Phylogenetic relationships between the coat protein gene nucleotide sequences of 29 Indian CVB isolates, a Russian CVB isolate (RU), Daphne virus S (DVS), Helenium virus S (HelVS) and Lily symptomless virus (LSV).  The tree was constructed using the maximum likelihood method (HKY model with transition/transversion ratios estimated from the data) implemented in PHYML [10] with 100 non-parametric bootstrap replicates.  Percentage bootstrap support for branches is displayed.  Branches with less than 50% bootstrap support have been collapsed.    Based on this tree topology the viruses have been sub-divided into three groups (I to III).  For purposes of clarity group I viruses have been tentatively further classified into seven subgroups (A through G).

Evidence that CVB isolate CHH is a recombinant virus resembling isolates HSR and JK1.  A. Maximum likelihood trees (model HKY, transition:transversion ratios estimated from the data, 100 non-parametric bootstrap replicates) constructed using different portions of the CVB coat protein gene (CP) sequence alignment.  In the ~750 5’-terminal nucleotides of the alignment CHH resembles HSR, a group I isolate, whereas it resembles JK1, a group II isolate, in the ~200  3’-terminal nucleotides (yellow regions in plots B and C).  In both trees branches with <50% bootstrap support were collapsed.  Virus names and/or origins are provided in Table 1. B. Estimation of the recombination breakpoints using the MAXIMUM CHI SQUARE method [18].  The approximate 5’ breakpoint for the recombination event lies between the blue peak (representing HSR) at alignment position 741 and the red peak (representing JK1) at alignment position 760.  Blue = HSR, red = JK1, grey = HP1, green = HP2, and purple = Lilly symptomless virus.  The broken line indicates a Bonferroni corrected chi square P-value = 0.05. C. Bootscan [15] evidence for the recombinant origins of different portions of the CHH CP.  Neighbor joining trees (1000 bootstrap replicates, Jukes Cantor distances) were constructed for 200 nucleotide sequence windows moved along the alignment one nucleotide at a time.  Plots represent the percentage bootstrap support in these trees (centered on the midpoint of each window) grouping CHH with various isolates (colors represent the same isolates as in B).

Description of recombination events: as can be seen in the figure, the RDP2 program identifies three recombination events with P value of 4.188x10-24. These events were detected by three programs viz GENECONV, MAXIMUM CHI SQUARE and CHIMAERA. The three recombination events can be described as follows:

Event 1: Recombination between an Indian CymMV isolate (AM055720), as major parent, and Korean type 2-CymMV isolate (AF016914), as minor parent. In this recombination event, a region (4973-6226 nt) of AM055720 genome were replaced with corresponding genome sequences of AF016914 (RDP P-value=4.188x10-24), which include N-terminal of TGB and C-terminal of coat protein.

Event 2: Recombination was detected with same P value as for event 1, between Singapore isolate (U62963), as major parent, and Taiwan isolate (AY571289) as minor parent. In this recombination event, a region (4337-5262 nt) of Singapore isolate (U62963), were replaced with corresponding genome sequence of Taiwan isolate (AY571289), which include N terminal of TGB1 and N terminal of TGB 3.

Event 3: Recombination event was also detected between AB197937 as major parent and Taiwan isolate (AY571289) as minor parent. In this case a region (5468-5753 nt) of Japanese isolate (AB197937) were replaced with corresponding genome sequence of AY571289.