WO1988001833A1 - Anticulicidian virus and compositions obtained therefrom for combating mosquitos - Google Patents

Anticulicidian virus and compositions obtained therefrom for combating mosquitos Download PDF

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WO1988001833A1
WO1988001833A1 PCT/FR1987/000351 FR8700351W WO8801833A1 WO 1988001833 A1 WO1988001833 A1 WO 1988001833A1 FR 8700351 W FR8700351 W FR 8700351W WO 8801833 A1 WO8801833 A1 WO 8801833A1
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virus
mav
aedes
culture
insects
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PCT/FR1987/000351
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French (fr)
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Michel Cornet
Vincent Deubel
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Institut Pasteur
Institut Français De Recherche Scientifique Pour L
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages

Definitions

  • the present invention relates to the isolation of a new anti-mosquito virus and to its application as a means of combating mosquitoes.
  • MAV Mosquito African Virus
  • the Inventors maintain in an Insectarium located at the Institut Pasteur in DAKAR (SENEGAL) a colony of Aedes aegypti which they use to obtain the replication of various strains of yellow fever virus; the inoculated mosquitoes are crushed and their crushed seeds are seeded on various insect cell cultures to obtain a quantity of yellow fever virus sufficient to carry out biochemical studies. It is on one of these cell lines (Ae. Aegypti, Clone C17, Deubel and Distill, Am. J. Trop. Med.
  • the inventors having been able to establish that the MAV virus which they have identified is highly pathogenic with regard to Aedes aegypti, it has therefore proved important to be able to have sufficiently large quantities of this virus to allow providing for means of fight against mites comprising the MAV virus and in particular high titer viral suspensions obtained by inoculation with insects or with continuous lines of insects.
  • the present invention therefore relates to a method for cultivating the virus MAV (Mosquito African Virus), which is characterized in that the virus is cultivated on insect cells, in an appropriate culture medium, in that, approximately at the 6th hour of culture, the culture supernatant is removed, concentrated and purified in a sucrose gradient, to isolate the virus.
  • MAV Mosquito African Virus
  • the insect cells are constituted by continuous lines of insect cells of the Aedes type, in particular of Ae. aegypti, albopictus or pseudoscutellaris.
  • the viral titer of the culture supernatant reaches a maximum of 8.8 PFU, at the 6th hour and that the The titer decreases in parallel with the appearance of the cytopathogenic effect, which is rapid on the abovementioned cells.
  • the culture supernatant is concentrated using high molecular weight polyethylene glycol, in particular polyethylene glycol 6000.
  • the culture medium is advantageously minimum essential medium supplemented with 2% fetal calf serum.
  • the culture medium is advantageously Leibovitz medium supplemented with 10% fetal calf serum, glutamine and antibiotics.
  • the purification technique in a sucrose gradient is adapted from the technique described by VEZZA et al.: "Characterization of the viral RNA species of prototype dengue viruses" Am. J. trop. Med. Hyg., 1980, 29, 643-652.
  • the MAV virus strain was the subject of a deposit with the National Collection of Culture of Microorganisms (CNCM) held by the INSTITUT PASTEUR of PARIS, under N ° 1-591 dated August 29, 1986.
  • CNCM National Collection of Culture of Microorganisms
  • the MAV virus isolated in accordance with the present invention is in a very heterogeneous form and not very usual for a virus, represented in the appended FIG. 1, which shows virions of 50 nm in diameter, flat, depressed at center and appearing from the front or in profile, which gives the impression of polymorphism mentioned above.
  • the photograph of the MAV virus shown in FIG. 1 was obtained under the electron microscope, in the cell culture supernatant, at a magnification of ⁇ 225,000, with a negative staining with sodium phosphotungstate.
  • the MAV virus is. characterized in. this. that its RNA is single-stranded, non-infectious when it is highly purified, with a sedimentation constant equal to 36S in a sucrose gradient, having an adenylated end and - in that it exerts a rapid cytopathogenic effect on Aedes cells, including Aedes albopictus.
  • the MAV virus is further characterized in that its virion has three major proteins, with apparent molecular weights of 50, 50.5 and 24.5 KDaltons.
  • the titer is obtained by the range method on C6 / 36 cells developed by IGARACHI (cf. cited), under agarose; the reading is done by coloring the cells with neutral red after 48 hours.
  • the MAV virus isolated in accordance with the present invention is therefore further characterized by the fact that it is sensitive to acidic pH, but resistant to ether, to nonionic detergents and to temperature.
  • the mosquito strain used is a colony of Ae. aegypti, domestic form, harvested in Ngoyé (Diourbel Region, Senegal) in 1981 and since maintained in an insectarium.
  • a series of 100 females (series B) was inoculated intrathoracically with a virus suspension; in parallel, a control series of 100 females was also inoculated with the diluent alone (series A). The number of surviving mosquitoes was noted each day until the 21st day by an observer who did not know which series was infected.
  • a third series (series C) consisted of 200 neonate larvae placed in water used to raise the larvae of series B and still containing 7 cadavers of larvae. Mortality was noted until hatching of adults.
  • - Figure 2 shows the number of insects Ae. aegypti adult survivors as a function of time
  • - Figure 3 represents the survival of the preimaginal stages of these insects, as a function of time.
  • the main conclusions are as follows: 1) The virus is highly pathogenic intrathoracically. 2 °) It is transmitted from one generation to another as shown by the mortality rates of the first generation. 3 °) The virus seems to cause a decrease in fertility, although this phenomenon is less clear than its role on longevity. 4o) The larvae can become infected by the oral route: series C.
  • the virus very resistant to the ambient temperature, has a high contaminating power: the non-significant results, substantially identical to those of series B, observed at the second generation are most likely due to contamination of the control series which was raised in the same room as the B series, but with different material. 6 °)
  • the virus was regularly found in 10 second generation females (series B). It appears from the results described in the foregoing that the MAV virus discovered by the inventors as an agent contaminating cultures of insect cells, and more particularly of Ae. aegypti, and isolated in accordance with the present invention, is capable of constituting an effective agent for combating certain insect vectors of epidemics and in particular the center of mosquitoes such as Ae. aegypti, which is a major vector of arbovirus.

Abstract

New anticulicidian virus known as MAV, isolated from cultures of insects, particularly of the Aedes type. Compositions for combating insects, particularly of the genus Aedes, of which the active constituent is the MAV virus. Viral anticulicidian combating suspensions obtained by inoculation of MAV virus into insects, particularly of the Aedes type.

Description

VIRUS ANTICULICIDIEN ET COMPOSITIONS DE LUTTE CONTRE LES MOUSTIQUES DE CELA.ANTICULICIDAL VIRUS AND MOSQUITO CONTROL COMPOSITIONS THEREOF.
La présente invention est relative à l'isolement d'un nouveau virus anticulicidien et à son application comme moyen de lutte contre les moustiques.The present invention relates to the isolation of a new anti-mosquito virus and to its application as a means of combating mosquitoes.
Un virus baptisé MAV : Mosquito African Virus, a été découvert par les Inventeurs sur une lignée cellulaire d'Aedes aegypti, dans les conditions suivantes :A virus called MAV: Mosquito African Virus, was discovered by the Inventors on a cell line of Aedes aegypti, under the following conditions:
Les Inventeurs maintiennent dans un Insectarium situé à l'Institut Pasteur à DAKAR (SENEGAL) une colonie d'Aedes aegypti qu'ils utilisent pour obtenir la réplication de diverses souches de virus amaril; les moustiques inoculés sont broyés et leurs broyats sont ensemencés sur diverses cultures de cellules d'insectes pour obtenir une quantité de virus amaril suffisante pour effectuer des études biochimiques. C'est sur une de ces lignées cellulaires (Ae. aegypti, Clone C17, Deubel et Digoutte, Am. J. Trop. Med. Hyg., 1981, 30, 1060-1070) que le virus MAV a été découvert : il se développait sur ces cellules conjointement avec le virus amaril : sa constante de sédimentation (36S) est voisine de celle du virus amaril et, de ce fait, les empreintes d'oligonucleotides T1 du virus amaril sont systématiquement contami- nées par ce virus.The Inventors maintain in an Insectarium located at the Institut Pasteur in DAKAR (SENEGAL) a colony of Aedes aegypti which they use to obtain the replication of various strains of yellow fever virus; the inoculated mosquitoes are crushed and their crushed seeds are seeded on various insect cell cultures to obtain a quantity of yellow fever virus sufficient to carry out biochemical studies. It is on one of these cell lines (Ae. Aegypti, Clone C17, Deubel and Digoutte, Am. J. Trop. Med. Hyg., 1981, 30, 1060-1070) that the MAV virus was discovered: developed on these cells jointly with the yellow fever virus: its sedimentation constant (36S) is close to that of the yellow fever virus and, as a result, the fingerprints of T1 oligonucleotides of the yellow fever virus are systematically contaminated. born by this virus.
L'importante diminution de la longévité des individus d'une colonie d' Aedes aegypti infectés par ce virus a conduit les Inventeurs â tester celui-ci pour en évaluer l'éventuelle pathogénicité, car si celle-ci était confirmée, ce virus pourrait constituer un nouveau moyen de lutte contre le redoutable vecteur que constitue Aedes aegypti.The significant decrease in the longevity of individuals in a colony of Aedes aegypti infected with this virus has led the inventors to test it to assess its possible pathogenicity, because if it were confirmed, this virus could constitute a new means of combating the formidable vector that constitutes Aedes aegypti.
Toutefois, pour que ce virus puisse être utilisé contre Ae. aegypti, il doit répondre â deux conditions : la dépendance de l'hôte ne doit pas être sous dépendance génétique, ce qui, comme pour les insecticides, aboutirait à la sélection de souches résistantes; il doit d'autre part être inoffensif pour les vertébrés.However, in order for this virus to be used against Ae. aegypti, it must meet two conditions: the dependence of the host must not be under genetic dependence, which, as with insecticides, would lead to the selection of resistant strains; on the other hand, it must be harmless to vertebrates.
Les Inventeurs ayant pu établir que le virus MAV qu'ils ont identifié est hautement pathogène vis-à-vis d'Aedes aegypti, il s'est avéré alors important de pouvoir disposer de quantités suffisamment grarrdes de ce virus pour permettre de pourvoir à des moyens de lutte anticulicidienne comprenant le virus MAV et notamment des suspensions virales de titre élevé obtenues par inoculation à des insectes ou à des lignées continues d'insectes.The inventors having been able to establish that the MAV virus which they have identified is highly pathogenic with regard to Aedes aegypti, it has therefore proved important to be able to have sufficiently large quantities of this virus to allow providing for means of fight against mites comprising the MAV virus and in particular high titer viral suspensions obtained by inoculation with insects or with continuous lines of insects.
La présente invention a en conséquence pour objet un procédé de culture du virus MAV (Mosquito African Virus), qui est caractérisé en ce que le virus est cultivé sur des cellules d'insectes, dans un milieu de culture approprié, en ce que, environ à la 6e heure de culture, on prélève le surnageant de culture, on le concentre et on le purifie dans un gradient de saccharose, pour isoler le virus.The present invention therefore relates to a method for cultivating the virus MAV (Mosquito African Virus), which is characterized in that the virus is cultivated on insect cells, in an appropriate culture medium, in that, approximately at the 6th hour of culture, the culture supernatant is removed, concentrated and purified in a sucrose gradient, to isolate the virus.
Conformément à l'invention, les cellules d'insectes sont constituées par des lignées continues de cellules d'insectes du type des Aedes, notamment d' Ae. aegypti, albopictus ou pseudoscutellaris.According to the invention, the insect cells are constituted by continuous lines of insect cells of the Aedes type, in particular of Ae. aegypti, albopictus or pseudoscutellaris.
Pour l'étude biochimique et morphologique du virus MAV, il s'est avéré opportun de séparer le surnageant de culture de la culture cellulaire environ à la 6e heure de la culture, car il a été observé par les Inventeurs qu'en condition de cycle unique dans des cellules d'insectes, telles qu'une lignée de cellules d'Aedes albopictus, clone C6/36 (obtenue par IGARACHI : "Isolation of a Singh's Aedes albopictus cell clone sensitive to dengue and chikungunya viruses" J. gen. Virol.; 1978, 40, 531-544), le titre viral du surnageant de culture atteint un maximum de 8,8 UFP, â la 6e heure et que le titre décroit parallèlement â l'apparition de l'effet cytopathogène, qui est rapide sur les cellules susdites.For the biochemical and morphological study of the MAV virus, it has been found expedient to separate the culture supernatant from the cell culture approximately at the 6th hour of the culture, because it has been observed by the inventors that in a single cycle condition in insect cells, such as a cell line of Aedes albopictus, clone C6 / 36 (obtained by IGARACHI: "Isolation of a Singh's Aedes albopictus cell clone sensitive to dengue and chikungunya viruses "J. gen. Virol .; 1978, 40, 531-544), the viral titer of the culture supernatant reaches a maximum of 8.8 PFU, at the 6th hour and that the The titer decreases in parallel with the appearance of the cytopathogenic effect, which is rapid on the abovementioned cells.
Selon un mode de mise en oeuvre préféré du procédé conforme â la présente invention, le surnageant de culture est concentré â l'aide de polyéthylèneglycol de poids moléculaire élevé, notamment de polyéthylèneglycol 6000. Selon un autre mode de mise en oeuvre préféré du procédé conforme à la présente invention, le milieu de culture est avantageusement du milieu minimum essentiel additionné de 2% de sérum de foetus de veau.According to a preferred embodiment of the method according to the present invention, the culture supernatant is concentrated using high molecular weight polyethylene glycol, in particular polyethylene glycol 6000. According to another preferred embodiment of the conforming method to the present invention, the culture medium is advantageously minimum essential medium supplemented with 2% fetal calf serum.
Selon encore un autre mode de mise en oeuvre préféré du procédé conforme à la présente invention, le milieu de culture est avantageusement du milieu de Leibovitz additionné de 10% de sérum de veau foetal, de glutamine et d'antibiotiques.According to yet another preferred embodiment of the method in accordance with the present invention, the culture medium is advantageously Leibovitz medium supplemented with 10% fetal calf serum, glutamine and antibiotics.
La technique de purification dans un gradient de saccharose est adaptée de la technique décrite par VEZZA et Al. : "Characterization of the viral RNA species of prototype dengue viruses" Am. J. trop. Med. Hyg., 1980, 29 , 643-652.The purification technique in a sucrose gradient is adapted from the technique described by VEZZA et al.: "Characterization of the viral RNA species of prototype dengue viruses" Am. J. trop. Med. Hyg., 1980, 29, 643-652.
La souche de virus MAV a fait l'objet d'un dépôt auprès de la Collection Nationale de Culture de Microorganismes (CNCM) tenue par l'INSTITUT PASTEUR de PARIS, sous le N° 1-591 en date du 29 Août 1986.The MAV virus strain was the subject of a deposit with the National Collection of Culture of Microorganisms (CNCM) held by the INSTITUT PASTEUR of PARIS, under N ° 1-591 dated August 29, 1986.
Le virus MAV isolé conformément à la présente invention, se présente sous une forme très hétérogène et peu usuelle pour un virus, représentée à la figure 1 annexée, qui montre des virions de 50 nm de diamètre, plats, déprimés au centre et apparaissant de face ou de profil, ce qui donne l'impression de polymorphisme évoquée plus haut. La photographie du virus MAV représentée à la figure 1 a été obtenue au microscope électronique, dans le surnageant de culture cellulaire, à un grossissement de x 225 000, avec une coloration négative au phosphotungstate de sodium.The MAV virus isolated in accordance with the present invention is in a very heterogeneous form and not very usual for a virus, represented in the appended FIG. 1, which shows virions of 50 nm in diameter, flat, depressed at center and appearing from the front or in profile, which gives the impression of polymorphism mentioned above. The photograph of the MAV virus shown in FIG. 1 was obtained under the electron microscope, in the cell culture supernatant, at a magnification of × 225,000, with a negative staining with sodium phosphotungstate.
Les Inventeurs ont étudié l ' ARN du virus MAV en utilisant les techniques mises au point par DEUBEL et Al. pour le virus amaril [DEUBEL et Al. "Preliminary characterization of the ribonucleic acid of yellow fever virus" Ann. Virol. (Inst. Pasteur), 1983, 134E, 581-588] et ils ont déterminé la composition protéique du virion par électrophorèse en gel de polyacrylamide selon la technique de LAEMMLI : "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", NATURE (Londres), 1970, 227, 680- 685.The inventors studied the RNA of the MAV virus using the techniques developed by DEUBEL et Al. For yellow fever virus [DEUBEL et Al. "Preliminary characterization of the ribonucleic acid of yellow fever virus" Ann. Virol. (Inst. Pasteur), 1983, 134E, 581-588] and they determined the protein composition of the virion by polyacrylamide gel electrophoresis according to the LAEMMLI technique: "Cleavage of structural proteins during the assembly of the head of bacteriophage T4" , NATURE (London), 1970, 227, 680- 685.
Le virus MAV est. caractérisé en. ce. que son ARN est monocaténaire, non infectieux lorsqu'il est hautement purifié, de constante de sédimentation égale à 36S en gradient de saccharose, possédant une extrémité adénylêe et- en ce qu'il exerce un effet cytopathogène rapide sur des cellules d'Aedes, et notamment d'Aedes albopictus.The MAV virus is. characterized in. this. that its RNA is single-stranded, non-infectious when it is highly purified, with a sedimentation constant equal to 36S in a sucrose gradient, having an adenylated end and - in that it exerts a rapid cytopathogenic effect on Aedes cells, including Aedes albopictus.
Le virus MAV est en outre caractérisé en ce que son virion possède trois protéines majeures, de masses moléculaires apparentes de 50, 50,5 et 24,5 KDaltons.The MAV virus is further characterized in that its virion has three major proteins, with apparent molecular weights of 50, 50.5 and 24.5 KDaltons.
Les masses moléculaires apparentes susdites ont été déterminées par la technique de LAEMMLI citée plus haut, les trois protéines majeures ayant été identifiées par marquage â la (35s) méthionine. Les Inventeurs ont mis en évidence les caractéristiques physico-chimiques du nouveau virus isolé conformément à la présente invention; ces caractéristiques sont réunies dans le Tableau I qui va suivre. TABLEAU IThe above apparent molecular weights were determined by the LAEMMLI technique cited above, the three major proteins having been identified by labeling with ( 35 s) methionine. The inventors have demonstrated the physicochemical characteristics of the new virus isolated in accordance with the present invention; these characteristics are brought together in Table I which follows. TABLE I
Caractères physico-chimiques du virus MAVPhysico-chemical characteristics of the MAV virus
Figure imgf000007_0001
Figure imgf000007_0001
* Le titre est obtenu par la méthode des plages sur cellules C6/36 mise au point par IGARACHI (cf. cité), sous agarose; la lecture se fait par coloration des cellules au rouge neutre après 48 heures.* The titer is obtained by the range method on C6 / 36 cells developed by IGARACHI (cf. cited), under agarose; the reading is done by coloring the cells with neutral red after 48 hours.
Le virus MAV isolé conformément à la présente invention, se caractérise donc en outre par le fait qu'il est sensible aux pH acides, mais résistant à l'éther, aux dêtergents non ioniques et â la température.The MAV virus isolated in accordance with the present invention is therefore further characterized by the fact that it is sensitive to acidic pH, but resistant to ether, to nonionic detergents and to temperature.
Les Inventeurs ont en outre procédé à une étude de la pathogénicitê du virus MAV sur une colonie d'Aedes aegypti. Le compte-rendu de cette étude expérimentale est donné ci-après. ETUDE EXPERIMENTALE SUR AEDES AEGYPTI : 1. Méthodologie :The inventors have also carried out a study of the pathogenicity of the MAV virus on a colony of Aedes aegypti. The account of this experimental study is given below. EXPERIMENTAL STUDY ON AEDES AEGYPTI: 1. Methodology:
La souche de moustiques utilisée est une colonie d'Ae. aegypti, forme domestique, récoltée à Ngoyé (Région de Diourbel, Sénégal) en 1981 et entretenue depuis en insectarium.The mosquito strain used is a colony of Ae. aegypti, domestic form, harvested in Ngoyé (Diourbel Region, Senegal) in 1981 and since maintained in an insectarium.
Une série de 100 femelles (série B) a été inoculée par voie intrathoracique avec une suspension de virus; parallèlement une série témoin de 100 femelles également a été inoculée avec le seul diluant (série A). Le nombre de moustiques survivants a été noté chaque jour jusqu ' au 21e jour par un observateur qui ignorait quelle était la série infectée.A series of 100 females (series B) was inoculated intrathoracically with a virus suspension; in parallel, a control series of 100 females was also inoculated with the diluent alone (series A). The number of surviving mosquitoes was noted each day until the 21st day by an observer who did not know which series was infected.
La descendance de ces deux séries a été suivie jusqu'à la deuxième génération, en s'attachant à évaluer la fécondité des femelles et la mortalité de chacun des stades.The progeny of these two series were followed until the second generation, endeavoring to assess the fertility of the females and the mortality of each of the stages.
Une troisième série (série C) a été constituée par 200 larves néonates placées dans de l'eau ayant servi à élever les larves de la série B et contenant encore 7 cadavres de larves. La mortalité a été notée jusqu'à l'eclosion des adultes. 2. Résultats :A third series (series C) consisted of 200 neonate larvae placed in water used to raise the larvae of series B and still containing 7 cadavers of larvae. Mortality was noted until hatching of adults. 2. Results:
Les principaux paramètres étudiés sont donnés au Tableau II ci-après.The main parameters studied are given in Table II below.
TABLEAU IITABLE II
Figure imgf000008_0001
Figure imgf000009_0001
Les résultats obtenus sont également illustrés par les figures 2 et 3 annexées dans lesquelles
Figure imgf000008_0001
Figure imgf000009_0001
The results obtained are also illustrated by Figures 2 and 3 attached in which
- la figure 2 représente le nombre d'insectes Ae. aegypti adultes survivants en fonction du temps, et - la figure 3 représente la survie des stades préimaginaux de ces insectes, en fonction du temps. Les principales conclusions sont les suivantes : 1°) Le virus est hautement pathogène par voie intrathoracique. 2°) Il est transmis d'une génération à l'autre comme le montrent les taux de mortalité de la première génération. 3°) Le virus semble entraîner une diminution de la fécondité, bien que ce phénomène soit moins net que son rôle sur la longévité. 4º) Les larves peuvent s'infecter par voie orale : série C. 5°) Le virus, très résistant à la températire ambiante, a un haut pouvoir contaminateur : les résultats non significatifs, sensiblement identiques à ceux de la série B, observés à la deuxième génération sont très probablement dus à une contamination de la série témoin qui a été élevêe dans la même salle que la série B, mais avec un matériel différent. 6°) Le virus a été régulièrement retrouvé chez 10 femelles de la deuxième génération (série B). II ressort des résultats décrits dans ce qui précède que le virus MAV découvert par les Inventeurs en tant qu'agent contaminant de cultures de cellules d'insectes, et plus particulièrement d'Ae. aegypti, et isolé' conformément à la présente invention, est propre à constituer un agent efficace de lutte contre certains insectes vecteurs d'épidémies et notamment centre des moustiques tels qu ' Ae. aegypti , qui est un vecteur majeur d'arbovirus.- Figure 2 shows the number of insects Ae. aegypti adult survivors as a function of time, and - Figure 3 represents the survival of the preimaginal stages of these insects, as a function of time. The main conclusions are as follows: 1) The virus is highly pathogenic intrathoracically. 2 °) It is transmitted from one generation to another as shown by the mortality rates of the first generation. 3 °) The virus seems to cause a decrease in fertility, although this phenomenon is less clear than its role on longevity. 4º) The larvae can become infected by the oral route: series C. 5 °) The virus, very resistant to the ambient temperature, has a high contaminating power: the non-significant results, substantially identical to those of series B, observed at the second generation are most likely due to contamination of the control series which was raised in the same room as the B series, but with different material. 6 °) The virus was regularly found in 10 second generation females (series B). It appears from the results described in the foregoing that the MAV virus discovered by the inventors as an agent contaminating cultures of insect cells, and more particularly of Ae. aegypti, and isolated in accordance with the present invention, is capable of constituting an effective agent for combating certain insect vectors of epidemics and in particular the center of mosquitoes such as Ae. aegypti, which is a major vector of arbovirus.
Ainsi que cela ressort de ce qui précède, l'invention ne se limite nullement â ceux de ces modes de mise en oeuvre de réalisation et d'application qui viennent d'être décrits de façon plus explicite; elle en embrasse au contraire toutes les variantes qui peuvent venir à l'esprit du technicien en la matière, sans s'écarter du cadre, ni de la portée, de la présente invention. As is apparent from the above, the invention is in no way limited to those of these embodiments and of application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.
Figure imgf000012_0001
Figure imgf000012_0001

Claims

REVENDICATIONS 1°) Nouveau virus anticulicidien, dénommé MAV, isolé de cultures d'insectes, notamment du type des Aedes. CLAIMS 1 °) New virus against mites, called MAV, isolated from insect cultures, especially of the Aedes type.
2°) Virus MAV anticulicidien, caractérisé en ce que son ARN est monocaténaire, en ce que cet ARN est non infectieux, en ce que sa constante de sédimentation est égale à 36S en gradient de saccharose, en ce qu'il possède une extrémité adênylêe et en ce que ce virus exerce un effet cytopathogène sur les cellules d' Aedes. 3°) Virus MAV anticulicidien selon la revendication 2, caractérisé, en outre, en ce que son virion possède trois protéines majeures, de masses moléculaires apparentes de 50, 50,5 et 24,5 KDaltons.2) MAV anticulicidal virus, characterized in that its RNA is single-stranded, in that this RNA is non-infectious, in that its sedimentation constant is equal to 36S in sucrose gradient, in that it has an adenylated end and in that this virus exerts a cytopathogenic effect on Aedes cells. 3 °) virus MAV anticulicidien according to claim 2, characterized, in addition, in that its virion has three major proteins, of apparent molecular masses of 50, 50.5 and 24.5 KDaltons.
4°) Virus MAV selon l'une quelconque des revendica- tions 1 à 3, également caractérisé par le fait qu'il est sensible aux pH acides, mais résistant à l'éther, aux détergents non ioniques et à la température.4) MAV virus according to any one of claims 1 to 3, also characterized in that it is sensitive to acid pH, but resistant to ether, to nonionic detergents and to temperature.
5°) Composition de lutte contre les insectes, et notamment contre les insectes du genre des Aedes, caractérisée en ce qu'elle comprend en tant que constituant actif, une quantité appropriée de virus MAV selon l'une quelconque des revendications 1 à 4.5) Composition for combating insects, and in particular against insects of the genus Aedes, characterized in that it comprises, as active ingredient, an appropriate quantity of MAV virus according to any one of Claims 1 to 4.
6º) Suspension virale de lutte anticulicidienne, caractérisée en ce qu'elle est obtenue par inoculation du virus selon l'une quelconque des revendications 1 à 4, à des insectes, notamment du type des Aedes, ou à des lignées continues de cellules desdits insectes.6º) Viral suspension for control of mites, characterized in that it is obtained by inoculation of the virus according to any one of claims 1 to 4, to insects, in particular of the Aedes type, or to continuous lines of cells of said insects .
7º) Procédé de culture du virus MAV (Mosquito African Virus), dont une souche a été déposée auprès de la Collection Nationale de Culture de Microorganismes (CNCM) tenue par l'INSTITUT PASTEUR de PARIS, sous le N° 1-591 en date du 29 Août 1986, caractérisé en ce que le virus est cultivé sur des cellules d'insectes, dans un milieu de culture approprié, en ce que, environ à la 6e heure de culture, on prélève le surnageant de culture, on le concentre et on le purifie dans un gradient de saccharose, pour isoler le virus.7º) Culture process of the virus MAV (Mosquito African Virus), of which a strain was deposited with the National Collection of Culture of Microorganisms (CNCM) held by the INSTITUT PASTEUR of PARIS, under N ° 1-591 in date of August 29, 1986, characterized in that the virus is cultivated on insect cells, in an appropriate culture medium, in that, approximately at the 6th hour of culture, the culture supernatant is removed, concentrated and we purify it in a sucrose gradient, to isolate the virus.
8°) Procédé selon la revendication 7, caractérisé en ce que le surnageant de culture est concentré à l'aide de polyéthylèneglycol de poids moléculaire élevé, notamment de polyéthylèneglycol 6000.8 °) Process according to claim 7, characterized in that the culture supernatant is concentrated using polyethylene glycol of high molecular weight, in particular polyethylene glycol 6000.
9º) Procédé selon l'une quelconque des revendications 7 et 8, caractérisé en ce que le milieu de culture est avantageusement un .milieu minimum essentiel additionné de serum de foetus de veau à concentration finale par exemple de 2%.9º) A method according to any one of claims 7 and 8, characterized in that the culture medium is advantageously a minimum essential .milieu added serum of calf fetus at a final concentration for example of 2%.
10º) Procédé selon l'une quelconque des revendications 7 à 9, caractérisé en ce que le milieu de culture est avantageusement du milieu de Leibovitz additionné de sérum de veau foetal à concentration finale par exemple de 10%, de glutaminé et d'antibiotiques.10º) Method according to any one of claims 7 to 9, characterized in that the culture medium is advantageously Leibovitz medium supplemented with fetal calf serum at a final concentration for example of 10%, glutamine and antibiotics.
11°) Procédé selon l'une quelconque des revendications 7 à 10, caractérisé en ce que les cellules d'insectes sont constituées par des lignées continues de cellules d'insectes du type des Aedes, notamment d'Ae. aegypti, albop ictus ou pseudoscutellaris. 11 °) Method according to any one of claims 7 to 10, characterized in that the insect cells are constituted by continuous lines of insect cells of the Aedes type, in particular of Ae. aegypti, albop ictus or pseudoscutellaris.
PCT/FR1987/000351 1986-09-11 1987-09-10 Anticulicidian virus and compositions obtained therefrom for combating mosquitos WO1988001833A1 (en)

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FR8612703A FR2603898B1 (en) 1986-09-11 1986-09-11 NOVEL ANTICULICIDAL VIRUS, METHOD FOR ISOLATING SAME, AND MOSQUITO CONTROL COMPOSITIONS CONTAINING SAME OR VIRUS PROVIDED THEREWITH
FR86/12703 1986-09-11

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WO1994004660A1 (en) * 1992-08-14 1994-03-03 Commonwealth Scientific & Industrial Research Organisation Insect viruses and their uses in protecting plants

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US4166112A (en) * 1978-03-20 1979-08-28 The United States Of America As Represented By The Secretary Of The Navy Mosquito larvae control using a bacterial larvicide
FR2552627A1 (en) * 1983-09-30 1985-04-05 Rhone Poulenc Agrochimie Oily pesticidal compositions based on entomopathogenic viruses.

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US4166112A (en) * 1978-03-20 1979-08-28 The United States Of America As Represented By The Secretary Of The Navy Mosquito larvae control using a bacterial larvicide
FR2552627A1 (en) * 1983-09-30 1985-04-05 Rhone Poulenc Agrochimie Oily pesticidal compositions based on entomopathogenic viruses.

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Publication number Priority date Publication date Assignee Title
WO1994004660A1 (en) * 1992-08-14 1994-03-03 Commonwealth Scientific & Industrial Research Organisation Insect viruses and their uses in protecting plants

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