Alternate hosts of African cassava mosaic virus and East African cassava mosaic Cameroon virus in Nigeria

Cassava mosaic disease (CMD) caused by African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) is the major constraint to cassava production in Nigeria. Sequences of the DNA-A component of ACMV and EACMCV isolates from leguminous plant species (Senna occidentalis, Leucana leucocephala and Glycine max), castor oil plant (Ricinus communis), a weed host (Combretum confertum) and a wild species of cassava (Manihot glaziovii) were determined. All ACMV isolates from these hosts showed 96-98% nucleotide sequence identity with cassava isolates from West Africa. EACMCV was found only in four hosts (S. occidentalis, L. leucocephala, C. confertum, M. glaziovii), and sequences of these isolates showed 96-99% identity with cassava isolates from West Africa. These results provide definitive evidence for the natural occurrence of ACMV and EACMCV in plant species besides cassava.     

Infectivity of African cassava mosaic virus clones to cassava by biolistic inoculation

Summary.  Clones of an African cassava mosaic virus isolate originating from Nigeria (ACMV-NOg) were shown to be infectious to cassava by biolistic inoculation. The production of pseudorecombinants between ACMV-NOg and clones of an ACMV isolate originating from Kenya (ACMV-K) indicated that the lack of infectivity of ACMV-K to cassava was due to defect(s) in the DNA B genomic component; this component encodes two proteins involved in cell-to-cell movement. This is the first demonstration of infectivity of a cloned geminivirus to cassava and conclusively proves that ACMV is the causative agent of cassava mosaic disease. The potential uses of infectious ACMV clones and the means by which to introduce them into cassava are discussed. Received January 18, 1998 Accepted May 27, 1998     

Efficient replication of cloned African cassava mosaic virus in cassava leaf disks

A transient viral replication assay for cloned African cassava mosaic virus (ACMV) was developed using cassava leaf disks. TMS60444 leaf disks were transfected using biolistic-mediated inoculation with ACMV clones pKACMVA and pKACMVB, which originate from West Kenya ACMV isolate 844 (ACMV-KE). Viral DNA synthesized de novo was monitored by Southern hybridization with an AV1 DNA probe. By using the methylation-sensitive restriction enzymes DpnI and MboI, it was possible to distinguish between the input DNA (dam-methylated) and the de novo synthesized viral DNA (not methylated). Different media used for pre- and post-culture of inoculated leaf disks significantly affected the efficiency of viral DNA accumulation. Without pre-culture, replicated viral DNA was not detectable. Culture time in optimized medium also affected the accumulation of nascent viral DNA, and the best results were obtained after 4 days pre-culture on CIM medium followed by 4-6 days post-culture in SH medium. Time-course analysis showed that viral DNA replication can persist for 5-6 days post-inoculation. Our results also confirmed that DNA B of ACMV could assist the accumulation of viral DNA in the leaf disks. The novel protocol described here has also been used successfully with other cassava cultivars (MCol22, MCol1505, TME282 and TMS92/0326) and ACMV clones from the ACMV Nigeria isolate (ACMV-NOg).     

African cassava mosaic virus DI DNA interferes with the replication of both genomic components.

Natural infections of the geminivirus African cassava mosaic virus (ACMV) are known to be associated with low levels of defective interfering (DI) DNAs. Recently it has been demonstrated that extrachromosomal copies of the DI DNA, mobilized and amplified from an integrated DI DNA dimer, can ameliorate ACMV symptoms in transformed Nicotiana benthamiana, providing a possible means for the control of cassava mosaic disease. To further understand the molecular basis of the interference phenomenon, we have compared the ability of ACMV and tomato golden mosaic virus (TGMV) genomic components to replicate in leaf discs derived from DI DNA-transformed and control plants. Results indicate that the ACMV DI DNA interferes with the replication of both genomic components of ACMV to a similar extent. TGMV DNA A replicates to normal levels in transformed leaf discs and plants because it is unable to mobilize and amplify ACMV DI DNA. Differences in the relative levels of ACMV genomic components in transformed leaf discs and plants are discussed in terms of DNA replication and the availability of the genomic components for spread throughout the plant.     

Molecular interaction between two cassava geminiviruses exhibiting cross-protection

There are increasing reports of geminivirus mixed infections of field plant hosts. These mixed infections have been suggested to result in recombinations, emergence of new viruses and new disease epidemics. We previously reported the occurrence of mixed infection between African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) resulting in severe symptoms in cassava fields in Cameroon. Here, we show that reassortment of DNA-A and DNA-B components of ACMV and EACMCV does not form viable recombinants. However, in the presence of both components of either virus, the DNA-A component of the other virus replicated and spread in the absence of its DNA-B component. This result suggests that failure of ACMV and EACMCV to form viable recombinants is due to the inability of each DNA-A component to trans-replicate the heterologous DNA-B component. This study also shows that ACMV DNA-A induces a resistance to ACMV and EACMCV as indicated by absence or late symptom development. Moreover, this resistance enabled plants to recover from severe symptoms caused by EACMCV in Nicotiana benthamiana, suggesting that the resistance induced is not specific to ACMV and is consistent with the phenomenon of cross-protection between related viruses.     

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.     

Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus

Two bipartite begomoviruses, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), have been isolated from mosaic-diseased cassava originating from central India and Sri Lanka, respectively. ICMV was transmitted with low efficiency from cassava to Nicotiana benthamiana by sap inoculation to give leaf curl symptoms. SLCMV was much more virulent in this host, producing severe stunting, leaf curl, and chlorosis. These symptoms were reproduced when their cloned genomic components (DNAs A and B) were introduced into N. benthamiana by either mechanical or Agrobacterium-mediated inoculation (agroinoculation). SLCMV is more closely related to ICMV (DNA A, 84%; DNA B, 94% nucleotide identity) than African cassava mosaic virus (ACMV) (DNA A, 74%; DNA B, 47% nucleotide identity). Sequence comparisons suggest that SLCMV DNA B originated from ICMV DNA B by a recombination event involving the SLCMV DNA A intergenic region. Pseudorecombinants produced by reassortment of the cloned components of ICMV and ACMV were not infectious in N. benthamiana, emphasising their status as distinct virus species. In contrast, a pseudorecombinant between ACMV DNA A and SLCMV DNA B was infectious. Consistent with these observations, iteron motifs located within the intergenic region that may be involved in the initiation of viral DNA replication are conserved between SLCMV and ACMV but not ICMV. When introduced into N. benthamiana by agroinoculation, SLCMV DNA A alone produced a severe upward leaf roll symptom, reminiscent of the phenotype associated with some monopartite begomoviruses. Furthermore, coinoculation of SLCMV DNA A and the satellite DNA beta associated with ageratum yellow vein virus (AYVV) produced severe downward leaf curl in N. glutinosa and yellow vein symptoms in Ageratum conyzoides, resembling the phenotypes associated with AYVV DNA A and DNA beta infection in these hosts. Thus, SLCMV DNA A has biological characteristics of a monopartite begomovirus, and the virus probably evolved by acquisition of a DNA B component from ICMV.     

Dual infection by cassava begomoviruses in two leguminous species (Fabaceae) in Yangambi, Northeastern Democratic Republic of Congo

A study on cassava mosaic begomoviruses was conducted around Yangambi (DR Congo) by sampling 10 different leguminous species with or without symptoms similar to cassava mosaic disease. DNA was isolated to amplify CMBs using primers targeting AC2 and AC4 genes for virus detection by PCR. The results showed a dual infection by ACMV and EACMV in two weed species, Centrosema pubescens and Pueraria javanica, associated with mosaic symptoms. The DNA-A genome component of ACMV and EACMV from the infested weeds was sequenced. Seven ACMV and four EACMV isolates are reported. The major ACMV strains were closely related to ACMV-NGogo, ACMV-IC and ACMV-UGMld, whereas all EACMV strains were closely related to a Uganda variant, the most prevalent virus. This study shows that whiteflies may transmit CMBs to non-cassava plants under high epidemic pressure.     

Genetic variability of East African cassava mosaic Cameroon virus under field and controlled environment conditions

Cassava geminiviruses occur in all cassava growing areas of Africa and are considered to be the most damaging vector-borne plant pathogens. At least seven species of these viruses have been identified. We investigated genetic variation in East African cassava mosaic cassava Cameroon virus (EACMCV) from naturally infected cassava and from experimentally infected Nicotiana benthamiana. Results showed that the populations of EACMCV in cassava and in N. benthamiana were genetically heterogeneous. Mutation frequencies in the order of 10⁻⁴, comparable to that reported for plant RNA viruses, were observed in both hosts. We also produced an EACMCV mutant that induces reversion and second site mutations, thus suggesting that a high mutation frequency facilitates the maintenance of genome structure and function. This is direct experimental evidence showing that cassava geminiviruses exhibit a high mutation frequency and that a single clone quickly transforms into a collection of mutant sequences upon introduction into the host.     

Molecular characterisation of a distinct South African cassava infecting geminivirus

Summary.  African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are whitefly-transmitted geminiviruses (WTGs) which are widespread in cassava in Africa and cause serious yield losses. Recently, a new geminivirus affecting cassava in South Africa (SACMV) has been reported. In this work SACMV was found to have DNA-A and DNA-B components. Comparisons of amino acid sequences of the putative coat protein, and nucleotide sequences of the common region and a 687-bp DNA B fragment of SACMV with other WTGs, showed that SACMV clustered with the Old World subgroup of the Begomovirus genus of geminiviruses. Despite its bipartite nature, SACMV was most closely related to monopartite TYLCVs, but was sufficiently different to justify designating it as a distinct virus. In serological studies, SACMV grouped biologically with EACMV isolates. Received January 22, 1998 Accepted May 21, 1998     

The use of biolistic inoculation of cassava mosaic begomoviruses in screening cassava for resistance to cassava mosaic disease

Inoculation of cassava with infectious clones of cassava mosaic geminiviruses (Geminiviridae: Begomovirus) and total DNA extracts from plants infected with well-characterised viruses was evaluated using the Bio-Rad Helios Gene Gun System. Total DNA extracts from infected plants and cloned viruses were produced for coating gold particles and bombardment onto new cassava genotypes, 96/1089A, 96/1039, 96/0160, 96/0304 and three local landraces TME 117, TME 3 and TME 4. Cloned DNA of a Kenyan isolate of the recombinant variant of East African cassava mosaic virus (EACMV-UG2-[Ka]), was only infectious to TME 117 (7/10 plants), 3 weeks post-inoculation with mild infection symptoms in the newly developing leaves. Biolistic inoculation with a chimeric pseudorecombinant virus between DNA A and B components from EACMV-[Ke-Kilifi] and EACMV-UG2-[Ka], respectively, was infectious to TME 117, 96/1039 and 96/0304 and developed very severe and persistent symptoms. TME 3 and TME 4 also developed symptoms, 12 days post-inoculation (d.p.i.). Total DNA extracts of ACMV and EACMV-[Ke-Kilifi] resulted in serious infections with symptoms already evident, 10d.p.i. In general, biolistic inoculation trials with total DNA extracts resulted in a higher number of infected plants expressing symptoms at a much earlier stage (10-12d.p.i.) compared with trials inoculated with virus clones.     

Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation

Coat proteins (CPs) of geminiviruses are multifunctional proteins. Using transient expression experiments, we have recently identified putative sequence motifs of African cassava mosaic virus (ACMV) CP involved in nuclear import (NLS) and export (NES) (Virology 286 (2001) 373). Here, we report on the effect of corresponding deletion mutants in the context of infecting viruses. Since NLS and NES may overlap with DNA binding and multimerisation domains, we have investigated their effect on viral infection, particularly, on particle formation. All deletion mutants were infectious in Nicotiana benthamiana when co-inoculated with DNA B, but poorly sap-transmissible. Some of the mutants showed reduced levels of viral single-stranded DNA (ssDNA), whereas the amount of double-stranded DNA (dsDNA) was not greatly affected. None of these CP mutants was able to produce stable virus particles. In contrast, viruses with CP fused to Flag epitopes at the N- or C-terminus (CP:Flag or Flag:CP) were readily sap-transmissible and formed amorphous nucleoprotein particles but only few geminate structures. The relevance of the identified sequences in replicating viruses with reference to nuclear import and export as well as to particle stability and DNA binding is discussed.     

Simultaneous Analysis of the Bidirectional African Cassava Mosaic Virus Promoter Activity Using Two Different Luciferase Genes

The expression of geminivirus genes is controlled by bidirectional promoters which are located in the large intergenic region of the circular DNA genomes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B promoters of African cassava mosaic virus (ACMV), they were cloned between two different luciferase genes with the firefly luciferase gene in complementary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replication-associated (Rep) proteins was assessed in tobacco and cassava protoplasts using dual luciferase assays. In addition, the regulation of the DNA A promoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and tobacco protoplasts, but not in transgenic tobacco plants. In contrast to this, DNA A encoded proteins activate virion-sense expression both in protoplasts and in transgenic plants. At the same time they reduce the expression of the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicating that the plant host also influences the promoter strength. Transient transformation experiments using linearized DNA indicate that the different regulation of the ACMV DNA A promoter in protoplasts and transgenic plants could be due to different DNA curvature in free plasmids and in genes integrated in plant genomic DNA.     

Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and exhibit high variability as assessed by PCR-RFLP

Summary. The biodiversity of geminiviruses associated with the Cassava Mosaic Disease (CMD) in India was investigated using PCR to specifically amplify the DNA of Indian cassava mosaic virus (ICMV) or Sri Lankan cassava mosaic virus (SLCMV) and also by using PCR to amplify specific viral genes, followed by digestion with different restriction endonucleases to obtain polymorphic patterns (PCR-RFLP). Results showed that both ICMV and SLCMV were present in mosaic-affected cassava; ICMV was geographically restricted to certain regions, whereas SLCMV was widespread. PCR-RFLP analysis showed that, in addition to ICMV-type and SLCMV-type patterns, a high proportion (40%) of the samples displayed novel patterns, some of which were localized in certain areas, whereas others were widely distributed.     

Rapid and biologically safe diagnosis of African swine fever virus infection by using polymerase chain reaction.

In order to circumvent the need for infectious virus for the diagnosis of African swine fever (ASF), we established the polymerase chain reaction (PCR) technique for the detection of ASF virus (ASFV) DNA. A 740-bp fragment that originated from the conserved region of the viral genome was partially sequenced. From this sequence, four PCR primers and one oligonucleotide probe were designed and synthesized. A specific 640-bp PCR product was amplified by using oligonucleotides 1 and 5 as primers and extracts of the following samples as templates: organs and plasma obtained from ASFV-infected pigs, ASFV-infected cell cultures, and cloned DNA fragments containing the same conserved genomic region as that in the original 740-bp clone. No specific reaction products were observed in the corresponding controls. The identities of the PCR products were confirmed either by a second amplification with nested primers or by hybridization with a specific, biotinylated oligonucleotide probe. PCR proved to be a quicker and more sensitive method than virus isolation followed by the hemadsorption test when spleen and plasma samples from experimentally ASFV-infected pigs were tested. Furthermore, cloned virus DNA could be used as a positive control in the place of a live virus control. This is advantageous whenever the use of live virus is undesirable.