icvg 2009 part I pp 1-131.pdf - Cornell University

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— 24 — 25 YEARS OF SEROLOGICAL IDENTIFICATION OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUSES: ANTISERUM AND MONOCLONAL ANTIBODIES TO GLRaV-1 TO GLRaV-9. Paul GUGERLI Agroscope Changins-Wädenswil Research Station ACW, Virology – Bacteriology, CH-1260 NYON, Switzerland Corresp. author : Paul Gugerli, Telephone: +41 22 366 27 29, Email: paul.gugerli@bluewin.ch ANTISERUM Antiserum to a grapevine leafroll-associated virus with up to 2200 nm long filamentous, Closterovirus like particles was first obtained 25 years ago (Gugerli et al., 1984). Subsequent studies rapidly revealed the presence of a second type of virus with serologically distinct but slightly shorter, up to 1800 nm long particles. We designated them provisionally leafroll-associated virus type I and II. A third, structurally similar but serologically distinct entity (type III) was soon after described (Zee et al., 1985, Rosciglione et al., 1986, Gugerli, 1987, Zee et al., 1987). Since then, at least another six related virus species have been discovered (Hu et al 1990b; Zimmermann et al., 1990a; Boscia et al., 1995; Gugerli et al., 1997; Choueiri et al., 1996; Monis, 2000; Alkowni et al., 2003, 2004; Maliogka et al., 2009). They are designated Grapevine leafroll-associated virus 1 to 9 (GLRaV-1 to GLRaV-9) and assigned to species of the genera Ampelovirus and Closterovirus within the family Closteroviridae (Martelli et al., 2002). Serology thus played a major role for the identification of GLRaVs since adequate molecular techniques were not available 25 years ago. In the eighties, a number of electron microscopical studies revealed Closterovirus like (Namba et al., 1979, Conti et al., 1980, Faoro et al., 1981, Von der Brelie et al., 1982) and phloemlimited isometric viruses (Castellano et al., 1983) frequently associated with grapevine leafroll disease. The complex infections in grapevine and the impossibility of assaying large number of samples made it however difficult to draw accurate conclusions. The production of antiserum to GLRaV-1 was therefore a methodological breakthrough. A number of premises were essential for this: (1) The initial experimental plant material was most crucial. Carefully indexed leafroll diseased Räuschling grapevine and its heat treated daughter plants served, respectively, as virus source and controls. Such virus-free controls were missing in an otherwise informative earlier study (Von der Brelie et al., 1982), where long Closterovirus like particles were also extracted from diseased but also from apparently healthy grapevines. (2) The definition of a reliable virus purification protocol was decisive for the extraction of reproducible amounts of fairly intact virus particles that could easily be analysed by electron microscopy. GLRaV-1 virions could therefore be distinguished from those shorter Closterovirus like particles reported earlier by Namba (1979) and Conti (1980). The partially purified viral nucleoprotein was also pure enough to be used for immunization of rabbits and mice to produce specific antibodies. (3) A further step was then the modification of the conditions for the large scale virus detection by enzyme-linked immunosorbent assay (ELISA), i.e. high strength buffer in order to stabilize and to suspend viral nucleoprotein in the otherwise inactivating, acid crude grapevine leaf or wood extract. (4) The development of highly specific monoclonal antibodies (Mabs) was vital to prove the unequivocal association of the physically defined filamentous virions, as seen by electron microscopy, the molecular weights of the their respective coat proteins as shown by Western immunoblots, the immunochemical reaction in ELISA and the sanitary status of the examined grapevine. (5) Finally, the world-wide exchange of antiserum stimulated collaborative research. The large-scale diagnostic application with commercialised antibody kits led to a further validation and fruitful feedbacks. Antiserum and Mabs were developed in various laboratories with a wide range of viral isolates from distinct leafroll diseased vines, as shown in Table 1. POLYCLONAL ANTIBODIES Reasonably useful antiserum to GLRaVs is easily obtained by immunisation of laboratory animals. Costs are low. Non-specific antibodies can partially been absorbed with antigens from healthy plants. Antiserum does generally not discriminate too stringently between virus variants. The production of antiserum requires however laboratory animals and the quality and specificity is hardly reproducible. Antiserum to GLRaVs also often cross-reacts with contaminating viruses, either closely related GLRaVs or frequently occurring viruses such grapevine fleck (GFkV). MONOCLONAL ANTIBODIES. The development of Mabs requires few experimental animals. Well selected Mabs have the desired specificity. If directed towards a well conserved antigenic determinant, Mabs may possess a higher and more regular affinity towards virus variants or related species than antiserum. Mabs are chemically and biologically homogenous and can be produced in large amounts when ever needed, with reproducible specificity and activity. They represent a longlasting memory of viral antigenic determinants. Some Mabs to GLRaVs were made more then 20 years ago and still react in the same way with well identified isolates maintained in grapevine virus collections. Mabs are reagents for the highly sensitive and specific
— 25 — immunoassays, either ELISA, ISEM, IPEM or Western blots. They can discriminate unequivocally between related viruses or variants in samples from mixed infections. As the evolution of viruses is often paralleled by changes of the coat protein, Mabs might well distinguish biological distinct variants. Mabs may be used to map epitopes on viral coat proteins (Zhou et al., 2003) or to partially decorate virus particles (Gugerli et al., 1993). They can easily be mixed with others to make efficient diagnostic tools. The development of Mabs to GLRaVs can however be laborious and costly. In some circumstances, Mabs may be too specific. This was eventually leading to an unnecessary segregation of provisional virus species, as in the case of GLRaV-4 or GLRaV-5 related viruses. As mentioned above, these disadvantages can at the same time be positive. As an example, specific Mabs to GLRaV-6 permitted to trace the intriguing massive occurrence of this virus species in the cultivar Cardinal (Boscia et al., 2000). Finally, some Mabs are difficult to handle. Table 1. Production of antiserum (As), monoclonal antibodies (Mabs) and recombinant antibodies (recAbs) to Grapevine leafroll-associated viruses (GLRaVs) Antibody Immunogen Cultivar Reference As; Mab 2-4 GLRaV-1 Räuschling 24 CH Gugerli et al., 1984, 1987 As GLRaV-2 Gamay RdL 356/r.37 CH Rosciglione et al., 1986 As; Mab 8 GLRaV-3 Frappato Mortillo I Gugerli et al., 1987, 1990 As GLRaV-1 Chardonnay F Legin et al., 1987 As; Mab GLRaV-3 Pinot noir NY-1 USA Zee et al., 1987; Madden et al., 1987 As GLRaV-1 Klevner Heiligenstein F Zimmermann et al., 1988 Mabs NY1.1 to 1.4 GLRaV-3 Pinot noir NY-1 USA Hu et al., 1989, 1990a, 1991 As GLRaV-2 Chaouch Rose TR Zimmermann et al., 1990a As; Mabs 1 to 3 GLRaV-3 Chardonnay F Zimmermann et al., 1990a, 1990b As GLRaV-5 1) White Emperor USA Zimmermann et al., 1990a As GLRaV-4 Thompson USA Hu et al., 1990b As GLRaV-2+3 Crouchen 2/1/20 ZA Engelbrecht et al., 1990 As; Mabs GLRaV-3 16G isolate I Faggioli F et al., 1991 Mab 29-1, 29-2 GLRaV-2 Chasselas 8/22 CH Gugerli et al., 1993 As; Mab 3-1, 6-3, 15-5 GLRaV-4 Thompson Seedless V.C.A2v22 USA Gugerli et al., 1993, Besse et al., 2009b As; Mab 43-1, 3-3, 8-2 GLRaV-5 Emperor V.C.A2v18 - White USA Gugerli et al., 1993, Besse et al., 2009 As GLRaV-1 to 5 CP Black Spanish 90/246 ZA Goszczynski et al., 1995 As GLRaV-7 AA42 unidentified AL Choueiri et al., 1996 As GLRaV-2 Muscat of Alexandria and N33 ZA Goszczynski et al., 1996a, 1996b As GLRaV-3 Zweigelt (Kecskemét) H Tobias et al., 1996 As; Mab 36-117 GLRaV-2+6 Chasselas 8/22 CH Gugerli et al.1997 As GLRaV-3 recCP Pinot noir NY-1 USA Ling et al., 1997, 2000 As; Mabs 3F76, 14F9, 15F1, 19A12 GLRaV-8 Thompson Seedless LR102 USA Monis et al., 1997, 2000 As GLRaV-1 CP French Colombart FC/2 USA Monis et al., 1997 As GLRaV-2 CP Malbec (incompatibility) USA Monis et al., 1997 As GLRaV-2 recCP N. benthamiana H4 USA Zhu et al., 1997 As; Mabs 1G10 3) ,1B7 4) , GLRaV-1 Houedi Y233 (F) 2) Seddas et al., 2000 1C4 5) , 2F11, 2F3 6) As GLRV-3 Raziki Y285 (F) 2) Seddas et al., 2000 Mab R19 GLRaV-2 N. benthamiana H4 (I) 2) Zhou et al., 2000 RecAb GLRaV-3 Nölke et al., 2003 Mabs Nig.A, B, C and.I GLRaV-3 Moscato giallo NIG3 I Zhou et al., 2003a, 2003b As GLRaV-2 Y252 IL Saldarelli et al., 2006 As GLRaV-2 Y253 TR Saldarelli et al., 2006 As ; Mabs 6-5, 37-15 GLRaV-7 Y276 (F) 2) Rigotti et al., 2006 As GLRaV-2 recCP Cabernet Franc CN Xu et al., 2006 As GLRaV-2 recCP Pinot noir USA Ling et al., 2007 As; Mab 5A5/C2, 8G5/H6 GLRaV-3 recCP Merlot Cl-766 RCH Engel et al., 2008 RecAb scFvLR3cp-1 GLRaV-3 (I) 2) Orecchia et al., 2008 As GLRaV-2 recCP LN33 BR Radaelli et al., 2008 RecAb C L -LR3 GLRaV-3 (I) 2) Cogotzi et al., 2009 As 1295 ; Mab 4-2 , Mab 8-2-3 GLRaV-2 variant Pinot noir 20/50 CH Besse et al., 2009a As; Mabs 62-4, 27-1 GLRaV-9 Cabernet Sauvignon AUS Gugerli et al., 2009 As GLRaV-Pr recCP Prevezaniko GR Maliogka et al., 2009 1) Originally named GLRaV-4; 2) Origin not stated; from collection (F) INRA Colmar or (I) University of Bari, Italia; 3) Cross-reacting with GLRaV-3; 4) Wide spectrum (32 out of 33 isolates tested); 5) Limited spectrum (25 out of 33 isolates tested); 6) Useful in diagnostic applications; RecCP: recombinant coat protein; As : polyclonal antibody (mostly from immunised rabbits); CP : coat protein; RecAb : recombinant antibody made from single-chain variable Fragment (scFv) . REC-CP AND REC-AB The use of recombinant coat proteins as immunogen (Ling et al., 1997, 2000; Zhu et al., 1997; Xu et al., 2006; Ling et al 2007; Engel et al., 2008; Radaelli et al., 2008) and in vitro engineered recombinant antibodies (rAb) with single-chain variable fragment (scFv) (Nölke et al., 2003; Orecchia et al., 2008; Cogotzi et al., 2009) reflect the development of exciting new technologies. Progrès Agricole et Viticole, 2009, Hors Série – Extended abstracts 16 th Meeting of ICVG, Dijon, France, 31 Aug – 4 Sept 2009
Page 3 and 4: 16th Meeting of the International C
Page 5 and 6: AVANT-PROPOS Nous sommes très heur
Page 7 and 8: Content - Sommaire Page Partners an
Page 9 and 10: Session IV - Epidemiology - Survey
Page 11 and 12: Poster 51 Poster 52 Poster 53 Poste
Page 13 and 14: Identification and distribution of
Page 15 and 16: — 15 — GRAPEVINE VIROLOGY HIGHL
Page 17 and 18: — 17 — were found, in that a va
Page 19 and 20: — 19 — Likewise, an antibody bi
Page 21 and 22: — 21 — LITERATURE ABOU GHANEM-S
Page 23: — 23 — ORECCHIA, M., NOELKE, G.
Page 27 and 28: — 27 — ENGELBRECHT, D.J. & KASD
Page 29 and 30: — 29 — NEW DEVELOPMENTS IN UNDE
Page 31 and 32: — 31 — LIU, Y.-P., PEREMYSLOV,
Page 33 and 34: — 33 — RESULTS AND DISCUSSION O
Page 35 and 36: — 35 — northeastern Oklahoma fr
Page 37 and 38: — 37 — viruses multiple primers
Page 39 and 40: — 39 — glass planar microarrays
Page 41 and 42: — 41 — Fig. 2. Part of a Micros
Page 43 and 44: — 43 — Complete sequencing of G
Page 45 and 46: — 45 — slightly bigger molecule
Page 47 and 48: — 47 — result suggests that the
Page 49 and 50: — 49 — ELISA. Rootstock samples
Page 51 and 52: — 51 — acquisition and analysis
Page 53 and 54: — 53 — identity at the protein
Page 55 and 56: — 55 — The sequences obtained w
Page 57 and 58: — 57 — MULLIS, K. & FALOONA, F.
Page 59 and 60: — 59 — qualitative level among
Page 61 and 62: — 61 — incidence of negative se
Page 63 and 64: — 63 — Madeira Islands. Eight a
Page 65 and 66: — 65 — stylet on actively growi
Page 67 and 68: — 67 — ANDRET-LINK, P. & FUCHS,
Page 69 and 70: — 69 — IDENTIFICATION OF PLASMO
Page 71 and 72: — 71 — SYMPTOM DETERMINANTS ON
Page 73 and 74: — 73 — RECOMBINATION EVENTS IN
Page 75 and 76:
— 75 — THE SPECIFICITY OF GFLV
Page 77 and 78:
— 77 — PARTIAL RNA-2 SEQUENCE O
Page 79 and 80:
— 79 — CHARACTERIZATION OF RASP
Page 81 and 82:
— 81 — ISOLATION OF MOVEMENT PR
Page 83 and 84:
— 83 — REAL-TIME PCR OF GRAPEVI
Page 85 and 86:
— 85 — FIRST RECORDS OF ARABIS
Page 87 and 88:
— 87 — primers allow us to quan
Page 89 and 90:
— 89 — transgenic grape lines,
Page 91 and 92:
— 91 — highest values were obse
Page 93 and 94:
— 93 — Imura et al., 2008, Loud
Page 95 and 96:
— 95 — GFLV-F13. Low levels of
Page 97 and 98:
— 97 — This is probably due to
Page 99 and 100:
— 99 — variation was distribute
Page 101 and 102:
— 101 — Figure 1. Proportion of
Page 103 and 104:
— 103 — were produced. The prod
Page 105 and 106:
— 105 — 2007 positive results w
Page 107 and 108:
— 107 — dehydrogenase (GAPDH) t
Page 109 and 110:
— 109 — detected, either as sin
Page 111 and 112:
— 111 — basal leaf tissue inclu
Page 113 and 114:
— 113 — GLRaV-3 incidence cover
Page 115 and 116:
— 115 — Figure 2. Frequency of
Page 117 and 118:
— 117 — Ljutun, Mladenka, Nincu
Page 119 and 120:
— 119 — ELISA reagent used in t
Page 121 and 122:
— 121 — Due the late season the
Page 123 and 124:
— 123 — RT-PCR & PCR: Primers f
Page 125 and 126:
— 125 — Table 2. Sanitary statu
Page 127 and 128:
— 127 — SANITARY STATUS OF AUTO
Page 129 and 130:
— 129 — THE VIRUS STATUS OF GRA
Page 131:
— 131 — PRESENCE OF GLRAV 1, 2,
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