Phylogenetic and recombination analysis of Tobacco bushy top virus in China

During the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates.

5’ and 3’ RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates.

The genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3’UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations.

The genome sizes of TBTV isolates were constant while different ORF-coding regions and 3’UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors.