Identification of a BTV-Strain-Specific Single Gene That Increases Culicoides Vector Infection Rate.

Since the 2000s, the distribution of bluetongue virus (BTV) has changed, leading to numerous epidemics and economic losses in Europe. Previously, we found a BTV-4 field strain with a higher infection rate of a Culicoides vector than a BTV-1 field strain has. We reverse-engineered parental BTV-1 and BTV-4 strains and created BTV-1/BTV-4 reassortants to elucidate the influence of individual BTV segments on BTV replication in both C. sonorensis midges and in KC cells. Substitution of segment 2 (Seg-2) with Seg-2 from the rBTV-4 significantly increased vector infection rate in reassortant BTV-14S2 (30.4%) in comparison to reverse-engineered rBTV-1 (1.0%). Replacement of Seg-2, Seg-6 and Seg-7 with those from rBTV-1 in reassortant BTV-41S2S6S7 (2.9%) decreased vector infection rate in comparison to rBTV-4 (30.2%). However, triple-reassorted BTV-14S2S6S7 only replicated to comparatively low levels (3.0%), despite containing Seg-2, Seg-6 and Seg-7 from rBTV-4, indicating that vector infection rate is influenced by interactions of multiple segments and/or host-mediated amino acid substitutions within segments. Overall, these results demonstrated that we could utilize reverse-engineered viruses to identify the genetic basis influencing BTV replication within Culicoides vectors. However, BTV replication dynamics in KC cells were not suitable for predicting the replication ability of these virus strains in Culicoides midges.

Full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction.

When rinderpest virus (RPV) was declared eradicated in 2011, the only remaining samples of this once much-feared livestock virus were those held in various laboratories. In order to allow the destruction of our institute's stocks of RPV while maintaining the ability to recover the various viruses if ever required, we have determined the full genome sequence of all our distinct samples of RPV, including 51 wild type viruses and examples of three different types of vaccine strain. Examination of the sequences of these virus isolates has shown that the African isolates form a single disparate clade, rather than two separate clades, which is more in accord with the known history of the virus in Africa. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the M and F protein coding sequences, and shown that, for more than half the genomes sequenced, translation of the F protein requires translational frameshift or non-standard translation initiation. Curiously, the clade containing the lapinised vaccine viruses that were developed originally in Korea appears to be more similar to the known African viruses than to any other Asian viruses.

Origin of Bluetongue Virus Serotype 8 Outbreak in Cyprus, September 2016.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2-5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42-98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July-September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus.

Carbon-13 Cross-Polarization Magic-Angle Spinning Nuclear Magnetic Resonance for Measuring Proanthocyanidin Content and Procyanidin to Prodelphinidin Ratio in Sainfoin ( Onobrychis viciifolia) Tissues.

A procedure based on 13C CPMAS NMR was developed to study procyanidins (PCs) and prodelphinidins (PDs) directly in milled sainfoin plant tissues. Blackcurrant and Tilia samples enabled reference spectra of purified proanthocyanidin (PA) fractions, crude extracts, and milled plant tissues, with characteristic resonances at 155, 144, and 132 ppm. PC/PD ratios were estimated from the I132/I155 intensity ratio and differed by 2.5 to 5.9% compared to thiolysis data. Normalization to the 155 ppm signal intensity from reference spectra enabled analysis of PA contents with an error of ca. 8 g PAs/100 g plant tissue. The procedure estimates the lignin contribution and allows for a correction of the PA content. In six sainfoin accessions, estimated PA contents were 1.6- to 20.8-fold higher than the thiolysis and 1.4- to 2.6-fold higher than the HCl-butanol-acetone results. Method differences may reflect the presence of unextractable, possibly high molecular weight PAs in sainfoin.

Complete Genome Sequence of Peste des Petits Ruminants Virus from Georgia, 2016.

We report here the complete genome sequence of a peste des petits ruminants virus (PPRV) from the first outbreak of the disease in Georgia in January 2016. Genome sequencing was performed using Illumina next-generation sequencing technology in conjunction with Sanger sequencing. This PPRV/Georgia/Tbilisi/2016 genome sequence clustered within lineage IV PPRV viruses.

Facile Purification of Milligram to Gram Quantities of Condensed Tannins According to Mean Degree of Polymerization and Flavan-3-ol Subunit Composition.

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or 1H-13C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.

Identification of Structural Features of Condensed Tannins That Affect Protein Aggregation.

A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r = -0.916; amw: r = -0.925; p<0.01; df = 27), and gelatin (mDP: r = -0.961; amw: r = -0.981; p<0.01; df = 12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r = 0.677; p<0.05; df = 9 and r = 0.887; p<0.01; df = 9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r = -0.941; p<0.01; df = 5) and increased the apparent ß-sheet content (r = 0.916; p<0.05; df = 5) of BSA.

Structure-Activity Relationship of Condensed Tannins and Synergism with trans-Cinnamaldehyde against Caenorhabditis elegans.

Parasitic gastrointestinal nematodes (GIN) of livestock are increasingly developing resistance to synthetic nematocidal drugs. Moreover, the use of nematocides can induce ecotoxicity by affecting free-living nematodes. Condensed tannins (CT) are a structurally diverse group of bioactive plant compounds possessing anthelmintic activity against GIN. We investigated the relationship between the chemical structure of contrasting, purified CT and nematocidal effects using Caenorhabditis elegans. We also explored whether the nematocidal activity of CT could synergize with trans-cinnamaldehyde (CIN). A nonsignificant correlation was evident between the ability of CT fractions to inhibit C. elegans motility and the molar proportion of prodelphinidin subunits in purified CT samples. Synergistic inhibition of motility was achieved by combinations of CT and CIN. Galloylation of procyanidins was also a key factor for synergy. To increase the nematocidal effect of CT, plant sources containing CT with specific structural features could be selected and combined with compounds acting in synergy.

Condensed tannins in extracts from European medicinal plants and herbal products.

Medicinal plant materials are not usually analysed for condensed tannins (CT). Thirty commercially available European medicinal plants and herbal products were screened for CT and fourteen CT samples were analysed in detail. This is also the first comprehensive CT analysis of pine buds, walnut leaves, heather flowers and great water dock roots. Acetone/water extracts contained between 3.2 and 25.9 g CT/100g of extract, had CT with mean degrees of polymerisation of 2.9 to 13.3, procyanidin/prodelphinidin ratios of 1.6/98.4 to 100/0 and cis/trans flavan-3-ol ratios of 17.7/82.3 to 97.3/2.7. The majority of samples contained procyanidins, four contained A-type linkages (blackthorn flowers, heather flowers, bilberry leaves and cowberry leaves) and one sample also had galloylated procyanidins (great water dock roots).

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes.

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity.

Anthelmintic activity of trans-cinnamaldehyde and A- and B-type proanthocyanidins derived from cinnamon (Cinnamomum verum).

Cinnamon (Cinnamomum verum) has been shown to have anti-inflammatory and antimicrobial properties, but effects on parasitic worms of the intestine have not been investigated. Here, extracts of cinnamon bark were shown to have potent in vitro anthelmintic properties against the swine nematode Ascaris suum. Analysis of the extract revealed high concentrations of proanthocyanidins (PAC) and trans-cinnamaldehyde (CA). The PAC were subjected to thiolysis and HPLC-MS analysis which demonstrated that they were exclusively procyanidins, had a mean degree of polymerization of 5.2 and 21% of their inter-flavan-3-ol links were A-type linkages. Purification of the PAC revealed that whilst they had activity against A. suum, most of the potency of the extract derived from CA. Trichuris suis and Oesophagostomum dentatum larvae were similarly susceptible to CA. To test whether CA could reduce A. suum infection in pigs in vivo, CA was administered daily in the diet or as a targeted, encapsulated dose. However, infection was not significantly reduced. It is proposed that the rapid absorption or metabolism of CA in vivo may prevent it from being present in sufficient concentrations in situ to exert efficacy. Therefore, further work should focus on whether formulation of CA can enhance its activity against internal parasites.

Anthelmintic Activities against Haemonchus contortus or Trichostrongylus colubriformis from Small Ruminants Are Influenced by Structural Features of Condensed Tannins.

Plants containing condensed tannins (CTs) may hold promise as alternatives to synthetic anthelmintic (AH) drugs for controlling gastrointestinal nematodes (GINs). However, the structural features that contribute to the AH activities of CTs remain elusive. This study probed the relationships between CT structures and their AH activities. Eighteen plant resources were selected on the basis of their diverse CT structures. From each plant resource, two CT fractions were isolated and their in vitro AH activities were measured with the larval exsheathment inhibition assay, which was applied to Haemonchus contortus and Trichostrongylus colubriformis. Calculation of mean EC50 values indicated that H. contortus was more susceptible than T. colubriformis to the different fractions and that the F1 fractions were less efficient than the F2 ones, as indicated by the respective mean values for H. contortus, F1 = 136.9 ± 74.1 µg/mL and F2 = 108.1 ± 53.2 µg/mL, and for T. colubriformis, F1 = 233 ± 54.3 µg/mL and F2 = 166 ± 39.9 µg/mL. The results showed that the AH activity against H. contortus was associated with the monomeric subunits that give rise to prodelphinidins (P < 0.05) and with CT polymer size (P < 0.10). However, for T. colubriformis AH activity was correlated only with prodelphinidins (P < 0.05). These results suggest that CTs have different modes of action against different parasite species.

¹H-¹³C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans-flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis.

Studies with a diverse array of 22 purified condensed tannin (CT) samples from nine plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans-flavan-3-ol ratios can be appraised by (1)H-(13)C HSQC NMR spectroscopy. The method was developed from samples containing 44-∼100% CT, PC/PD ratios ranging from 0/100 to 99/1, and cis/trans ratios ranging from 58/42 to 95/5 as determined by thiolysis with benzyl mercaptan. Integration of cross-peak contours of H/C-6' signals from PC and of H/C-2',6' signals from PD yielded nuclei-adjusted estimates that were highly correlated with PC/PD ratios obtained by thiolysis (R(2) = 0.99). cis/trans-Flavan-3-ol ratios, obtained by integration of the respective H/C-4 cross-peak contours, were also related to determinations made by thiolysis (R(2) = 0.89). Overall, (1)H-(13)C HSQC NMR spectroscopy appears to be a viable alternative to thiolysis for estimating PC/PD and cis/trans ratios of CT if precautions are taken to avoid integration of cross-peak contours of contaminants.

Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum.

BACKGROUND: Plant-derived condensed tannins (CT) show promise as a complementary option to treat gastrointestinal helminth infections, thus reducing reliance on synthetic anthelmintic drugs. Most studies on the anthelmintic effects of CT have been conducted on parasites of ruminant livestock. Oesophagostomum dentatum is an economically important parasite of pigs, as well as serving as a useful laboratory model of helminth parasites due to the ability to culture it in vitro for long periods through several life-cycle stages. Here, we investigated the anthelmintic effects of CT on multiple life cycle stages of O. dentatum. METHODS: Extracts and purified fractions were prepared from five plants containing CT and analysed by HPLC-MS. Anthelmintic activity was assessed at five different stages of the O. dentatum life cycle; the development of eggs to infective third-stage larvae (L3), the parasitic L3 stage, the moult from L3 to fourth-stage larvae (L4), the L4 stage and the adult stage. RESULTS: Free-living larvae of O. dentatum were highly susceptible to all five plant extracts. In contrast, only two of the five extracts had activity against L3, as evidenced by migration inhibition assays, whilst three of the five extracts inhibited the moulting of L3 to L4. All five extracts reduced the motility of L4, and the motility of adult worms exposed to a CT-rich extract derived from hazelnut skins was strongly inhibited, with electron microscopy demonstrating direct damage to the worm cuticle and hypodermis. Purified CT fractions retained anthelmintic activity, and depletion of CT from extracts by pre-incubation in polyvinylpolypyrrolidone removed anthelmintic effects, strongly suggesting CT as the active molecules. CONCLUSIONS: These results suggest that CT may have promise as an alternative parasite control option for O. dentatum in pigs, particularly against adult stages. Moreover, our results demonstrate a varied susceptibility of different life-cycle stages of the same parasite to CT, which may offer an insight into the anthelmintic mechanisms of these commonly found plant compounds.

Cell-penetrating conjugates of coproporphyrins with oligoarginine peptides: rational design and application for sensing intracellular O2.

A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetra-substituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O(2). This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O(2) and ratiometric pH-sensing, respectively. The results produced and the structure-activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications.

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor.

Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.