 148 bp longer than the full length genome segment 11 sequence reported previously. This paper reports the first complete consensus genome sequence for the tissue culture adapted DS-1 strain free from cloning bias and the limitations of Sanger sequencing. Sequence differences in previous publications reporting on DS-1 rotavirus genome segment sequencing, were identified and discussed.

Whole genome analysis of multiple rotavirus strains from a single stool specimen using sequence-independent amplification and 454® pyrosequencing reveals evidence of intergenotype genome segment recombination.

Infection of a single host cell with two or more different rotavirus strains creates conditions favourable for evolutionary mechanisms like reassortment and recombination that can generate novel strains. Despite numerous reports describing mixed rotavirus infections, whole genome characterisation of rotavirus strains in a mixed infection case has not been reported. Double-stranded RNA, exhibiting a long electropherotype pattern only, was extracted from a single human stool specimen (RVA/Human-wt/ZAF/2371WC/2008/G9P[8]). Both short and long electropherotype profiles were however detected in the sequence-independent amplified cDNA derived from the dsRNA, suggesting infection with more than one rotavirus strain. 454® pyrosequencing of the amplified cDNA revealed co-infection of at least four strains. Both genotype 1 (Wa-like) and genotype 2 (DS-1-like) were assigned to the consensus sequences obtained from the nine genome segments encoding NSP1-NSP5, VP1-VP3 and VP6. Genotypes assigned to the genome segments encoding VP4 were P[4] (DS-1-like), P[6] (ST3-like) and P[8] (Wa-like) genotypes. Since four distinct genotypes [G2 (DS-1-like), G8, G9 (Wa-like) and G12] were assigned to the four consensus nucleotide sequences obtained for genome segment 9 (VP7), it was concluded that at least four distinct rotaviruses were present in the stool. Intergenotype genome recombination events were observed in genome segments encoding NSP2, NSP4 and VP6. The close similarities of some of the genome segments encoding NSP2, VP6 and VP7 to artiodactyl rotaviruses suggest that some of the infecting strains shared common ancestry with animal strains, or that interspecies transmission occurred previously. The sequence-independent genome amplification technology coupled with 454® pyrosequencing used in this study enabled the characterisation of the whole genomes of multiple rotavirus strains in a single stool specimen that was previously assigned single genotypes, i.e. G9P[8], by sequence-dependent RT-PCR.

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase.

The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.

Initiation of viral RNA-dependent RNA polymerization.

This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3' terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.