Contribution à l'étude de virus de mollusques marins apparentés ...

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1994 : P. Maffart, R.M. Le Deuff, T. Renault, N. Cochennec et B. Chollet, " Situation épidémiologique de Crassos/rea gigas sur les côtes françaises ". Journées Conchylicoles du Département Recherche Aquacole, IFREMER, Nantes, France. 1995 : R.M. Le Deuff. " Contribution à l'étude de virus de mollusques marins : iridovirus-like et herpesvirus-like. Description et caractérisation biochimique, cycle de multiplication viral, diagnostic et étude épidémiologique. ". Journées de présentation des travaux des thèses de la Direction des Ressources Vivantes, IFREMER, Nantes, France. 189
Publication 1 : Production of monoclonal antibodies against Iymphocystis disease virus (LDV, /ridoviridae). Application to early and reliable LDV titration. Journal of Fish Diseases, sous presse. 1 2 3 2 R.M. LE DEUFF , B. DESPRES , S. H. MORTENSEN, V. BOULO, E. BACHERE 2 & E. MIALHE 4 1 Laboratoire de Génétique, Aquaculture et Pathologie, Unité de Recherche en Pathologie et Immunologie Générales, IFREMER, BP133, F- 17390 La Tremblade, France, 2Défenses et Résistances des Invertébrés Marins, lFREMER-CNRS UM2, CP71, F-34000 Montpellier, France, 3/nstitute of Marine Research, Department of Aquaculture, PO Box 1870, N-5024 Bergen-Nordnes, Norway and 4CENAIM ESPOL, Campus Politecnico, P.o. Box 09-01-4519, Guayaquil, Ecuador, South America Correspondence : R.M. Le Deuff, Laboratoire de Génétique, Aquaculture et Pathologie, Unité de Recherche en Pathologie et Immunologie Générales, IFREMER, BP133, F-17390 La Tremblade, France. Tel: (33) 46.36.98.36. Fax: (33) 46.36.37.51 Abstract. Monoclonal antibodies against Lymphocystis disease virus (LDV) were prepared using infected BF2 cell cultures as antigen source for immunisation of mice and hybridoma screening. The screening was performed by indirect immunofluorescent assay (lIFA) on non-infected (negative screening test), and on early (3 days) and delayed early (9 days) infected BF2 cells (positive screening tests). Ten LDV specific antibody producing clones were selected. Clones were characterized according to the immunofluorescent patterns of antibodies produced. Sensitive and reliable titration of LDV was possible with Iwo of the monoclonal antibodies obtained (II A2 and 15D II C9). Introduction Lymphocystis disease is a contagious viral infection described from a variety of fresh, brackish and sea water teleost fish species (Anders, 1989). This disease is caused by Lymphocystis disease virus (LDV), an lridovirus which composition, structure and biological properties are weil described (Mid lige & Malsberger, 1968 ; Berthiaurne, Alain, & Robin, 1984 ; Samalecos, 1986 ; Flügel, 1985). LDVs have been classified in the Iridoviridae family as a separate genus, Lymphocystivirus, associated to fishes (Francki, Fauquet, Knudson & Brown, 1991), and Iwo different LDV types have been proposed on the basis of restriction enzyme DNA cleavage patterns (Darai, Anders, Koch, Delius, Gelderblom, Samalecos & FlUgel, 1983). ln addition to LDV, sorne of the Iridoviridae, such as Frog Virus 3 (FV3) and Chilo Iridescent Virus (CIV) have been thoroughly studied and characterized (Essani & Granoff, 1989 ; Devauchelle, Attias, Monnier, Barray, Cerutti, Guerillon & Orange Balange, 1985) due to the availability of susceptible cell lines. Others, have only been described structurally. These are mainly associated with invertebrates, and their impact on host populations are not weil known. Jt is also presurned that viruses morphologically similar to LDV, were involved in gill disease which caused a total disappearance of the Portuguese oyster, Crassostrea angulata (Lmk) from French waters between 1967 and 1973 (Comps, Bonami, Vago & Campillo, 1976). 190
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1 1 1 1 Année 1995 UNIVERSITE DE B
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à mes parents à Stéphane
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1 1 ) TABLE DES MATIERES INTRODUCTI
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3 - Essais d'infection expérimenta
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INTRODUCTION
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d'approche pour contrôler les mala
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1 1 1 1 1 1 1 1 j Un premier chapit
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DONNEES BIBLIOGRAPHIQUES 1 - Rappel
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1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
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et Wray, 1952 ; Mackin, 1962 ; Burr
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1 1 1 1 1 1 1 1 des zones indemnes
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3 - Procaryotes Les mollusques biva
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Tableau 4 : Infections à Chlamydie
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cheptels de C. angulala, a conduit
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t t 1 1 1 1 t 1 1 t 1 1 t 1 1 t 1 1
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i 1 1 1 1 1 1 1 1 1 1 1 1 1 J 1 int
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1 1 1 1 1 1 1 ! 1 1 1 1 ! 1 1 1 1 1
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III - Caractéristiques principales
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1 1 1 1 1 1 1 1 1 1 1 J 3.2 - Entr
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1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
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i 1 1 1 1 1 1 1 1 1 1 1 1 1 J 1 int
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1 1 1 1 1 1 1 1 1 1 1 PREAMBULE Pub
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Chapitre 1 : Caractérisation d'un
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l 1 1 1 1 1 1 1 1 1 1 1 1 PUBLICATI
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References AllIle . w .. SchlOl fc
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Platichtys flesus, plie, Pleuronect
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Figure 1 : Marquage de coupes de do
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CHAPITRE 2 Comparaison antigénique
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Ces électrophorèses ont égalemen
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1\6 , HMW LMW Figure 1 : Caractéri
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a 2 3 4 5 6 7 8 9 10 --- - - - - Il
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Tableau 2 : Comparaison anti géniq
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Figure 4 : Coupe histologique d'hû
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Chapitre 1 : Mise en évidence et d
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Les virions observés chez les larv
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• •• Figure 2 : Cliché en mi
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2 - Participation à la mise en év
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Figure 10 : Lésions histologiques
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Bu ll. Eur. Ass. Fi sh Palhal.. 14(
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736 Material and methods Samples of
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740 RENAULT (T.) ET COLLABORATEURS
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742 15. - MURP HY (F.A.) and K INGS
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Chapitre Il : Essais de reproductio
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2 - Essais d'infection expérimenta
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CHAPITRE 3 Effet de la température
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26°C), de détecter la présence d
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présentent des lésions histologiq
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Introduction The Pacific oyster, 'C
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(Table 1). 100% mortality in this g
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than at high temperature (25-26°C)
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Our plans for testing this la st hy
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LEGEND TO FIGURES Figure 1 : Origin
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CHAPITRE IV : Essais de propagation
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Figure 1 : Principales caractérist
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2 - Essais d'infection de primocult
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(SVF) et/ou d'hémolymphe de Crassa
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68 0.2 g/I, KCI, No. P 1300' 2.77 g
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72 2. Cousserans F, Bonami lR, Comp
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2.2. Essais d'infection de primocul
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Chapitre V : Mise au point d'un pro
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Page 154 and 155: Chapitre VI : Mise au point de mét
Page 156 and 157: chromatine est rejetée en périph
Page 159 and 160: 528 RM Le DeuN et al ,GD Fig 2. Imm
Page 161 and 162: 1.2 - Recherche de séquences conse
Page 163 and 164: DJBE16 DJBE2L DJBECI DJBE16 DJBE2L
Page 165 and 166: 1.2.2 - Alignement des séquences D
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Page 175 and 176: CONCLUSION L'ensemble de ce travail
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Page 179 and 180: REFERENCES BIBLIOGRAPHIQUES Ahne W.
Page 181 and 182: Cahour A., 1979. Mar/eilia refringe
Page 183 and 184: Darlington R.W. et Moss L.H., 1969.
Page 185 and 186: Girard M. et Hirth L., 1989b. Méth
Page 187 and 188: Johnson D.C. et Spear P.G., 1982. M
Page 189 and 190: Lemaster S. et Roizman B., 1980. He
Page 191 and 192: Ohba M., Kanda K. et Aizawa K., 199
Page 193 and 194: Read G.S. et Frenkel N., 1978. Herp
Page 195: Shuster C.L. et Hillman T.E., 1963.
Page 203: days, according to Reed and Muench'
Page 206 and 207: detect a single infected cell in a
Page 211 and 212: Vet Res (1995) 26. 539·543 © Else
Page 216 and 217: Annexe 2 : Méthodes et techniques
Page 218 and 219: minutes, puis les coupes sont réhy
Page 220 and 221: 2.1 - Préparation des solutions -
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Page 226: Gel de concentration 4% H 20 Tampon
Page 229 and 230: le tampon de transfert et superpos
Page 231: NaCI NaN) Ajuster le pH à 7 Tampon
Page 235: Epuisement des anticorps 1 - Utilis
Page 238 and 239: 6 - Prélever la phase supérieure
Page 241: Tris-HCl pH8 1 ml Préparer extempo
Page 245: 2 - Prélever 1 ml de tampon d'hybr
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