Molecular identification and phylogenetic characterization of A-strain isolates of maize streak virus from western Ethiopia.

The A-strain of maize streak virus (MSV) causes maize streak disease (MSD), which is a major biotic threat to maize production in sub-Saharan Africa. Previous studies have described different MSV strains of economic importance from southern and eastern African countries and how eastern African regions are hubs for MSV diversification. Despite these efforts, due to a lack of extensive sampling, there is limited knowledge about the MSV-A diversity in Ethiopia. Here, field sampling of maize plants and wild grasses with visible MSD symptoms was carried out in the western Ethiopian regions of Gambela, Oromia, and Benishangul-Gumuz during the maize-growing season of 2019. The complete genomes of MSV isolates (n = 60) were cloned and sequenced by the Sanger method. We used a model-based phylogenetic approach to analyse 725 full MSV genome sequences available in the GenBank database together with newly determined genome sequences from Ethiopia to determine their subtypes and identify recombinant lineages. Of the 127 fields accessed, MSD prevalence was highest, at 96%, in the Gambela region and lowest in Oromia, at 66%. The highest mean symptom severity of 4/5 (where 5 is the highest and 1 the lowest) was observed in Gambela and Benishangul-Gumuz. Our results show that these newly determined MSV isolates belong to recombinant lineage V of the A1 subtype, with the widest dissemination and greatest economic significance in sub-Saharan Africa and the adjacent Indian Ocean islands.

How virulent are emerging maize-infecting mastreviruses?

Maize streak disease (MSD) is one of the most significant biotic constraints on the production of Africa's most important cereal crop. Until recently, the only virus known to cause severe MSD was the A-strain of maize streak virus (MSV/A), a member of the genus Mastrevirus, family Geminiviridae. However, over the past decade, two other mastreviruses, MSV/C and maize streak Réunion virus (MSRV), have been repeatedly found in the absence of MSV/A in maize plants displaying severe MSD symptoms. Here, we report on infectious clones of MSV/C and MSRV and test their ability to cause severe MSD symptoms. Although cloned MSV/C and MSRV genomes could cause systemic symptomatic infections in MSD-sensitive maize genotypes, these infections yielded substantially milder symptoms than those observed in the field. The MSV/C and MSRV isolates that we have examined are therefore unlikely to cause severe MSD on their own. Furthermore, mixed infections of MSRV and MSV/C with other mild MSV strains also consistently yielded mild MSD symptoms. It is noteworthy that MSRV produces distinctive striate symptoms in maize that are similar in pattern, albeit not in severity, to those seen in the field, showing that this virus may contribute to the severe MSD symptoms seen in the field. Therefore, despite not fulfilling Koch's postulates for MSV/C and MSRV as causal agents of severe MSD, we cannot exclude the possibility that these viruses could be contributing to currently emerging maize diseases.

Sorghum mastrevirus-associated alphasatellites: new geminialphasatellites associated with an African streak mastrevirus infecting wild Poaceae plants on Reunion Island.

Nine complete nucleotide sequences of geminialphasatellites (subfamily Geminialphasatellitinae, family Alphasatellitidae) recovered from the wild Poaceae Sorghum arundinaceum collected in Reunion are described and analyzed. While the helper geminivirus was identified as an isolate of maize streak virus (genus Mastrevirus, family Geminiviridae), the geminialphasatellite genomes were most closely related to, and shared ~63% identity with, clecrusatellites. Even though the geminialphasatellite molecules lack an adenine rich-region, they have the typical size of geminialphasatellites, encode a replication-associated protein in the virion sense, and have probable stem-loop structures at their virion-strand origins of replication. According to the proposed geminialphasatellite species and genus demarcation thresholds (88% and 70% nucleotide identity, respectively), the genomes identified here represent a new species (within a new genus) for which we propose the name "Sorghum mastrevirus-associated alphasatellite" (genus "Sorgasalphasatellite").

Identification and molecular characterization of a novel sugarcane streak mastrevirus and an isolate of the A-strain of maize streak virus from sugarcane in Nigeria.

Sugarcane and maize plants showing symptoms typical of those described for the so-called "African streak viruses" (AfSVs) were encountered during field surveys conducted from February to July 2015 to document viruses infecting both crops across the northern Guinea savannah region of Nigeria. As part of this study, two categories of complete mastrevirus-like genome sequences were obtained from nine samples (maize = 2; sugarcane = 7). In pairwise comparisons, the full-length genomes of the first sequence category (2,687 nt each; maize = 2; sugarcane = 2) shared 96 to 99% identity with global isolates of the A-strain of maize streak virus (MSV-A), indicating that sugarcane may also serve as a reservoir host to MSV-A. Analysis of the complete genomes belonging to the second sequence category (2,757 nt each; sugarcane = 5) showed that they shared 42 to 67% identity with their closest AfSV relatives, thus indicating that they represent sequences of a novel mastrevirus. Both sequence categories shared 61-62% sequence identity with each other. Further analysis revealed that the novel sugarcane-infecting virus, tentatively named as sugarcane chlorotic streak virus (SCSV), arose from a putative interspecific recombination event involving two grass-infecting mastreviruses, eragrostis streak virus and urochloa streak virus, as putative parental sequences. The results of this study add to the repertoire of diverse AfSVs present in cereal and sugarcane mixed cropping landscapes in the northern Guinea savannah region of Nigeria, with implications for disease epidemiology.

Extensive recombination-induced disruption of genetic interactions is highly deleterious but can be partially reversed by small numbers of secondary recombination events.

Although homologous recombination can potentially provide viruses with vastly more evolutionary options than are available through mutation alone, there are considerable limits on the adaptive potential of this important evolutionary process. Primary among these is the disruption of favorable coevolved genetic interactions that can occur following the transfer of foreign genetic material into a genome. Although the fitness costs of such disruptions can be severe, in some cases they can be rapidly recouped by either compensatory mutations or secondary recombination events. Here, we used a maize streak virus (MSV) experimental model to explore both the extremes of recombination-induced genetic disruption and the capacity of secondary recombination to adaptively reverse almost lethal recombination events. Starting with two naturally occurring parental viruses, we synthesized two of the most extreme conceivable MSV chimeras, each effectively carrying 182 recombination breakpoints and containing thorough reciprocal mixtures of parental polymorphisms. Although both chimeras were severely defective and apparently noninfectious, neither had individual movement-, encapsidation-, or replication-associated genome regions that were on their own "lethally recombinant." Surprisingly, mixed inoculations of the chimeras yielded symptomatic infections with viruses with secondary recombination events. These recombinants had only 2 to 6 breakpoints, had predominantly inherited the least defective of the chimeric parental genome fragments, and were obviously far more fit than their synthetic parents. It is clearly evident, therefore, that even when recombinationally disrupted virus genomes have extremely low fitness and there are no easily accessible routes to full recovery, small numbers of secondary recombination events can still yield tremendous fitness gains. Importance: Recombination between viruses can generate strains with enhanced pathological properties but also runs the risk of producing hybrid genomes with decreased fitness due to the disruption of favorable genetic interactions. Using two synthetic maize streak virus genome chimeras containing alternating genome segments derived from two natural viral strains, we examined both the fitness costs of extreme degrees of recombination (both chimeras had 182 recombination breakpoints) and the capacity of secondary recombination events to recoup these costs. After the severely defective chimeras were introduced together into a suitable host, viruses with between 1 and 3 secondary recombination events arose, which had greatly increased replication and infective capacities. This indicates that even in extreme cases where recombination-induced genetic disruptions are almost lethal, and 91 consecutive secondary recombination events would be required to reconstitute either one of the parental viruses, moderate degrees of fitness recovery can be achieved through relatively small numbers of secondary recombination events.

Mapping the distribution of maize streak virus genotypes across the forest and transition zones of Ghana.

Throughout sub-Saharan Africa, maize streak virus strain A (MSV-A), the causal agent of maize streak disease (MSD), is an important biological constraint on maize production. In November/December 2010, an MSD survey was carried out in the forest and transition zones of Ghana in order to obtain MSV-A virulence sources for the development of MSD-resistant maize genotypes with agronomic properties suitable for these regions. In 79 well-distributed maize fields, the mean MSD incidence was 18.544 % and the symptom severity score was 2.956 (1 = no symptoms and 5 = extremely severe). We detected no correlation between these two variables. Phylogenetic analysis of cloned MSV-A isolates that were fully sequenced from samples collected in 51 of these fields, together with those sampled from various other parts of Africa, indicated that all of the Ghanaian isolates occurred within a broader cluster of West African isolates, all belonging to the highly virulent MSV-A1 subtype. Besides being the first report of a systematic MSV survey in Ghana, this study is the first to characterize the full-genome sequences of Ghanaian MSV isolates. The 51 genome sequences determined here will additionally be a valuable resource for the rational selection of representative MSV-A variant panels for MSD resistance screening.

A high degree of African streak virus diversity within Nigerian maize fields includes a new mastrevirus from Axonopus compressus.

The A-strain of maize streak virus (MSV-A; genus Mastrevirus, family Geminiviridae), the causal agent of maize streak disease, places a major constraint on maize production throughout sub-Saharan Africa. In West-African countries such as Nigeria, where maize is not cultivated year-round, this MSV strain is forced to overwinter in non-maize hosts. In order to both identify uncultivated grasses that might harbour MSV-A during the winter season and further characterise the diversity of related maize-associated streak viruses, we collected maize and grass samples displaying streak symptoms in a number of Nigerian maize fields. From these we isolated and cloned 18 full mastrevirus genomes (seven from maize and 11 from various wild grass species). Although only MSV-A isolates were obtained from maize, both MSV-A and MSV-F isolates were obtained from Digitaria ciliaris. Four non-MSV African streak viruses were also sampled, including sugarcane streak Reunion virus and Urochloa streak virus (USV) from Eleusine coacana, USV from Urochloa sp., maize streak Reunion virus (MSRV) from both Setaria barbata and Rottboellia sp., and a novel highly divergent mastrevirus from Axonopus compressus, which we have tentatively named Axonopus compressus streak virus (ACSV). Besides the discovery of this new mastrevirus species and expanding the known geographical and host ranges of MSRV, we have added D. ciliaris to the list of uncultivated species within which Nigerian MSV-A isolates are possibly able to overwinter.

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.

Reconstructing the history of maize streak virus strain a dispersal to reveal diversification hot spots and its origin in southern Africa.

Maize streak virus strain A (MSV-A), the causal agent of maize streak disease, is today one of the most serious biotic threats to African food security. Determining where MSV-A originated and how it spread transcontinentally could yield valuable insights into its historical emergence as a crop pathogen. Similarly, determining where the major extant MSV-A lineages arose could identify geographical hot spots of MSV evolution. Here, we use model-based phylogeographic analyses of 353 fully sequenced MSV-A isolates to reconstruct a plausible history of MSV-A movements over the past 150 years. We show that since the probable emergence of MSV-A in southern Africa around 1863, the virus spread transcontinentally at an average rate of 32.5 km/year (95% highest probability density interval, 15.6 to 51.6 km/year). Using distinctive patterns of nucleotide variation caused by 20 unique intra-MSV-A recombination events, we tentatively classified the MSV-A isolates into 24 easily discernible lineages. Despite many of these lineages displaying distinct geographical distributions, it is apparent that almost all have emerged within the past 4 decades from either southern or east-central Africa. Collectively, our results suggest that regular analysis of MSV-A genomes within these diversification hot spots could be used to monitor the emergence of future MSV-A lineages that could affect maize cultivation in Africa.

Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution.

BACKGROUND: Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. RESULTS: Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV-MatA. Although the MSV-sensitive maize genotype gave rise to the greatest variety of recombinants, the measured fitness of each of these recombinants correlated with their similarity to MSV-MatA. CONCLUSIONS: The mechanistic predispositions of different MSV genomic regions to recombination can strongly influence the accessibility of high-fitness MSV recombinants. The frequency with which the fittest recombinant MSV genomes arise also correlates directly with the escalating selection pressures imposed by increasingly MSV-resistant maize hosts.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay.

Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicative-form-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepΔI(662)) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepΔI(662) inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepΔI(662) inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic maize against diverse MSV-A genotypes found throughout sub-Saharan Africa.

Replicative intermediates of maize streak virus found during leaf development.

Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombination-dependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR.

A quick and sensitive diagnostic tool for detection of Maize streak virus.

Maize streak virus disease (MSVD), caused by Maize streak virus (MSV; genus Mastrevirus), is one of the most severe and widespread viral diseases that adversely reduces maize yield and threatens food security in Africa. An effective control and management of MSVD requires robust and sensitive diagnostic tests capable of rapid detection of MSV. In this study, a loop-mediated isothermal amplification (LAMP) assay was designed for the specific detection of MSV. This test has shown to be highly specific and reproducible and able to detect MSV in as little as 10 fg/µl of purified genomic DNA obtained from a MSV-infected maize plant, a sensitivity 105 times higher to that obtained with polymerase chain reaction (PCR) in current general use. The high degree of sequence identity between Zambian and other African MSV isolates indicate that this LAMP assay can be used for detecting MSV in maize samples from any region in Africa. Furthermore, this assay can be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories across Africa strengthening diagnostic capacity in countries dealing with MSD.

Dating the origins of the maize-adapted strain of maize streak virus, MSV-A.

Maize streak virus (MSV), which causes maize streak disease (MSD), is one of the most serious biotic threats to African food security. Here, we use whole MSV genomes sampled over 30 years to estimate the dates of key evolutionary events in the 500 year association of MSV and maize. The substitution rates implied by our analyses agree closely with those estimated previously in controlled MSV evolution experiments, and we use them to infer the date when the maize-adapted strain, MSV-A, was generated by recombination between two grass-adapted MSV strains. Our results indicate that this recombination event occurred in the mid-1800 s, approximately 20 years before the first credible reports of MSD in South Africa and centuries after the introduction of maize to the continent in the early 1500 s. This suggests a causal link between MSV recombination and the emergence of MSV-A as a serious pathogen of maize.

A novel sugarcane-infecting mastrevirus from South Africa.

The sugarcane-infecting streak viruses (SISVs) are a diverse collection of mastreviruses (family Geminiviridae) within the African streak virus group. Four SISVs have currently been described, including the well-characterized maize streak virus. Here, we present a full annotated sequence record of an isolate of a new SISV species, Saccharum streak virus (SacSV), isolated in South Africa. The isolate shares less than 66% identity with any other mastrevirus, but is most closely related to Urochloa streak virus (USV), a mastrevirus from Nigeria that has until now been an outlier in the African streak virus phylogenetic tree. As with USV, the SacSV isolate we have characterized bears no obvious evidence of inter-species recombination.

Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias.

BACKGROUND: Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of approximately 10(-4) substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10,000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered. RESULTS: We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 x 10(-4) and 7.9 x 10-4 subs/site/year. CONCLUSION: These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.

Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions.

BACKGROUND: A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses in the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. RESULTS: Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. CONCLUSION: Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity.

Viral protein-nucleic acid interaction: South (North)-Western blot.

Maize streak virus (MSV) genome has four open reading frames. C1 and C2 encoded by the complementary sense are required for virus replication, while V1 and V2 encoded by virion sense are required for infectivity. V1 encodes movement protein (MP), while V2 encodes coat protein (CP). Deletion or mutation of MSV CP does not prevent virus replication in single cells or protoplasts but leads to a loss of infectivity in the inoculated plant suggesting that MSV CP is required for virus movement. Towards understanding the role of MSV CP and MP in virus movement, the interaction of MSV CP and MP with viral DNA was investigated using the South-western assay. Wild type and truncated MSV CPs and MP were expressed in E. coli and the expressed CPs and MP were used to investigate interaction with single-stranded (ss) and double-stranded (ds) DNA. The results showed MSV MP does not bind DNA in the assay while MSV CP bound ss and ds viral and uidA DNA in a sequence non-specific manner.

The evolutionary value of recombination is constrained by genome modularity.

Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV), we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general.

Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.