Characterization and Population Genetics of a New Virus Infecting Blackberry

A new virus belonging to the genus Ampelovirus in the family Closteroviridae was identified in blackberry in several states in the Southern United States (U.S.). Experiments were conducted to characterize the virus,; investigate its distribution in the U.S., and study its population genetics. Virus-specific dsRNAs were extracted and genome sequences were obtained by random-primed cloning and ‘next-generation’ sequencing. Molecular analysis of sequences revealed a genome organization resembling GLRaV-3, a representative member of the subgroup I in the genus Ampelovirus. The sequenced genome contains 10 open reading frames (ORFs), which encode closterovirid signature replication and quintuple gene block proteins in addition to four proteins of unkown function. Genetic variation within the population was analyzed by amplifying and sequencing portions of the polyprotein (region between methyl transferase and helicase domains), HSP70 homolog (HSP70h) and minor coat protein (CPm) genes. Nucleotide and predicted amino acid sequences revealed significant diversity in the polyprotein (23%) and CPm (14%) compared to the HSP70h region (1%). The ratio of non-synonymous substitution per non-synonymous site and synonymous substitution per synonymous site indicated that these proteins are under stringent purifying selections. No predicted recombinational events were observed. Sensitive detection assays were developed based on a highly conserved region for both conventional and real-time reverse transcription PCR and were able to detect all sequenced isolates. Efforts are underway to identify potential transmission vector(s) for this new virus.