WINETECH Technical Yearbook 2019

regions, a section encompassing the coat protein gene (‘ CPreg ’) and a part of the replicase gene (‘ pREP ’), were determined and used to determine which GRSPaV variant(s) were present in infected plants. To investigate the impact of recombination on variant classification and the success of the GRSPaV coat protein and replicase doma i ns to co r rec t l y c l a s s i f y v i r us variants, the full genome sequences of the 18 representative GRSPaV isolates from GenBank were used. The sequences were screened for the presence of recombination using a recombination detection programme (RDP4) (Martin, et al ., 2015). The 18 isolates were then classified both by their full genomes, and by considering only the CPreg or pREP areas of their genomes. While a low incidence of GRSPaV was observed in the MB survey (6.25%), the prevalence of GRSPaV was much higher in the OV vineyards (29.06%), indicating a decrease of GRSPaV in plants screened using biological indexing. Isolates from five of the six currently recognised variant groups (I, IIa, IIb, IIc and III) were detected. A distinct variant group that is not represented by any of the full genome representative sequences was detected and subsequently labelled group IId. In most cases, the classification of variants from a single sample based on both the CPreg and pREP regions was consistent. The majority of isolates from the surveys clustered with groups IIa, IIb, IIc and IId.

Infections by more than one GRSPaV variant per plant were detected in both surveys. Genetic diversity was lowest in the MB survey and all but one positive sample originated from the same vineyard, which could be the result of failed elimination of the virus from source material. Isolates from this block fall within group IIa or IIb, which are known to elicit mild or no symptoms on indicator plants (Meng, et al ., 2005). This could indicate that hardwood indexing is less effective than RT- PCR at detecting asymptomatic or mildly symptomatic GRSPaV variants. Recombination was detected in three of the 18 full genome sequences used to classify GRSPaV isolates found in the field. Most notably, classification of reference isolates for variant group IIc based on the CPreg and pREP regions was not consistent. Because a recombination event occurred between the CPreg and pREP regions of these isolates, variants from group IIc were classified as group III based on sequences of the CPreg area. These results were reflected in the survey, where isolates from the same samples for which the pREP sequences fall within group IIc, seemed to cluster with group III based on the sequence of their coat protein. This demonstrates the possibility of incorrect classification if only one genomic region is considered. This study illustrates the broad genetic diversity of GRSPaV in South Africa. A

Parental variant 1

Parental variant 2

Recombination event

Recombinant sequence

Breakpoint position

FIGURE 1. Graphical representation of a recombination event between two virus variants resulting in a recombinant sequence containing fragments from both parental variants. Breakpoint position indicated with a red arrow.

Recombination is a process during which one virus variant exchanges parts of its genome with that of another variant. The resulting virus, also referred to as the ‘recombinant,’ is a ‘hybrid’ of the two parental variants and may possess a mixture of the parental traits. Recombination in viruses is a mechanism to generate genetic variability and has been linked to an increase in virulence, evasion of host immunity, and an increase in resistance to antiviral agents (Figure 1) (Simon-Loriere & Holmes, 2011). If a recombination event occurred between or inside the regions used to categorise variants, classification may be incorrect. It is therefore important to take recombination into consideration when classifying virus variants.

The influence of recombination and the use of two different genomic regions for classification of virus variants were also assessed. A survey (OV) was carried out on South African vineyards that were established prior to the implementation of current sanitary protocols. A second survey (MB) was conducted on mother blocks that, based on results from biological indexing, previously conformed to South African certification requirements, but are no longer used for vine propagation. Diagnostic reverse transcription polymerase chain reactions (RT-PCR) were used to identify plants infected with GRSPaV. The nucleotide sequences of two genomic