RT-PCR-based detection and partial genome sequencing indicate high genetic diversity between CBSV and UCBSV in Kenya

Abstract

Cassava is grown by small-scale farmers and consumed by over 200 million people in sub-Saharan Africa mainly as staple food. An emerging viral disease Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), has severely affected cassava production and resulted in poor quality tubers. The disease severely affecting root quality both for domestic use and marketing and has become a real threat to the livelihoods of the millions of poor in both eastern and southern African countries. There is also increase in the number of CBSV and UCBSV sequences available now, and a need to have primers that would detect all isolates of CBSV and UCBSV is required, particularly for field surveys. Two sets of virus-specific primers, CBSVF2 & CBSVR7 and CBSVF2 & CBSVR8, were developed to specifically detect CBSV and UCBSV respectively on six glass house maintained isolates and field collected samples from coastal Kenya. Positive samples produced expected PCR products of 345 bp and 441 bp using CBSVF2 & CBSVR7 and CBSVF2 & CBSVR8, respectively. In addition, our study further confirmed the genetic diversity of these viruses by sequencing over 41 new samples from Kenya as well as Tanzania and Zanzibar. The coat protein, partial HAM1 and 3' UTR regions of CBSV and UCBSV were amplified using CBSVF2 & CBSVR1 and CBSVF2 & CBSVR2, respectively. The PCR products of ~1.6 Kb obtained were extracted from gel, cloned and sequenced. The sequences of the UCBSV and CBSV isolates varied in length; 1678 nucleotide for UCBSV isolates and 1615 nucleotide for CBSV isolates. CBSV and UCBSV sequences were used to the construct phylogenetic trees by comparing with other sequences from GenBank. The phylogenetic tree clustered the CBSD isolates in two groups reflecting the two virus species causing CBSD. Based on the available sequences (~1600 bases), the CBSV group shared 93.7% nucleotide identities, UCBSV 93.1%, and there was ~70% identity between the two groups. However, the percentage nucleotide identities for coat protein nucleotide sequences were slightly greater than those observed for the sequences involving partial HAM1 and 3'UTR region. CBSV group shared 94.4% nucleotide identity, UCBSV 93.5% and ~74% identity between the two groups. In conclusion, this study has shown the genetic diversity between CBSV and UCBSV isolates collected from Kenya with those from Tanzania and Zanzibar.

Keywords: Brown streak virus, cassava, Kenya