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WO2013026994A1 - Moustiques présentant une résistance accrue aux agents pathogènes - Google Patents

Moustiques présentant une résistance accrue aux agents pathogènes Download PDF

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WO2013026994A1
WO2013026994A1 PCT/GB2011/051600 GB2011051600W WO2013026994A1 WO 2013026994 A1 WO2013026994 A1 WO 2013026994A1 GB 2011051600 W GB2011051600 W GB 2011051600W WO 2013026994 A1 WO2013026994 A1 WO 2013026994A1
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pathogen
wolbachia
virus
mosquito
mosquitoes
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Steven Paul SINKINS
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Oxford University Innovation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • A01K67/68—Genetically modified insects
    • 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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated

Definitions

  • the present invention relates to mosquitoes with enhanced resistance to pathogens, and to the uses of mosquitoes as biological agents for the reduction of pathogen transmission, and to biological agents for such use.
  • the invention also relates to methods of producing mosquitoes, and to methods of modifying populations of mosquitoes.
  • Dengue virus causes dengue fever in infected humans. It is estimated that between 50 million and 100 million people are infected with the dengue virus yearly, and the virus is endemic in more than 1 10 countries worldwide.
  • the Asian tiger mosquito Aedes albopictus a native of south-east Asia that in recent decades has invaded Africa, the Americas and southern Europe, is an important vector of dengue virus in rural and semi-urban areas across the tropics. It is probably involved in maintaining sylvatic cycles of transmission and acting as a bridge vector from these to urban epidemic cycles; it also transmits other Flaviviruses such as yellow fever and West Nile, and the Alphavirus chikungunya. Like the primary urban dengue vector Aedes aegypti, Ae. albopictus is a day biting species and therefore not amenable to control or prevention using insecticide-treated bednets.
  • the invention provides a mosquito of the species Aedes albopictus comprising a Wolbachia bacterium of the strain i i Mel, wherein the mosquito has enhanced resistance to a pathogen.
  • the pathogen may be a viral pathogen, such as dengue virus.
  • Mosquitoes in accordance with the present invention are unable to transmit dengue virus, thus making them suitable agents for use to prevent dengue virus transmission.
  • the mosquitoes of the invention may be crossed with native mosquito populations to reduce the total pool of virus-carrying mosquitoes in a locality. To the best of the inventor's knowledge, these are the first such examples of Aedes albopictus, which is normally an important vector of dengue virus, that are unable to propagate the virus.
  • the present invention is based, at least in part, on the inventor's surprising finding that infection of Aedes albopictus mosquitoes with the i i Mel strain of Wolbachia gives rise to mosquitoes that have a markedly enhanced resistance to pathogens, such as pathogenic viruses.
  • Aedes albopictus is normally host to Wolbachia strains designated wAlbA and wAIbB, while the i i/Mel strain is naturally found in Drosophila melanogaster. Since Wolbachia bacteria, such as wMel, are obligate intracellular bacteria they would not be expected to transfer between species without artificial intervention (since no breeding or other natural propagation can occur between Aedes albopictus and Drosophila melanogaster).
  • cytoplasmic incompatibility is unidirectional in the case of mating between a mosquito comprising wMel and a mosquito in which no form of Wolbachia is present, and is bidirectional in cases in which a mosquito comprising wMel mates with a mosquito comprising another Wolbachia strain.
  • mosquitoes of the invention comprising wMel also comprise wAlbA and/or wAIbB.
  • wMel As described in more details elsewhere in the specification, the ability of wMel to induce bidirectional cytoplasmic incompatibility between mosquitoes of the invention and other mosquito populations is highly beneficial, since this provides a method by which wMel can extensively permeate throughout a local mosquito population. Since such mosquitoes (mosquitoes in accordance with the invention) are resistant to pathogens, and thus provide biological agents capable of inhibiting transmission of such pathogens, this provides great benefits to humans or other animals. When pathogen transmission is inhibited, incidences of infection, and so illness, among human populations are also reduced.
  • mosquitoes comprising wMel exhibit bidirectional cytoplasmic incompatibility with mosquitoes comprising other Wolbachia strains is surprising, since there is nothing among previously published reports that would lead the skilled person to believe that this would be the case.
  • a previous report showed that the i i MelPop strain when transferred from Drosophila melanogaster into Ae. albopictus, did not produce bidirectional cytoplasmic incompatibility with mosquitoes comprising the naturally occurring ⁇ ⁇ AlbA and i i/AIbB strains.
  • a mosquito in accordance with the present invention for use as a biological agent for the reduction of pathogen transmission.
  • the invention provides a biological agent for the reduction of pathogen transmission comprising a mosquito according to the present invention.
  • the mosquitoes of the invention which comprise wMel bacteria, may be used as agents that can mate with, and thus modify biological properties of, other mosquito populations within an area.
  • the invention also provides, in a fourth aspect, a method of modifying a population of Aedes albopictus mosquitoes, the method comprising introducing an Aedes albopictus mosquito according to the first aspect of the invention into the population, wherein the introduction of the Aedes albopictus comprising the wMel strain of Wolbachia modifies one or more biological properties of the Aedes albopictus population.
  • introducing may comprise release, or any other suitable mechanism, of mosquitoes according to the present invention in an area where they are able to mate with the Aedes albopictus population to be modified.
  • One of the biological properties of the Aedes albopictus population that may be modified by the introduction of mosquitoes according to the invention is the tendency of mosquitoes of the population to transmit a pathogen (which may optionally be achieved by enhancing resistance of the population of mosquitoes to the pathogen).
  • embodiments of the methods of the invention may be used to inhibit transmission of a pathogen by the Aedes albopictus population.
  • Pathogens, the transmission of which is to be inhibited, or resistance to which is to be enhanced include viral or nematode pathogens and may be selected from those considered elsewhere in the specification.
  • mosquitoes of the invention comprising wMel do not appear to suffer from reduced lifespan, and this represents a preferred embodiment of the invention.
  • This provides an advantage in that the spread dynamics of Wolbachia are not adversely affected.
  • Previous attempts to produce mosquitoes suitable for use as biological agents to reduce pathogen transmission have tended to yield short-lived insects.
  • a mosquito comprising Wolbachia may readily be produced by infection of a suitable mosquito species using a desired Wolbachia strain. Examples of suitable techniques that may be used to infect mosquitoes with Wolbachia are described elsewhere in the specification, including the Experimental Results section.
  • the invention also provides a method of producing an Aedes albopictus mosquito comprising i i Mel, the method comprising treating a native population of Aedes albopictus to remove native Wolbachia bacteria; and infecting the population of treated mosquitoes with the i i Mel strain of Wolbachia.
  • the native Wolbachia bacteria may suitably be selected from ⁇ ⁇ /AlbA and/or i i/AlbB.
  • a "native population" of mosquitoes may be a population present in an area where it is desired to influence biological activity of mosquitoes, for example by inhibiting transmission of pathogens by the mosquitoes.
  • Use of a “native population” in this manner may be beneficial in that the mosquitoes may be expected to be well adapted to the environment found in the area where they are to be used.
  • “native bacteria” may be the bacteria, such as Wolbachia or other bacterial parasites, naturally occurring within the mosquito population.
  • the treatment to remove native bacteria comprises antibiotic treatment of the native mosquitoes (such as Aedes albopictus).
  • antibiotic treatment comprises injection of antibiotics, such as rifampicin, into mosquito embryos. More details of these methods are provided in the Materials and Methods section of the Experimental Results described below.
  • the present invention in many of its' aspects and embodiments, relates to mosquitoes, methods of producing mosquitoes and also uses of such mosquitoes.
  • a number of the aspects of the invention specify that the mosquitoes in question are of the species Aedes albopictus, and this species represents a preferred form of mosquito in accordance with most aspects and embodiments of the invention. As described elsewhere this species is of major importance due to its role as a vector of pathogens harmful to humans and other animals, and the finding that resistance of this species to pathogens can be enhanced by the wMel strain of Wolbachia is both surprising and highly beneficial.
  • the inventor does not believe that the advantages noted are limited solely to this species of mosquito, and believe that, to a great extent, it is the wMel strain of Wolbachia that has most importance in mediating enhanced pathogen resistance, rather than the mosquito species infected by i i Mel. Accordingly, the invention provides aspects and embodiments that are not limited to this species. Certain aspects and embodiments of the invention are applicable to mosquitoes more generically, and a sixth aspect of the invention provides a mosquito comprising a Wolbachia bacterium of the strain i i Mel, wherein the mosquito has enhanced resistance to a pathogen. In a seventh aspect the invention provides a mosquito according to the sixth aspect of the invention for use as a biological agent for the reduction of pathogen transmission.
  • the invention provides a biological agent for the reduction of pathogen transmission comprising a mosquito in accordance with the sixth aspect of the invention.
  • a ninth aspect of the invention provides a method of producing a mosquito comprising a bacterium of interest, the method comprising treating a native population of mosquitoes to remove native bacteria; and crossing the population of treated mosquitoes with a line carrying the bacterium of interest.
  • the invention provides a method of modifying a population of mosquitoes, the method comprising introducing a mosquito comprising a bacterium of interest into the population, wherein the introduction of the mosquito comprising the bacterium of interest modifies one or more biological properties of the population of mosquitoes.
  • the "bacterium of interest” may be one (such as i i/Mel) that confers a desirable property to the mosquitoes, such as enhanced resistance to a pathogen.
  • An eleventh aspect of the invention provides a mosquito comprising a bacterium of interest, wherein the bacterium of interest confers upon the mosquito enhanced resistance to a pathogen.
  • the resistance to pathogen(s) of a mosquito in accordance with this aspect of the invention, or other aspects of the invention may be assessed with reference to resistance to the same pathogen(s) exhibited by a mosquito that lacks i i Mel (or another bacterium of interest).
  • particular embodiments may make use of species of mosquito such as Aedes polynesiensis.
  • Suitable pathogens in the context of these preceding aspects of the invention may be the same as those described elsewhere in the specification, and include viral pathogens such as dengue virus and Chikungunya virus.
  • any of the aspects of the invention mentioned above may make use of details of the various embodiments described in connection with aspects of the invention utilising Aedes albopictus and/or i i/Mel.
  • a reference to "a mosquito comprising a bacterium” or "a mosquito infected with a bacterium” in the context of the present invention should, except for where the context requires otherwise, be taken as encompassing such a mosquito at any stage of its life cycle, and also as encompassing any isolated part or parts of such a mosquito.
  • a reference to a mosquito should, generally, be taken as encompassing a reference to an adult mosquito, but also to an egg, embryo, larval or juvenile form of such a mosquito. Any such reference should also be taken as encompassing isolated tissues, organs or cells of such a mosquito, so long as the parts in question contain the bacterium (which may be expected to be located in the cytoplasm).
  • references should also be taken as encompassing the reproductive material of such mosquitoes, since it is these tissues that mediate cytoplasmic incompatibility. While sperm of mosquito comprising a wMel bacterium will not in themselves comprise the bacterium, they will be characteristically modified as a result of the bacterium's presence in the mosquito. Except for where the context requires otherwise references to "a mosquito comprising a bacterium” or references to "a mosquito infected with a bacterium” in the various aspects and embodiments of the invention should also be taken as encompassing sperm comprising such modifications characteristic of wMel infection. i i Mel wMel is a strain of Wolbachia found in Drosophila melanogaster.
  • mosquitoes in accordance with the present invention exhibit enhanced resistance to pathogens, and may be used as biological agents for the reduction of pathogen transmission. Except for where the context requires otherwise, the following considerations will be applicable to any reference to a pathogen, or pathogens, in relation to any aspect or embodiment of the invention.
  • the pathogen may be a viral pathogen.
  • viral pathogens include, but are not limited to, those selected from the group consisting of: dengue virus; Chikungunya virus; West Nile virus; St. Louis encephalitis virus; Rift Valley fever virus; and yellow fever virus.
  • the mosquitoes of the present invention may have enhanced resistance to a nematode pathogen.
  • a nematode pathogen include Dirofilaria immitis (which gives rise to damaging infections in hosts such as dogs or humans), and Wuchereria bancrofti (which can cause elephantitis in infected individuals).
  • Dirofilaria immitis which gives rise to damaging infections in hosts such as dogs or humans
  • Wuchereria bancrofti which can cause elephantitis in infected individuals.
  • an Aedes albopictus mosquito in accordance with the invention may have enhanced resistance to Dirofilaria immitis
  • an Aedes polynesiensis mosquito in accordance with the invention may have enhanced resistance to Wuchereria bancrofti.
  • references to enhance resistance to a single pathogen or class of pathogens should generally be taken as encompassing the same activity in respect of one or more pathogens, or indeed in respect of a range of pathogens.
  • a mosquito in accordance with the present invention may have enhanced resistance to two or more viral pathogens, and additionally, or alternatively, may have enhanced resistance to two or more nematode pathogens.
  • a mosquito of the invention may be able to reduce transmission of two or more viral pathogens, and additionally, or alternatively, may be able to reduce transmission of two or more nematode pathogens. Cytoplasmic incompatibility
  • Wolbachia pipientis is a maternally inherited intracellular bacterial symbiont of invertebrates that is common in insects. It can manipulate insect reproduction by inducing sterility when individuals of different Wolbachia carrier status mate with each other, known as cytoplasmic incompatibility or CI.
  • CI cytoplasmic incompatibility
  • the basic form of CI is observed when females that do not carry Wolbachia when they mate with males that do, leading to early embryonic death of the offspring. Although it is not itself transmitted through sperm, Wolbachia modifies these sperm of carrier males so that they are unable to complete the process of fertilization.
  • Wolbachia is able to spread spatially using this mechanism of unidirectional CI.
  • a second form known as bidirectional CI is only seen in crosses between individuals that both carry Wolbachia, but of different strains. In this case both directions of the cross are incompatible, and produce no hatching eggs. If populations carrying such mutually incompatible strains came into contact in nature, an unstable equilibrium would result, and whichever formed the local majority would be expected to reach 100% frequency while the other strain would be eliminated, because its females would more frequently encounter and mate with males with which they are compatible (see fig. 2).
  • Bidirectional CI therefore provides a method to stably introduce Wolbachia into target populations, and once fixed it would be stable to further immigration of individuals carrying the strain with which they are incompatible.
  • bidirectional CI gives spatial control over the process of population replacement with an introduced strain, which in some circumstances would be a very useful feature. If bidirectionally incompatible Wolbachia strains are combined to form strain superinfections, superinfected males will be incompatible with females that lack one or more of the Wolbachia strains in the male, due to the absence of the rescue factor in the egg for the strain it lacks. Because the reciprocal cross is fully compatible, these superinfections are expected to spread through populations that lack one of the strains, exactly in the same way as Wolbachia spreads through a population that does not carry the bacterium at all.
  • the i i Mel strain occurs naturally in the fruit fly Drosophila melanogaster and its genome sequence has been published (Wu et al. 2003).
  • the i i/MelPop strain is also naturally found in Drosophila melanogaster, and i i/Mel and i i MelPop are phylogenetically quite closely related (Sun et at. 2003). They were thought to produce the same crossing type, since lines carrying these two Wolbachia strains were compatible with each other when crossed in Drosophila (McGraw et at. 2002).
  • the i i MelPop strain did not produce CI when introduced into Ae.
  • Figure 1 illustrates Uju.i i Mel crossing type.
  • Experiments to characterize the crossing type of the Uju. i i/Mel line at G 6 were performed using UjuT and Ascoli strains. Error bars represent the standard error of the mean of hatch rates between females; fifteen adult males and fifteen females were used for each mass cross.
  • a second round of crossing experiments was performed two generations later (G 8 ) using Uju.i i/Mel x Uju.wMel, Ascoli x Uju. i i Mel, Uju. i i/Mel x Ascoli and Uju.i i Mel x UjuT according to the same procedure as the previous experiment.
  • FIG. 2 illustrates transmission capacity (A) and titer of dengue virus in mosquito saliva (B).
  • A transmission capacity
  • B titer of dengue virus in mosquito saliva
  • Three Ae. albopictus strains Uju.wAlbA/wAIbB, UjuT, and Uju.wMel were orally infected with dengue 2 virus using glass feeders covered with a chicken skin membrane containing the infectious blood-meal at a titer of 10 7 FFU/mL. After blood-feeding, mosquitoes were transferred in cardboard containers and maintained in BSL3 insectaries at 28°C.
  • FIG. 3 illustrates immune gene expression in Wolbachia infected and uninfected mosquitoes.
  • the expression of four Ae. albopictus orthologs for Ae. aegypti immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, PGRPS1; cecropin D, CECD; CLIP-domain serine protease, CLIPB37; and a thioester-containing protein, TEP20.
  • Fig. 3B Adult females were transiently infected with Wolbachia using intrathoracic injections with either i i MelPop or ⁇ ⁇ AlbB, or controls of either heat killed E. coli, or the buffer alone, approximately three days post-eclosion. RNA was extracted five days after injection. The expression of four Ae. aegypti immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, PGRPS1; cecropin D, CECD; CLIP-domain serine protease, CLIPB37; and a thioester-containing protein, TEP20. Orthologs for these genes in Ae. albopictus were also analyzed by qRT-PCR.
  • FIG. 5 (from Sinkins and Gould 2005) Unidirectional CI between insects carrying Wolbachia (red) and not carrying Wolbachia (green). By providing a frequency-dependent reproductive advantage to carrier females, which unlike their counterparts without Wolbachia can mate successfully with any male in this mixed population, Wolbachia can rapidly increase in frequency.
  • Figure 6 Bidirectional CI can only occur between insects carrying different Wolbachia strains; these strains cannot stably co-exist within a population, and whichever strain is in the majority will replace the other.
  • the i i/MelPop strain also causes chronic immune upregulation in Ae. aegypti.
  • the Toll pathway some components of which are upregulated in Ae. aegypti in the presence of i i MelPop, has previously been shown to play a role in the control of dengue dissemination in Ae. aegypti.
  • a general role of immune upregulation in pathogen inhibition is also supported by the knockdown of the major immune gene TEP1 which partly rescued the inhibitory effect of the presence of i i/MelPop on Plasmodium berghei development in transiently infected Anopheles gambiae.
  • the i i/MelPop strain over-replicates and can approximately halve the lifespan of both its D. melanogaster and Ae. aegypti hosts.
  • a i i/MelPop transinfection into Ae. albopictus also produced a greatly reduced egg hatch from intra-strain matings, and this appeared to preclude its application to disease control in Ae. albopictus.
  • the i i Mel strain which is phylogenetically close to the i i MelPop variant, does not produce the life- shortening phenotype of the latter in its native Drosophila melanogaster host.
  • i i/Mel can significantly delay the accumulation of RNA viruses such as Drosophila C virus in D. melanogaster. Therefore i i/Mel was selected for experimental transfer into Ae. albopictus, in order to examine whether this strain is capable of producing dengue inhibition and CI in this new host background.
  • the Wolbachia uninfected Ae. albopictus strain UjuT was generated by tetracycline treatment by Dr Yasushi Otsuka, Oita Medical University, Japan; the Ascoli strain of Ae. albopictus was colonized from San Benedetto del Tronto, Italy in 2006 by G. Favia and colleagues; and the Ae. aegypti Rockefeller strain originated in the Caribbean in the 1930's. All colonies and lines were kept at 27°C and 70% relative humidity, and a 12 hour light/dark cycle.
  • the i i/AlbA and ⁇ ⁇ /AlbB strains of Wolbachia were introgressed into the UjuT background for four generations by removing all male pupae from one colony of the Ascoli strain and providing an approximately equal number of UjuT males.
  • the resulting line was thus approximately 94% UjuT nuclear background, and contained both ⁇ ⁇ AlbA and ⁇ ⁇ /AlbB. This line was only used for dengue infection, in order to partly control for any effects of host background.
  • the i i Mel strain of Wolbachia was transferred from Drosophila melanogaster i i/ 1118 embryos into Ae. albopictus (UjuT) by the transfer of cytoplasm.
  • Adult Drosophila were encouraged to oviposit using apple-juice agar plates and yeast paste.
  • Eggs were collected approximately 30mins post-oviposition.
  • Ae. albopictus were encouraged to lay eggs by placing around fifteen females, blood-fed seven days earlier, into a small (3cm diameter, 10cm height) plastic vial with moist filter paper on the bottom. Eggs were collected approximately 30mins post-oviposition.
  • Ae. albopictus eggs were allowed to desiccate for 15-30mins.
  • Both donor and recipient eggs were aligned on a nitrocellulose membrane and transferred to a glass slide using double sided tape. The eggs were then covered with Voltalef oil ready for injection. Cytoplasm was aspirated from the posterior of the donor eggs using a FemtoJet microinjector (Eppendorf) and injected into the posterior of recipient eggs. After a short incubation time, eggs were transferred onto wet filter paper, stored at 100% humidity at 27 5 C for five days, and then hatched in deoxygenated water. G 0 larvae were reared using standard conditions. Females were separated 1 - 2 days after blood feeding into small plastic vials with moist filter paper on the bottom.
  • Eppendorf FemtoJet microinjector
  • qPCR Quantitative PCR
  • DNA was extracted from adult mosquitoes using the Livak method. DNA was diluted to 100 ng/ ⁇ using a Nanodrop spectrophotometer. Thermocycler conditions and reaction chemistry followed the same protocol as for qRT-PCR. Primer pairs used for qPCR and qRT-PCR are listed in table 1 .
  • Ae. albopictus primers were designed using sequence data generated using either degenerate primers based on Ae. aegypti sequence data (from Vectorbase) or from Ae. albopictus EST data from NCBI.
  • Engorged mosquitoes were transferred in cardboard containers, provided with sucrose solution and maintained in BSL-3 insectaries at 28 °C for 14 days.
  • Saliva was collected using the forced salivation technique, which consists of inserting a capillary tube containing fetal calf serum in the proboscis of females whose legs and wings had been removed. After 45 min, saliva was collected and titrated by focus fluorescent assay on C6/36 Aedes albopictus cell culture. The transmission capacity was estimated as the percentage of mosquitoes with infectious saliva among tested mosquitoes.
  • FBS fetal bovine serum
  • 69 nl_ of Wolbachia suspension (or 69 nl_ Schneider's/ E. coli suspension for the controls) was microinjected into the thorax of around three day old Ae. aegypti Rockefeller / Ae. albopictus UjuT strains using a Nanoject microinjector (Drummond).
  • the mosquitoes were supplied with water and sucrose and left for five days prior to qRT-PCR experiments.
  • the Wolbachia strain present was confirmed to be i i Mel by sequencing the wsp ( Wolbachia surface protein) gene, which showed 100% identity with published i i Mel wsp sequence.
  • the transinfected strain initially showed reduced hatch rates compared to wild-type females, 46.1 ⁇ 7.8% hatch for Uju.i i Mel in G 6 compared to 65.1 ⁇ 10.7% for UjuT. Following two successive generations of selection for high hatch rates, the Uju.i i/Mel x Uju.i i Mel hatch rate had risen to 56.4 ⁇ 9.4% by G 8 (figure 1 ).
  • G 8 figure 1
  • a similar effect has previously been observed in newly transinfected Drosophila and Ae. aegypti. The effects of i i/Mel on fecundity of Ae.
  • the transinfected Uju.wMel strain was challenged with dengue 2 virus provided in an artificial blood-meal and after fourteen days showed a complete inhibition of dengue transmission capacity, with no infectious viral particles detected in the saliva of any tested mosquito.
  • transient somatic infections of i i MelPop and ⁇ ⁇ /AlbB were also created in Ae. aegypti and Ae. albopictus using intrathoracic inoculation as described in and.
  • Adult females were injected with suspensions of Wolbachia purified from Ae. albopictus cell lines (Aa23) approximately three days after eclosion.
  • the transcription of immune genes was measured by qRT-PCR at five days post injection (figure 3b). Strong immune upregulation was observed in Ae.
  • the concentration of i i Mel in Uju.wMel was compared to the combined concentration of both i i/AlbA and ⁇ ⁇ /AlbB in the Ascoli strain using adults 11 days post eclosion.
  • the ratio between Wolbachia WSP DNA and host S17 DNA was used to estimate the concentration of Wolbachia.
  • the concentration of i i/Mel was found to be approximately seven times that of the total concentration of Wolbachia in the superinfected Ascoli strain (figure 4). Novel method for removing Wolbachia infections in Ae. albopictus
  • Crossing the lVo/£>ac/7/ ' a-cured males with i i/Mel-carrying females for six or more generations allows the introduction of the i i Mel Wolbachia into the Ascoli (Italy) genetic background, or any other genetic background of a target wild Ae. albopictus population, in order to improve the local competitiveness of the insects to be released.
  • Uju. i i Mel produces complete bidirectional CI with the wild-type Ascoli line (containing a i i/AlbA and ⁇ ⁇ AlbB superinfection, as do all known wild populations of this species).
  • the i i/Mel crossing type unexpectedly differs from the crossing type of a i i/MelPop transinfection into Ae. albopictus, given the phylogenetic similarity between i i Mel and i i MelPop.
  • Bidirectional CI provides a method to stably introduce Wolbachia into populations, since bidirectionally incompatible crossing types cannot stably co-exist; whichever strain is at a local majority would be at a reproductive advantage, because its females would more frequently encounter and mate with males with which they are compatible. Assuming the complete bidirectional CI will also be produced under field conditions, once the i i Mel infection reaches a population majority, it would then be expected to go to local fixation and be stable to further immigration of ⁇ ⁇ /AlbA / i i AlbB- infected (wildtype) individuals with which they are incompatible.
  • albopictus line that may provide the basis of a viable new option for dengue control in this species. It also demonstrates that both of the two main vectors of dengue globally are amenable to a lVo/£>ac/7/ ' a-based strategy - improving the prospects for elimination of the disease.

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Abstract

La présente invention concerne des moustiques de l'espèce Aedes albopictus abritant une bactérie Wolbachia de souche w Mel, lesdits moustiques présentant une résistance accrue à un agent pathogène. Ledit agent pathogène peut être un virus pathogène, par exemple le virus de la dengue, le virus du chikungunya, le virus du Nil occidental, le virus de l'encéphalite de Saint-Louis, le virus de la fièvre de la vallée du Rift ou le virus de la fièvre jaune. Ledit agent pathogène peut être un nématode pathogène, par exemple Dirofilaria immitis ou Wuchereria bancrofti. Ladite bactérie peut induire une incompatibilité cytoplasmique et, notamment, une incompatibilité cytoplasmique bidirectionnelle. L'invention concerne également l'utilisation desdits moustiques et des procédés de génération et d'utilisation desdits moustiques.
PCT/GB2011/051600 2011-08-24 2011-08-24 Moustiques présentant une résistance accrue aux agents pathogènes Ceased WO2013026994A1 (fr)

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WO2015170322A2 (fr) 2014-05-04 2015-11-12 Forrest Innovations Ltd. Compositions et leurs procédés d'utilisation pour accroître la résistance des moustiques infectés
CN108377976A (zh) * 2018-03-02 2018-08-10 广州威佰昆生物科技有限公司 电动蚊卵定量器、蚊卵定量孵化器及蚊卵定量方法
WO2019000028A1 (fr) * 2017-06-28 2019-01-03 Monash University Bactérie adaptée aux moustiques contre des virus hautement infectieux transmis par les moustiques
CN111317733A (zh) * 2020-03-24 2020-06-23 中国人民解放军海军军医大学 利福霉素类抗生素在制备抗黄热病毒感染药物中的应用
WO2021189444A1 (fr) * 2020-03-24 2021-09-30 中国人民解放军海军军医大学 Utilisation d'antibiotiques du type rifamycine dans la préparation de médicaments contre des infections par le virus de la fièvre jaune

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015170322A2 (fr) 2014-05-04 2015-11-12 Forrest Innovations Ltd. Compositions et leurs procédés d'utilisation pour accroître la résistance des moustiques infectés
WO2019000028A1 (fr) * 2017-06-28 2019-01-03 Monash University Bactérie adaptée aux moustiques contre des virus hautement infectieux transmis par les moustiques
CN108377976A (zh) * 2018-03-02 2018-08-10 广州威佰昆生物科技有限公司 电动蚊卵定量器、蚊卵定量孵化器及蚊卵定量方法
CN108377976B (zh) * 2018-03-02 2024-03-15 广州威佰昆生物科技有限公司 电动蚊卵定量器、蚊卵定量孵化器及蚊卵定量方法
CN111317733A (zh) * 2020-03-24 2020-06-23 中国人民解放军海军军医大学 利福霉素类抗生素在制备抗黄热病毒感染药物中的应用
WO2021189444A1 (fr) * 2020-03-24 2021-09-30 中国人民解放军海军军医大学 Utilisation d'antibiotiques du type rifamycine dans la préparation de médicaments contre des infections par le virus de la fièvre jaune
CN111317733B (zh) * 2020-03-24 2022-07-12 中国人民解放军海军军医大学 利福霉素类抗生素在制备抗黄热病毒感染药物中的应用

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