Molecular diagnostics, genetic diversity and generating infectious clones for cassava brown streak viruses

Musa, Muawiya Abarshi (2012) Molecular diagnostics, genetic diversity and generating infectious clones for cassava brown streak viruses. PhD thesis, University of Greenwich.

Preview

PDF - Published Version Available under License Creative Commons Attribution Non-commercial No Derivatives. Download (4MB) | Preview

Abstract

Cassava brown streak disease (CBSD) threatens cassava production in eastern and southern African countries. Diagnostic protocols currently available for the causal agents of CBSD, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV), were unreliable but were urgently needed. In this study, sampling procedures and diagnostic protocols were developed for accurate and reliable detection of both CBSV and CBSUV. The cetyltrimethylammonium bromide (CTAB) method of RNA extraction was optimized for sample preparation from infected cassava plants and compared with the commercial kit RNeasy (Qiagen) for sensitivity and reproducibility. Results showed that both protocols were reliable but CTAB was more cost-effective and ideal for resource-poor laboratories. Mixed infections of cassava mosaic begomoviruses (CMBs) that cause cassava mosaic disease (CMD), CBSV and CBSUV have become more common with the recent spread of CBSD at mid-altitudes. A multiplex PCR for the simultaneous detection of viruses that cause both diseases, the first of its kind for cassava, was therefore developed to detect CBSV and CBSUV along with the three commonly occurring CMBs (African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), and East African cassava mosaic virus-Uganda (EACMVUG) in eastern Africa. Similarly, a duplex PCR was developed for the simultaneous detection of CBSV and CBSUV, both viruses being detected in field-collected samples from Tanzania and Kenya. The genetic diversity of more than 40 CBSD isolates from Kenya, Tanzania, Uganda, and Mozambique was further examined by sequencing the coat protein (CP) gene and partial HAM1 gene sequences. The phylogenetic tree clustered the CBSD isolates into two groups reflecting the two virus species causing CBSD. In this study, various strategies were carried out for generating infectious clones of CBSV; gateway cloning, in vivo and in vitro transcription methods, and amplification of the viral genome in three fragments. Although 3 overlapping CBSV fragments were successfully cloned, the presence of an unexpected mutation at one of the cloning sites unfortunately did not allow reassembling of the fragments to construct the full-length cDNA.

Actions (login required)