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.

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.

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.

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.

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.

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.

Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen.

Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus), the causal agent of maize streak disease, ranks amongst the most serious biological threats to food security in subSaharan Africa. Although five distinct MSV strains have been currently described, only one of these - MSV-A - causes severe disease in maize. Due primarily to their not being an obvious threat to agriculture, very little is known about the 'grass-adapted' MSV strains, MSV-B, -C, -D and -E. Since comparing the genetic diversities, geographical distributions and natural host ranges of MSV-A with the other MSV strains could provide valuable information on the epidemiology, evolution and emergence of MSV-A, we carried out a phylogeographical analysis of MSVs found in uncultivated indigenous African grasses. Amongst the 83 new MSV genomes presented here, we report the discovery of six new MSV strains (MSV-F to -K). The non-random recombination breakpoint distributions detectable with these and other available mastrevirus sequences partially mirror those seen in begomoviruses, implying that the forces shaping these breakpoint patterns have been largely conserved since the earliest geminivirus ancestors. We present evidence that the ancestor of all MSV-A variants was the recombinant progeny of ancestral MSV-B and MSV-G/-F variants. While it remains unknown whether recombination influenced the emergence of MSV-A in maize, our discovery that MSV-A variants may both move between and become established in different regions of Africa with greater ease, and infect more grass species than other MSV strains, goes some way towards explaining why MSV-A is such a successful maize pathogen.

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.

Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant.

Maize streak virus (MSV) contributes significantly to the problem of extremely low African maize yields. Whilst a diverse range of MSV and MSV-like viruses are endemic in sub-Saharan Africa and neighbouring islands, only a single group of maize-adapted variants - MSV subtypes A(1)-A(6) - causes severe enough disease in maize to influence yields substantially. In order to assist in designing effective strategies to control MSV in maize, a large survey covering 155 locations was conducted to assess the diversity, distribution and genetic characteristics of the Ugandan MSV-A population. PCR-restriction fragment-length polymorphism analyses of 391 virus isolates identified 49 genetic variants. Sixty-two full-genome sequences were determined, 52 of which were detectably recombinant. All but two recombinants contained predominantly MSV-A(1)-like sequences. Of the ten distinct recombination events observed, seven involved inter-MSV-A subtype recombination and three involved intra-MSV-A(1) recombination. One of the intra-MSV-A(1) recombinants, designated MSV-A(1)UgIII, accounted for >60 % of all MSV infections sampled throughout Uganda. Although recombination may be an important factor in the emergence of novel geminivirus variants, it is demonstrated that its characteristics in MSV are quite different from those observed in related African cassava-infecting geminivirus species.

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.