Characterization of 21 Cacao Swollen Shoot Viral Genomes Reveals Multiple Species with Diverse Genome Organizations

Cacao swollen shoot virus (CSSV) is a cacao-infecting badnavirus, endemic to West Africa. Infected cacao trees exhibit foliar symptoms and shoot swelling, reduced yield, and eventual death. Recently, CSSV-like foliar and swollen shoot symptoms, accompanied by atypical, rapid decline symptoms with death ensuing 1-2 years post-infection, has occurred in Cote d’Ivoire and Ghana. At the same time, serological and molecular assays have failed to detect CSSV in 40-50% of symptomatic trees, coincident with the spread of the atypical severe decline/death syndrome. The collective evidence points to the possible emergence of a previously unrecognized CSSV-like virus(es). To test this hypothesis, the genomic variability of CSSV isolates from West Africa was investigated using Illumina HiSeq and Sanger DNA sequencing validation of recovered genomes. Pairwise nucleotide (nt) analysis of 14 full-length badnaviral genomes determined herein and 7 CSSV genomes available in GenBank, and of the RT-RNase H locus used for badnavirus species demarcation, at >80% nt identity, indicated four distinct CSSV-like groups. Phylogenetic analysis of sequences for the 21 full-length genome and 87 badnaviral RT-RNase loci, each resolved one well-supported, major CSSV clade consisting of four sister/subclades, while excluding all previously reported, non-cacao-infecting badnaviruses. The full-length genome sequence for members of the CSSV clade encoded either 3, 4, or 5 open reading frames (ORFs) containing homologous as well as distinct functional protein domains. Unexpectedly, CSSV species grouped phylogenetically within the same sister clade, but differed by ORF arrangement and/or functional domain architecture.