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Keywords:

  • cassava;
  • Cassava brown streak virus;
  • Ipomovirus;
  • Potyviridae

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A partial sequence of 1114 nucleotides of a virus from cassava brown streak diseased (CBSD) material was obtained. Alignment of the predicted amino acid sequence with those of other members of the Potyviridae showed closest identity with the coat protein of Sweet potato mild mottle virus (genus Ipomovirus). The predicted amino acid sequence has one open reading frame with a 3′ untranslated region of 144 nucleotides and a poly(A) tail. The expressed protein was shown to cross-react with an antiserum raised previously to a virus isolated from CBSD material. Evidence presented suggests that CBSD is caused by Cassava brown streak virus, a tentative member of the genus Ipomovirus, as this virus is consistently found associated with CBSD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cassava (Mannihot esculenta) is a major food resource in sub-Saharan Africa. Historically grown in all parts, production in eastern and southern regions has increased in recent years. Cassava is a drought-hardy crop and can survive and produce a yield where cereal crops fail.

Cassava brown streak disease (CBSD) was first reported in Tanzania in 1936 (Storey, 1936). The name brown streak was given to the disease because brown lesions sometimes appear on the young green stems. Many of the roots also exhibit necrosis and, once harvested, the infected roots deteriorate rapidly in storage and cannot be consumed (Hillocks et al., 1996). In the absence of any visible parasite and the ability of the disease to be transmitted by grafting, the agent of infection was assumed to be viral. Cassava is propagated through cuttings and as such is particularly prone to virus disease problems because infection tends to build up in selected clones. Natural spread has been reported to be at a very low rate and it has been proposed that the vector is a whitefly (Storey, 1939; Bock, 1994). A recent report proposed that Bemisia afer, and not the previously suggested B. tabaci, was the vector responsible (Legg & Raya, 1998), although this has not been confirmed. Diagnosis of CBSD is difficult because immature leaves of infected cassava are symptomless, and symptoms of the disease vary greatly with the cultivar of cassava (Hillocks et al., 1996) and environmental conditions (Nichols, 1950; Lister, 1959). Symptoms of CBSD have been observed to be more severe during the cool ‘winter’ months, especially in upland sites, with recovery occurring during the hot season (Storey, 1936). During the dry season cassava plants lose their leaves and new growth often shows no signs of the disease. CBSD has been reported from Kenya, Tanzania, Uganda, Malawi and Mozambique (Nichols, 1950; Bock, 1994; Thresh et al., 1994), although with the difficulty in diagnosing the disease it may be more widespread.

There has been some uncertainty over the virus responsible for CBSD. In previous reports it has been suggested to be a potyvirus, a carlavirus or a virus complex of both (Brunt, 1996). The confusion has arisen from conflicting results of purified virus particles of carlavirus length (650–690 nm) from infected material and the observation of pinwheel inclusion bodies in sections of infected material (Lennon et al., 1985). At the time of these observations, pinwheel structures had only been found associated with potyviruses, thus suggesting the presence of a second virus. Attempts to separate the two suspect viruses using herbaceous host plants were not successful. The only reported serologically related virus is the Cowpea mild mottle virus, a whitefly-transmitted carlavirus (Lennon et al., 1985). An antiserum was raised to the carlavirus-length virus from cassava and this antiserum detected the virus readily in Nicotiana benthamiana but only erratically in cassava (Lennon et al., 1985).

In the work described in this paper, a virus associated with CBSD was purified, cloned and sequenced and found to be related to Sweet potato mild mottle virus (SPMMV; genus Ipomovirus, family Potyviridae) but distinct from any virus previously reported. It is proposed that this virus alone be called Cassava brown streak virus (CBSV) as it has attributes necessary to account for previous observations of this disease.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Host material

Cassava sticks from Tanzania were grown under quarantine glasshouse conditions. Leaf material showing typical symptoms of cassava brown streak disease was harvested, and macerated in water with carborundum powder. The extract was rubbed onto N. benthamiana leaves and these inoculated plants maintained at 24°C.

Virus isolation protocol

Leaves showing symptoms were harvested from 30 to 40 plants, 8–10 days after inoculation (approximately 40 g of material). The tissue was homogenized in 400 mL of 0·5 m borate buffer (pH 7·8), containing EDTA (0·005 m) and thioglycolic acid (0·1% v/v). The buffer was clarified with an equal volume of chloroform and the phases separated by centrifugation (4000 g) at 4°C. The top aqueous phase was removed and 6% polyethylene glycol 8000 was added to this and stirred at 4°C overnight. The resulting precipitate was collected by centrifugation (4000 g) and resuspended in 0·5 m borate buffer containing triton X−100 (0·5% v/v), approximately 4–8 mL. The virus preparation was first centrifuged at low speed (4000 g) and the supernatant then transferred to a high-speed centrifuge where the virus was sedimented at 30 000 g for 90 min. The resulting pellet was resuspended thoroughly in 2–4 mL of 0·05 m borate buffer containing 0·5% triton X−100. Further low- and high-speed centrifugations were performed and the virus pellet resuspended in 0·5 mL of 0·05 m borate buffer containing 1% triton X−100.

RNA extraction

RNA was extracted from virus particles in a microfuge tube, with an equal volume of cracking solution (40 mm Tris-HCl, pH 9·0, 2 mm EDTA, 2% SDS). This was vortexed and RNAase inhibitor (Promega, Southampton, UK) was added, followed by incubation at 60°C for 5 min. An equal volume of phenol was added, and the preparation was vortexed and incubated for a further 1 min at 60°C. The phases were separated in a microfuge (maximum speed for 5 min). The aqueous phase was extracted with an equal volume of phenol/chloroform (1 : 1, pH 5·2) and the RNA was precipitated with ethanol. The RNA was resuspended in 50 µL of DEPC-treated water.

Total RNA was isolated from the leaves of cassava and N. benthamiana using the RNeasy plant RNA isolation kit (Qiagen, Crawley, UK).

cDNA synthesis

Oligo-(dT)-primed single-stranded cDNA was synthesized from virus and plant RNA with the Expand reverse-transcription kit (Boehringer Mannheim, Lewes, UK). Second-strand cDNA was made using the riboclone cDNA synthesis kit (Stratagene Ltd, Cambridge, UK) and cloned into Sma I digested pUC18 (Amersham Pharmacia Biotech, Little Chalfont, UK).

Polymerase chain reaction (PCR)

Amplification from cDNA was performed in a MJ Research mini-cycler. The 20-µL reaction mix contained 80 ng of each primer, 1·8 µL of 11 × buffer (500 mm Tris-Cl pH 8·8, 125·0 mm (NH4)2SO4, 50 mm MgCl2, 75 mm 2-mercaptoethanol, 50 µm EDTA pH 8·0, 11 mm each dNTP, 1·26 mg mL−1 BSA), 1 µL cDNA and 0·5 U Taq polymerase (Promega). The PCR programme was 94°C for 1 min, 50°C for 1 min and 72°C for 1 min 30, repeated for 30 cycles.

A control primer set for the RT-PCR of cassava, rbc1 and rbc2, was designed to the ribulose bisphosphate carboxylase gene (Mak & Ho, 1995; rbc1: 5′-CTA-CTA-TGG-TGG-CTC-CGT-TC-3′ and rbc2: 5′-CCG-TTC-AGT-CGG-AGA-AAC-TC-3′). This primer pair generates a 619-bp product with cassava cDNA.

Sequence analysis

Sequencing was performed using an automatic ABI sequencer. The nucleotide sequence of the virus was obtained from three independent cDNA clones generated from RNA from a virus purification. The sequence of the 5′ end of the longest clone was confirmed with RT-PCR. Products were generated from RNA of infected material with specific primers made to the virus sequence. Computer analysis was carried out using GeneJockey II (BioSoft, Cambridge, UK) and available World Wide Web software (Clustal W, from http://www.clustalw.genome.ad.jp, and the boxshade server http://www.ch.embnet.org/software/BOX_form.html). The phylogenetic tree was generated with the phylip package.

Protein expression, extraction and Western analysis

Total protein was extracted from 0·3 g of plant tissue by grinding the tissue in liquid nitrogen, to which an equal volume of 2 × SDS loading buffer (100 mm Tris-Cl, pH 6·8, 200 mm dithiothreitol, 4% SDS, 0·2% bromophenol blue, 20% glycerol) was added. Protein samples were denatured by heating at 100°C for 5 min. Debris was pelleted by microfuge (maximum speed for 5 min) and the supernatant containing the proteins collected. Bacterial proteins were extracted similarly but 1·5 mL of bacterial culture was first pelleted by microfuge (maximum speed for 2 min) and 100 µL of the loading buffer added.

Proteins were separated on a 10% acrylamide SDS-polyacrylamide gel and stained with coomassie brilliant blue according to Sambrook et al. (1989). Proteins were blotted onto Immobilon-P transfer membrane (Millipore Corporation, Bedford, MA., USA), using a semidry transfer cell (Bio-Rad Laboratories, Hemel Hempstead, UK) according to the manufacturer's instructions. Westerns were performed using alkaline phosphatase-conjugated antibodies and visualized using the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium.

Expression of the virus coat protein was achieved through induction of the LacZ promoter with isopropylthio-β-d-galactoside (IPTG) (Foster, 1998).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

RT-PCR analysis

The possibility that a carlavirus or a potyvirus or both were involved in CBSD was first investigated using universal PCR primers in RT-PCR analysis. Carla-uni for carlaviruses (Badge et al., 1996) and CN48 for potyviruses (Pappu et al., 1993) were tried with the primer oligo-(dT); the primer NGDD (Badge et al., 1997a), designed to bymo- and macluraviruses, was also tried. However, none of these primers gave a product from CBSD-infected cassava or N. benthamiana material (results not presented). All appropriate positive and negative controls for the cDNA and primers were included.

Sequence analysis of the 3′ end of CBSV

The lack of success using the RT-PCR approach suggested that this virus was unusual and a more traditional method of identification through virus purification was used. Virus was partially purified and RNA extracted. Double-stranded cDNA was generated using an oligo(dT) primer and ligated into SmaI-digested pUC18. Three recombinant plasmids that contained an insert in excess of 500 bp were sequenced: pdT1, pdT3 and pdT4 with inserts of 1114 bp, 911 bp and 592 bp, respectively. The longest clone, pdT1, differed from the other two by one substitution in the untranslated region. At the 5′ end of the pdT1 sequence a region of approximately 200 bp was available only with the pdT1 clone; to verify this sequence a PCR product was generated. A PCR primer was designed to the sequence at the 5′ end of pdT1 (CBSV9: 5′-ATG-CTG-GGG-TAC-AGA-CAA-G-3′). This primer was used with the primer CBSV8 (5′-GGC-ACA-TAC-GCT-AGA-AAG-3′), on cDNA generated from CBSV-infected cassava material, to generate a 300-bp RT-PCR product. This product was cloned and sequenced, and confirmed the sequence of the pdT1 clone. Computer analysis revealed one open reading frame open at the 5′ end, followed by an untranslated region (UTR) of 144 nucleotides and a poly(A) tail (Fig. 1, GenBank AY007597). Submission of the predicted amino acid sequence to the GenBank database showed sequence identity to the coat protein of known members of the Potyviridae family; closest identity was with the only sequenced member of the genus Ipomovirus, Sweet potato mild mottle virus (SPMMV).

image

Figure 1. Sequence of 1114 nucleotides at the 3′ end of CBSV cDNA. The predicted amino acid sequence is shown (GenBank accession No. AY007597).

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Sequence comparison between CBSV and other Potyviridae

The deduced CBSV amino acid sequence was aligned with the corresponding sequences of different genera of the Potyviridae family. The viruses chosen were those members of the genera that showed closest identity to CBSV on a search of the GenBank database, namely two bymoviruses, Barley mild mosaic virus (BaMMV) (Kashiwazaki et al., 1992) and Barley yellow mosaic virus (BaYMV) (Kashiwazaki et al., 1989), two macluraviruses, Maclura mosaic virus (MacMV) (Badge et al., 1997b) and Narcissus latent virus (NLV) (Badge et al., 1997b), two tritimoviruses, Wheat streak mosaic virus (WSMV) (Niblett et al., 1991) and Brome streak virus (BStV) (Gotz & Maiss, 1995), one potyvirus, Potato virus Y (PVY) (Robaglia et al., 1989) and one ipomovirus, SPMMV (Colinet et al., 1996). The conserved cores of the coat proteins equivalent to D2829 to R3044 in PVY (GenBank D00441), I80-R307 in CBSV, are shown in Fig. 2. This core region between pairs of viruses within the Potyviridae has been shown to accurately reflect the genome identity (Ward & Shukla, 1991). Pairwise percentage sequence identities between the coat protein cores of CBSV and selected members of the Potyviridae (equivalent to D2829 to R3044 in PVY (D00441)) are shown in Table 1. CBSV shows a 43·2% sequence identity to SPMMV, the only sequenced member of the genus Ipomovirus. CBSV also shows quite high sequence identity to the genus Tritimovirus, 27·3% with both BStV and WSMV. The phylogenetic tree generated from the alignment of the coat protein core of CBSV and selected members of the Potyviridae is shown in Fig. 3. The two macluraviruses, bymoviruses and tritimoviruses form pairs within the tree, as do SPMMV and CBSV.

image

Figure 2. Multiple alignment of the C-terminal core amino acids (equivalent to D2829–R3044 in PVY) of CBSV (GenBank AY007597) with corresponding protein sequences of BaMMV (GenBank D10949), BaYMV (GenBank D00544), MacMV (GenBank U58771), NLV (GenBank U58770), PVY (GenBank D00441), BStV (GenBank Z48506), WSMV (GenBank AF057533) and SPMMV (GenBank Z48058). Residues conserved in all sequences are in bold type.

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Table 1.  Pairwise percentage amino acid sequence identities between the coat protein cores (equivalent to D2829– R3044 in PVY (D00441)) of CBSV and other selected members of the Potyviridae
 BaYMVMacMVNLVPVYBStVWSMVRGMVSPMMVCBSV
  1. BaMMV, Barley mild mosaic virus; BaYMV, Barley yellow mosaic virus; MacMV, Maclura mosaic virus; NLV, Narcissus latent virus; PVY, Potato virus Y; BStV, Brome streak virus; WSMV, Wheat streak mosaic virus; RGMV, Ryegrass mosaic virus; SPMMV, Sweet potato mild mottle virus.

BaMMV31·422·424·913·811·811·912·211·413·4
BaYMV 22·323·113·312·211·811·810·012·7
MacMV  53·219·311·410·918·515·013·7
NLV   21·112·214·222·314·510·5
PVY    17·016·153·718·814·7
BStV     52·714·529·927·3
WSMV      15·429·527·3
RGMV       17·313·0
SPMMV        43·2
image

Figure 3. Phylogenetic tree generated from the alignment of the C-terminal core amino acid residues of the coat protein of CBSV and other members of the Potyviridae family (equivalent to D2829–R3044 in PVY (D00441)): BaMMV (GenBank D10949), BaYMV (GenBank D00544), MacMV (GenBank U58771), NLV (GenBank U58770), PVY (GenBank D00441), WSMV (GenBank AF057533), BStV (GenBank Z48506), RGMV (GenBank Y09857) and SPMMV (GenBank Z48058). The tree was produced using Clustal X output, re-sampled 100-fold with SEQBOOT. The bootstrapped data was used as input for PROTPARS part of the phylip package. The numbers at each branch indicate the percentage of bootstrap analysis that supports the grouping at that node.

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Detection of CBSV within infected tissue

Infected and uninfected N. benthamiana plant sap was spotted onto a nitro-cellulose membrane and probed with radioactively labelled CBSV pdT1. CBSV pdT1 hybridized only to the infected sap (results not shown). A duplicate dot blot with the CBSV-infected cassava from Tanzania failed to detect the virus.

The PCR primers CBSV6 (5′-GTA-TAC-AAG-CAT-TGA-AAA-TAA-G-3′) and CBSV7 (5′-TCC-GGA-ATA-TAT-CTT-GGC-3′) generated from the sequence of CBSV (Fig. 1) produced a RT-PCR product from the infected cassava material from Tanzania, but not from uninfected cassava (result not shown). When sequenced, this was found to be identical to the corresponding region of CBSV (Fig. 1). Primer sets designed to the sequence have since been used successfully in RT-PCR tests on numerous samples from CBSD-affected cassava from Tanzania.

Western blots were performed with infected N. benthamiana and cassava material with the universal potyvirus antiserum made to the conserved core of the coat protein of the virus (Agdia Inc., Elkhart, IN, USA); no cross-reaction was observed (results not shown). A polyclonal antiserum previously raised to carlavirus-length virus particles isolated from CBSD material was obtained from SCRI (Invergowrie, Dundee, Scotland). This antiserum produced a strong reaction with a protein of approximately 45 kDa in the CBSV-infected N. benthamiana and to a much lesser degree with the CBSV-infected cassava. Antiserum raised to purified virus particles of Cowpea mild mottle virus (supplied by A. Brunt, HRI Wellesbourne, UK) was also found to cross-react with the 45 kDa protein in the infected N. benthamiana (results not shown).

Protein was extracted from partially purified CBSV and separated by SDS-PAGE. Coomassie blue staining revealed one strong protein band of approximately 45 kDa. This protein was shown to cross-react with the SCRI CBSV antiserum (Fig. 4).

image

Figure 4. Western blot of the coat protein from partially purified CBSV with the SCRI antiserum previously raised to a carlavirus-length virus from cassava.

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E. coli expression of CBSV coat protein

The 1114-nucleotide clone of CBSV (pdT1) had been cloned into the multiple cloning site of pUC18. The orientation and reading frame of the insert was such that expression of the protein was possible. Clone pdT1 was induced to give a product with IPTG and the total protein was extracted and separated by SDS-PAGE. Total protein was also extracted from noninduced pdT1 and E. coli containing unmodified pUC18, induced with IPTG and noninduced. The result of a Western blot with these proteins and the SCRI antiserum raised to CBSV is shown in Fig. 5. The CBSV antiserum reacted with a protein of approximately 40 kDa in both the induced (lane 1) and noninduced (lane 2) pdT1, but no reaction was seen with the control bacterial proteins (lanes 3 and 4). The cross-reaction was stronger with the induced bacteria.

image

Figure 5. Western blot of the E. coli-expressed coat protein of CBSV encoded by the clone pdT1 with antiserum previously raised to a carlavirus-length virus from cassava. Lane 1, pdT1 induced to express protein; lane 2, pdT1 not induced; lane 3, pUC 18 induced to express protein; lane 4, pUC 18 not induced.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The suggestion by Lennon et al. (1985) that CBSD was caused by a potyvirus together with a carlavirus has been carried through to some reviews about the disease. However, there is no strong identity of the amino acid sequence of the CBSV coat protein core with the corresponding sequences of members of the Potyvirus genus. Moreover, all attempts to locate a potyvirus through PCR with the universal potyvirus primer (Pappu et al., 1993) failed. This degenerate primer is based on the conserved motif WCIEN found within sequenced potyviruses. As the corresponding amino acid motif within CBSV sequenced in this paper is NCIVN, it is not surprising that this universal potyvirus primer was not successful at amplifying cDNA from CBSV. The other conserved motif found with Potyvirus members reported by Pappu et al. (1993) is QMKAAA, which for the CBSV is QILAAN. An antiserum raised to the conserved core of the potyvirus coat protein was also unsuccessful in producing a reaction with CBSD material. This evidence would suggest that there is no potyvirus associated with cassava brown streak disease.

The suggestion that a potyvirus was involved was based mainly on the observation of pinwheel inclusion bodies in sections of infected material (Lennon et al., 1985). However, the ipomovirus SPMMV was shown later to produce cytoplasmic inclusion bodies in infected material (Colinet et al., 1996). As the greatest sequence identity of the CBSV core coat protein sequence was found with SPMMV (43·2%), it is proposed that CBSV falls best within the genus Ipomovirus of the Potyviridae. A further similarity between SPMMV and CBSV is that they both have larger than ‘normal’ coat proteins of 37·7 kDa (Colinet et al., 1996) and approximately 45 kDa, respectively.

The suggestion that a carlavirus was involved with CBSD came from carlavirus-like virus particles isolated from infected material (Lennon et al., 1985). Attempts to PCR a carlavirus from infected material using the conserved universal carlavirus primer Carla-uni (Badge et al., 1996) were not successful. However, antiserum raised to the carlavirus-length virus particles (SCRI, Scotland) did cross-react with the expressed coat-protein of the virus in the present tests. It is therefore suggested that the ipomovirus in this paper is the carlavirus-length virus isolated by SCRI and that this virus alone causes CBSD.

Although direct evidence is lacking, it has been suggested that CBSV is transmitted naturally by the whitefly B. afer (Legg & Raya, 1998). The fact that CBSV has close identity to SPMMV, a whitefly transmitted virus, would support this idea. Although whitefly is quite clearly the strongest candidate for the natural virus vector, the relatively high sequence identity with the mite-transmitted tritimoviruses, Wheat streak mosaic virus and Brome streak virus, may suggest a possible role for mites in the transmission of CBSV.

The virus is at a lower titre in cassava than artificially inoculated N. benthamiana, as shown by the virus being detectable only in radioactively probed dot blots of infected N. benthamiana, and not in infected cassava. Antiserum previously raised to the virus was found to be unreliable with cassava (Lennon et al., 1985); almost certainly the result of low virus levels in the plant.

Western analysis with an antiserum raised to Cowpea mild mottle virus and CBSD material confirmed a serological relationship between these viruses, which was also reported previously (Brunt, 1996). There are some characteristics that these viruses are believed to share, i.e. the length of virus particle, the ability to produce inclusion bodies and the whitefly as the agent of transmission. The sequence of Cowpea mild mottle virus has now been published (Badge et al., 1996; Naidu et al., 1998); it contains all the characteristic genome organization found with carlaviruses. Sequence analysis between CBSV and that published for Cowpea mild mottle virus failed to show any motifs in common that could account for the serological relationship, which remains a mystery.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to thank Dr Rory Hillocks and Professor Mike Thresh for comments on this manuscript; together with Mrs Kay Mtunda and Dr Frances Kimmins for providing CBSV-infected cassava sticks and Mr Tim Colborn for photographic work. This publication is a result of a research project funded by the Department for International Development of the United Kingdom (DFID project number R6617, Crop Protection Programme). However, the Department for International Development cannot accept responsibility for any information provided or views expressed.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Badge J, Brunt A, Carlson R, Dagless E, Karamagioli M, Phillips S, Seal S, Turner R, Foster GD, 1996. A carlavirus-specific PCR primer and partial nucleotide sequence provides further evidence for the recognition of cowpea mild mottle virus as a whitefly-transmitted carlavirus. European Journal of Plant Pathology 102, 30510.
  • Badge JL, Kashiwazaki S, Lock S, Foster GD, 1997a. A bymovirus PCR primer and partial nucleotide sequence provides further evidence for the recognition of rice necrosis mosaic virus as a bymovirus. European Journal of Plant Pathology 103, 7214.
  • Badge JL, Robinson DJ, Brunt AA, Foster GD, 1997b. 3′- End terminal sequences of the RNA genomes of narcissus latent and Maclura mosaic viruses suggest that they represent a new genus of the Potyviridae. Journal of General Virology 78, 2537.
  • Bock KR, 1994. Studies on cassava brown streak virus disease in Kenya. Tropical Sciences 34, 13445.
  • Brunt AA, 1996. Cassava brown streak-associated(?) carlavirus, cassava brown streak potyvirus. In: CrabtreeK, DallwitzMJ, GibbsAJ, WatsonL, ZurcherEJ, eds. Plant Viruses Online: Descriptions and Lists from the VIDE Database. [http://biology.anu.edu.au/Groups/MES/vide/;.
  • Colinet D, Kummert J, Lepoivre P, 1996. Molecular evidence that the whitefly-transmitted sweet potato mild mottle virus belongs to a distinct genus of the Potyviridae. Archives of Virology 141, 12535.
  • Foster GD, 1998. Expression library screening. In: FosterGD, TaylorSC, eds. Plant Virology Protocols from Virus Isolation to Transgenic Resistance Methods in Molecular Biology, vol. 81. Totowa, N J, USA: Humana Press, 290.
  • Gotz R & Maiss E, 1995. The complete nucleotide sequence and genome organization of the mite-transmitted brome streak mosaic rymovirus in comparison with those of potyviruses. Journal of General Virology 76, 203542.
  • Hillocks RJ, Raya M, Thresh JM, 1996. The association between root necrosis and above-ground symptoms of brown streak virus infection of cassava in southern Tanzania. International Journal of Pest Management 42, 2859.
  • Kashiwazaki S, Hayano Y, Minobe Y, Omura T, Hibino H, Tsuchizaki T, 1989. Nucleotide sequence of the capsid protein gene of barley yellow mosaic virus. Journal of General Virology 70, 301523.
  • Kashiwazaki S, Nomura K, Kuroda H, Ito K, Hibino H, 1992. Sequence analysis of the 3′-terminal halves of RNA 1 of two strains of barley mild mosaic virus. Journal of General Virology 73, 217381.
  • Legg JP & Raya MP, 1998. Survey of cassava virus diseases in Tanzania. International Journal of Pest Management 44, 1723.DOI: 10.1080/096708798228473
  • Lennon AM, Aiton MM, Harrison BD, 1985. Cassava Viruses from Africa. Report of the Scottish Crop Research Institute, 168. Dundee, Scotland: SCRI.
  • Lister RM, 1959. Mechanical transmission of cassava brown streak virus. Nature 183, 15889.
  • Mak YM & Ho KK, 1995. Sequence of cassava ribulose-1,5-bisphosphate carboxylase small subunit precursor cDNA. DNA Sequence 5, 22932.
  • Naidu RA, Gowda S, Satyanarayana T, Boyko V, Reddy AS, Dawson WO, Reddy DVR, 1998. Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus. Archives of Virology 143, 76980.DOI: 10.1007/s007050050328
  • Niblett CL, Zagula KR, Calvert LA, Kendall TL, Stark DM, Smith CE, Beachy RN, Lommel SA, 1991. cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene. Journal of General Virology 72, 499504.
  • Nichols RFW, 1950. The brown streak disease of cassava: distribution, climatic effects and diagnostic symptoms. East African Agricultural Journal 15, 15460.
  • Pappu SS, Brand R, Pappu HR, Rybicki EP, Gough KH, Frenkel MJ, Niblett CL, 1993. A polymerase chain reaction method adapted for selective amplification and cloning of 3′ sequences of potyviral genomes: application to Dasheen mosaic virus. Journal of Virological Methods 41, 920.
  • Robaglia C, Durand-Tardif M, Tronchet M, Boudazin G, Astier-Manifacier S, Casse-Delbart F, 1989. Nucleotide sequence of potato virus Y (N strain) genomic RNA. Journal of General Virology 70, 93547.
  • Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A Laboratory Manual. New York, USA: Cold Spring Harbor Laboratory Press.
  • Storey HH, 1936. Virus diseases of East African plants. VI-A progress report on studies of the disease of cassava. East African Agricultural Journal 2, 349.
  • Storey HH, 1939. Report of the Plant Pathologist. Mugua, Kenya: Report of the East African Agricultural Research Station, 9.
  • Thresh JM, Fargette D, Otim-Nape GW, 1994. The viruses and virus diseases of cassava in Africa. African Crop Science Journal 2, 45978.
  • Ward CW & Shukla DD, 1991. Taxonomy of potyviruses: current problems and some solutions. Intervirology 32, 26996.