Alberdina A. van Dijk

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. J. Med. Virol. 83:2018–2042, 2011.

Structural insight into African horsesickness virus infection

ABSTRACT 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 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.

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.

A new perspective on the importance of glycine conjugation in the metabolism of aromatic acids

Abstract A number of endogenous and xenobiotic organic acids are conjugated to glycine, in animals ranging from mosquitoes to humans. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, the recently proposed glycine deportation hypothesis states that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilized as neurotransmitters in the central nervous systems of animals. This hypothesis is based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. In this review it will be argued that the major role of glycine conjugation is to dispose of the end products of phenylpropionate metabolism. Furthermore, glucuronidation, which occurs in the endoplasmic reticulum, would not be ideal for the detoxification of free benzoate, which has been shown to accumulate in the mitochondrial matrix. Glycine conjugation, however, prevents accumulation of benzoic acid in the mitochondrial matrix by forming hippurate, a less lipophilic conjugate that can be more readily transported out of the mitochondria. Finally, it will be explained that the glycine conjugation of benzoate, a commonly used preservative, exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated.

Glycine conjugation: importance in metabolism, the role of glycine N-acyltransferase, and factors that influence interindividual variation

Introduction: Glycine conjugation of mitochondrial acyl-CoAs, catalyzed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterized in detail. Here, we review the causes and possible consequences of interindividual variation in the glycine conjugation pathway. Areas covered: The authors review the importance of CoASH in metabolism, formation and toxicity of xenobiotic acyl-CoAs, and mechanisms for restoring levels of CoASH. They focus on GLYAT, glycine conjugation, how genetic variation in the GLYAT gene could influence glycine conjugation, and the emerging roles of glycine metabolism in cancer and musculoskeletal development. Expert opinion: The substrate selectivity of GLYAT and its variants needs to be further characterized, as organic acids can be toxic if the corresponding acyl-CoA is not a substrate for glycine conjugation. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability, and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development, and mitochondrial energy metabolism.

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.

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.

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

BackgroundHuman 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.ResultsWe 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.ConclusionOur 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.