WO2017030449A1 - Virus pour la lutte biologique contre les fourmis d'argentine - Google Patents

Virus pour la lutte biologique contre les fourmis d'argentine Download PDF

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WO2017030449A1
WO2017030449A1 PCT/NZ2016/050131 NZ2016050131W WO2017030449A1 WO 2017030449 A1 WO2017030449 A1 WO 2017030449A1 NZ 2016050131 W NZ2016050131 W NZ 2016050131W WO 2017030449 A1 WO2017030449 A1 WO 2017030449A1
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virus
seq
sequence
nucleic acid
acid molecule
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PCT/NZ2016/050131
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Philip John LESTER
Alexandra SEBASTIEN
Monica Alexandra Maria GRUBER
Richard James Hall
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Lester Philip John
Sebastien Alexandra
Gruber Monica Alexandra Maria
Richard James Hall
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Publication of WO2017030449A1 publication Critical patent/WO2017030449A1/fr

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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

  • This invention relates to invertebrate viruses, nucleic acids characterising invertebrate viruses, biocontrol compositions, and methods of using the viruses and/or compositions in the control of invertebrate populations, in particular social insect populations such as ants and wasps.
  • Argentine ants Linepithema humile are one of the six most globally widespread, abundant and damaging invasive ant species (Holway et al. 2002). They frequently form large colonies with the interchange of workers between nests over a wide area. Such behaviours could substantially increase the probability of disease transmission and facilitate rapid epidemics (Ugelvig & Cremer 2012). Population collapse has been observed with Argentine ants and pathogens were
  • U.S. Patent No. 7,332,176 and U.S. Patent No. 8034333 disclose a virus effective against fire ants (Solenopsis invicta).
  • the present invention relates to novel viruses identified in Argentine ant (Linepithema humile) populations. These viruses have been identified by analysis of the metagenome of various populations of Argentine Ants. Analysis of the RNA metagenome has in particular resulted in several nucleotide sequences which characterise viruses present in the ants. [0009]
  • the present invention particularly relates to the virus named herein as Linepithema humile virus -1, or LHUV-1, nucleic acid sequences derived therefrom, compositions comprising LHUV-1 and methods and uses of LHUV-1.
  • the present invention relates to an isolated nucleic molecule.
  • the nucleic acid molecule may comprise a sequence which is at least about 50%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5.
  • the nucleic acid molecule is 100% identical to SEQ ID NO: 5.
  • the nucleic acid is at least about 50%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more preferably 100% identical to SEQ ID NO: 1.
  • the nucleic acid molecule is at least about 50%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more preferably 100% identical to SEQ ID NO: 6.
  • the invention relates to a virus, preferably an isolated virus, comprising a nucleic acid molecule as described herein. The virus preferably infects Linepithema humile.
  • the virus is an NA virus, more preferably a single stranded RNA virus.
  • the virus may be of the Dicistroviridae family. Still more preferably the virus does not infect honey bees.
  • the viruses described herein can be formulated for use as a biocontrol agent against invasive or undesirable invertebrates, in particular against social insects such as ants and wasps.
  • the invention relates to a composition comprising the virus described herein and a carrier.
  • the composition comprises a bait matrix, particularly a bait matrix which is attractive to the invertebrate to be controlled and is preferably unattractive to other animals.
  • the matrix is attractive to ants, and in particular Argentine ants but not to honey bees.
  • the matrix is XstinguishTM.
  • the composition may comprise one or more additional active ingredient.
  • additional active ingredients include biological compounds, such as other viruses, bacteria or fungi, inorganic insecticides or organic insecticides such as fipronil.
  • the present invention provides for a method of controlling or eradicating an invertebrate population comprising administering an effective amount of a virus or composition as described herein.
  • the virus is administered by way of a bait system, which is preferably set in the vicinity of the invertebrate population.
  • the invertebrate population is a social insect population, such as an ant population, more particularly an Argentine ant population or a wasp population.
  • the present invention provides for the use of a virus or composition as described herein for controlling or eradicating an invertebrate population, in particular a social insect population.
  • the population is an ant population, more particularly an Argentine ant population.
  • the present invention is also directed to tools for analysing the presence and replication of the viruses described herein.
  • the invention provides for primers suitable for amplifying a nucleic acid with a sequence of SEQ ID NO: 1 and SEQ ID NO: 5.
  • the primers are of SEQ ID NO: 7, 8 , 9, 10 , 11 or 12, 13 or 14.
  • the primers may be tagged primers suitable for use in a T-PC protocol.
  • the invention provides a kit comprising a forward and reverse primer according to the above.
  • the invention provides for a virus identifiable by a primer having a sequence of SEQ ID NO: 7, 8 , 9, 10 , 11 or 12, 13 or 14.
  • nucleic acid molecules preferably isolated nucleic acid molecules, comprising a nucleotide sequence which is at least about 50% identical to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the nucleotide sequence is at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • isolated viruses comprising a nucleic acid molecule comprising those sequence, compositions comprising the viruses and methods and uses thereof in accordance with the descriptions provided herein.
  • DWV Deformed wing virus
  • FIG. 1 shows (a) the percentage of the RNA metagenome classified into each domain
  • FIG. 2 shows the distribution of the honey bee DWV, the novel RNA dicistrovirus LHUV-1 (contig n6409), and the three contigs nlOOO, nl905 and nl050, in (a) Argentina, (b) Australia, and (c) New Zealand.
  • FIG. 3 shows the maximum likelihood tree of Linepithema humile virus -1 (LHUV-1) protein sequences after 1000 bootstraps of the WAG model with Uniform rates in MEGA 6.06.
  • LHUV-1 partial contig n6409
  • Sequences obtained from one step RT-PCRs in this study are in bold black. Sequences with* were the best hits of LHUV-1 after BLASTN search on GenBank and corresponded to two dicistroviruses, Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV).
  • Country of origin New Zealand- NZ, Canada, and United States- USA
  • host organisms Argentine ants- L.
  • FIG. 4 shows the maximum likelihood tree of n 1000 nucleotide sequences after 1000 bootstraps of the Tamura-3 parameter model with Uniform rates (T92) in MEGA 6.06. The original (partial) contig nlOOO from the preliminary metagenomics analysis is uppermost. Sequences obtained from one step RT-PCRs in this study are in bold black. Sequences with* were the best hits of nlOOO after BLASTN search on GenBank and corresponded to the dicistrovirus Israeli acute paralysis virus (IAPV). Country of origin (New Zealand- NZ, Australia, and Korea) and host organisms (Argentine ants- L. humile, and honey bees- Apis mellifera) are indicated for each sequence.
  • IAPV dicistrovirus Israeli acute paralysis virus
  • FIG. 5 shows the maximum likelihood tree of nl050 nucleotide sequences after 1000 bootstraps of the Tamura-3 parameter model with Uniform rates (T92) in MEGA 6.06.
  • the original (partial) contig nl050 from the preliminary metagenomics analysis is the fourth from the top.
  • Sequences obtained from one step RT- PCRs in this study are in bold black. Sequence with* was the best hits of nlOOO after BLASTN search on GenBank and corresponded to the dicistrovirus Solenopsis invicta virus 2 (SINV-2). Country of origin (New Zealand- NZ, Australia, and United States- USA) and host organisms (Argentine ants- L. humile, and red imported fire ants- S. invicta) are indicated for each sequence.
  • Fig. 6 shows the maximum likelihood tree of RNA metagenome (partial) contig nl905 nucleotide sequences with 1000 bootstraps of the Tamura-3 parameter model with uniform rates (T92) in MEGA 6.06.
  • Argentine ant and “Linepithema humile” are used interchangeably to describe the Argentine ant, originating in South America, but now widespread throughout the world including New Zealand.
  • the term "isolated” in the context of a virus is defined as separated from other viruses found in naturally occurring organisms.
  • the term “isolated” is defined as separated from other nucleic acid molecules and other non-nucleic acid structures found in a cell or virus.
  • nucleic acid molecule means a biopolymer made from nucleotide monomers and includes but is not limited to DNA and RNA.
  • sequence it is to be understood that the well understood modifications to alter a DNA sequence to an RNA sequence or vice versa are also within the scope of the nucleic acid molecule.
  • reference to a nucleic acid molecule defined by a DNA sequence also refers to an RNA molecule of the same sequence, but with uracil (u) in the place of thymine (t).
  • the term "effective amount” or “amount effective for” as used herein means that minimum amount of a virus or composition needed to control, reduce, or substantially eradicate ants in an ant colony when compared to the same colony or other colony which is untreated. The precise amount needed will vary in accordance with the particular virus or composition used; the colony to be treated; and the environment in which the colony is located. The exact amount of virus composition needed can easily be determined by one having ordinary skill in the art given the teachings of the present specification.
  • carrier when used in the context of the compositions described herein refers to any component which enables delivery of the virus to an invertebrate or invertebrate population.
  • a carrier may be solid or liquid.
  • a carrier may perform a dual purpose. For instance it may also serve as an attractant for the invertebrate to be controlled or eradicated, or a deterrent for species not desired to be controlled.
  • carrier is not intended to exclude such additional purposes.
  • sequence identity refers to a pairwise sequence identity (%). the relationship between two or more amino acid or nucleic acid sequences, as determined by aligning the sequences.
  • sequence identity with a particular sequence preferably means sequence identity over the entire length of said particular sequence in optimal alignment.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • Optimal alignment is the alignment in which the percentage sequence identity is the highest possible. It can be achieved in various ways using computer programs.
  • One preferable method is the Needleman-Wunsch Global Alignment method (Needleman & Wunsch, 1970).
  • Needleman & Wunsch, 1970 A computer algorithm running Needle-Wunsch, such as that available from the National Center for Needle-Wunsch, such as that available from the National Center for Needle-Wunsch, such as that available from the National Center for
  • an "active ingredient”, as used herein, refers to a compound which, when administered in an effective amount has a deleterious effect on an invertebrate population, thereby resulting in control or eradication of the population.
  • LHUV-1 Linepithema humile virus-1
  • SEQ ID NO: 1 SEQ ID NO: 1.
  • T-PC has further confirmed the region of LHUV-1 encoded by SEQ ID NO: 5 Genbank ID: KT713624.2.
  • RNA viral species identified by contigs nlOOO (SEQ ID NO: 2), nl905 (SEQ ID NO: 3) and nl050 (SEQ ID NO: 4). Similar to LHUV-1, nlOOO, nl050 and nl905 have been found to be present in multiple populations (Figs 4, 5 and 6 respectively).
  • LHUV-1 and the other viruses can be constructed using the RACE system (Invitrogen, Carlsbad, CA) as described by Valles et al. 2016, using the identified contigs.
  • viruses described above have been identified as being present in Argentine ants, it is expected that other ants, or indeed other invertebrates, will also be susceptible to infection with these viruses.
  • One of the other viruses identified in the Argentine ant populations was the Deformed wing virus, which is a common virus found in insects such as honey bees, wasps and bumble bees.
  • the RNA viruses described herein may show similar host specificity
  • DWV Deformed wing virus
  • the formulation of the viruses for administration can be undertaken by various methods known in the art.
  • One method is to grind up known infected ants, and administer the ground up ants to the population to be controlled.
  • the ground up ants can be included in a food media or other bait composition, which is fed to the population to be controlled.
  • One approach is demonstrated by In Valles et al. 2013, in which viral extractions of the Solenopsis invicta virus-3 (SINV-3) were obtained by homogenising virus-positive ant colonies in a blender. The blended colonies were suspended in either a sugar or oil solution, and filtered. The filtrates were then added to either a 10% sugar solution, an insect paste consisting of homogenised crickets, and soybean oil adsorbed to defatted corn grit. These three baits were then feed to virus-negative colonies. All three baits delivered the virus and successfully infected ants.
  • Another method of preparing the viruses is to prepare them in an insect cell line.
  • insect cell lines There are a variety of insect cell lines available that can be grown in the laboratory and commercial environment (Goblirsch et al. 2013, Boyapelle et al. 2007). Cell lines for the culture of viruses disclosed herein could be investigated using the methods of Goblirsch et al. 2013. These authors developed a honey bee cell culture, based on the collection and homogenisation of honey bee eggs. They used a basal solution of Leibovitz's L15 medium (Life Technologies, Grand Island, NY) modified to include the addition of glucose, organic acids, vitamins, trace minerals and amino acids.
  • the virus is introduced into a cell line, allowed to reproduce, and the cells harvested and incorporated into a composition, which can then be spread into the environment. As many related viruses are spread between hosts by foraging on the same flowers, the virus is expected to have a reasonable viability outside of the host.
  • Another method of preparing and isolating the viruses of the present invention is by way of transgenic expression system.
  • a baculovirus expression system is particularly suitable for expression of viruses of this type (Pal et al. 2007).
  • Various methods of isolating the viruses are known in the art, for instance by sucrose gradient centrifugation (Krishna et al. 2003), or by isopycnic centrifugation using CsCI (Valles et al. 2016).
  • viruses described herein can be used as biocontrol agents against invertebrate species and especially ants, honey bees, wasps, bumble bees etc.
  • the invention provides methods of controlling or eradicating an invertebrate population comprising administering an effective amount of a virus or composition as described herein.
  • Viruses related to LHUV-1 can devastate insect populations, including those associated with colony collapse disorder in honey bees (Schroeder et al. 2012). Relatives of LHUV-1 are known to exert substantial effects in the widespread and invasive Red imported fire ant, Solenopsis invicta. Six viruses have been described from the Red imported fire ants and are currently being considered as potential biological control agents (Valles 2007, 2014). LHUV-1 and the other viruses disclosed herein are therefore possible causes for the population declines that have been observed in Argentine ants (Cooling et al. 2012) and have potential as a biological control agents.
  • DWV may also have potential uses as a biological control agent against invertebrates, and in particular against ants such as Argentine ant.
  • the collapse of ant populations is relatively common, with evidence they occur in nearly all invasive ant populations.
  • One proposed model for invasive ant collapse is the acquisition of pathogens over time, combined with issues of low genetic diversity, which contribute to this collapse (Lester and Gruber 2016).
  • the viruses may not need to be lethal in order to be considered for biocontrol.
  • Viruses of Solenopsis invicta can cause direct mortality or have been associated with mortality when infected ant colonies experience stress (Valles et al. 2007, 2014). Interspecific interactions, including competition, are frequently thought to be a major driver of ant community assembly and population dynamics. Populations infected with pathogens may be less competitive, obtaining less food, and be excluded from other resources, resulting in reduced or negative colony growth rates.
  • Species such as Argentine ants are known to have a low competitive ability in interactions with native species, with competitive dominance achieved only when the invader is present in overwhelming numbers (Sagata & Lester 2009).
  • factors, such as a pathogen that lower the competitive dominance of Argentine ants or other invertebrates could have a dramatic effect on ant population dynamics.
  • This effect may be the equivalent of the 'extinction vortex' dynamics observed in conservation biology, whereby low genetic diversity results in low breeding success that leads to a further reduction in genetic diversity, and so on (Gilpin & Soule 1986).
  • an effective amount of a virus as described herein need not be enough to kill the invertebrate population, but rather enough to reduce an invasive population's competitive advantage over other ant species present in the community.
  • the reduced competitive ability of the infected ant population may limit the ability to acquire resources and result in reduced or negative colony growth rates.
  • the method may further comprise administration of one or more further active ingredients.
  • Administration of the further active ingredient may be separate, simultaneous or sequential with the administration of the virus. These active ingredients may have a deleterious effect on the invertebrate population in their own right, or they may stimulate an improvement in the
  • the further active ingredient may act as a stressor which stimulates the viral replication necessary to achieve the control or eradication, thereby resulting in a synergistic effect.
  • Suitable additional active ingredients include include Chlorfenapyr, Imidacloprid, Fipronil, Hydramethylnon, Sulfluramid, Hexaflumuron, Pyriproxyfen, methoprene, lufenuron, dimilin, Chlorpyrifos, and their active derivatives, Neem, azadiractin, and boric acid based toxins.
  • the virus and the other active ingredient are combined into a single delivery composition.
  • the mode of administration of the viruses will depend on the nature of the invertebrate to be targeted.
  • Various modes of administration of insecticides are known in the art, including direct application to a colony, aerosol spray, bait system, incorporation into watering or irrigation systems, distribution of solid granules, etc.
  • Known modes of administration of insecticides with formulation changes should be suitable for delivery of the viruses.
  • the viruses of the invention have the ability to replicate and thereby infect other members of the population. Accordingly, unlike conventional insecticides, it is only necessary that the delivery mechanism allows for infection of one or a small number of individuals who will subsequently spread it to the remainder of the population. This is particularly true for invertebrates who live in a hive- or nest- type colony where the queen is primarily responsible for reproduction but takes little or no part in foraging activities. In these circumstances infection of a worker individual can result in subsequent infection of the entire hive or nest, and especially the queen, thereby resulting in efficient control, or even eradication of the population. This makes bait delivery systems particularly suitable for this type of invertebrate.
  • An alternative approach of pathogen delivery is to infect an insect with a pathogen and introduce it directly back into a population thereby making a 'living insect bomb' (Gumus et al.
  • Another method of delivery of the viruses of the present invention is to infect an invertebrate with the virus and reintroduce it back into the population, thereby facilitating the spread of the virus.
  • the host range of the virus is broad, it may be necessary to tailor the mode of administration to ensure only the undesirable or invasive species is targeted.
  • Argentine ants are typically found in massive densities that exclude other ant species, especially native ant species. Therefore, it is possible to target the Argentine ants directly, with few non-target effects.
  • honey bees are responsible not only for a substantial apiculture industry, but in New Zealand are also important pollinators of native plant communities.
  • the Deformed Wing Virus is shown to be present in both Argentine ants and honey bees and has been implicated in population collapse of honey bees. DWV may thus not be suitable for biocontrol of Argentine ant populations which a co-located with honey bee populations.
  • the LHUV-1 virus has not been observed in honey bees, despite it being present in co-located ant populations. It is therefore likely LHUV-1 does not infect honey bees. The use of LHUV-1 in a biocontrol mechanism would therefore avoid non-target effects in honey bees.
  • the formulation of the delivery composition will also be relevant for both effective delivery and host targeting.
  • the viruses described herein are formulated with a carrier into a composition.
  • the carrier can be a liquid or a solid material and is an inert, non-repellent (to the species desired to be controlled) carrier for delivering the composition to a desired site.
  • the composition comprises a bait matrix.
  • a suitable bait matrix should be one to which the foraging insects are attracted, and which they will feed on and carry back to the nest and feed it to larvae, queens and nest mates.
  • the bait matrix should be tailored to the target species and can comprise a balance of protein, carbohydrate and lipids suited to the target insect's seasonal food requirements.
  • a bait matrix comprising soybean oil on corn grits has been used in against S. invicta since the 1960s (Lofgren et al. 1963; Williams et al. 2001).
  • An attractant in a bait matrix or composition can be targeted to the invertebrate in question and may comprise lipids such as soybean oil, proteins such as ground silkworm pupae,
  • carbohydrates for example in the form of sugar water solutions, or a combination of these.
  • Solid or granular bait matrices are particularly suitable for large scale ground or aerial broadcast of the virus, while liquid baits are more suitable for small infestations.
  • Liquids suitable as carriers include water, and any liquid which will not affect the viability of the viruses of the present invention.
  • the bait matrix is one which has been developed for Argentine ants, such as the Xstinguish ® matrix.
  • the Xstinguish ® matrix is a wet matrix which comprises cooked egg and sucrose. Importantly, this matrix is unappealing to bees and highly appealing to Argentine ants and other species previously thought difficult to attract with baits other than sweet liquids. Species such as invasive social wasps are also attracted to this matrix.
  • the composition may further include such active ingredient.
  • Further active ingredients include biological compounds, organic insectides or inorganic insecticides.
  • the composition or bait matrix may also include toxins or further active ingredients such as Chlorfenapyr, Imidacloprid, Fipronil, Hydramethylnon, Sulfluramid, Hexaflumuron, Pyriproxyfen, methoprene, lufenuron, dimilin, Chlorpyrifos, and their active derivatives, Neem, azadiractin, and boric acid based toxins.
  • the formulation of the composition will necessarily take into account the properties of the additional active ingredient.
  • Argentine ant workers were collected in 2013 from two nests in Wellington, New Zealand (41.2218°S, 174.8724°E). Each sample included 30 ants homogenised using pestles in Eppendorf tubes containing 400 ⁇ iPrep PureLink Virus kit lysis buffer (Life Technologies, Carlsbad, CA, USA). 400 ⁇ of molecular grade water and 50 ⁇ proteinase K was then added to each sample and incubated at 50°C for 1 hour, followed by 96°C for 5 minutes. Samples were then centrifuged at 12,000 x g for 5 minutes.
  • the supernatant was removed and used in the extraction along with the iPrep Purelink Virus kit (Life Technologies, Carlsbad, CA, USA), eluting into 50 ⁇ of RT-PCR molecular grade water (Ambion, Austin, TX, USA).
  • the extractions were combined into a single sample to increase nucleic acid amount.
  • RNA virus DNA was removed using DNAse treatment by Ambion DNA- free (Life Technologies) from half of the combined extractions. 8 ⁇ of DNA-free RNA was incorporated into first-strand cDNA synthesis (Life Technologies) including RNase H digestion. To ensure > 1 ⁇ g of DNA for library preparation, the DNA and cDNA were amplified by multiple displacement amplification using the Whole Transcriptome Amplification kit (Qjagen). Sequencing libraries were then prepared with the lllumina TruSeq DNA library preparation kit followed by sequencing on an lllumina MiSeq instrument producing 250 bp paired-end reads. [00071] The quality of the sequence data was examined using Fast QC. Reads were trimmed when the average Phred score was ⁇ 30.
  • SINV-2 EF428566 Vims in fire ants nl050 56.4 56.4 6 0.002 74
  • Table 2 gives 10 closest matches, ordered by the highest score for each contig of interest after BLASTX search on Genbank. Abbreviations correspond to: Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV) and Solenopsis invicta virus-2 (SINV2).
  • ABSPV Acute bee paralysis virus
  • IAPV Israeli acute paralysis virus
  • KBV Kashmir bee virus
  • SINV2 Solenopsis invicta virus-2
  • IAPV 384 384 95 6e- 44 ABY57950 Virus structural n6409
  • IAPV 383 383 95 l e- 44 ACD01400 Virus structural
  • IAPV 383 383 95 2e ⁇ 44 ABY71757 Virus structural
  • SINV2 452 le- 50 YP_001285729 Virus non-structural
  • Argentine ants were collected between 2012 and 2015 in either 100% ethanol or Ambion RNAIater (Life Technologies). Samples were collected from 30 sites distributed among nine regions throughout the ant's invaded range in New Zealand, two sites in Australia, and three sites in the ant's Argentinian native range. RNA was extracted for each site from a pool of 30 ants using either an iPrep PureLink Virus kit (Life Technologies) as for example 1 above, or a GeneJET Viral DNA & RNA Purification Kit (Thermo Scientific), as described below.
  • DNA and RNA were extracted from Argentine ant samples using a GeneJET Viral DNA & RNA Purification Kit (Thermo Scientific, Waltham, MA, USA) following modified protocol. Ants from each sample were homogenised using pestles in Eppendorf tubes containing 250 ⁇ GeneJET Viral DNA & RNA Purification Kit lysis buffer, and then 5 ⁇ RNA carrier and 50 ⁇ protein K were added. Samples were incubated for 1 hour at 56°C. Samples were briefly centrifuged, before the supernatant was removed and used in the standard manufacturer's protocol. Amplicons were visualized by gel electrophoresis and purified using USB ExoSAP-IT (Affymetrix).
  • the purified amplicons were sent for Sanger sequencing on a capillary sequencer at Massey University Genome Service (New Zealand). The duplicate extractions were then combined into a single sample to maximise nucleic acid content.
  • Sanger sequencing was used to confirm the presence of the contigs of interest in a number of samples.
  • the partial n6409 contig was detected in all sampling sites, with the exception of one site in New Zealand (figure 2).
  • the partial nlOOO contig sequence was present in all sites in Argentina and Australia but in less than half the sites in New Zealand (13 out of 27 sites).
  • the partial nl905 and nl050 contigs were only detected in Australia and New Zealand (figure 2). All Sanger-sequencing data was identical to the partial metagenomic contig, with the exception of a few nucleotides differences ( ⁇ 1%).
  • RNA metagenome (partial) contig n6409 matched the structural polyprotein (capsid region) of KBV and therefore was suitable for a phylogenetic analysis for provisional taxonomic assignment of the putative virus.
  • Phylogenetic analysis of this contig positioned it with other dicistroviruses (figure lc). This sequence is from a novel virus, provisionally named Linepithema humile virus 1 (LHUV-1).
  • Example 5 Analysis of DWV and LHUV-1 replication in Argentine Ants
  • a modified RT-PCR was used to examine for DWV and LHUV-1 replication, and thus whether there was an active infection within the ants.
  • Tagged primers were designed to only allow reverse transcription of the negative-sense strand of the viral genome.
  • tag primers were used in a modified one step RT-PCR as described by Yue & Genersch (2005).
  • a pool containing an equal amount of RNA from each site from one country was created for each country.
  • the 10- nucleotide long tags for the primers were generated by BARCRAWL (Frank, 2009) using the default settings of the program and one pair of tags was selected for each pair of primers (see electronic supplementary material).
  • a modified one step RT-PCR was realized using a Superscript III One Step RT-PCR system with Platnium Taq DNA polymerase kit (Life Technologies).
  • Reaction mixes consisted of a 24.5 ⁇ mix containing ⁇ 20 ng RNA, IX Reaction mix (final concentration), 0.8 ⁇ of reverse tagged primer (either tag- DWVrtR or tag-n6409-A1055R - final concentration), 5.5 ⁇ ddH20 and 1 ⁇ Superscript ® III RT/Platinum ® Taq Mix. After the reverse transcription step at 45°C for 30 min, and 0.8 ⁇ forward tagged primer (tag-DWVrtF or tag-n6409-A1055R) was added to obtain a final 25.0 ⁇ mix.
  • the PCR thermal cycling continued with an initial denaturation step at 94 ° C for 2 min, followed by 35 cycles of 94 ° C for 15 s, 55 ° C for 15 s and 68 ° C for 30 s, with a final extension step of 68 ° C for 5 min.
  • the amplification of the positive strain of the viruses was realized in parallel.
  • the first step of the RT-PCR only the tagged forward primers (either tag-DWVrtF or tag- n6409-A1055F) were added and in the second step, the tagged reverse primers tag- DWVrtR or tag- n6409-A1055R) were added.
  • RT-PCR products were visualized by gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide.
  • Amplicons were purified using USB ExoSAP-IT (Affymetrix) and sent for Sanger sequencing on a capillary sequencer to Massey University (Genome Service, Palmerston North, New Zealand).
  • Table 4 shows the tagged primers used to amplify the RNA negative strand of Deformed wing virus (DWV) and L. humile virus-1 (LHUV-1) using a modified one step RT-PCR protocol. The tag sequence is in lower case.
  • Valles SM, Hashimoto Y. 2009 Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta. Virology 388, 354- 361.
  • Valles SM Porter SD, Choi M, Oi DH 2013 Successful transmission of Solenopsis invicta virus 3 to Solenopsis invicta fire ant colonies in oil, sugar, and cricket bait formulations J. Invertebr. Pathol. 113, 138-204 Valles SM, Strong CA. 2005 Solenopsis invicta virus-lA (SINV-1A): distinct species or genotype of SINV-1? 7. Invertebr. Pathol. 88, 32-237 ' .
  • Valles SM Strong CA, Hashimoto Y. 2007.
  • Nylanderia fulva virus 1 a positive-sense, single- stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva, Virology 496:244-254

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Abstract

L'invention concerne des virus contre la fourmi d'Argentine, des molécules d'acide nucléique les identifiants, des compositions les comprenant et des procédés pour les utiliser en lutte biologique.
PCT/NZ2016/050131 2015-08-20 2016-08-19 Virus pour la lutte biologique contre les fourmis d'argentine WO2017030449A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009060429A2 (fr) * 2007-11-07 2009-05-14 Beeologics, Llc Compositions pour conférer une tolérance à une maladie virale dans des insectes sociaux, et leur utilisation
WO2011021171A1 (fr) * 2009-08-21 2011-02-24 Beeologics, Llc Prévention et traitement de maladies des insectes bénéfiques par l’intermédiaire de molécules transcrites végétales

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009060429A2 (fr) * 2007-11-07 2009-05-14 Beeologics, Llc Compositions pour conférer une tolérance à une maladie virale dans des insectes sociaux, et leur utilisation
WO2011021171A1 (fr) * 2009-08-21 2011-02-24 Beeologics, Llc Prévention et traitement de maladies des insectes bénéfiques par l’intermédiaire de molécules transcrites végétales

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ÉBASTIEN, A ET AL.: "Invasive ants carry novel viruses in their new range and form reservoirs for a honeybee pathogen.", BIOLOGY LETTERS, vol. 11, 2015, XP055365134 *

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