Strategies for the Construction of Cassava Brown Streak Disease Viral Infectious Clones.

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.

Simultaneous virus-specific detection of the two cassava brown streak-associated viruses by RT-PCR reveals wide distribution in East Africa, mixed infections, and infections in Manihot glaziovii.

The expanding cassava brown streak disease (CBSD) epidemic in East Africa is caused by two ipomoviruses (genus Ipomovirus; Potyviridae), namely, Cassava brown streak virus (CBSV), and Ugandan cassava brown streak virus (UCBSV) that was described recently. A reverse transcription polymerase chain reaction (RT-PCR) based diagnostic method was developed in this study for simultaneous virus-specific detection of the two viruses. Results showed that CBSV and UCBSV are distributed widely in the highlands (> 1000 m above the sea level) of the Lake Victoria zone in Uganda and Tanzania and also in the Indian Ocean costal lowlands of Tanzania. Isolates of UCBSV from the Lake Victoria zone were placed to two phylogenetic clusters in accordance with their origin in Uganda or Tanzania, respectively. Mixed infections with CBSV and UCBSV were detected in many cassava plants in the areas surveyed. CBSV was also detected in the perennial species Manihot glaziovii (DNA-barcoded in this study) in Tanzania, which revealed the first virus reservoir other than cassava. The method for detection of CBSV and UCBSV described in this study has important applications for plant quarantine, resistance breeding of cassava, and studies on epidemiology and control of CBSD in East Africa.

First Report of Sweetpotato symptomless virus 1 and Sweetpotato virus A in Sweetpotatoes in Tanzania.

Sweetpotato (Ipomea batatas L.) is grown widely from tropical to temperate regions and is an important food security crop in tropical countries. In Africa, sweetpotato is infected by RNA viruses of many taxa (4), but DNA viruses, such as the genus Begomovirus (family Geminiviridae), infecting sweetpotatoes in the Americas have been reported only in Kenya (3). A caulimo-like DNA virus (family Caulimoviridae) has been detected in sweetpotatoes in Uganda (1). Recently, two novel badnaviruses (genus Badnavirus, family Caulimoviridae) and a new mastrevirus (genus Mastrevirus, family Geminiviridae) were discovered in a local sweetpotato cultivar maintained in a germplasm collection in Peru (2) but were not reported elsewhere. This study examined the possible existence of these novel viruses in landrace sweetpotato varieties grown in Tanzania. Nine landrace sweetpotato varieties and one introduced cultivar (NIS 91 from the International Potato Centre, Peru) were sampled from six regions of Tanzania. DNA was extracted (2) and amplified by PCR using primers (MastvKF: 5'-GACAGACCCCTAGGGTGA-3'; MastvsR 5'-ACTGCATATAGTACATGCCACA-3') designed in this study to amplify partial, putative movement and coat protein gene sequences of Sweetpotato symptomless virus 1 (SPSMV-1) (GenBank Accession No. FJ560945) (2). Products of the expected size were detected in seven samples (varieties Ex-London, Ex-Lyawaya, Gairo, Hombolo, Kagole white, Mbeya, and Shangazi) representing four regions surveyed (Dodoma, Mbeya, Morogoro, and Kagera). PCR products from five samples were sequenced (396 nt; GenBank Accession Nos. HQ316938 to HQ316942) and found to be identical to each other and the isolate described originally in Peru (2). Amplification with primers (BadnaBKF: 5'-CAAATTAGGAGGCAGATAAATG-3'; BadnaBsR: 5'-GGTCTTCTTATGTTCCACCTT-3') designed in this study according to the sequence of Sweetpotato virus B (SPBV-B) (GenBank Accession No.FJ560944) resulted in products of the expected size in three samples (varieties Ex-Lyawaya, Gairo, and Hombolo collected in Mbeya, Morogoro, and Dodoma, respectively) that were positive also for SPSMV-1. Sequences of the products (787 nt; HQ316935 to HQ316937) were nearly identical (99.4%). They were 96.8 to 96.9% similar to a region (nts 830-1616) of Sweetpotato virus A (SPBV-A; FJ560943) (2), whereas they were only 83.2 to 83.6 % similar to the corresponding region (1,486 to 2,272 nt) of SPBV-B (FJ560944) (2). No virus was detected in cv. NIS 91. All plants sampled exhibited mild mottling or mosaic symptoms, but a contribution to the symptoms by other untested viruses cannot be excluded because few of the large number of sweetpotato viruses have been studied in Africa (4). To our knowledge, this is the first report of SPSMV-1 and SPBV-A outside South America and in sweetpotatoes grown in the field. The results show that the two viruses are distributed widely in local sweetpotato varieties in Tanzania, which suggests that they may be found in other sweetpotato-growing areas where they have not been studied. While the yield losses caused by SPSMV-1 and SPBV-A remains to be studied, the data from this study are of practical importance in terms of regional and international exchange of sweetpotato germplasm. References: (1) V. Aritua et al. Plant Pathol. 56:324, 2007. (2) J. F. Kreuze et al. Virology 388:1, 2009. (3) T. Paprotka et al. Virus Res. 149:224, 2010. (4) F. Tairo et al. Mol. Plant Pathol. 6:199, 2005.

Evolution of cassava brown streak disease-associated viruses.

Cassava brown streak disease (CBSD) has occurred in the Indian Ocean coastal lowlands and some areas of Malawi in East Africa for decades, and makes the storage roots of cassava unsuitable for consumption. CBSD is associated with Cassava brown streak virus (CBSV) and the recently described Ugandan cassava brown streak virus (UCBSV) [picorna-like (+)ssRNA viruses; genus Ipomovirus; family Potyviridae]. This study reports the first comprehensive analysis on how evolution is shaping the populations of CBSV and UCBSV. The complete genomes of CBSV and UCBSV (four and eight isolates, respectively) were 69.0-70.3 and 73.6-74.4% identical at the nucleotide and polyprotein amino acid sequence levels, respectively. They contained predictable sites of homologous recombination, mostly in the 3'-proximal part (NIb-HAM1h-CP-3'-UTR) of the genome, but no evidence of recombination between the two viruses was found. The CP-encoding sequences of 22 and 45 isolates of CBSV and UCBSV analysed, respectively, were mainly under purifying selection; however, several sites in the central part of CBSV CP were subjected to positive selection. HAM1h (putative nucleoside triphosphate pyrophosphatase) was the least similar protein between CBSV and UCBSV (aa identity approx. 55%). Both termini of HAM1h contained sites under positive selection in UCBSV. The data imply an on-going but somewhat different evolution of CBSV and UCBSV, which is congruent with the recent widespread outbreak of UCBSV in cassava crops in the highland areas (>1000 m above sea level) of East Africa where CBSD has not caused significant problems in the past.

Genetically distinct strains of Cassava brown streak virus in the Lake Victoria basin and the Indian Ocean coastal area of East Africa.

Six isolates of Cassava brown streak virus (CBSV, genus Ipomovirus; Potyviridae) from the Lake Victoria basin in Uganda and Tanzania were characterized. Virus particles were 650 nm long. The complete coat protein (CP)-encoding sequences (1,101 nucleotides, nt) were 90.7-99.5 and 93.7-99.5% identical at the nt and amino acid (aa) levels, respectively. The 3' untranslated region was 225, 226 or 227 nt long. These eight isolates were only 75.8-77.5% (nt) and 87.0-89.9% (aa) identical when compared to the partial CP sequences (714 nt) of six CBSV isolates characterized previously from the costal lowlands of Tanzania and Mozambique. Hence, two genetically different and geographically separated populations of CSBV exist in East Africa.