The Spread of Tomato Yellow Leaf Curl Virus from the Middle East to the World

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV; Genus Begomovirus, Family Geminiviridae) represents a serious looming threat to tomato production in all temperate parts of the world. Whereas determining where and when TYLCV movements have occurred could help curtail its spread and prevent future movements of related viruses, determining the consequences of past TYLCV movements could reveal the ecological and economic risks associated with similar viral invasions. Towards this end we applied Bayesian phylogeographic inference and recombination analyses to available TYLCV sequences (including those of 15 new Iranian full TYLCV genomes) and reconstructed a plausible history of TYLCV's diversification and movements throughout the world. In agreement with historical accounts, our results suggest that the first TYLCVs most probably arose somewhere in the Middle East between the 1930s and 1950s (with 95% highest probability density intervals 1905–1972) and that the global spread of TYLCV only began in the 1980s after the evolution of the TYLCV-Mld and -IL strains. Despite the global distribution of TYLCV we found no convincing evidence anywhere other than the Middle East and the Western Mediterranean of epidemiologically relevant TYLCV variants arising through recombination. Although the region around Iran is both the center of present day TYLCV diversity and the site of the most intensive ongoing TYLCV evolution, the evidence indicates that the region is epidemiologically isolated, which suggests that novel TYLCV variants found there are probably not direct global threats. We instead identify the Mediterranean basin as the main launch-pad of global TYLCV movements

Fungal enzymes as animal feed additives

Thesis (MSc)--Stellenbosch University, 2001.ENGLISH ABSTRACT: The use of fungal enzymes as ruminant feed digestibility enhancers was investigated. Currently, ruminants may not digest 38 to 80 % of fibrous forages' content. A renewed interest in the potential of feed enzymes for ruminants was prompted by the high costs of livestock production, together with the availability of newer enzyme preparations. Direct application of enzyme preparations can improve in vitro dry matter (DM) and neutral detergent fibre (NDF) degradation, indicating that direct-fed fibrolytic enzymes may be effective in enhancing in vivo digestion of forages. Two commercial enzyme products, Fibrozyme and Celluclast, and fungal extracellular enzyme extracts from Aureobasidium pullulans, Trichoderma reesei, Aspergillus aculeatus, and Thermomyces lanuginosus were evaluated for enhancing in vitro feed digestibility. Fibrozyme addition to both wheat straw and lucerne hay did not improve their in vitro digestibilities, even after a two hour pre-incubation period. The four fungal enzyme extracts did not enhance wheat straw's digestibility, but marginal increases were evident for lucerne hay. Celluclast addition resulted in marginal increases in the digestibility of both oat hay and oat silage, with no enhanced effect on lucerne hay and NaOH-treated wheat straw. No relationship could be found between the level of enzyme activity and the degree of feed digestion in the in vitro assay. Enzyme hydrolysis with Celluclast, in the absence of rumen fluid, gave more conclusive results. All the feed samples tested showed a positive response to Celluclast addition, even the less digestible feeds, namely sugarcane bagasse and wheat straw. In vitro results show that the assays were unsuccessful, because almost all of the experiments conducted showed inconclusive results. Alternative feed evaluation assays, which include the in vivo, in sacco and in situ methods of analysis, as well as gas production measurement and in vitro analysis with the DAISyII system, should be evaluated. A more detailed study of feed digestibility should be motivated by determining which feeds are hydrolysable, their chemical composition, i.e. how accessible the feeds are, and also evaluation of feed mixtures. The enzyme supplements also need to be evaluated for optimum temperature and pH, as well as the compilation of enzyme cocktails.AFRIKAANSE OPSOMMING: Die gebruik van swamensieme om die verteerbaarheid van herkouervoere te verhoog, is ondersoek. Tussen 38 en 80 % van veselagtige voere se inhoud is tans onverteerbaar. 'n Hernieude belangstelling in die potensiaal van voerensieme vir herkouers word deur die hoë koste van veeproduksie, asook die beskikbaarheid van nuwe ensiempreparate gedryf Direkte byvoeging van ensiempreparate kan die in vitro droëmateriaal (DM) en neutrale onoplosbare vesel (NOV) vertering verbeter, wat daarop dui dat fibrolitiese ensieme wat direk gevoer word, effektief mag wees tydens die in vivo vertering van voer. Twee kommersiële ensiemprodukte, Fibrozyme en Celluclast, en die vier ekstrasellulêre ensieme van vier swamme, naamlik Aureobasidium pullulans, Trichoderma reesei, Aspergillus aculeatus, en Thermomyces lanuginosus is vir hul vermoë om die in vitro verteerbaarheid van voere te verbeter getoets. Byvoeging van Fibrozyme by beide koringstrooi en lusernhooi het geen verbetering in hulonderskeie in vitro verteerbaarheid tot gevolg gehad nie, selfs nie eens na 'n twee uur vooraf inkubasieperiode nie. Koringstrooi se verteerbaarheid is nie verbeter deur die byvoeging van die vier swam-ensiempreparate nie, maar 'n minimale verbetering is wel waargeneem in die verteerbaarheid van lusernhooi. Byvoeging van Celluclast het 'n minimale verbetering in beide hawerhooi en hawerkuilvoer se verteerbaarheid tot gevolg gehad, maar geen effek op lusernhooi of NaOH-behandelde koringstrooi se verteerbaarheid nie. Geen verwantskap is tussen die vlak van ensiemaktiwiteit en die mate van vertering tydens die in vitro toets gevind nie. Ensiematiese afbraak met Celluclast, in die afwesigheid van rumenvloeistof, het meer konkrete resultate gelewer. Al die voermonsters het 'n positiewe respons op die byvoeging van Celluclast getoon, selfs ook die minder verteerbare voere, nl. suikerrietbagasse en koringstrooi. In die wyer konteks was die resulate van die in vitro verteringstoetse egter onbeduidend as gevolg van groot variasie in die metings. Alternatiewe voerontledingstoetse, wat moontlik beter resultate mag lewer, sluit in in vivo, in sacco en in situ analises, asook die meting van gasproduksie en in vitro analise met die DAISyII sisteem. 'n Meer uitgebreide studie van voerverteerbaarheid wat die bepaling van die afbraak van voere, hul chemiese samestelling, met ander woorde toeganklikheid van voere, en die ondersoek van voermengsels behels, behoort aandag te geniet. Die ensiemmengsels behoort ook ten opsigte van samestelling, optimum temperatuur en pH ondersoek teword

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product

Adaptive evolution by recombination is not associated with increased mutation rates in Maize streak virus

BACKGROUND: Single-stranded (ss) DNA viruses in the family Geminiviridae are proving to be very useful in real-time evolution studies. The high mutation rate of geminiviruses and other ssDNA viruses is somewhat mysterious in that their DNA genomes are replicated in host nuclei by high fidelity host polymerases. Although strand specific mutation biases observed in virus species from the geminivirus genus Mastrevirus indicate that the high mutation rates in viruses in this genus may be due to mutational processes that operate specifically on ssDNA, it is currently unknown whether viruses from other genera display similar strand specific mutation biases. Also, geminivirus genomes frequently recombine with one another and an alternative cause of their high mutation rates could be that the recombination process is either directly mutagenic or produces a selective environment in which the survival of mutants is favoured. To investigate whether there is an association between recombination and increased basal mutation rates or increased degrees of selection favoring the survival of mutations, we compared the mutation dynamics of the MSV-MatA and MSV-VW field isolates of Maize streak virus (MSV; Mastrevirus), with both a laboratory constructed MSV recombinant, and MSV recombinants closely resembling MSV-MatA. To determine whether strand specific mutation biases are a general characteristic of geminivirus evolution we compared mutation spectra arising during these MSV experiments with those arising during similar experiments involving the geminivirus Tomato yellow leaf curl virus (Begomovirus genus). RESULTS: Although both the genomic distribution of mutations and the occurrence of various convergent mutations at specific genomic sites indicated that either mutation hotspots or selection for adaptive mutations might elevate observed mutation rates in MSV, we found no association between recombination and mutation rates. Importantly, when comparing the mutation spectra of MSV and TYLCV we observed similar strand specific mutation biases arising predominantly from imbalances in the complementary mutations G->T: C->A. CONCLUSIONS: While our results suggest that recombination does not strongly influence mutation rates in MSV, they indicate that high geminivirus mutation rates are at least partially attributable to increased susceptibility of all geminivirus genomes to oxidative damage while in a single stranded state

Identification and control for the effects of bioinformatic globin depletion on human RNA-seq differential expression analysis

Abstract When profiling blood samples by RNA-sequencing (RNA-seq), RNA from haemoglobin (Hgb) can account for up to 70% of the transcriptome. Due to considerations of sequencing depth and power to detect biological variation, Hgb RNA is typically depleted prior to sequencing by hybridisation-based methods; an alternative approach is to deplete reads arising from Hgb RNA bioinformatically. In the present study, we compared the impact of these two approaches on the outcome of differential gene expression analysis performed using RNA-seq data from 58 human tuberculosis (TB) patient or contact whole blood samples–29 globin kit-depleted and 29 matched non-depleted—a subset of which were taken at TB diagnosis and at six months post-TB treatment from the same patient. Bioinformatic depletion of Hgb genes from the non-depleted samples (bioinformatic-depleted) substantially reduced library sizes (median = 57.24%) and fewer long non-coding, micro, small nuclear and small nucleolar RNAs were captured in these libraries. Profiling published TB gene signatures across all samples revealed inferior correlation between kit-depleted and bioinformatic-depleted pairs when the proportion of reads mapping to Hgb genes was higher in the non-depleted sample, particularly at the TB diagnosis time point. A set of putative “globin-fingerprint” genes were identified by directly comparing kit-depleted and bioinformatic-depleted samples at each timepoint. Two TB treatment response signatures were also shown to have decreased differential performance when comparing samples at TB diagnosis to six months post-TB treatment when profiled on the bioinformatic-depleted samples compared with their kit-depleted counterparts. These results demonstrate that failure to deplete Hgb RNA prior to sequencing has a negative impact on the sensitivity to detect disease-relevant gene expression changes even when bioinformatic removal is performed

Primer sequences.

1<p>Underlined letters highlight engineered restriction enzyme (RE) sites (names of the introduced RE sites are incorporated in the primer names).</p><p>Primer sequences.</p

Vertical box-and-whisker plots summarising real-time PCR data on all constructs bombarded at a 5∶1 weight ratio with infectious clones of diverse MSV strains and another mastrevirus species.

<p>A) MSV-Kom. The plots were constructed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone-0105932-g006" target="_blank">Figure 6</a>. The number of replicates (i.e. the number of bombarded samples) were as follows: pSK, 34; pSPLITGusUbi, 18; pSPLIT<i>rep</i>^1-219Rb-UbiΔI, 18; pSPLIT<i>rep</i>^1-219Rb-Ubi, 14; pSPLIT<i>rep</i>^III-Rb-Ubi, 8; p<i>rep</i>^1-219Rb-, 9; pMSV-<i>Pst</i>I, 11. Plots in B-C) were constructed as described for A), but this time either pSPLIT<i>rep</i>^III-Rb-Ubi or pSK were co-bombarded with infectious clones of: B) the MSV-B strain isolate VW; and C) the PanSV strain A isolate Kar. The number of replicates for B) were: pSK, 11; pSPLIT<i>rep</i>^III-Rb-Ubi, 14. The number of replicates for C) were: pSK, 16; pSPLIT<i>rep</i>^III-Rb-Ubi, 25. All real-time PCRs were performed on total DNA extracted from BMS cells four days post-bombardment.</p

Schematic diagram of synthesised constructs, with restriction enzyme sites incorporated for subsequent cloning.

<p>A) pSPLIT<i>rep</i>^1-219Rb-35S containing “modules” that could be removed and replaced with other sequences by restriction digest. B) Illustration showing how the <i>rep</i>^1-219Rb- transgene was split at the first AGGC (nucleotides 155, 156, 157 and 158 with respect to the start codon). The exon 2, cloned at the 5′ terminus of the split gene cassette in A) therefore began with GC, and the exon 1, cloned at the 3′ terminus, ended in AG. C) The synthesised <i>rep</i>^III-Rb- (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone-0105932-g001" target="_blank">Fig. 1A</a> for the full-length gene product) exon 2, preceded by the 3′-terminal half of the syntron, flanked by <i>Swa</i>I and <i>Spe</i>I RE sites. The 3′-terminal syntron/<i>rep</i>^1-219Rb- exon 2 in pSPLIT<i>rep</i>^1-219Rb-35S was replaced by the 3′-terminal syntron/<i>rep</i>^III-Rb- exon 2 to create pSPLIT<i>rep</i>^III-Rb-35S. Exon 1 remained the same for both constructs since they share the same 5′-terminal 156 bp. Similarly, other modules were exchanged to create further constructs, such as the CaMV 35S promoter for the maize ubiquitin promoter etc (see text for details).</p

Schematic representation of the INPACT system.

<p>MSV-inducible expression from a “split gene cassette” using pSPLIT<i>rep</i>^III-Rb-Ubi is used as an example. NosT = nopaline synthase terminator; UbiP = maize ubiquitin promoter. MSVLIR²⁴¹– =  truncated MSV long intergenic region.</p