Dynamical analysis of a stochastic maize streak virus epidemic model with logarithmic Ornstein-Uhlenbeck process.

To describe the transmission dynamics of maize streak virus infection, in the paper, we first formulate a stochastic maize streak virus infection model, in which the stochastic fluctuations are depicted by a logarithmic Ornstein-Uhlenbeck process. This approach is reasonable to simulate the random impacts of main parameters both from the biological significance and the mathematical perspective. Then we investigate the detailed dynamics of the stochastic system, including the existence and uniqueness of the global solution, the existence of a stationary distribution, the exponential extinction of the infected maize and infected leafhopper vector. Especially, by solving the five-dimensional algebraic equations corresponding to the stochastic system, we obtain the specific expression of the probability density function near the quasi-endemic equilibrium of the stochastic system, which provides valuable insights into the stationary distribution. Finally, the model is discretized using the Milstein higher-order numerical method to illustrate our theoretical results numerically. Our findings provide a groundwork for better methods of preventing the spread of this type of virus.

Symptom evolution following the emergence of maize streak virus.

For pathogens infecting single host species evolutionary trade-offs have previously been demonstrated between pathogen-induced mortality rates and transmission rates. It remains unclear, however, how such trade-offs impact sub-lethal pathogen-inflicted damage, and whether these trade-offs even occur in broad host-range pathogens. Here, we examine changes over the past 110 years in symptoms induced in maize by the broad host-range pathogen, maize streak virus (MSV). Specifically, we use the quantified symptom intensities of cloned MSV isolates in differentially resistant maize genotypes to phylogenetically infer ancestral symptom intensities and check for phylogenetic signal associated with these symptom intensities. We show that whereas symptoms reflecting harm to the host have remained constant or decreased, there has been an increase in how extensively MSV colonizes the cells upon which transmission vectors feed. This demonstrates an evolutionary trade-off between amounts of pathogen-inflicted harm and how effectively viruses position themselves within plants to enable onward transmission.

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.

Virus-independent and common transcriptome responses of leafhopper vectors feeding on maize infected with semi-persistently and persistent propagatively transmitted viruses.

BACKGROUND: Insects are the most important epidemiological factors for plant virus disease spread, with >75% of viruses being dependent on insects for transmission to new hosts. The black-faced leafhopper (Graminella nigrifrons Forbes) transmits two viruses that use different strategies for transmission: Maize chlorotic dwarf virus (MCDV) which is semi-persistently transmitted and Maize fine streak virus (MFSV) which is persistently and propagatively transmitted. To date, little is known regarding the molecular and cellular mechanisms in insects that regulate the process and efficiency of transmission, or how these mechanisms differ based on virus transmission strategy. RESULTS: RNA-Seq was used to examine transcript changes in leafhoppers after feeding on MCDV-infected, MFSV-infected and healthy maize for 4 h and 7 d. After sequencing cDNA libraries constructed from whole individuals using Illumina next generation sequencing, the Rnnotator pipeline in Galaxy was used to reassemble the G. nigrifrons transcriptome. Using differential expression analyses, we identified significant changes in transcript abundance in G. nigrifrons. In particular, transcripts implicated in the innate immune response and energy production were more highly expressed in insects fed on virus-infected maize. Leafhoppers fed on MFSV-infected maize also showed an induction of transcripts involved in hemocoel and cell-membrane linked immune responses within four hours of feeding. Patterns of transcript expression were validated for a subset of transcripts by quantitative real-time reverse transcription polymerase chain reaction using RNA samples collected from insects fed on healthy or virus-infected maize for between a 4 h and seven week period. CONCLUSIONS: We expected, and found, changes in transcript expression in G. nigrifrons feeding of maize infected with a virus (MFSV) that also infects the leafhopper, including induction of immune responses in the hemocoel and at the cell membrane. The significant induction of the innate immune system in G. nigrifrons fed on a foregut-borne virus (MCDV) that does not infect leafhoppers was less expected. The changes in transcript accumulation that occur independent of the mode of pathogen transmission could be key for identifying insect factors that disrupt vector-mediated plant virus transmission.

Replication modes of Maize streak virus mutants lacking RepA or the RepA-pRBR interaction motif.

The plant-infecting mastreviruses (family Geminiviridae) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize streak virus (MSV) mutants that either fail to express RepA (RepA(-)), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA(-) viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA-pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication.

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.

Behavioral responses of the leafhopper, Cicadulina storeyi China, a major vector of maize streak virus, to volatile cues from intact and leafhopper-damaged maize.

The chemical ecology of the leafhopper, Cicadulina storeyi China (Homoptera: Cicadellidae), an important vector of Maize Streak Virus (MSV), was studied with a view to developing novel leafhopper control strategies in sub-Saharan Africa. Choice tests using a Y-tube olfactometer revealed that odors from uninfested maize seedlings (Zea mays cv. Delprim) were significantly more attractive to C. storeyi than odors from C. storeyi-infested seedlings. Headspace samples of volatile organic compounds (VOCs) collected from 10 to 12 day-old uninfested seedlings were more attractive than those collected from infested seedlings. While VOCs collected from uninfested maize seedlings were attractive, VOCs collected from C. storeyi-infested seedlings were significantly repellent. Analysis of the collected VOCs by gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) led to the identification of myrcene, linalool, (E)-2-decen-1-ol, and decanal from uninfested seedlings, and (Z)-3-hexenyl acetate, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), methyl salicylate, benzyl acetate, indole, geranyl acetate, (E)-caryophyllene, α-bergamotene, (E)-ß-farnesene, ß-sesquiphellandrene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) from infested seedlings. Of these, methyl salicylate, (E)-caryophyllene, (E)-ß-farnesene, and TMTT were identified previously as volatile semiochemicals involved in plant defense against other sucking insect pests. When tested individually for behavioral activity, all compounds were repellent for C. storeyi. Moreover, when these induced VOCs were added to the blend of VOCs from uninfested maize seedlings, a shift from attraction to repellency was observed. Addition of methyl salicylate, (E)-ß-farnesene, or TMTT resulted in a choice for the solvent control (i.e., repulsion), whereas addition of (E)-caryophyllene resulted in no reduction in host VOC attractiveness. These results show that VOCs induced in maize have the potential to be exploited in the control of viruliferous leafhoppers in 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.

Rapid host adaptation by extensive recombination.

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature.

Large accumulations of maize streak virus in the filter chamber and midgut cells of the leafhopper vector Cicadulina mbila.

Maize streak virus (MSV, Mastrevirus, Geminiviridae) is persistently transmitted by Cicadulina mbila, apparently without propagation in its leafhopper vector. MSV was shown earlier by quantitative PCR to accumulate in the alimentary canal of C. mbila. We examined the alimentary canals of C. mbila leafhoppers that acquired MSV from diseased plants for various acquisition access periods (AAP) by immunofluorescence confocal laser scanning microscopy (iCLSM) and by immunogold labelling transmission electron microscopy (iTEM). Following a 7-day AAP and a 7-day inoculation period (IP) on healthy seedlings, MSV was detected by iCLSM mainly in the filter chamber and anterior midgut. Using iTEM, large accumulations of MSV particles, usually enclosed in membranous vesicles, were detected only in cells of the midgut, inside and outside the filter chamber, following 14- or 30-day AAPs, and also following 7-day AAP and 7-day IP on healthy plants. No virus was detected in the control non-vector species C. chinaï. Coated pits or vesicles, typical of clathrin-mediated endocytosis, were not observed. We discuss an alternative endocytosis pathway and suggest that the MSV accumulations are stored in endosomes in the midgut epithelial cells.

Identification of long intergenic region sequences involved in maize streak virus replication.

The main cis-acting control regions for replication of the single-stranded DNA genome of maize streak virus (MSV) are believed to reside within an approximately 310 nt long intergenic region (LIR). However, neither the minimum LIR sequence required nor the sequence determinants of replication specificity have been determined experimentally. There are iterated sequences, or iterons, both within the conserved inverted-repeat sequences with the potential to form a stem-loop structure at the origin of virion-strand replication, and upstream of the rep gene TATA box (the rep-proximal iteron or RPI). Based on experimental analyses of similar iterons in viruses from other geminivirus genera and their proximity to known Rep-binding sites in the distantly related mastrevirus wheat dwarf virus, it has been hypothesized that the iterons may be Rep-binding and/or -recognition sequences. Here, a series of LIR deletion mutants was used to define the upper bounds of the LIR sequence required for replication. After identifying MSV strains and distinct mastreviruses with incompatible replication-specificity determinants (RSDs), LIR chimaeras were used to map the primary MSV RSD to a 67 nt sequence containing the RPI. Although the results generally support the prevailing hypothesis that MSV iterons are functional analogues of those found in other geminivirus genera, it is demonstrated that neither the inverted-repeat nor RPI sequences are absolute determinants of replication specificity. Moreover, widely divergent mastreviruses can trans-replicate one another. These results also suggest that sequences in the 67 nt region surrounding the RPI interact in a sequence-specific manner with those of the inverted repeat.

Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants.

Maize streak disease is a severe agricultural problem in Africa and the development of maize genotypes resistant to the causal agent, Maize streak virus (MSV), is a priority. A transgenic approach to engineering MSV-resistant maize was developed and tested in this study. A pathogen-derived resistance strategy was adopted by using targeted deletions and nucleotide-substitution mutants of the multifunctional MSV replication-associated protein gene (rep). Various rep gene constructs were tested for their efficacy in limiting replication of wild-type MSV by co-bombardment of maize suspension cells together with an infectious genomic clone of MSV and assaying replicative forms of DNA by quantitative PCR. Digitaria sanguinalis, an MSV-sensitive grass species used as a model monocot, was then transformed with constructs that had inhibited virus replication in the transient-expression system. Challenge experiments using leafhopper-transmitted MSV indicated significant MSV resistance--from highly resistant to immune--in regenerated transgenic D. sanguinalis lines. Whereas regenerated lines containing a mutated full-length rep gene displayed developmental and growth defects, those containing a truncated rep gene both were fertile and displayed no growth defects, making the truncated gene a suitable candidate for the development of transgenic MSV-resistant maize.

An intact RBR-binding motif is not required for infectivity of Maize streak virus in cereals, but is required for invasion of mesophyll cells.

The replication-associated protein (RepA) of Maize streak virus interacts in yeast with retinoblastoma-related protein (RBR), the negative regulator of cell-cycle progression. This may allow geminiviruses to subvert cell-cycle control to provide an environment that is suitable for viral DNA replication. To determine the importance of this interaction for MSV infection, the RBR-binding motif, LxCxE, was mutated to IxCxE or LxCxK. Whilst RBR binding in yeast could not be detected for the LxCxK mutant, the IxCxE protein retained limited binding activity. Both mutants were able to replicate in maize cultures and to infect maize plants. However, whereas the wild-type virus invaded mesophyll cells of mature leaves, the LxCxK mutant was restricted to the vasculature, which is invaded prior to leaf maturity. Mature leaves contain high levels of RBR and it is suggested that the MSV RepA-RBR interaction is essential only in tissues with high levels of active RBR.