Development of Methodologies for Rapid Detection of Grapevine Viruses

Antisera produced to grapevine leafroll associated virus (GLRaV) -2 and -4 were evaluated. Our evaluation showed the first 4 bleeding had moderate specific titer with low non-specific background. However, later bleeding (those after the boost injection) developed high non¬specific titer and were not useful for ELISA test. This problem resulted in modifying our virus purification protocol to obtain virus preparations with higher purity. We have found grapevine sources which are singly infected with GLRaV-1, -4 and, -5. Virions have been purified from these sources using modified purification procedure and are ready for immunizing rabbits after an additional density gradient centrifugation (in order to highly purify). A source of grapevine virus B (GVB, a virus isolate which is associated with corky bark disease) has been identified and increased in a tobacco species (Nicotiana occidentalis). This source will be used for virus purification and antiserum production. Cloning and sequencing of grapevine virus RNAs will enable us to both improve serological tests for these viruses and develop nucleic acid-based detection methods. Previously we had developed improved double stranded (ds) RNA purification methods and used them to create a GLRaV-4 clone. We had also improved our ability to identify whether our clones are of plant or viral origin and which portion of viral chromosomes we have cloned. We had confirmed our GLRaV-4 clone. We were in the process of confirming our previous GLRaV-2 clone. We had begun development of polymerase chain reaction (PCR) detection methods for GLRaV-4. We had repeated our previous successes with colorimetric PCR detection of GLRaV-3, GVA, and GVB. Conventional PCR detection of GLRaV-3 still needed some improvement before its results consistently agreed with colorimetric PCR for this virus. From March through May, we have determined that our previous GLRaV-2 clone is unlikely to be of viral origin. We have since created another, sequenced it, and confirmed that this new clone does indeed contain closterovirus sequences. We are currently using data from this clone to develop IC-PCR for this virus. We have also made cDNA to GLRaV types 3 and 5. This is the first step in cloning these viruses. We are in the process of finishing the cloning of these cDNAs in order to sequence them. When this is done, we will be able to confirm the viral origins of these clones and, should they check out, begin developing IC-PCR detection techniques for GLRaV-5 and additional detection techniques for GLRaV-3. We have also determined the cause of the inconsistent results gained from previous experiments involving conventional PCR detection of GLRaV-3 and remedied the situation. We can now get consistent conventional and colorimetric PCR results for this virus. Additional experiment was completed to compare the efficiency of ELISA for detecting grapevine leafroll associated viruses with bioassay index on field indicator Cabernet Franc. The test results revealed a good correlation between ELISA and field index.