FruitDisease - VirusResearch, characterisation of viruses

Characterisation of viruses

Plant viruses are categorized according to their physical properties, such as their nucleic acid content (DNA or RNA, single-stranded or double-stranded, messenger-sense or negative-sense or both [ambisense]) and their particle structure (rod-shaped, spherical, bullet-shaped, enveloped or other configurations). Knowing these and other characteristics allows us to group similar viruses into genera, and then to organise related genera into families. This process is overseen by the International Committee on Taxonomy of Viruses (ICTV), and is continually updated as new viruses are discovered. This is useful because when only a small amount of information is known about a particular virus it may be possible to make further assumptions about the virus based on the properties of related viruses. This, in turn, may suggest approaches to obtain more information about the uncharacterised virus.

Mechanical Transmission

One problem in studying viruses of soft fruit plants is that they are present only at very low concentrations making it very difficult, if not impossible, to isolate pure preparations of virus from these plants. Fortunately, in some cases, the virus can be transferred to other species of herbaceous plants by grinding the infected raspberry leaves in an extraction buffer (containing nicotine to neutralise inhibitory compounds), mixing the extract with some abrasive powder and rubbing it onto the leaves of the herbaceous (indicator) plants. Commonly used indicator plants include: Chenopodium quinoa, Chenopodium amaranticolor, Nicotiana clevelandii, Nicotiana benthamiana, Nicotiana tabacum and Cucumis sativa. Viruses generally accumulate to high levels in these plants, in addition the plants are not woody and do not contain large amounts of inhibitory substances, allowing pure preparations of virus to be isolated from them. Purified virus can be used to raise virus-specific antibodies, and to obtain sequence information from the virus (nucleic acid) genome.

RuCMV in C quinoaSoft fruit viruses that can also infect herbaceous plants include: (for more information on soft fruit viruses, see our database)

Rubus (Raspberry/Blackberry) viruses - Apple mosaic virus, Arabis mosaic virus, Blackberry chlorotic ringspot virus, Bramble yellow mosaic virus, Cherry leaf roll virus, Cherry rasp leaf virus, Cucumber mosaic virus, Raspberry bushy dwarf virus, Raspberry ringspot virus, Strawberry latent ringspot virus, Tobacco ringspot virus, Tobacco streak virus, Tomato black ring virus, Tomato ringspot virus, Wineberry latent virus.

Fragaria (Strawberry) viruses – Arabis mosaic virus, Fragaria chiloensis latent virus, Raspberry ringspot virus, Strawberry latent ringspot virus, Strawberry mottle virus, Strawberry necrotic shock virus, Tobacco necrosis virus, Tomato black ring virus, Tomato ringspot virus.

Vaccinium (Blueberry/Cranberry) viruses – Blueberry leaf mottle virus, Blueberry scorch virus, Peach rosette mosaic virus, Tobacco ringspot virus, Tomato ringspot virus.

Ribes (Blackcurrant/Redcurrant/Gooseberry) viruses – Alfalfa mosaic virus, Arabis mosaic virus, Cucumber mosaic virus, Raspberry ringspot virus, Strawberry latent ringspot virus, Tobacco rattle virus, Tomato ringspot virus.

Graft Transmission

Characterisation of viruses also often proceeds by grafting of the infected plant to particular indicator species or varieties. If the virus is not sap transmitted, then grafting may be the only available testing procedure. The indicator plants may then develop specific symptoms of infection that can point to the presence of particular viruses. For raspberries, indicators often include the red raspberry (Rubus idaeus) varieties “Malling Landmark”, “Lloyd George” and “Norfolk Giant”, and the black raspberry (Rubus occidentalis) but may sometimes include other Rubus species. Depending on the grafting technique it is possible to make interspecific and even intergeneric grafts, for example raspberry leaves can be grafted to alpine strawberry (Fragaria vesca), as reported for the examination of Raspberry leaf curl virus and Raspberry yellow net virus. Complications arise when plants are infected with mixtures of viruses, which will not be separated by the grafting process, making it difficult to pin down particular symptoms with specific viruses.

tip necrosis

Soft fruit viruses that are tested for by grafting (and cannot be sap transmitted) include:

 Rubus (Raspberry/Blackberry) viruses – Blackberry calico virus, Raspberry leaf curl virus, Raspberry leaf mottle virus, Raspberry leaf spot virus, Raspberry vein chlorosis virus, Rubus yellow net virus

Fragaria (Strawberry) viruses – Strawberry chlorotic fleck virus, Strawberry crinkle virus, Strawberry mild yellow-edge virus, Strawberry vein banding virus

Vaccinium (Blueberry/Cranberry) viruses – Blueberry shoestring virus, Blueberry red ringspot virus

Ribes (Blackcurrant/Redcurrant/Gooseberry) viruses – Blackcurrant reversion virus, Gooseberry vein banding virus

Sequencing of Viruses

The most definitive information about a virus is its nucleic acid sequence. Several approaches can be taken to obtain sequence information from uncharacterised viruses. When the virus can be purified in sufficient amounts from herbaceous plants, virus nucleic acid can be isolated free from contaminating plant DNA and RNA and can be cloned and sequenced. For viruses than cannot be transferred to herbaceous plants, and are usually present at extremely low concentration in the soft fruit plant an alternative strategy may be used. If the physical characteristics of the virus show that it is closely related to other viruses then PCR primers can be designed that correspond to highly conserved regions of the virus genome – often part of the virus replication (polymerase) protein. This method was successfully used to clone part of the Strawberry crinkle virus (negative-strand RNA virus, rhabdovirus) as well as Gooseberry vein banding virus (double-stranded DNA, caulimovirus).

Sequnce trace

A characteristic of virus infection is the production of double-stranded RNA molecules, which is mostly absent from healthy plants. This can be purified by binding to granular cellulose (commonly Whatman CF-11 cellulose powder) in the presence of a low concentration of ethanol (visit for more information and practical tips). The pattern of dsRNA bands, when visualised by gel electrophoresis and staining, can give clues to the identity of the virus. Otherwise, the dsRNA is itself a template for cDNA cloning and sequencing.