A 3-methylcrotonyl-CoA carboxylase deficient human skin fibroblast transcriptome reveals underlying mitochondrial dysfunction and oxidative stress.

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive inherited metabolic disease of leucine catabolism with a highly variable phenotype. Apart from extensive mutation analyses of the MCCC1 and MCCC2 genes encoding 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4), molecular data on MCC deficiency gene expression studies in human tissues is lacking. For IEMs, unbiased '-omics' approaches are starting to reveal the secondary cellular responses to defects in biochemical pathways. Here we present the first whole genome expression profile of immortalized cultured skin fibroblast cells of two clinically affected MCC deficient patients and two healthy individuals generated using Affymetrix(®)HuExST1.0 arrays. There were 16191 significantly differentially expressed transcript IDs of which 3591 were well annotated and present in the predefined knowledge database of Ingenuity Pathway Analysis software used for downstream functional analyses. The most noticeable feature of this MCCA deficient skin fibroblast transcriptome was the typical genetic hallmark of mitochondrial dysfunction, decreased antioxidant response and disruption of energy homeostasis, which was confirmed by mitochondrial functional analyses. The MCC deficient transcriptome seems to predict oxidative stress that could alter the complex secondary cellular response that involve genes of the glycolysis, the TCA cycle, OXPHOS, gluconeogenesis, ß-oxidation and the branched-chain fatty acid metabolism. An important emerging insight from this human MCCA transcriptome in combination with previous reports is that chronic exposure to the primary and secondary metabolites of MCC deficiency and the resulting oxidative stress might impact adversely on the quality of life and energy levels, irrespective of whether MCC deficient individuals are clinically affected or asymptomatic.

Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes.

This paper reports significant improvements in the efficacy of sequence-independent amplification and quality of sequencing of viruses with segmented double-stranded RNA (dsRNA) genomes. We demonstrate that most remaining bottlenecks in dsRNA virus genome characterization have now been eliminated. Both the amplification and sequencing technologies used require no previous sequence knowledge of the viral dsRNA, there is no longer a need to separate genome segments or amplicons and the sequence-determined bias observed in cloning has been overcome. Combining very efficient genome amplification with pyrophosphate-based 454 (GS20/FLX) sequencing enabled sequencing of complete segmented dsRNA genomes and accelerated the sequence analysis of the amplified viral genomes. We report the complete consensus sequence of seven viruses from four different dsRNA virus groups, which include the first complete sequence of the genome of equine encephalosis virus (EEV), the first complete sequence of an African horsesickness virus (AHSV) genome determined directly from a blood sample and a complete human rotavirus genome determined from faeces. We also present the first comparison between the complete consensus sequence of a virulent and an attenuated strain of AHSV1. Ultra-deep sequencing (>400-fold coverage) of the AHSV1 reference and attenuated strains revealed different ratios of reassortants in the reference strain and allowed quasispecies detection in the plaque-purified attenuated strain of AHSV1. This approach amounts to a paradigm shift in dsRNA virus research, since it is sensitive and specific enough for comprehensive investigations of the evolution and genetic diversity in dsRNA virus populations.

Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of a novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China.

A diarrhoeal outbreak among adults in China was caused by a new rotavirus, termed ADRV-N, that does not react with antisera directed against group A, B or C rotaviruses [Zhonghua Liu Xing Bing Xue Za Zhi (Chin. Epidemiol.) 19 (1998) 336]. ADRV-N can be propagated in cell cultures [Zhonghua Yi Xue Za Zhi (Natl. Med. J. China) 82 (2002) 14]. We present the complete sequences for ADRV-N genome segments 5 and 6, and a full ORF sequence of genome segment 7. The deduced amino acid sequences suggest that these segments encode NSP1, VP6 and NSP3, respectively. These three ADRV-N genome segments have a unique -ACCCC-3' terminal sequence. The 5'-GG- terminus of segments 5 and 6 is the same as that of other rotaviruses. The amino acid similarity between VP6 and NSP3 of ADRV-N and the cognate sequences of their closest counterpart, group B IDIR, was 37 and 35%, respectively. The ADRV-N NSP1 has a double-stranded RNA binding motif (DSRM) and a putative autoproteolytic cleavage motif upstream from the DSRM. The putative ADRV-N NSP3 has a truncated C-terminus compared to the cognate protein of group B rotaviruses. All the available data demonstrate that ADRV-N differs significantly from the known rotaviruses and strongly suggest that ADRV-N is the first recognized member of a new group of rotaviruses infecting humans.

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera.

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.

VP2 gene phylogenetic characterization of field isolates of African horsesickness virus serotype 7 circulating in South Africa during the time of the 1999 African horsesickness outbreak in the Western Cape.

We present the first VP2-gene phylogenetic analysis of African horsesickness (AHS) viruses within a serotype. Thirteen AHSV 7 isolates were obtained from cases that occurred in South Africa during 1998-1999, and three were historical AHSV 7 isolates. The goals were to start a database of isolates of known location and time of isolation and to determine if we could identify the origin of an AHS outbreak in the surveillance area in the Western Cape. We prepared full-length cDNA copies of the VP2-genes of the isolates. Nucleic acid sequence data of a 786 bp region was used to characterize the genetic relationships between the isolates. The nucleic acid identities between the isolates ranged from 95.5 to 100%. Isolates from common geographical regions grouped together. Characterization of field isolates revealed the presence of two AHSV 7 lineages in South Africa during this period. The grouping of the viruses into two clades accurately reflected the geographical groupings of the isolates. The average nucleic acid divergence between the clades was 4.3%. Within the clades the divergence was 0.5 and 0.1%, respectively. The data suggests that the AHS outbreak in the Western Cape could have been an incursion from the Kwazulu Natal Province.

A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes.

The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.

Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes.

Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Phi12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0.8 to 6.8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5' GUU and UAC 3') and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22-30 cycles compared to 30-35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.

The protective efficacy of a recombinant VP2-based African horsesickness subunit vaccine candidate is determined by adjuvant.

We previously demonstrated that soluble baculovirus-expressed African horsesickness virus (AHSV) serotype 5 VP2 protein (AHSV5 rVP2) elicits neutralising antibodies in guinea pigs. We have now determined the immunogenicity of soluble AHSV5 rVP2 in horses when administered in three different adjuvant types, ISA-50, aluminium phosphate and different saponin preparations. Doses of 10 and 50microg of rVP2 administered with saponin induced full protection to a lethal challenge, albeit with dose-related side effects. The results establish that soluble rVP2 is the biologically active form and that it can induce complete protection when it is delivered with saponin adjuvants. We conclude that the use of the soluble biologically active form of AHSV rVP2 and the choice of adjuvant will be crucial factors in determining efficacy, safety and the production cost of recombinant AHSV subunit vaccines.

Variation of African horsesickness virus nonstructural protein NS3 in southern Africa.

NS3 protein sequences of recent African horsesickness virus (AHSV) field isolates, reference strains and current vaccine strains in southern Africa were determined and compared. The variation of AHSV NS3 was found to be as much as 36.3% across serotypes and 27.6% within serotypes. NS3 proteins of vaccine and field isolates of a specific serotype were found to differ between 2.3% and 9.7%. NS3 of field isolates within a serotype differed up to 11.1%. Our data indicate that AHSV NS3 is the second most variable AHSV protein, the most variable being the major outer capsid protein, VP2. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters. The only exception was AHSV-8 NS3, which clustered into different groups than previously described. No obvious sequence markers could be correlated with virulence. Our results suggest that NS3 sequence variation data could be used to distinguish between field isolates and live attenuated vaccine strains of the same serotype.

Development of probes for typing African horsesickness virus isolates using a complete set of cloned VP2-genes.

A set of cloned full-length VP2-genes from the reference strain of each of the nine serotypes of African horsesickness virus (AHSV) was used to develop probes for typing AHSV isolates. The VP2-gene probes hybridised serotype-specific to purified viral dsRNA from its corresponding serotype. No cross-hybridisation was observed between the different AHSV serotypes or with RNA from equine encephalosis virus or bluetongue virus (BTV) which are related viruses within the genus Orbivirus that co-circulate with AHSV in South Africa. The probes were able to detect AHSV isolates from recent field cases of AHS in South Africa, despite being derived from historical reference strains. With regard to sensitivity and time considerations: radioactive 32P-labelling resulted in a marginal increase in sensitivity over digoxigenin-labelled probes. By infecting cell cultures at different multiplicities of infection (m.o.i.) and harvesting at various times post infection, it was established that AHSV RNA could be detected 16 h post infection (p.i.) at a m.o.i. of 1.00 pfu per cell and 48 h p.i. at a m.o.i. of 0.01 pfu per cell. Typing of AHSV isolates by means of VP2-gene probe hybridisation can be completed in 4 days, which is less than half the time required for conventional isolation and serotyping. This report on the use of a complete set of cloned AHSV VP2-gene probes is the first demonstration of typing for a whole specie (serogroup) in a genus of the family Reoviridae.

Charge modification of plasma and milk proteins results in antiviral active compounds.

Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti-HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available epsilonNH2 groups were anionized yielded the most potent anti-HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti-HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120-CD4 mediated virus/cell binding. The relative contribution of these effects to the anti-HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti-HIV activity but a clearly increased anti-HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti-HIV and anti-HCMV activity, respectively. The N-terminal portion of Lf appeared essential for its anti-HCMV effect: N-terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications.

Vector competence of Culicoides bolitinos and C. imicola for South African bluetongue virus serotypes 1, 3 and 4.

The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis. The concentration of the virus per millilitre of bloodmeal was 10(5.0) and 10(6.0)TCID50 for BLU 4 and 10(7.2)TCID50 for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log10 TCID50 titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola. The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola. However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.

Protein aggregation complicates the development of baculovirus-expressed African horsesickness virus serotype 5 VP2 subunit vaccines.

This paper describes the expression of a cloned African horsesickness virus (AHSV) serotype 5 VP2-gene by a baculovirus recombinant that was generated by the BAC-TO-BAC system. Immunization of horses with crude cell lysates containing recombinant baculovirus-expressed AHSV5 VP2 did induce neutralizing antibodies, but afforded only partial protection against virulent virus challenge. Further analysis of partially protective crude cell lysates revealed that baculovirus-expressed AHSV5 VP2 was predominantly present in the form of insoluble aggregates. Only approximately 10% of VP2 was present in a soluble form. Immunization of guinea-pigs with aggregated and soluble forms of AHSV5 VP2 established that only soluble VP2 was capable of inducing neutralizing antibodies. This finding adds a new dimension to the development of AHSV VP2s as subunit vaccines. Further investigation is needed to limit formation of insoluble aggregates and optimize conditions for producing VP2 in a form capable of inducing protective immunity.

Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation.

A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3-4 kb genes of dsRNA viruses. The method of single primer amplification (Lambden et al., 1992) was adapted by the inclusion of a 3' poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro.

The processing, transport and heterologous expression of Epstein-Barr virus gp110.

Epstein-Barr virus (EBV) glycoprotein gp110 has substantial structural and sequence homology with herpes simplex virus (HSV) gB and gBs of other alpha- and betaherpesviruses but unlike HSV gB localizes differently in infected cells and is absent from virions. To facilitate the analysis of EBV gp110, antisera were raised to fragments of gp110 expressed in a bacterial system. They recognized a protein of the predicted size in recombinant bacterial lysates, in lymphoblastoid cells and in recombinant vaccinia virus-gp110 infected cells. gp110 from all sources possessed a high-mannose type of N-glycosylation implying that gp110 has not passed through the Golgi. Immunofluorescence and immuno-electron microscopy confirmed this conclusion and demonstrated that, in contrast to HSV gB, the majority of immunoreactive gp110 was present at the nuclear membrane or endoplasmic reticulum (ER) but not at the cell membrane. Unexpectedly, a truncated version of gp110 lacking the hydrophobic C-terminal region, despite forming dimers analogous to HSV dimers, was transported in a similar manner to full-length gp110. Two chimeric proteins constructed by replacing the N- and C-terminal domains of gp110 with corresponding regions of gp340/220 were also transported to the nuclear membrane/ER. These data suggest that unlike HSV gB both the N- and C-terminal portions of EBV gp110 contain independent signals sufficient to direct the molecule to the ER/nuclear membrane. Specific transport of gammaherpesvirus gB homologues to the nuclear membrane, from where herpesviruses bud, suggests that they may be involved in the egress of virus from the nucleus.

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.

Detection of bluetongue virus and African horsesickness virus in co-infected cell cultures with NS1 gene probes.

The serogroup specificity of the bluetongue virus (BTV) NS1 and VP3 gene probes was confirmed by means of northern blot hybridization. Under high-stringency conditions both probes hybridized to 22 BTV serotypes (18 South African serotypes, BTV3 from Cyprus and BTV16 from Pakistan) but not to serotypes that originate from Australia and India. Furthermore, NS1 gene probes of BTV and African horsesickness virus (AHSV) were used in a dot-spot in situ hybridization procedure to differentiate between BTV and AHSV in co-infected cell cultures. The method detects viral RNA directly i glutaraldehyde-fixed infected cell cultures without prior nucleic-acid extraction or purification. AHSV could be detected in cells infected with AHSV at a multiplicity of infection of 10(-4) PFU/cell in the presence of a hundred excess of co-infecting BTV. The method may have an application in epidemiological surveys to detect different orbiviruses in the same Culicoides population.

Differentiation between minus- and plus-strand synthesis: polymerase activity of dsRNA bacteriophage phi 6 in an in vitro packaging and replication system.

Empty procapsids of the segmented dsRNA virus phi 6, produced in Escherichia coli from a cloned L genome segment, package plus-strand phi 6 ssRNA genomic segments, synthesize minus strands, and transcribe the newly formed dsRNA templates. Procapsids can be restricted to minus-strand synthesis by high concentrations of CaCl2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) RNA-dependent RNA polymerase activities in vitro. Reaction conditions for minus-strand synthesis were optimized. Plus-strand synthesis by procapsids could be activated by binding of purine nucleoside triphosphates to a low-affinity NTP-binding site. The second 5'-terminal nucleotide of the phi 6 plus-sense ssRNA L genomic segment is important for determining the level of transcription of that segment and the generation of infectious procapsids.

Mutational effects on the spectroscopic properties and biological activities of oxidized bovine adrenodoxin, and their structural implications.

Of the aromatic 1H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through-space ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation. As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-450(11 beta) (CYP11B1)-dependent and P-450scc (CYP11A1)-dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin. The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule.