ANTI-VIRAL DEFENSE PATHWAYS

This International PCT Application claims the benefit of and priority to U.S. Provisional Application No. 62/757,844, filed November 9, 2018. The entire specification and figure of the above-referenced application is hereby incorporated, in its entirety by reference.

TECHNICAL FIELD

Generally, the inventive technology relates to novel systems, methods and compositions for the bio-control of vector-borne pathogens. The inventive technology includes novel systems, methods and compositions to deliver bioactive molecules to the mosquito that can activate or enhance the endogenous RNAi response, or silence insect vectors genes potentially involved in controlling viral replication within the mosquito.

BACKGROUND OF THE INVENTION

Virus transmitting insect vectors uptake viral particles upon feeding, being it blood meals in the case of Aedes aegypti , or plant sap in the case of aphids, family Aphididae , and thrips, family Thripidae , for example. In the case of the former, following the blood meal, and once inside the insect vector’s midgut, viruses will replicate in the hosts cells and disseminate throughout the body and potentially reach the salivary glands of the mosquito, at which stage the pathogen can now be transmitted as the insect takes another blood meal.

Endogenous RNAi pathways control viral replication within the host to the extent that the virus is not deleterious to the insect vector but nevertheless still able to move from the point of entry in the organism, the midgut, to the salivary glands, where it can be transmitted to a new host. Recent evidence indicates that the virus escapes the hosts RNAi in the midgut of A. aegypti due to the tissue-specific transcriptional silencing of loquacious2 ( loqs2 ), a double-strand RNA (dsRNA) binding protein functioning downstream of smRNA biogenesis. Transcriptional silencing of loqs2 is not virus induced but naturally occurring in the mosquito. Knockdown of Argonaute2 (AG02) in A. aegypti , the core protein of the RNA-induced silencing complex involved in anti-viral defense in animals and plants, did not affect dengue virus (DENV) accumulation in the midgut.

In contrast, virus titer and number of mosquitoes displaying DENV infection in the carcass were significantly increased upon knock-down of AG02, indicating that RNAi anti-viral defense pathways in the midgut likely fails to load available AG02 with the 2lnt anti-viral siRNAs and, hence, no RISC is formed to control viral replication. loqs2 functionally lies between siRNA biogenesis and RISC loading and is specific to anti-viral RNAi pathways in Aedes sp. mosquitoes, being directly implicated in DENV and Zika virus control. Ectopic expression of loqs2 in midgut cells of transgenic A. aegypti reduced overall viral load of DENV in both midgut and carcass of infected mosquitoes 8 days post feeding on a DENV infected blood meal, demonstrating the role of loqs2 in controlling viral replication and dissemination throughout the host. Thus, targeted expression of Loqs2 in mosquito midgut is anticipated to suppress viral replication.

Translational control of host genes as well as viral genes is an important determinant of viral pathogenesis. A screen of small non-coding RNAs expressed in Aedes aegypti strains that showed varying susceptibility to dengue virus in the context of co-infection with the bacteria Wolbachia identified a substantial upregulation (2.7 log fold change, 500X) of small RNAs Feature_26588l (SEQ ID NO. 9) and Feature_266l0l (SEQ ID NO. 9) with homology to a guanine nucleotide binding protein domain within the RACK1 gene (SEQ ID NO. 3) in mosquitoes that were better able to control viral infection. The RACK1 protein (SEQ ID NO. 3) has been implicated in the control of molecular signaling cascades, translational control, kinase inhibition, and apoptosis. Although RACK1 has not been directly shown to be involved in controlling viral infection in mosquitoes, the role of apoptosis in controlling viral infection in Ae. aegypti has been demonstrated and considered an important antiviral response.

The role of protein kinase inhibitors in controlling dengue replication has also been demonstrated. The RACK1 protein is a receptor of protein kinase C and inhibition of protein kinase C has been shown to promote dengue virus replication. In addition, there is interplay between protein kinase C activity and apoptosis. Of greatest importance, the RACK1 gene has been shown to be required for dengue replication in humans by mediating the glycosylation of nonstructural protein 1 and 4B (NS1 and NS4B). The Feature_26588l and Feature_266l0l below are the sequences of the small RNAs that mapped to the guanine nucleotide binding domain of the RACK1 gene in Ae. aegypti and would be the potential sequence targets to design the dsRNA against.

SUMMARY OF THE INVENTION One aspect of the present invention may include novel endogenous paratransgenic biocontrol strategies for vector-borne diseases. In this aspect, the inventive technology includes various cross-kingdom mechanisms for the expression of endogenous effector genes to activate or enhance endogenous RNAi viral-defense pathways in the midgut of an insect host, and to control viral replication and limit or eliminate spread of the virus to the carcass and salivary glands of the host and, ultimately, to eliminate dissemination of the virus to the environment.

Another aspect of the present invention may include novel RNAi-mediated paratransgenic biocontrol strategies. In this embodiment, the inventive technology includes various cross-kingdom mechanisms for the expression of specific inhibitory RNA molecules that may downregulate and/or suppress selected endogenous host genes that may suppress viral replication, transmission and/or pathogenicity.

Another aspect of the present invention may include the bacterial expression of one or more eukaryotic-like mRNAs. In a preferred embodiment, expression of a eukaryotic-like mRNA may include expression of a eukaryotic endogenous effector gene configured to be expressed in a bacterial system and transported to a target host, preferably a mosquito. In further embodiments, such a eukaryotic-like mRNA may activate or enhance RNAi viral-defense pathways in the host.

Another aim of the present invention may couple the expression of one or more endogenous effector genes to activate RNAi viral-defense pathways in the midgut of the insect host with the expression of one or more interfering RNA molecules, such as dsRNA polynucleotides configured to down-regulated expression of one or more host genes that may be responsible for inhibiting viral infection, replication, or transmission.

Another aim of the inventive biocontrol systems may be to optimize dsRNA survival and/or delivery to a disease-carrying vector, such as a mosquito, by co-expression of helper genes that may enhance the uptake, stabilization, and intercellular transfer and/or mobilization of dsRNA or other endogenous effector molecules.

Yet other aims of the present invention may include the expression of endogenous effector genes to activate RNAi viral-defense pathways in the midgut of the insect host with the expression of one or more interfering RNA molecules, such as double stranded RNA (dsRNA) polynucleotides configured to down-regulated expression of one or more viral genes or endogenous host genes in a functional RNAselll mutants - configured to cleave dsRNA precursors to 2l-23nt smRNA duplexes that are functional in RNAi - competent to form functional RISC complex, such as those identified in by the inventors in Sayre et al., in PCT/US 19/40747, the sequences and methods of expression of such specific RNAselll mutants being incorporated herein reference).

Another embodiment may include the generation of genetically modified bacteria that may express one or more inhibitory RNA polynucleotides, such as dsRNA that may be configured to inhibit viral replication in a target mosquito host by suppressing expression of one or more of the following endogenous mosquito genes selected from the group: chymotrypsin-like serine protease; RACK 1 /guanine nucleotide-binding protein; Cadherin 87 A, identified by Ford et al., as being important in viral replication and vector-borne disease propagation [22] In one preferred embodiment, such genetically modified bacteria may include enteric, symbiotic, probiotic, or endosymbiotic bacteria isolated from Aedes aegypti , or other insect species such as from the family Aphididae (aphids), and Thripidae (thrips), that are able to colonize the gut and other tissue to express dsRNA targeted to suppress expression of one or more target genes.

One aspect of the invention may include the co-delivery of Loqs2 protein (SEQ ID NO. 6) and dsRNA to enhance RNAi response in the mosquito midgut enhancing the endogenous RNAi response or silence insect vectors genes, as well as viral genes, involved in controlling viral replication or pathogenicity within the mosquito. In this preferred aspect, a Loqs2 nucleotide sequence (SEQ ID NO. 5) encoding a Loqs2 protein (SEQ ID NO. 6) may be incorporated into an expression vector that is also configured to express one or more dsRNA targeting a specific gene. This expression vector may be used to generate genetically modified bacteria, and preferably symbiotic, endosymbiotic, or probiotic bacteria such that the heterologously expressed Loqs2 protein and targeted dsRNA may be introduced to a mosquito and colonize the midgut where they are co-expressed. In this aspect, mosquitos that expressed a corresponding target for the dsRNA, such as an endogenous gene or viral pathogen gene that may be subject to RNAI-mediated downregulation. This RNAi response may be enhanced through the co-expression of Loqs2.

Another aspect of the invention may include the co-delivery of a eukaryotic Loqs2 mRNA, expressed in a prokaryotic organism, such as a bacterium, and a targeted dsRNA to enhance the RNAi response in the mosquito midgut. In this embodiment, a nucleotide sequence encoding a eukaryotic-like Loqs2 mRNA may be heterologously expressed in bacteria, and preferably symbiotic, endosymbiotic, or probiotic bacteria, and subsequently transported to the mosquito midgut for translation. (In this embodiment, the eukaryotic-like Loqs2 mRNA may be generated by the systems and methods of engineering a eukaryotic mRNA configured to be expressed in a prokaryotic organism, and exported to a eukaryotic target for translation as generally described by the inventors in Sayre et ah, in PCT/US 19/40747, such systems methods and compositions being specifically incorporated herein by reference). In this specific aspect, the Loqs2 coding sequence may be under the control of an internal ribosomal entry site such that it may be expressed by a bacteria, but may not be translated, but will instead be exported and ultimately translated inside the mosquito host after bacterial ingestion.

Another aspect of the invention may include bacterial delivery of dsRNA to regulate the expression of one or more endogenous mosquito genes that may control viral replication. In this preferred aspect, genetically engineered bacteria, and preferably symbiotic, endosymbiotic, or probiotic bacteria, may be configured to expressed one or more dsRNA targeting an endogenous mosquito gene, and preferably an endogenous gene, or viral pathogen gene, involved in controlling viral replication in the mosquito. This genetically engineered bacteria having such a dsRNA construct may be introduced to a mosquito population or to an environment where mosquitos are known to be vectors for viral disease.

Another aspect of the invention may include bacterial co-delivery of helper gene loquacious (SEQ ID NO. 7) and a targeted dsRNA to enhance the RNAi response in the mosquito. In this preferred aspect, a nucleotide sequence encoding a loquacious mRNA may be heterologously expressed in bacteria and preferably symbiotic, endosymbiotic, or probiotic bacteria, and subsequently transported to the mosquito midgut, and further co-expressed with a targeted dsRNA. The translated loquacious protein (SEQ ID NO. 8) may work synergistically with the dsRNA in the midgut of a mosquito to produce an enhance RNAi response that may more effectively control viral replication/pathogenicity.

In another preferred aspect, a nucleotide sequence encoding a loquacious protein may be heterologously expressed in bacteria, and preferably symbiotic, endosymbiotic, or probiotic bacteria, and subsequently transported to the mosquito midgut for translation. This loquacious protein may further be co-expressed with a targeted dsRNA. The translated loquacious protein may work synergistically with the dsRNA in the midgut of a mosquito to produce an enhance RNAi response that may more effectively control viral replication/pathogenicity. Additional aspects of the current invention may include one or more of the following embodiments:

1. A paratransgenic method of downregulating expression of an endogenous gene in a mosquito comprising the steps of:

- introducing into the mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding a heterologous double stranded RNA (dsRNA) polynucleotide that is expressed in the bacteria and delivered to the mosquito and inhibits expression of an endogenous gene in the mosquito; and

- wherein the inhibition of said endogenous gene in the mosquito disrupts one or more viral pathogens.

2. The method of embodiment 1 wherein said endogenous gene comprises and endogenous gene according to the nucleotide sequence SEQ ID NO. 2.

3. The method of embodiment 2 wherein said endogenous gene comprises the rackl gene.

4. The method of embodiment 3 wherein said nucleotide sequence encoding a heterologous dsRNA polynucleotide comprises the heterologous nucleotide sequence according to SEQ ID NO. 2.

5. The method of embodiment 1 wherein said nucleotide sequence encoding a heterologous dsRNA polynucleotide comprises the heterologous nucleotide sequence encoding a dsRNA configured to target one or more guanine nucleotide binding domains of RACK1 according to the nucleotide sequences SEQ ID NOs. 9, and 10.

6. The method of embodiment 1 wherein the disrupting of one or more viral pathogens comprises inhibiting viral replication of one or more viral pathogens.

7. The method of embodiment 1 wherein said one or more viral pathogens comprises one or more viral pathogens selected from the group consisting of: a Zika virus, La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick-borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, and a Flock House virus.

8. The method of embodiment 1 wherein the genetically modified bacteria is obtained from a bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27.

9. The method of embodiment 1 wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

10. A method of enhancing the endogenous RNA-interference (RNAi) response in a host mosquito comprising the steps of:

- introducing into the host mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding:

- at least one double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito, or an essential gene of a viral pathogen carried by the mosquito;

- a nucleotide sequence overexpressing an endogenous Loquacious2 (Loqs2) polypeptide;

- co-expressing and transporting said dsRNA polynucleotide and said Loqs2 polypeptide to the host mosquito; and

- wherein the dsRNA polynucleotide and said Loqs2 polypeptide enhance said host mosquito’s endogenous RNAi response toward said endogenous gene in a host mosquito, or an essential gene of a viral pathogen.

11. The method of embodiment 10 wherein said nucleotide sequence overexpressing an endogenous Loquacious polypeptide comprises an endogenous Loqs2 polypeptide according to amino acid sequence SEQ ID NO. 6. 12. The method of embodiment 10 wherein the endogenous gene in the mosquito comprises an endogenous gene according to the nucleotide sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 11, and SEQ ID NO. 12.

13. The method of embodiment 10 wherein said endogenous gene comprises the rackl gene.

14. The method of embodiment 13 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito the nucleotide sequence according to SEQ ID NO. 2.

15. The method of embodiment 13 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to target the guanine nucleotide binding domain of the rackl gene, identified as SEQ ID NOs. 9, 10.

16. The method of embodiment 10 wherein said one or more viral pathogens comprises one or more viral pathogens selected from the group consisting of: a Zika virus, La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick-borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, and a Flock House virus.

17. The method of embodiment 10 wherein the genetically modified bacteria is obtained from a bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27.

18. The method of embodiment 10 wherein said transporting comprises the step of transporting said dsRNA polynucleotide and said Loqs2 polypeptide to the host mosquito through an outer membrane vesical (OMV).

19. The method of embodiment 10 and further comprising the step of introducing into the host mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding a nucleotide sequence overexpressing an endogenous Loquacious polypeptide according to nucleotide sequence SEQ ID NO. 7. 20. The method of embodiment 19 and further comprising the step of co-expressing and transporting said dsRNA polynucleotide, and said Loqs2 polypeptide and Loquacious polypeptide to the host mosquito wherein the dsRNA polynucleotide, Loqs2 polypeptide, and Loquacious polypeptide enhance the host mosquito’s endogenous RNAi response disrupting replication of said viral pathogen.

21. The method of embodiment 10 wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

22. The method of embodiment 10 wherein the step of enhancing the host mosquito’s endogenous RNAi response comprises the step of enhancing the host mosquito’s endogenous RNAi response in the midgut disrupting replication of said viral pathogen in said midgut.

23. A method of enhancing the endogenous RNA-interference (RNAi) response in a host mosquito comprising the steps of:

- introducing into the host mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding:

- at least one double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito, or an essential gene of a viral pathogen carried by the mosquito;

- a heterologous nucleotide sequence overexpressing an eukaryotic Loquacious2 (Loqs2) messenger RNA (mRNA);

- co-expressing and transporting said dsRNA polynucleotide and said eukaryotic Loqs2 mRNA to the host mosquito wherein said eukaryotic Loqs2 mRNA is translated to generate a Loqs2 polypeptide; and

- wherein the dsRNA polynucleotide and said Loqs2 polypeptide enhance said host mosquito’s endogenous RNAi response toward said endogenous gene in a host mosquito, or an essential gene of a viral pathogen. 24. The method of embodiment 23 wherein said Loqs2 polypeptide comprises an amino acid sequence according to SEQ ID NO. 6.

25. The method of embodiment 23 wherein said Loqs2 mRNA comprises a Loqs2 mRNA having an internal ribosomal entry site (IRES) and a poly- A tail.

26. The method of embodiment 25 wherein said IRES comprises an mRNA encoded by a

IRES portion according to nucleotide sequence SEQ ID NO. 37 coupled with a Loqs2 portion according to nucleotide sequence SEQ ID NO. 5

27. The method of embodiment 23 wherein the endogenous gene in the mosquito comprises an endogenous gene according to the nucleotide sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 11, and SEQ ID NO. 12.

28. The method of embodiment 23 wherein said endogenous gene comprises the rackl gene.

29. The method of embodiment 28 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito the nucleotide sequence according to SEQ ID NO. 2.

30. The method of embodiment 29 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to target the guanine nucleotide binding domain of the rackl gene, identified as SEQ ID NOs. 9, 10.

31. The method of embodiment 23 wherein said one or more viral pathogens comprises one or more viral pathogens selected from the group consisting of: a Zika virus, La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick-borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, and a Flock House virus.

32. The method of embodiment 23 wherein the genetically modified bacteria is obtained from a bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27. 33. The method of embodiment 23 wherein said transporting comprises the step of transporting said dsRNA polynucleotide and said Loqs2 polypeptide to the host mosquito through an outer membrane vesical (OMV).

34. The method of embodiment 23 and further comprising the step of introducing into the host mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding a nucleotide sequence overexpressing an endogenous Loquacious polypeptide according to nucleotide sequence SEQ ID NO. 7.

35. The method of embodiment 34 and further comprising the step of co-expressing and transporting said dsRNA polynucleotide, and said Loqs2 polypeptide and Loquacious polypeptide to the host mosquito wherein the dsRNA polynucleotide, Loqs2 polypeptide, and Loquacious polypeptide enhance the host mosquito’s endogenous RNAi response disrupting replication of said viral pathogen.

36. The method of embodiment 23 wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

37. The method of embodiment 23 wherein said step of enhancing the host mosquito’s endogenous RNAi response comprises the step of enhancing the host mosquito’s endogenous RNAi response in the midgut disrupting replication of said viral pathogen in said midgut.

38. A method of enhancing the RNA-interference (RNAi) response in a host mosquito comprising the steps of:

- introducing into the host mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding:

- at least one double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito, or an essential gene of a viral pathogen carried by the mosquito; - a nucleotide sequence overexpressing an endogenous Loquacious polypeptide;

- co-expressing and transporting said dsRNA polynucleotide and said Loquacious polypeptide to the host mosquito; and

- wherein the dsRNA polynucleotide and said Loquacious polypeptide enhance the host enhance said host mosquito’s endogenous RNAi response toward said endogenous gene in a host mosquito, or an essential gene of a viral pathogen.

39. The method of embodiment 38 wherein said nucleotide sequence overexpressing an endogenous Loquacious polypeptide comprises an endogenous Loquacious polypeptide according to amino acid sequence SEQ ID NO. 8.

40. The method of embodiment 38 wherein the endogenous gene in the mosquito comprises an endogenous gene according to the nucleotide sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 11, and SEQ ID NO. 12.

41. The method of embodiment 38 wherein said endogenous gene comprises the rackl gene.

42. The method of embodiment 41 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito the nucleotide sequence according to SEQ ID NO. 2.

43. The method of embodiment 42 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to target the guanine nucleotide binding domain of the rackl gene, identified as SEQ ID NOs. 9, 10.

44. The method of embodiment 38 wherein said one or more viral pathogens comprises one or more viral pathogens selected from the group consisting of: a Zika virus, La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick-borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, and a Flock House virus.

45. The method of embodiment 38 wherein the genetically modified bacteria is obtained from a bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27. 46. The method of embodiment 38 wherein said transporting comprises the step of transporting said dsRNA polynucleotide and said Loquacious polypeptide to the host mosquito through an outer membrane vesical (OMV).

47. The method of embodiment 38 and further comprising the step of introducing into the host mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding a nucleotide sequence overexpressing an endogenous Loqs2 polypeptide according to amino acid sequence SEQ ID NO. 6.

48. The method of embodiment 47 and further comprising the step of co-expressing and transporting said dsRNA polynucleotide, and said Loqs2 polypeptide and Loquacious polypeptide to the host mosquito wherein the dsRNA polynucleotide, Loqs2 polypeptide, and Loquacious polypeptide enhance the host mosquito’s endogenous RNAi response disrupting replication of said viral pathogen.

49. The method of embodiment 38 wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

50. The method of embodiment 38 wherein the step of enhancing the host mosquito’s endogenous RNAi response comprises the step of enhancing the host mosquito’s endogenous RNAi response in the midgut disrupting replication of said viral pathogen in said midgut.

51. A genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding at least one double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in a host mosquito, or an essential gene of a viral pathogen carried by the mosquito, and at least one of the following:

- a nucleotide sequence overexpressing an endogenous Loquacious polypeptide according to amino acid sequence SEQ ID NO. 8; - a nucleotide sequence overexpressing an endogenous Loquacious2 (Loqs2) polypeptide;

- a heterologous nucleotide sequence overexpressing an eukaryotic Loquacious2 (Loqs2) messenger RNA (mRNA) that is translated into a Loqs2 polypeptide in said host mosquito; and

- wherein the dsRNA polynucleotide, and said Loqs2 polypeptide, or said Loqs2 mRNA enhance said host mosquito’s endogenous RNAi response toward said endogenous gene in a host mosquito, or an essential gene of a viral pathogen.

52. The genetically modified bacteria of embodiment 51 wherein the endogenous gene in the mosquito comprises an endogenous gene according to the nucleotide sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 11, and SEQ ID NO. 12.

53. The genetically modified bacteria of embodiment 51 wherein said endogenous gene comprises the rackl gene.

54. The genetically modified bacteria of embodiment 53 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to inhibit expression of an endogenous gene in the mosquito the nucleotide sequence according to SEQ ID NO. 2.

55. The genetically modified bacteria of embodiment 54 heterologous double stranded RNA (dsRNA) polynucleotide that is configured to target the guanine nucleotide binding domain of the rackl gene, identified as SEQ ID NOs. 9, 10.

56. The genetically modified bacteria of embodiment 51 wherein said one or more viral pathogens comprises one or more viral pathogens selected from the group consisting of: a Zika virus, La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick-borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, and a Flock House virus.

57. The genetically modified bacteria of embodiment 51 wherein the genetically modified bacteria is obtained from a bacteria selected from the group consisting of: an endosymbiotic bacteria that persists through the life-cycle of the mosquito, Enterobacter strain Ae073, and E. coli strain HT27. 58. The genetically modified bacteria of embodiment 51 wherein said transporting comprises the step of transporting to the host mosquito through an outer membrane vesical (OMV).

59. The genetically modified bacteria of embodiment 51 wherein said Loqs2 polypeptide comprise the amino acid sequence according to SEQ ID NO. 6.

60. The genetically modified bacteria of embodiment 59 wherein said nucleotide sequence overexpressing an endogenous Loquacious polypeptide according to amino acid sequence SEQ ID NO. 8.

61. The genetically modified bacteria of embodiment 51 wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.

62. The genetically modified bacteria of embodiment 51 wherein the step of enhancing the host mosquito’s endogenous RNAi response comprises the step of enhancing the host mosquito’s endogenous RNAi response in the midgut disrupting replication of said viral pathogen in said midgut.

Additional aspect may become apparent from the description and figures provided below. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B: (A) Plasmid maps of the Loqs2 construct with the flag-tag on the N- terminus; or plasmid maps of the Loqs2 construct with the flag-tag on the C-terminus.

FIG. 2: Gene expression conformation of Loqs2 with the flag-tag on the N-terminus or with the flag-tag on the C-terminus. Western blot of cell pellet of HT27 E. coli cells expressing Loqs2 and dsRNA-GFP with the FLAG tag on either the N or C terminus. Expected size of Loqs2 is 42 kDa.

FIG. 3: Gene expression conformation of Loqs2 with the flag-tag after stable integration into symbiotic Enterobacter strain Ae073. Western blot of cell pellet of Enterobacter delivering Loqs2 protein and dsZikal in Enterobacter Ae073. Blot was probed with FLAG antibody. Positive control: flag-Loqs2 in HT27 cells. Expected size of Loqs2 is 42 kDa. FIG. 4: HT27/ELF Paratransgenically-expressed aLoqs2 is delivered to and expressed in mosquito midguts. Poza Rica Ac. aegypti adults were treated with strains as indicated for 1 day, then midguts were dissected 4 days post-treatment. Blue, nuclei, red, dsRNA. Midguts were treated with primary antibody anti-dsRNA mAh J2 (1 :200) and rabbit anti -FLAG at 1 :500 (ThermoFisher cat# 740001) for one hour, then with the secondary antibodies goat anti mouse Alexa Fluor 555 (1 : 1000) and Goat anti-rabbit: Alexa Fluor 647 (1 : 1,000) for 40 minutes. DAPI (ThermoFisher cat# R37606), which stains nuclear DNA, was also added at this time. Slides were viewed on an Olympus 1X81 Inverted Confocal Laser Scanning Microscope using the lOOx oil immersion objective. DIC, differential interference contrast. Scale bar indicates 10 pm.

FIG. 5: Quantification of GFP transcripts in the mosquito following bacteria feed delivery Loqs2 protein and dsRNA-GFP. Adult mosquitoes which express GFP in the midgut following a bloodmeal were fed a solution without bacteria or with E. coli cells delivering dsRNA-GFP or E. coli cells delivering dsRNA-GFP and the Loqs2 protein. The mosquitoes either received a bloodmeal (BF) or not (NBF) 48 hours post bacteria feed and the mosquitoes were harvested 24 hours later. RNA was extracted from pools of 5 mosquitoes and the copies ofGFP cDNA (Copies GFP cDNA/ul) were quantified by digital droplet PCR. Each sample above represents a pool of 5 mosquitoes. Based on these results we conclude that the co expression of Loqs2 enhances the silencing of GFP by dsRNA-GFP compared to delivery of just the dsRNA-GFP alone

FIG. 6: Plasmid map of construct delivering dsRNA-RACKl.

FIG. 7: Confirmation of dsRNA-RACKl expression in HT27 cells. HT27 E.coli cells expressing dsRNA-RACKl or dsRNA-GFP were grown in liquid culture overnight and an aliquot of the culture was centrifuged to pellet the bacterial cells. Total RNA was extracted from the bacterial cells using Trizol and total RNA was converted to cDNA using the Invitrogen Superscript IV kit. The RACK1 transcript was amplified using Q5 DNA polymerase from NEB and the expected size is 300 bp.

FIG. 8: Quantification of RACK1 knockdown following a bacterial feed. The expression of the RACK1 gene was measured in females 7 days after being fed E.coli delivering dsRNA- GFP or dsRNA- RACK1 using the digital droplet PCR machine which measured the copies/ul of RACK1 cDNA . Silencing the RACK1 gene should help mosquitoes control DENY replication. This plot represents 2 replicates of 5 pooled individuals for dsRNA-GFP and 5 replicates of 5 pooled individuals for the dsRNA- RACK1. Based on these results we conclude that mosquitoes fed E.coli delivering dsRNA- RACK1 had fewer copies/ul of RACK1 transcripts indicating that we are able to reduce the expression of RACK1 in the mosquito through bacterial delivery of dsRNA. Welch Two Sample t-test data: Copies by Treatment t = 32.58, df = 4.0459, p-value = 4.74e-06 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: 2857.731 and 3387.570. Sample estimates: mean in group dsRNA GFP mean in group dsRNA RACK 1 were 3710.7157 and 588.0654.

FIG. 9: Processing of long dsRNA into small siRNAs in the mosquito. Total RNA was extracted from a pool of 12 mosquitoes 3, 5, or 7 days post feeding on HT27 cells delivering dsRNA GFP or dsRNA RACK1 and a Northern Blot was performed. The Northern blot was probed with 16 probes of 40nt that span the sequence of the dsRNA delivered.

FIG. 10: Gene expression confirmation of loquacious gene in HT27 cells. Western blot of cell pellet of HT27 E. coli cells expressing FLAG tagged loquacious and dsRNA-GFP. Expected size of Loqs2 is 42 kDa.

FIG. 11: Quantification of GFP transcripts in the mosquito following bacteria feed delivery loquacious protein and dsRNA-GFP. Adult mosquitoes which express GFP in the midgut following a bloodmeal fed a solution without bacteria or with E. coli cells delivering dsRNA-GFP or E. coli cells delivering dsRNA-GFP and the Loqs protein. The mosquitoes either received a bloodmeal (BF) or not (NBF) 48 hours post bacteria feed and the mosquitoes were harvested 24 hours later. RNA was extracted from pools of 5 mosquitoes and the copies of GFP cDNA (Copies GFP cDNA/ul) was quantified by digital droplet PCR. Each sample above represents a pool of 5 mosquitoes. Based on these results we conclude that the co-expression of the Loqs protein enhances the silencing of GFP by dsRNA-GFP compared to delivery of just the dsRNA-GFP alone.

FIG. 12: Quantification of GFP transcripts in the mosquito following bacteria feed delivering eukaryotic Loqs2 mRNA and dsRNA-GFP. Adult mosquitoes which express GFP in the midgut following a bloodmeal were fed a solution without bacteria or with E. coli cells delivering dsRNA-GFP, E. coli cells delivering dsRNA-GFP and the Loqs2 protein, or E. coli cells delivering dsRNA-GFP and the Loqs2 eukaryotic mRNA. The mosquitoes either received a bloodmeal (BF) or not (NBF) 48 hours post bacteria feed and the mosquitoes were harvested 24 hours later. RNA was extracted from pools of 3 mosquitoes and the copies GFP cDNA (Copies GFP cDNA/ul) was quantified by digital droplet PCR. Each sample above represents a pool of 3 mosquitoes.

FIG. 13: Plasmid map of final cloning product of the construct delivering eukaryotic mRNA and dsRNA-GFP.

FIG. 14: Detection of Loqs2 mRNA in the cell pellet and outer membrane vesicles. Reverse transcription reaction to determine if Loqs2 RNA was being transcribed from the engineered constructs. HT27 E. coli cells containing a plasmid that does not have a cassette expressing Loqs2 mRNA (negative control) and from the intermediate cloning product that contains the cassette expressing Loqs2 eukaryotic mRNA but not dsRNA-GFP, and the final cloning product which contains the cassette expressing Loqs2 mRNA and dsRNA-GFP were grown for 36 hours. Total RNA was extracted from the cell pellet and from isolated outer membrane vesicles and converted to cDNA. The Loqs2 sequence was amplified with an expected size of450 bp.

FIG. 15: Detection of Loqs2 protein, Loqs2 mRNA, and Loquacious protein in the mosquito by mass spectrophotometry. Protein extracts were prepared from whole mosquitoes five days post ingestion of HT27 E. coli cells delivering dsRNA-GFP, Loqs2 protein + dsRNA- GFP, Loqs2 mRNA, or loquacious protein + dsRNA-GFP. The protein extracts were digested with trypsin and analyzed my mass spectrophotometry. Peptides aligning to the protein of interest were found in mosquitoes that ingested bacteria delivering the protein of interest.

FIG. 16: Coverage map of peptides detected by mass spectrophotometry aligning the protein of interest. Protein sequence and % coverage of Loq2 protein and Loquacious protein obtained from analysis of protein digest by LC-MS/MS. Eight peptides were identified for the Loqs2 protein and four peptides were identified for the Loquacious protein.

FIG. 17: Detection of dsRNA-GFP in bacterial cells. Expression of dsGFP was confirmed in bacterial cell pellets. HT27 E. coli cells containing constructs that deliver the Loqs2 protein + dsRNA-GFP or the Loquacious protein + dsRNA-GFP were grown overnight and total RNA was extracted. Total RNA was converted to cDNA and GFP transcripts were amplified.

FIG. 18: Confirmation of Loqs2 protein export in outer membrane vesicles exported from HT27 cells. Western blot to detect Loqs2 or Loquacious protein delivered from constructs containing the Loqs2 protein + dsRNA-GFP or the Loquacious protein + dsRNA-GFP in either in the cell pellet or outer membrane vesicle (OMV) derived from HT27 E. coli cells. Blot was probed with FLAG antibody. Positive control: Ha-tagged protein that was probed with a different antibody (data not shown). Expected size of Loqs2 is 42 kDa.

DETAILED DESCRIPTION OF THE INVENTION(S)

As noted above, in one embodiment the present invention includes a novel paratransgenic system which may further include a novel method for implementation of endogenous and/or RNAi-based expression strategies in which natural mosquito symbiotic bacteria configured to express endogenous genes or interfering RNA polynucleotides that are targeted to mosquito or viral genes resulting in the inhibition of viral infection, colonization, replication or transmission.

In one preferred embodiment, the invention may include novel paratransgenic vector- borne pathogen biocontrol strategies. In this embodiment, the inventive technology includes various cross-kingdom mechanisms for the expression of endogenous effector genes to activate RNAi viral-defense pathways in the midgut of the insect host, and to control viral replication and transmission. In one preferred embodiment, the invention may include systems, methods and compositions for the generation of genetically modified enteric or symbiotic bacteria that may express one or more endogenous effector gene including loquacious2 ( loqs2 ). In this preferred embodiment, expression of loqs2 may activate RNAi viral-defense pathways in the midgut of the insect host, to control viral replication and limit or eliminate spread of the virus transmission to a susceptible host, such as a human.

In another embodiment, the present invention may include the bacterial expression of one or more eukaryotic-like mRNAs. In a preferred embodiment, expression of a eukaryotic-like mRNA may include expression of a eukaryotic loqs2 gene configured to be expressed in a bacterial system and transported to a target host, preferably a mosquito. In further embodiments, such eukaryotic-like mRNA, such as a eukaryotic-like loqs2 mRNA, may activate RNAi viral- defense pathways in the host as generally described herein.

In one embodiment, the present invention may couple the expression of at least one endogenous effector gene that may activate RNAi viral-defense pathways in the midgut of the insect host, such as a loqs2 gene, with the expression of one or more interfering RNA molecules, such as dsRNA polynucleotides configured to down-regulated expression of one or more viral and/or host genes. The synergistic effect of the co-expression of a loqs2 and dsRNA, for example, may more effectively prevent viral infection, replication, and/or transmission.

Another embodiment may include the generation of genetically modified bacteria that may express one or more inhibitory RNA polynucleotides, such as dsRNA that may be configured to inhibit viral replication in a target mosquito host by suppressing expression of one or more of the following endogenous genes selected from the group comprising: chymotrypsin- like serine protease; RACK1; one or more guanine nucleotide-binding proteins; one or more proteins having a guanine nucleotide-binding domains; cadherin 87A; and one or more uncharacterized hydrolase enzymes. In one prefer embodiment, such genetically modified enteric and/or symbiotic bacteria may be isolated from Aedes aegypti , or other mosquito species, that are able to colonize the gut and other tissue to express dsRNA targeted to suppress expression of one or more target genes.

As noted above, mosquitos, such as the in Ae. aegypti species possess natural anti- arboviral RNA interference (RNAi) defense mechanisms. Briefly, by use of the exo-siRNA RNAi pathway, Ae aegypti recognizes arboviral long double-stranded RNA generated during virus replication, digests it to 21 -bp short interfering RNA (siRNA) segments with an RNase III family enzyme called Dicer 2, and uses these as effectors to identify, cleave and inactivate replicating virus genomes. Thus, according to one aspect of the present invention there is provided a method of controlling a pathogenically infected mosquito, the method comprising administering to a mosquito a nucleic acid agent comprising a nucleic acid sequence which specifically downregulates the expression of at least one mosquito gene product, wherein downregulation inhibits viral infection, replication, pathogenicity, or transmission.

The present invention may further include one or more vectors for modulating multiple host genes, wherein the expression vector encodes one or plurality of effector genes or dsRNAs that correspond to one or more select host or viral genes. This embodiment may include the use of a plasmid expression system or stable genome integration. In some embodiments, this expression vector may have one or more expression cassettes, including: at least one gene cassette containing a polynucleotide operably-linked to a promoter sequence, wherein the polynucleotide encodes an effector gene or interfering RNA molecule, such as a dsRNA, that reduces expression of a target gene by RNA interference. For example, each gene cassette can encode a dsRNA molecule that targets an mRNA sequence of two or more different genes. Each gene or gene cassette may be operable linked to a promotor. Examples of suitable promoters for gene suppressing cassettes include, but are not limited to, T7 promoter, bla promotor, EG6 promoter, pol II promoter, Ell promoter, and CMV promoter and the like. Optionally, each of the promoter sequences of the gene promoting cassettes and the gene cassettes can be inducible and/or tissue-specific.

Another embodiment of the present invention may include novel methods of generating a paratransgenic system directed to suppressing arbovirus pathogens, where the arbovirus may be selected from the group consisting of an alphavirus, a flavivirus, a bunyavirus and an orbivirus. According to some embodiments of the invention, the arbovirus is selected from the group consisting of a La Crosse encephalitis virus, an Eastern equine encephalitis virus, a Japanese encephalitis virus, a Western equine encephalitis virus, a St. Louis encephalitis virus, a Tick- borne encephalitis virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Chikungunya virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Bluetongue disease virus, a Sindbis Virus, a Rift Valley Fever virus, a Colorado tick fever virus, a Murray Valley encephalitis virus, an Oropouche virus, a Flock House virus and a Zika virus and the like.

Another embodiment of the inventive biocontrol systems may be to optimize dsRNA survival and/or delivery to a disease-carrying vector, such as a mosquito, by co-expression of helper genes that may enhance the uptake, stabilization, and intercellular transfer and/or mobilization of dsRNA or other endogenous effector molecules, such as loqs2 , in mosquitoes.

Yet another embodiment of the present invention may include the expression of endogenous effector genes to activate RNAi viral-defense pathways in the midgut of the insect host and/or the expression of one or more interfering RNA molecules, such as double stranded RNA (dsRNA) polynucleotides configured to down-regulated expression of one or more viral genes or endogenous host genes in a functional RNAselll mutants - configured to cleave dsRNA precursors to 2l-23nt smRNA duplexes that are functional in RNAi - competent to form functional RISC complex.

In another embodiment, the invention provides for methods of environmental dispersal of one or more engineered microorganisms. Such methods may utilize aerial (e.g., crop dusters) spraying to cover the greatest land area at the lowest cost. Additional methods of dispersion known within the field may also be contemplated within the inventive technology. Significantly, many microorganisms can persist dried in the soil for multiple years allowing for the re- population of dry areas after precipitation. This is particularly suitable for areas where larval habitats appear and vanish alternatively between dry and rainy seasons.

As noted above, translational control of host, as well as viral genes is an important determinant of viral pathogenesis. A screen of long non-coding RNAs expressed in Aedes aegypti strains that showed varying susceptibility to dengue virus in the context of co-infection with the bacteria Wolbachia identified a substantial upregulation (2.7 log fold change, 500X) of long non-coding RNAs (Feature_26588l and Feature_266l0l below) with homology to a guanine nucleotide binding protein domain within the RACK1 gene in mosquitoes that were better able to control viral infection.

RACK1 protein (SEQ ID NO. 3), which in this example is merely an example of a larger class of guanine nucleotide binding proteins, or proteins having a guanine nucleotide binding protein domain, has been implicated in the control of molecular signaling cascades, translational control, kinase inhibition, and apoptosis. Although RACK1 has not been directly shown to be involved in controlling viral infection in mosquitoes, the role of apoptosis in controlling viral infection in Ae. aegypti has been demonstrated and considered an important antiviral response. The role of protein kinase inhibitors in controlling dengue replication has also been demonstrated. The RACK1 protein is a receptor of protein kinase C and inhibition of protein kinase C has been shown to promote dengue virus replication in vertebrates.

In addition, there is interplay between protein kinase C activity and apoptosis. Of greatest importance, the RACK1 gene has been shown to be required for dengue replication in humans by mediating the glycosylation of nonstructural protein 1 and 4B (NS1 and NS4B). The Feature_26588l (SEQ ID NO. 9) and Feature_266l0l (SEQ ID NO. 9) disclosed herein are the sequences of the long-coding RNAs that mapped to the guanine nucleotide binding domain of the RACK1 gene in Ae. aegypti and may be sequence targets to design the dsRNA against as a method of viral bio-control.

Serine proteases constitute a large proportion of the proteolytic enzymes found in the midguts of Aedes aegypti. Research has shown that these proteases are associated with the infection and dissemination rates of flaviviruses by the mosquito. In some cases, inhibition of serine protease have been positively correlated with viral infectivity suggesting that when active they inhibit the ability of the virus to infect the host gut epithelium. Other research shows that inhibition of serine protease activity was correlated with lower viral titer and dissemination leading to the hypothesis that serine proteases function in proteolytic viral processing.

Furthermore, expression of serine proteases in A. aegypti has been shown to be influenced by viral infection as well as microbiome composition. Analysis of non-coding RNA in a study of Wolbachia infected mosquitoes supports the findings that serine proteases are highly varied in their expression when exposed to symbionts. In this analysis a plurality of particular long non-coding RNA that demonstrated apparent suppressed expression of a serine protease with analogy to a Chymotrypsin-like serine protease showed consistent enhanced expression (Log fold changes ranging from 2.6 - 5.1) in Wolbachia infected mosquitoes that have reduced (5% of controls) viral titers relative to strains of Wolbachia infected mosquitoes that have viral titers identical to uninfected mosquitoes having very high viral titers. As such, viral replication may inhibited by suppressing expression of chymotrypsin-like serine protease (SEQ ID NO. 11). As such, in one embodiment, the invention may include the target inhibition of chymotrypsin-like serine protease (SEQ ID NO. 11) in a mosquito through the bacterial delivery of a targeted dsRNA to control replication and transmission. Such inhibition may be enhanced through the co-expression and delivery of Loqs2 and/or Loquacious proteins as generally described herein.

Cadherins (Calcium-dependent adhesion molecules) are important in the formation of adherens junctions that bind cells with each other. Cadherins are transmembrane proteins that require calcium for their function. Cell-cell adhesion is mediated by extracellular cadherin domains, whereas the intracellular cytoplasmic tail associates with a large number of cytoplasmic adaptor and signaling proteins as well as with the actomyosin cytoskeleton Cadherins have been recently implicated as important factors in viral infections. E-cadherin has been found to regulate entry of HCV (flaviviridae family) and its silencing inhibited HCV infection. Cadherin has also been found as a receptor molecule critical for DENV (flaviviridae family) entry in Ae. Aegypti. Thus suppression of cahedrin expression may substantially reduce or inhibit Dengue residence in the mosquito gut reducing replication rates and transmission of viral particles to the salivary glands and associated viral transmission to humans. As such, in one embodiment, the invention may include the target inhibition of cadherin 87A (SEQ ID NO. 12) in a mosquito through the bacterial delivery of a targeted dsRNA to control replication and transmission. Such inhibition may be enhanced through the co-expression and delivery of Loqs2 and/or Loquacious proteins as generally described herein. In one preferred embodiment, exemplary bacterial endosymbionts are identified at Tables lAbelow. In one preferred embodiment, HT27 E. coli, and. /or symbiotic Enterobacter strain Ae073 may be preferred. In this embodiment, such genetically engineered bacteria may express and translate d aegypti Loqs2 (also referred to as Loqs2) (according to nucleotide sequence SEQ ID NO. 65; according to amino acid sequence SEQ ID NO. 6). Exogenous delivery of bacterially expressed Loqs2 may rescue transcriptional silencing phenotype of Loqs2 in midgut, and activate/enhance endogenous anti-viral RNAi defense pathway and control/eliminate virus from the insect host. In a preferred embodiment, the Loqs2 coding region may be codon optimized for bacterial expression and can be further modified to include secretion peptides, for example as shown below as Table 2 below, to facilitate transfer of bacterially expressed Loqs2 from a bacteria to host cells via inclusion in outer membrane vesicles (OMVs) or direct secretion to the media / epithelial cells by T3SS (e.g., but not limited to, OmpA, HylA signaling peptides, respectively).

Another embodiment may include the co-expression of Loqs2 and pathogen targeted dsRNA molecules, such a pathogen target dsRNA directed to ZIKA of DENV as identified by the inventors Sayre et al., in ETS Pat. Application No. 16/031607, such sequences being specifically incorporated herein by reference). In another embodiment, penetrance and efficiency of trans-kingdom anti -viral RNAi mediated control in the hosts midgut by delivery of Loqs2 can be further improved by engineering RNAselll null mutant strains of symbiotic or enteric bacteria to co-expressed Loqs2 and dsRNA molecules homologous to the viral genome to be targeted by RNAi. In one embodiment, a target viral genome may be a viral gene of an arbovirus pathogen such as Alphaviruses pathogens (e.g. Eastern Equine encephalitis virus, Western Equine encephalitis virus, Venezuelan Equine encephalitis virus, Ross River virus, Sindbis Virus and Chikungunya virus), Flavivirus pathogens (e.g. Zika virus, Japanese Encephalitis virus, Murray Valley Encephalitis virus, West Nile Fever virus, Yellow Fever virus, Dengue Fever virus, St. Louis encephalitis virus, and Tick-borne encephalitis virus), Bunyavirus pathogens (e.g. La Crosse Encephalitis virus, Rift Valley Fever virus, and Colorado Tick Fever virus), Orthobunyavirus pathogens (e.g. Oropouche virus), and Orbivirus (e.g. Bluetongue disease virus)).

In further embodiments, dsRNA molecules, either transcribed from convergent promoters or folding into hpRNA, may be a substrate for mosquito Dicer enzymes giving rise to mature smRNA duplexes to be loaded into Argonaute2 protein - a step requiring Loqs2 function. This embodiment may allow pre-loading of RISC complexes with siRNAs homologous to targeted viral genomes allowing for a rapid response of host’s defense to viral particle uptake through feeding.

Another preferred embodiment may include the co-expression of Loqs2 and pathogen targeted dsRNA molecules in RNAselll functional mutants. In this embodiment, penetrance and efficiency of trans-kingdom anti -viral RNAi mediated control in the host’s midgut by delivery of Loqs2 can be further improved by engineering RNAselll functional mutant enteric bacterial strains to co-expressed Loqs2 and dsRNA molecules homologous to the viral genome to be targeted by RNAi (e.g. but not limited to, dengue, Zika, Yellow Fever, Chikungunya among other noted herein). In this embodiment, RNaselll functional mutants cleave dsRNA precursors to 2l-23nt smRNA duplexes directed to one or more viral or host endogenous genes that are functional in RNAi - competent to form functional RISC complex. (Exemplary RNaselll enzymes and their characteristics are described in co-owned No. PCT/US19/40747, exemplary sequences are provided below and are specifically incorporated herein by reference) This strategy may allow pre-loading of RISC complexes with siRNAs homologous to targeted viral genomes allowing for a rapid response of host’s defense to viral particle uptake through feeding. Efficiency of RISC loading can be further increased in some embodiments by bacterial co- expression of r2d2 dsRNA binding protein (SEQ ID NO. 13) known to interact with Loqs2.

Additional embodiments may include the expression of eukaryotic-like mRNA encoding a Loqs2 protein for translation in a mosquito host’s midgut cells. In an alternative embodiment, to export of bacterially produced Loqs2 protein, midgut complementation of anti-viral RNAi pathway with trans-kingdom delivery of Loqs2, can be achieved by bacterial transcription of eukaryotic-like mRNA encoding Loqs2 protein for translation in host’s midgut cells. Such coding regions may be designed to exclude bacterial ribosome binding sequences and include 5’CAP independent eukaryotic translation drivers and poly A tails to promote eukaryotic translation. (In this embodiment, a eukaryotic-like Loqs2 mRNA may be generated by the systems and methods of engineering a eukaryotic mRNA configured to be expressed in a prokaryotic organism, and exported to a eukaryotic target for translation as generally described by the inventors in Sayre et ah, in PCT/US 19/40747, such systems methods and compositions being specifically incorporated herein by reference). In this preferred embodiment, the eukaryotic-like Loqs2mRNA may include paired termini translation competent constructs (ptRNA), stabilized by pairing of 5’ and 3’- end regions and including IRES sequences for ribosome recruitment and poly-adenylation recognition signals. Translation of circular RNAs (circRNAs) formed by 5’ and 3’ joining through back-splicing, with recruitment of ribosomes associated with RNA methylation before the start codon. In this embodiment, the IRES sequence(s) (Internal Ribosome Entry Sites) (SEQ ID NO. 37) may facilitate recruitment of ribosomes to an engineered gene construct, such as Loqs2 encoding ptRNA and mimic the role of RNA methylation in driving translation of circRNAs. IRES sequences are common in viral genomes and part of the strategy to sequester the host’s ribosomes for 5’CAP independent translation of viral proteins. Export of circRNA-Loqs2 coding mRNA can be facilitated by co- expression of dsRNA binding proteins tagged with secretion peptides - as indicated above.

Additional embodiments may include one or more synergistic genetic modifications to facilitate smRNA biogenesis or RISC loading with nucleotide sequences homologous to viral genome in combination with bacterial expression of eukaryotic-like mRNA encoding Loqs2 protein competent for translation in a mosquito host’s midgut cells. In this embodiment, mosquito midgut complementation of anti-viral RNAi pathway with trans-kingdom delivery of Loqs2, can be achieved by bacterial transcription of eukaryotic-like mRNA encoding Loqs2 protein for translation in host’s midgut cells. Such coding regions will be designed to exclude bacterial ribosome binding sequences and include 5’CAP independent eukaryotic translation drivers and polyA tails to promote eukaryotic translation. In this preferred embodiment, the eukaryotic-like mRNA may include paired termini translation competent constructs (ptRNA), stabilized by pairing of 5’ and 3’- end regions and including IRES sequences for ribosome recruitment and poly-adenylation recognition signals. Translation of circular RNAs (circRNAs) formed by 5’ and 3’ joining through back-splicing has been demonstrated previously in the art, with recruitment of ribosomes associated with RNA methylation before the start codon. Export of circRNA-Loqs2 coding mRNA from bacteria to host can be facilitated by co-expression of dsRNA binding proteins tagged with secretion peptides - as indicated above.

In this embodiment, the genetically engineered bacteria described above may further be modified to express RNAselll functional mutants cleave dsRNA precursors to 2l-23nt smRNA duplexes that are functional in RNAi - competent to form functional RISC complex. (Exemplary RNAselll enzymes and their characteristics are described in co-owned PCT/US19/40747, exemplary sequences are provided below and are specifically incorporated herein by reference) This strategy may allow pre-loading of RISC complexes with siRNAs homologous to targeted viral genomes allowing for a rapid response of host’s defense to viral particle uptake through feeding. Efficiency of RISC loading can be further increased in some embodiments by bacterial co-expression of r2d2 dsRNA binding protein known to interact with Loqs2.

In this embodiment, again the genetically engineered bacteria described above may further be modified to include expression of eukaryotic-like mRNA encoding Loqs2 protein for translation in host’s midgut cells. Such combination or genetic modifications may facilitate smRNA biogenesis or RISC loading with nucleotide sequences homologous to viral genome.

In another preferred embodiment, one or more chymotrypsin-like serine protease targeted dsRNA molecules can be transcribed in RNaselll mutant enteric and/or endosymbiotic bacteria strains or bacterial strains engineered to express functional RNaselll mutant proteins with a 21- 22nt dsRNA cleavage register. (Exemplary RNaselll enzymes and their characteristics are described in co-owned PCT US 19/40747, which is incorporated herein by reference) For example, dsRNA produced in the former may trigger RNAi in mosquito hosts. In the latter embodiment, is expected to be readily bound by mosquito Argonaute proteins forming a functional RNA induced silencing complex (RISC) which in turn will target cleavage and subsequent degradation of chymotrypsin-like serine protease coding RNA. This inhibition of serine protease expression/activity may result in lower viral titer and dissemination in the host mosquito.

Additional embodiments may include the delivery of bacterially produced smRNA duplexes by co-expressing R2D2 RISC loading co-factor protein in the bacterial host. R2D2 (SEQ ID NO. 13) is known to bind smRNA duplexes and to interact with Dcr-2 in vivo , functioning in smRNA strand selection and RISC assembly. In this embodiment, dsRNA binding proteins will also be co-expressed in order to facilitate export and Dcr-2 processing in mosquito cells of long dsRNA molecules transcribed in engineered bacteria. Drosophila sp., and its A. aegypti homologous, Loquacious (Loqs) and (Loqs2) dsRNA binding protein, isoform Loquacious, may be used for this purpose and tagged with a secretion peptide ( See Table 2 from US Provisional Application No. 62/757,844) to maximize secretion from bacteria to host cells. Once uploaded, Loquacious may recruit R2D2/Dcr-2 complex, which in turn may process the dsRNA precursor into 2l-22nt small interfering RNAs and promote RISC assembly. Such embodiment may more efficiently induce an RNA interference mechanism to downregulate expression of chymotrypsin-like serine protease. This inhibition of serine protease expression/activity may result in lower viral titer and dissemination in the host mosquito.

Additional embodiments may include the dsRNA-mediated downregulation of racAT/guanine nucleotide-binding protein gene expression. In one preferred embodiment, the present inventors may generate genetically engineered symbiotic bacteria that are able to colonize the gut and other tissues (endosymbionts) of a target mosquito. Exemplary endosymbionts are identified at Table 1A below. As noted above, in one embodiment an endosymbiont may be genetically engineered to express one or more RNA interfering molecules, such as a dsRNA (according to the nucleotide sequence SEQ ID NO. 1), that may downregulate expression of RACK1. It should be noted that RACK1 is an exemplary target only, and not intended to be limiting on the other guanine nucleotide-binding proteins, and/or proteins having a guanine nucleotide-binding domain.

In a preferred embodiment, an endosymbiont may be genetically engineered to express one or more nucleotides that heterologously express a dsRNA targeting expression of RACK1 gene, such as the nucleotide sequence identified at SEQ ID NO. 1. In another preferred embodiment, dsRNA may be generated that target the guanine nucleotide binding domain of the RACK 1 gene, identified as SEQ ID NOs. 9, 10. Such dsRNA may be secreted into the target mosquito host and inhibit RACK1 expression through an induced RNA interference mechanism, which as discussed above may be enhanced by co-expression and delivery of Loqs2 or Louacious. This inhibition of RACK 1 expression/activity may result in improved viral biocontrol in the host mosquito.

Additional embodiments may include the dsRNA-mediated downregulation of Cadherin 87A gene expression in a mosquito host. The present inventors conducted a genetic screen to identify single-nucleotide polymorphisms (SNPs) linked to genes blocking dengue viral replication carried out in 3 different mosquito lines infected with Wolbachia which exhibited differential efficiency in blocking DENV3 (high blocking, low blocking and randomly selected). Replication revealed that Cadherin 87A was closely linked to SNPs that were highly correlated with the failure of DENV3 to replicate in mosquitoes. In addition, a comparison of expression levels of Cadherin87A from wild-type mosquitoes and those infected with Wolbachia showed that Cadherin87A is statistical significantly down-regulated in Wolbachia- infected mosquitoes that do not support DENV3 replication. Furthermore, comparative analyses of Cadherin87A expression levels in the above mentioned mosquito lines showed that Cadherin 87A is down- regulated in the high-blocking mosquito group in comparison to the low-blocking group.

As such, in one preferred embodiment, the present inventors may generate genetically engineered bacteria, that are able to colonize the gut and other tissues of a target mosquito. As noted above, in one embodiment an endosymbiont may be genetically engineered to express one or more RNA interfering molecules that may downregulate expression of Cadherin 87A according to nucleotide sequence identified as SEQ ID NO. 12. In a preferred embodiment, an endosymbiont may be genetically engineered to express one or more dsRNA targeting expression of cadherin 87A gene. Such dsRNA may be secreted into the target mosquito host and inhibit Cadherin 87A expression through an induced RNA interference mechanism. This suppression of Cadherin 87A expression may substantially reduce or inhibit Dengue residence in the mosquito gut reducing replication rates and transmission of viral particles to the salivary glands and associated viral transmission to humans. Such dsRNA may be secreted into the target mosquito host and inhibit Cadherin 87A expression through an induced RNA interference mechanism, which as discussed above may be enhanced by co-expression and delivery of Loqs2 or Louacious. This inhibition of Cadherin 87A expression/activity may result in improved viral biocontrol in the host mosquito.

In another preferred embodiment, one or more Cadherin 87A targeted dsRNA molecules can be transcribed in RNAselll mutant enteric and/or endosymbiotic bacteria strains or bacterial strains engineered to express functional RNAselll mutant proteins with a 2l-22nt dsRNA cleavage register. (Exemplary RNAselll enzymes and their characteristics are described in co owned PCT/US 19/40747, which is incorporated herein by reference) For example, dsRNA produced in the former may trigger RNAi in mosquito hosts. In the latter embodiment, is expected to be readily bound by mosquito Argonaute proteins forming a functional RNA induced silencing complex (RISC) which in turn will target cleavage and subsequent degradation of cahedrin coding RNA. This suppression of Cadherin 87A expression may substantially reduce or inhibit Dengue residence in the mosquito gut reducing replication rates and transmission of viral particles to the salivary glands and associated viral transmission to humans.

Additional embodiments of the invention may include systems and methods for enhanced Outer Membrane Vesicles (OMVs) formation. The present invention may further include systems, methods and compositions to enhance secretion of one or more target molecules from a genetically engineered bacterium. In one preferred embodiment, a mosquito enteric, symbiotic and/or endosymbiotic may be genetically engineered to express one or more exogenous or heterologous RNA molecules or other targets polypeptides. Such genetically engineered bacteria may be further modified to exhibit enhanced or greater than wild-type levels of Outer Membrane Vesicles (OMVs) formation. As a result of this enhanced OMV formation, the secretion of any expressed dsRNAs, miRNAs, peptides, proteins or other target macromolecules is increased from the aforementioned genetically engineered bacteria to the host may be increased.

In one embodiment, upregulation of OMV formation may be accomplished through genetically modifying a target bacterium, preferably a symbiotic or endosymbiotic bacteria, to inhibit or eliminate expression of VacJ/Yrb genes. Inhibition or deletion of VacJ/Yrb genes may result in asymmetric accumulation of phospholipids in the outer leaflet of the outer membrane. This asymmetric expansion may further initiate an outward bulging which in turns results in increased secretion.

In another embodiment, upregulation of OMV formation may be accomplished through genetically modifying a target bacteria, preferably a symbiotic or endosymbiotic bacteria, to express mutated forms of Lpp and/or the Tol-Pal system of bacteria that may result in inducing the loss of anchoring structures, which in turn increases the rate of OMV secretion.

In still another embodiment, upregulation of OMV formation may be accomplished through genetically modifying a target bacteria, preferably a symbiotic or endosymbiotic bacteria, to express mutated forms of one or more enzymes involved in peptidoglycan degradation which may induce OMV formation.

As noted above, in some embodiment, the systems, methods and compositions may incorporate paratransgenic strategies and bacteria. Paratransgenesis, as used herein may be generally understood as a technique that attempts to control vector populations through transgenesis of a symbiont of the vector. The goal of this technique is to control vector-borne diseases. The first step is to identify proteins that may endogenously or exogenously disrupt viral pathogens. The genes coding for these proteins are then introduced into the symbiont, so that they can be expressed in the vector. The final step in the strategy is to introduce these transgenic symbionts into vector populations in the wild. Characteristics of a successful paratransgenesis system may include: i) The symbiotic bacteria can be grown in vitro easily; ii) They can be genetically modified, such as through transformation with a plasmid containing the desired gene; The engineered symbiont is stable and safe; The association between vector and symbiont cannot be attenuated; and Field delivery is easily handled. A paratransgenic system is a system that can achieve paratransgenesis in a target organism.

Identification of suitable commensal bacteria that are non-pathogenic to humans or animals among the many organisms that insects harbor, particularly in their digestive systems, is paramount for the success of a paratransgenic system. In mosquitoes, these bacteria are involved in various biological functions associated with digestion, primarily in the midgut. There is a close association between blood-dependent insects and symbiotic microorganisms that help the anabolic processes of vitellogenesis and ovogenesis. Eradication of these bacteria leads to a decline in fecundity and a slower growth rate. Interference with the digestion of proteins in mosquito blood-meals can reduce fecundity and may represent a new approach for controlling mosquito populations and preventing the transmission of pathogens.

For example, the chosen bacteria should be capable of colonizing a wide variety of mosquito species so that they can be deployed in different species and isolated strains. Furthermore, the number of bacteria increases dramatically (100 to 1000 of times) after ingestion of blood, resulting in a proportional increase in the amount of effector molecules expressed and secreted by GM bacteria, leading to various possible outcomes: obstructing pathogen transmission, reducing the mosquito’s vector capacity, preventing fertilization of eggs, interfering with embryogenesis and causing the death of the mosquito. These technical and physiological challenges make the development of paratransgenic systems extremely difficult. Importantly, these technical issues are such that many paratransgenic systems are neither effective nor appropriate as an effective biocontrol strategy. These difficulties may also prevent many paratransgenic systems from being appropriately scaled-up to be effective for environmental deployment. Generally, biocontrol means utilizing disease-suppressive microorganisms to eliminate, control or prevent infection, expression and/or transmission of selected pathogens.

Each of the aforementioned systems may be embodied in genetic constructs that may include transcription regulation elements such as promoters, terminators, co-activators and co- repressors and other control elements at may be regulated in prokaryotic as well as eukaryotic systems. Such systems may allow for control of the type, timing and amount of, inhibitory RNA molecules or other proteins, expressed within the system. Additional embodiments may include genetic constructs that may be induced through additional outside and/or environmental factors, such as the presence of a specific protein or compound, such as stress related proteins generated in response to a pathogen or even proteins and other precursor compounds generated by pathogens and the like. Such beneficial co-expressed polypeptides may generally be referred to as“helper” genes or“helper” proteins which may be outlined below.

Another embodiment of the present invention may include a cell comprising the isolated nucleic acid agent, such as a dsRNA, or the nucleic acid construct, such as a plasmid, or stable genome integration, of some embodiments of the invention. The present invention may further include a cell comprising the isolated nucleic acid agent, such as a dsRNA, or the nucleic acid construct, such as a plasmid. Some embodiments of the invention that may further include one or more“helper” genes that may aid in gene suppression, dsRNA survival, dsRNA uptake, dsRNA secretion and the like. Another aim of the present invention may include the use of autotrophic bacteria, as well as an RNase III deficient strain of bacteria as a nucleic acid agent (e.g., dsRNA) transmission vector.

The term“mosquito” or“mosquitoes” as used herein, refers to an insect of the family Culicidae. The mosquito of the invention may include an adult mosquito, a mosquito larva, a pupa or an egg thereof. Typically, a mosquito’s life cycle includes four separate and distinct stages: egg, larva, pupa, and adult. Thus, a mosquito’s life cycle begins when eggs are laid on a water surface (e.g. Culex, Culiseta, and Anopheles species) or on damp soil that is flooded by water (e.g. Aedes species). Most eggs hatch into larvae within 48 hours. The larvae live in the water, feeding on microorganisms and organic matter and come to the surface to breathe. They shed their skin four times, growing larger after each molting, and on the fourth molt the larva changes into a pupa. The pupal stage is a resting, non-feeding stage of about two days. At this time the mosquito turns into an adult. When development is complete, the pupal skin splits and the mosquito emerges as an adult.

According to one embodiment, the mosquitoes are of the sub-families Anophelinae and Culicinae. According to one embodiment, the mosquitoes are of the genus Culex, Culiseta, Anopheles and Aedes. Exemplary mosquitoes include, but are not limited to, Aedes species e.g. Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cantator, Aedes cinereus, Aedes rusticus, Aedes vexans', Anopheles species e.g. Anopheles gambiae, Anopheles freeborni, Anopheles arabiensis, Anopheles funestus, Anopheles gambiae Anopheles moucheti, Anopheles balabacensis, Anopheles baimaii, Anopheles culicifacies, Anopheles dims, Anopheles latens, Anopheles leucosphyrus, Anopheles maculatus, Anopheles minimus, Anopheles fluviatilis sd., Anopheles sundaicus Anopheles superpictus, Anopheles farauti, Anopheles punctulatus, Anopheles sergentii, Anopheles stephensi, Anopheles sinensis, Anopheles atroparvus, Anopheles pseudopunctipennis, Anopheles bellator and Anopheles cruzii', Culex species e.g. C. annulirostris, C. antennatus, C. jenseni, C. pipiens, C. pusillus, C. quinquefasciatus, C. rajah, C. restuans, C. salinarius, C. tarsalis, C. territans, C. theileri and C. tritaeniorhynchus; and Culiseta species e.g. Culiseta incidens, Culiseta impatiens, Culiseta inornata and Culiseta particeps.

The term“nucleic acid” as used herein, refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically,“nucleic acid or“nucleic acid agent” polymers occur in either single or double-stranded form, but are also known to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein.

The terms “genetically modified” “bio-transformed,” “transgenic”, “transformed”, “transformation”, and“transfection” is similar in meaning to“recombinant”.“Transformation”, “transgenic”, and“transfection” refer to the transfer of a polynucleotide into the genome of a host organism or into a cell. Such a transfer of polynucleotides can result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining extra-chromosomally (not integrated into the chromosome of the cell). Genetically stable inheritance may potentially require the transgenic organism or cell to be subjected for a period of time to one or more conditions which require the transcription of some or all of the transferred polynucleotide in order for the transgenic organism or cell to live and/or grow. Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell. One may need to grow the cell under certain growth or environmental conditions in order for the expression vector to remain in the cell or the cell's progeny. Further, for expression to occur the organism or cell may need to be kept under certain conditions. Host organisms or cells containing the recombinant polynucleotide can be referred to as“transgenic” or“transformed” organisms or cells or simply as“transformants” as well as recombinant organisms or cells.

As used herein, the term“essential” with respect to a gene, means a gene that is involve in viral replication, pathogenicity, or transmission. The term“enhanced,” for example as used to describe a response, such as an RNAi response, means a greater than wild-type, or greater then without oen or more enhancer elements, such as a Loqs2 proteins in one embodiment.

A genetically altered organism is any organism with any change to its genetic material, whether in the nucleus or cytoplasm (organelle). As such, a genetically altered organism can be a recombinant or transformed organism. A genetically altered organism can also be an organism that was subjected to one or more mutagens or the progeny of an organism that was subjected to one or more mutagens and has changes in its DNA caused by the one or more mutagens, as compared to the wild-type organism (i.e., organism not subjected to the mutagens). Also, an organism that has been bred to incorporate a mutation into its genetic material is a genetically altered organism.

The term“vector” refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host. The polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; can be regulatory in nature; etc. There are various types of vectors including virus, plasmid, bacteriophages, cosmids, and bacteria.

An“expression vector” is a nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, expression vectors are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an“expression cassette”. In contrast, as described in the examples herein, a“cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of the cassette assists in the assembly of the expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).

A polynucleotide sequence is“operably linked to an expression control sequence(s)” (e.g., a promoter and, optionally, an enhancer as well as a terminator) when the expression control sequence controls and regulates the transcription and/or translation of that polynucleotide sequence. As used herein, the phrase“gene product” refers to an RNA molecule or a protein.

This invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include Green and Sambrook, 4th ed. 2012, Cold Spring Harbor Laboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual (1993); and Ausubel et ah, eds., Current Protocols in Molecular Biology, l994-current, John Wiley & Sons. Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology maybe found in e.g., Benjamin Lewin, Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632); Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0- 632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569- 8)·

As used herein, the term“controlling” and/or“bio-control” refers to reducing and/or regulating pathogen/disease progression and/or transmission.

As used herein, the phrase“host” refers to an organism carrying a disease-causing pathogen, an organism susceptible to a disease-causing pathogen, an organism population where members are carrying a disease-causing pathogen, or an organism population where members are susceptible to a disease-causing pathogen. A preferred host may be a mosquito.

Homology (e.g., percent homology, sequence identity + sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment. As used herein, “sequence identity” or“identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are to have“sequence similarity” or“similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9]

According to a specific embodiment, homolog sequences are at least 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95% or even identical to the sequences (nucleic acid or amino acid sequences) provided herein. Homologs of the sequences provided herein of between 50%-99% may be included in certain embodiments of the present invention.

As generally noted above, according to some aspects of the invention, there is provided an isolated nucleic acid agent comprising a nucleic acid sequence, which specifically downregulates the expression of at least one pathogen or host gene product. According to one embodiment, the agent is a polynucleotide agent, such as an RNA silencing agent. In a preferred embodiment, agent is a polynucleotide agent, such as dsRNA, configured to induce RNA interference.

As used herein, the term“interfering RNA molecules” or“interfering RNA” refers to an RNA which is capable of inhibiting or“silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g. the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

In some embodiments of the invention, the nucleic acid agent is a double stranded RNA (dsRNA). As used herein the term “dsRNA” relates to two strands of anti-parallel polyribonucleic acids held together by base pairing. The two strands can be of identical length or of different lengths provided there is enough sequence homology between the two strands that a double stranded structure is formed with at least 60%, 70% 80%, 90%, 95% or 100% complementary over the entire length. According to an embodiment of the invention, there are no overhangs for the dsRNA molecule. According to another embodiment of the invention, the dsRNA molecule comprises overhangs. According to another embodiment of the invention, a dsRNA may have a loop in one end when transcribed from the cassette. According to other embodiments, the strands are aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (i.e., for which no complementary bases occur in the opposing strand) such that an overhang of 1, 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed.

It will be noted that the dsRNA can be defined in terms of the nucleic acid sequence of the DNA encoding the target gene transcript, and it is understood that a dsRNA sequence corresponding to the coding sequence of a gene comprises an RNA complement of the gene’s coding sequence, or other sequence of the gene which is transcribed into RNA.

The inhibitory RNA sequence can be greater than 90% identical or even 100% identical, to the portion of the target gene transcript. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript under stringent conditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 60 degrees C hybridization for 12- lb hours; followed by washing). The length of the double-stranded nucleotide sequences complementary to the target gene transcript may be at least about 18, 19, 21, 25, 50, 100, 200, 300, 400, 491, 500, 550, 600, 650, 700, 750, 800, 900, 1000 or more bases. In some embodiments of the invention, the length of the double-stranded nucleotide sequence is approximately from about 18 to about 530, or longer, nucleotides in length.

The present teachings relate to various lengths of dsRNA, whereby the shorter version i.e., x is shorter or equals 50 bp (e.g., 17-50), is referred to as siRNA or miRNA. Longer dsRNA molecules of 51-600 are referred to herein as dsRNA, which can be further processed for siRNA molecules. According to some embodiments, the nucleic acid sequence of the dsRNA is greater than 15 base pairs in length. According to yet other embodiments, the nucleic acid sequence of the dsRNA is 19-25 base pairs in length, 30-100 base pairs in length, 100-250 base pairs in length or 100-500 base pairs in length. According to still other embodiments, the dsRNA is 500- 800 base pairs in length, 700-800 base pairs in length, 300-600 base pairs in length, 350-500 base pairs in length or 400-450 base pairs in length. In some embodiments, the dsRNA is 400 base pairs in length. In some embodiments, the dsRNA is 750 base pairs in length.

The term“siRNA” refers to small inhibitory RNA duplexes (generally between 17-30 base pairs, but also longer e.g., 31-50 bp) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 2lmers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a lOO-fold increase in potency compared with 2lmers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (2lmer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC. It has been found that position of the 3 '-overhang influences potency of a siRNA and asymmetric duplexes having a 3'-overhang on the antisense strand are generally more potent than those with the 3 '-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.

In certain embodiments, dsRNA can come from 2 sources; one derived from gene transcripts generated from opposing gene promoters on opposite strands of the DNA and 2) from fold back hairpin structures produced from a single gene promoter but having internal complimentary. For example, strands of a double-stranded interfering RNA (e.g., a siRNA) may be connected to form a hairpin or stem -loop structure (e.g., a shRNA). Thus, as mentioned the RNA silencing agent may also be a short hairpin RNA (shRNA). The term“shRNA”, as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'- UUC AAGAGA-3 ' (Brummelkamp, T. R. et al. (2002) Science 296: 550, and 5'-UUUGUGUAG- 3' (Castanotto, D. et al. (2002) RNA 8: 1454. Notably, a loops as generally described herein can be 20, 50 or 200-300nt long, depending on the length of the complimentary strands. It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem- loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.

As used herein, the phrase“microRNA (also referred to herein interchangeably as “miRNA” or“miR”) or a precursor thereof’ refers to a microRNA (miRNA) molecule acting as a post-transcriptional regulator. Typically, the miRNA molecules are RNA molecules of about 20 to 22 nucleotides in length which can be loaded into a RISC complex and which direct the cleavage of another RNA molecule, wherein the other RNA molecule comprises a nucleotide sequence, essentially complementary to the nucleotide sequence of the miRNA molecule. Typically, a miRNA molecule is processed from a“pre-miRNA” or as used herein a precursor of a pre-miRNA molecule by proteins, such as DCL proteins, and loaded onto a RISC complex where it can guide the cleavage of the target RNA molecules. Pre-microRNA molecules are typically processed from pri-microRNA molecules (primary transcripts). The single stranded RNA segments flanking the pre- microRNA are important for processing of the pri-miRNA into the pre-miRNA. The cleavage site appears to be determined by the distance from the stem- ssRNA junction (Han et al. 2006, Cell 125, 887-901, 887-901).

As used herein, a“pre-miRNA” molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides, which can adopt a secondary structure comprising an imperfect double stranded RNA stem and a single stranded RNA loop (also referred to as“hairpin”), and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem. According to a specific embodiment, the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem. The length and sequence of the single stranded loop region are not critical and may vary considerably, e.g. between 30 and 50 nucleotides in length. The complementarity between the miRNA and its complement need not be perfect, and about 1 to 3 bulges of unpaired nucleotides can be tolerated. The secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFOLD. The particular strand of the double stranded RNA stem from the pre- miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand, which at its 5' end, is the least involved in hydrogen bonding between the nucleotides of the different strands of the cleaved dsRNA stem, is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation. However, if empirically the miRNA molecule from a particular synthetic pre-miRNA molecule is not functional (because the“wrong” strand is loaded on the RISC complex), it will be immediately evident that this problem can be solved by exchanging the position of the miRNA molecule and its complement on the respective strands of the dsRNA stem of the pre-miRNA molecule. As is known in the art, binding between A and U involving two hydrogen bonds, or G and U involving two hydrogen bonds is less strong that between G and C involving three hydrogen bonds.

Naturally occurring miRNA molecules may be comprised within their naturally occurring pre-miRNA molecules, but they can also be introduced into existing pre- miRNA molecule scaffolds by exchanging the nucleotide sequence of the miRNA molecule normally processed from such existing pre-miRNA molecule for the nucleotide sequence of another miRNA of interest. The scaffold of the pre-miRNA can also be completely synthetic. Likewise, synthetic miRNA molecules may be comprised within, and processed from, existing pre-miRNA molecule scaffolds or synthetic pre- miRNA scaffolds. Some pre-miRNA scaffolds may be preferred over others for their efficiency to be correctly processed into the designed microRNAs, particularly when expressed as a chimeric gene wherein other DNA regions, such as untranslated leader sequences or transcription termination and polyadenylation regions are incorporated in the primary transcript in addition to the pre-microRNA. According to the present teachings, the dsRNA molecules may be naturally occurring or synthetic. The dsRNA can be a mixture of long and short dsRNA molecules such as, dsRNA, siRNA, siRNA+dsRNA, siRNA+miRNA, or a combination of same.

The nucleic acid agent can be further comprised within a nucleic acid construct comprising additional regulatory elements. For example, transcription from an expression cassette, a regulatory region (e.g., promoter, enhancer, silencer, leader, terminator, intron and polyadenylation) may be used to modulate the transcription of the RNA strand (or strands). Therefore, in one embodiment, there is provided a nucleic acid construct comprising the nucleic acid agent. The nucleic acid construct can have polynucleotide sequences constructed to facilitate transcription of the RNA molecules of the present invention operably linked to one or more promoter sequences functional in a mosquito cell. The polynucleotide sequences may be placed under the control of an endogenous promoter normally present in the mosquito genome. The polynucleotide sequences of the present invention, under the control of an operably linked promoter sequence, may further be flanked by additional sequences that advantageously effect its transcription and/or the stability of a resulting transcript. Such sequences are generally located upstream of the promoter and/or downstream of the 3' end of the expression construct. The term “operably linked,” as used in reference to a regulatory sequence and a structural nucleotide sequence, means that the regulatory sequence causes regulated expression of the linked structural nucleotide sequence.

In a further embodiment, a composition including a genetically modified bacteria configured to express dsRNA may be formulated as a water dispersible granule or powder that may further be configured to be dispersed into the environment. In yet a further embodiment, the compositions of the present invention may also comprise a wettable powder, spray, emulsion, colloid, aqueous or organic solution, dust, pellet, or colloidal concentrate. Dry forms of the compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner. Alternatively or additionally, the composition may comprise an aqueous solution. Such aqueous solutions or suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready -to-apply. Such compositions may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (silicone or silicon derivatives, phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations or compositions containing paratransgenic bacteria may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.

As mentioned, the dsRNA of the invention may be administered as a naked dsRNA. Alternatively, the dsRNA of the invention may be conjugated to a carrier known to one of skill in the art, such as a transfection agent e.g. PEI or chitosan or a protein/ lipid carrier or coupled to nanoparticles. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, microencapsulated, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. Suitable agricultural carriers can be solid, semi-solid or liquid and are well known in the art. Such compositions may be considered “agriculturally-acceptable carriers”, which may covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology.

As mentioned, the nucleic acid agents can be delivered to the mosquito larva in various ways. Thus, administration of the composition to the mosquito larva may be carried out using any suitable or desired manual or mechanical technique for application of a composition comprising a nucleic acid agent, including, but not limited to, spraying, soaking, brushing, dressing, dripping, and coating, spreading, applying as small droplets, a mist or an aerosol. According to one embodiment, the composition is administered to the larvae by soaking or by spraying.

As used herein, the term“gene” or“polynucleotide” refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs, and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example, a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition, are nucleic acid polymers that have been modified, whether naturally or by intervention.

Peptides and proteins for use within the methods and compositions of the invention thus include native or“wild-type” peptides and proteins and naturally occurring variants of these molecules, e.g., naturally occurring allelic variants and mutant proteins. Also included are synthetic, e.g., chemically or recombinantly engineered, peptides and proteins, as well as peptide and protein“analogs” and chemically modified derivatives, fragments, conjugates, and polymers of naturally occurring peptides and proteins. As used herein, the term peptide or protein“analog” is meant to include modified peptides and proteins incorporating one or more amino acid substitutions, insertions, rearrangements or deletions as compared to a native amino acid sequence of a selected peptide or protein, or of a binding domain, fragment, immunogenic epitope, or structural motif, of a selected peptide or protein. Peptide and protein analogs thus modified exhibit substantially conserved biological activity comparable to that of a corresponding native peptide or protein, which means activity (e.g., specific binding to a peptide YY protein, or to a cell expressing such a protein, specific ligand or receptor binding activity, etc.) levels of at least 50%, typically at least 75%, often 85%-95% or greater, compared to activity levels of a corresponding native protein or peptide.

For purposes of the present invention, the term peptide or protein“analog” further includes derivatives or synthetic variants of a native peptide or protein, such as amino and/or carboxyl terminal deletions and fusions, as well as intrasequence insertions, substitutions or deletions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.

As used herein, the term“gene” or“polynucleotide” refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs, and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example, a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition, are nucleic acid polymers that have been modified, whether naturally or by intervention. All nucleotide sequences, including primers, are described in their standard 5’ to 3’ prime orientation unless otherwise noted.

As used herein the terms“approximately” or“about” refer to ± 10%. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases“ranging/ranges between” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean“including but not limited to”. The term“consisting of means“including and limited to”. The term“consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form“a”,“an” and“the” include plural references, unless the context clearly dictates otherwise. For example, the term“a compound” or“at least one compound” may include a plurality of compounds, including mixtures thereof.

As used herein the term“method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It should be noted that for all sequence provided herein, such sequences include all corresponding sequences, so an amino acid sequence include its corresponding DNA or RNA sequence as generally understood by one of ordinary skill in the art. The invention may be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXAMPLES

Example 1 : Co-delivery of Loqs2 protein and dsRNA to enhance RNAi response in the mosquito midgut.

Cloning of expression cassette expressing Loqs2 protein and dsRNA targeting GFP:.

In this embodiment, a 500bp region of the GFP RNA sequence was cloned into an OX19 backbone from Oxford genetics. The 500bp sequence was cloned in between two convergent Ptac promoters to generate dsRNA targeting the GFP region, generally identified herein as SEQ ID NO. 4. The Loqs2 protein coding sequence was codon optimized for E.coli and synthesized by GenScript. This sequence was PCR amplified off of the GeneScript construct and cloned into a backbone from Oxford genetics that contained the dsRNA-GFP region and under the control of the OX19 promoter. Two versions of the construct were made, one with the flag-tag on the N- terminus of the protein and one with the flag-tag on the C-terminus of the protein. The construct was originally cloned into Dh5a E. coli cells and then transferred to an RNaselll mutant E.coli line HT27. Expression of either C-terminus flagged or N-terminus flagged Loqs2 was measured by Western Blot (Figure la and Figure lb). Expression of dsRNA-GFP was confirmed by RT- PCR (Figure 17). Furthermore, the Loqs2 protein was detected in outer membrane vesicles from HT27 cells. (Figure 18).

Cloning the construct expressing Loqs2 protein and dsRNA targeting Zika virus (ZIKV) into symbiotic bacteria Enterobacter: To demonstrate that we see expression of the Loqs2 gene in a symbiotic bacteria, the expression cassette from the OX19 construct containing the 0X19 promoter, multiple cloning site, Loqs2 protein coding sequence and dsRNA-GFP coding region under the control of 2 convergent ptac promoters was stably integrated into Enterobacter Ae073 genome. The cassette is first transferred into to the Tn7 backbone and then moved into Enterobacter as generally described in the methods section. Expression of Loqs2 from this construct was verified by Western blot (Figure 3).

Detection of the Loqs2 protein in the mosquito midgut: The expression cassette described above with the flag-tag on the N-terminus in the RNaselll mutant E. colt line (HT27) was fed to mosquitoes. Poza Rica Ae. aegypti adults were treated with strains as indicated for 1 day, then midguts were dissected 4 days post-treatment. (Blue, nuclei, red, dsRNA). Midguts were treated with primary antibody anti-dsRNA mAb J2 (1 :200) and rabbit anti -FLAG at 1 :500 (ThermoFisher cat# 740001) for one hour, then with the secondary antibodies goat anti mouse Alexa Fluor 555 (1 : 1000) and Goat anti-rabbit: Alexa Fluor 647 (1 : 1,000) for 40 minutes. DAPI (ThermoFisher cat# R37606), which stains nuclear DNA, was also added at this time. Slides were viewed on an Olympus 1X81 Inverted Confocal Laser Scanning Microscope using the lOOx oil immersion objective DIC, differential interference contrast.

To determine if the protein of interest is in fact being delivered to the mosquito following ingestion of HT27, E. colt cells delivering the protein of interest, protein extracts of whole mosquitoes were harvested 5 days after ingestion of bacteria were analyzed by mass spectrophotometry. The present inventors detected 8 peptides aligning to the Loqs2 protein in mosquitoes fed HT27 cells delivering Loqs2 protein and dsRNA-GFP. (Figure 15)

Enhancement of RNAi response in the mosquito midgut by co-delivery of Loqs2 protein and dsRNA targeting GFP: The expression cassette described above was fed to a transgenic line of mosquitoes generated by Alexander Franz at LTniversity of Missouri. This transgenic line of mosquitoes has been engineered to express GFP only in midgut tissue for 16- 36 hours following a bloodmeal. The GFP protein is controlled by a carboxypeptidase promoter which is activated by a bloodmeal. HT27 containing the Loqs2 construct with the flag tag on the C -terminus were grown overnight and the bacteria cells were pelleted by centrifugation and washed. Adult mosquitoes were fed a feeding solution (see adult bacterial feeding protocol) containing the Loqs2 construct for 24 hrs. After 24hrs, the bacteria were removed and the mosquitoes were allowed to sugar feed for 24 hours and then starved for 24 hours. The mosquitoes were then offered a bloodmeal through the Hemotek feeding system and bloodied females were separated from non-bloodfed females. Approximately 20 hours after bloodfeeding, the mosquitoes were harvested to assess the level of GFP expression. The treatments in this experiment are mosquitoes not fed bacteria and not bloodfed (negative control), mosquitoes not fed bacteria but allowed to bloodfeed (positive control), mosquitoes fed a bacterial construct delivering dsRNA targeting GFP and bloodfed, and mosquitoes fed a bacterial construct delivery dsRNA targeting GFP and the Loqs2 protein and bloodfed. The present inventors expect to see the mosquitoes fed the construct delivering dsRNA targeting GFP to have fewer GFP transcripts compared to mosquitoes that did not receive bacteria and that were bloodfed. If the Loqs2 protein is enhancing the RNAi response in the mosquito midgut, we expected to see even fewer GFP transcripts in midgut compared to the mosquitoes fed the construct delivering dsRNA targeting GFP alone. Indeed this is what the data demonstrated. (Figure 5). Copies of GFP transcripts in the mosquito were measured by digital droplet PCR. (Figure 5).

Example 2: Co-delivery of eukaryotic Loqs2 mRNA and dsRNA to enhance the RNAi response in the mosquito midgut.

Cloning the construct delivering eukaryotic Loqs2 mRNA and dsRNA targeting

GFP:

To determine if delivery of Loqs2 eukaryotic mRNA is more efficient than delivering Loqs2 protein at enhancing the RNAi response, a construct was cloned that contains a dsRNA- GFP cassette under convergent OX11 promoters from Oxford genetics and expresses a RNA that mimics a eukaryotic mRNA. Briefly, the Loqs2 coding sequence is downstream of the Tobacco etch virus internal ribosome entry site (IRES) and contains a poly-A tail (Figure 13). The IRES sequence is meant to disallow the mRNA from being translated in the bacteria cell and allow it to be translated in the host mosquito cells. Transcription of the Loqs2 mRNA was confirmed by RT-PCR of the cell pellet of HT27 cells containing either the intermediate construct that just contains the Loqs2 mRNA cassette or the final construct that contains the Loqs2 mRNA and dsRNA-GFP cassette (Figure 14). Export of the mRNA from the bacterial cells was determined by RT-PCR of the outer membrane vesicles of the same constructs (Figure 14).

To determine if the protein of interest is being delivered to the mosquito following ingestion of HT27 E. coli cells delivering the protein of interest, protein extracts of whole mosquitoes harvested 5 days after ingestion of bacteria were analyzed by mass spectrophotometry. The present inventor were able to detect 8 peptides aligning to Loqs2 protein in mosquitoes fed HT27 cells delivering the intermediate cloning product which expresses eukaryotic Loqs2 mRNA. (Figure 15 and 16). Enhancement of RNAi in the mosquito midgut by co-delivery of Loqs2 mRNA and dsRNA targeting GFP:

The construct described above was fed to a transgenic line of mosquitoes generated by Alexander Franz at University of Missouri. This transgenic line of mosquitoes has been engineered to express GFP only in midgut tissue for 16-36 hours following a bloodmeal. The GFP protein in controlled by a carboxypeptidase promoter which is activated by a bloodmeal. HT27 cells expressing dsRNA-GFP, Loqs2 protein and dsRNA-GFP, or the Loqs2 mRNA and dsRNA-GFP were grown over night and the bacteria cells were pelleted by centrifugation and washed. Adult mosquitoes were fed a feeding solution containing no bacteria or bacteria delivering the construct of interest for 24 hrs. After 24hrs, the bacteria were removed and the mosquitoes were allowed to sugar feed for 24 hours and then starved for 24 hours. The mosquitoes were then offered a bloodmeal through the Hemotek feeding system and bloodied females were separated from non-bloodfed females. Approximately 20 hours after bloodfeeding, the mosquitoes were harvested to assess the level of GFP expression. The controls in this experiment are mosquitoes not fed bacteria and not bloodfed (negative control), mosquitoes not fed bacteria but allowed to bloodfeed (positive control), mosquitoes fed a bacterial construct delivering dsRNA-GFP and bloodfed.

The present inventors expect to see the mosquitoes fed the construct delivering dsRNA- GFP to have fewer GFP transcripts compared to mosquitoes that did not receive bacteria and that were bloodfed. Indeed, the present inventors expected to see delivery of the Loqs2 protein and dsRNA-GFP to enhance the silence of GFP constructs, and if delivery of Loqs2 eukaryotic mRNA further enhances the RNAi response in the mosquito midgut, it would further be expected to see even fewer GFP transcripts in midgut compared to the mosquitoes fed the construct delivering dsRNA-GFP alone and the mosquitoes fed the construct delivering Loqs2 protein and dsRNA-GFP. Copies of GFP transcripts in the mosquito were measured by digital droplet PCR. Indeed this is what the data showed (Figure 12). Based on these results we conclude that the co-expression of Loqs2 mRNA enhances the silencing of GFP by dsRNA-GFP compared to delivery of Loqs2 protein plus dsRNA-GFP just the dsRNA-GFP alone.

Example 3: Bacterial delivery of dsRNA to regulate the expression of a mosquito gene to potentially control viral replication. Cloning the construct delivering dsRNA targeting the mosquito gene RACK1: A 478 bp region of the RACK1 gene sequence was chosen from the 5’ region of the gene. This 478 bp region of RACK1 was amplified from genomic DNA of mosquitoes originating from south Texas called the MLM colony and inserted into an OX19 backbone from Oxford genetics by Gibson Assembly. The RACK1 sequence was cloned between 2 convergent ptac promoters to create dsRNA (Figure 6). Expression of the dsRNA-RACKl RNA sequence was verified by RT- PCR (Figure 7).

Knockdown of RACK1 RNA transcripts in the mosquito: A liquid culture of HT27 cells expressing a heterologous nucleotide sequence encoding dsRNA-RACKl (SEQ ID NO. 1) was grown at 37° overnight. The liquid culture was centrifuged to pellet the cells and the cells were washed once with LB media. Adult Ae. aegypti mosquitoes belonging to the MLM colony were fed a feeding solution (see adult bacterial feeding protocol) containing HT27 cells expressing dsRNA-RACKl. The mosquitoes were allowed access to the bacteria feeding solution for 24 hours and then the bacteria was removed and the mosquitoes were allowed to sugar feed. Mosquitoes were harvested at 3, 5, and 7 days post bacterial feeding and the mosquitoes collected at day 7 were processed for RNA expression levels of RACK1 compared to mosquitoes that were fed a feeding solution containing HT27 cells delivering dsRNA-GFP. Expression of RACK1 was measured by digital droplet PCR in both the dsRNA-GFP fed and dsRNA-RACKl fed lines. Two pools of 5 individuals were analyzed for the dsRNA-GFP fed control and five pools of 5 individuals were analyzed for the dsRNA-RACKl fed treatment. As shown in Figure 5, the present inventors demonstrated a significant reduction of RACK1 expression in the mosquito following ingestion of HT27 cells delivering dsRNA-RACKl (Figure

5)·

Processing of long dsRNA into small siRNAs in the mosquito: To confirm that the long dsRNAs expressed by the bacteria are being processed into the appropriate sized 22nt small siRNAs within the mosquito to initiate the RNAi response, total RNA extracted from MLM mosquitoes fed HT27 delivering either dsRNA-GFP or dsRNA-RACKl 3, 5, or 7 days post feeding and probed for RACK1 fragments by Northern Blot. We can see a band at the expected 22nt size 3 days post bacterial feed, but the band is not detected 5 or 7 days post feeding. These results indicate the dsRNA is being processed into siRNA in the mosquito since it is being delivered by an RNaselll mutant of bacteria that should not be able to cut the dsRNA up into smaller pieces.

Example 4: Co-delivery of loquacious protein and dsRNA to enhance the RNAi response in the mosquito midgut.

Cloning the construct that will co-delivery the Loquacious protein and dsRNA targeting GFP: A 500bp region of the GFP RNA sequence was cloned into an OX19 backbone from Oxford genetics. The 500bp sequence was cloned in between two convergent ptac promoters to generate dsRNA targeting the GFP region. The RA isoform of the loquacious protein, which is the isoform of loquacious implicated in the siRNA pathway in mosquitoes, was codon optimized for E. coli and synthesized on a plasmid by GenScript. The sequence was then PCR amplified and cloned under the OX19 promoter in the OX backbone containing the dsRNA- GFP sequence. Expression of the Loquacious protein was verified by Western blot (Figure 10). Expression of dsRNA-GFP was confirmed by RT-PCR (Figure 17).

Enhancement of RNAi in the mosquito midgut by co-delivery of the Loquacious protein and dsRNA targeting GFP: The construct described above was fed to a transgenic line of mosquitoes generated by Alexander Franz at ETniversity of Missouri. This transgenic line of mosquitoes has been engineered to express GFP only in midgut tissue for 16-36 hours following a bloodmeal. The GFP protein is controlled by a carboxypeptidase promoter which is activated by a bloodmeal. HT27 cells expressing the loquacious protein and dsRNA-GFP were grown over night and the bacteria cells were pelleted by centrifugation and washed. Adult mosquitoes were fed a feeding solution (see adult bacterial feeding protocol) containing the loquacious construct for 24 hrs. After 24hrs, the bacteria were removed and the mosquitoes were allowed to sugar feed for 24 hours and then starved for 24 hours. The mosquitoes were then offered a bloodmeal through the Hemotek feeding system and bloodied females were separated from non-bloodfed females. Approximately 20 hours after bloodfeeding, the mosquitoes were harvested to assess the level of GFP expression. The treatments in this experiment are mosquitoes not fed bacteria and not bloodfed (negative control), mosquitoes not fed bacteria but allowed to bloodfeed (positive control), mosquitoes fed a bacterial construct delivering dsRNA targeting GFP and bloodfed, and mosquitoes fed a bacterial construct delivery dsRNA targeting GFP and the loquacious protein and bloodfed. The present inventors expect to see the mosquitoes fed the construct delivering dsRNA targeting GFP to have fewer GFP transcripts compared to mosquitoes that did not receive bacteria and that were bloodfed. If the loquacious protein is enhancing the RNAi response in the mosquito midgut, it would be expected to see even fewer GFP transcripts in midgut compared to the mosquitoes fed the construct delivering dsRNA targeting GFP alone. Indeed this is what the data demonstrates (Figure 11). Copies of GFP transcripts in the mosquito were measured by digital droplet PCR.

Example 5: Materials and Methods.

Cloning the Loqs2 protein construct

Flag on N-terminus: The Loqs2 sequence was codon optimized for E. coli and synthesized by GenScript. The Loqs2 sequence was PCR amplified from the synthesized plasmid using the following primers

Lq2F GB forward:

CGAATTCAAAGGAGGTACCCACCATGGACTACAAAGACGATGACGACAAGG

GcGGcGGcGGcGGcGGcGGGCGGACAAATTCGCCGTCA (SEQ ID NO. 14)

Lq2 GB reverse:

CAGAAATCGATTGTATCAGTCAGTCATTATATTGTAGCATTGGTCAATTCGCG

C (SEQ ID NO. 15)

An existing construct that contained a 500 bp dsRNA-GFP region was used as the backbone to insert the Loqs2 sequence into. The backbone containing the dsRNA-GFP was amplified using the primers:

pQX OmpAHA GB Fwd: TGACTGACTGATACAATCGATTTCTG (SEQ ID NO.

16)

OX MCS GB Rev: GGTGGGTACCTCCTTTGAATTC (SEQ ID NO. 17)

The two fragments were joined together by Gibson Assembly using the HiFi enzyme from New England Biosystem. The plasmid was transformed into electrocompetent Dh5a E. coli cells. Colonies were screened by Sanger sequencing and the presence of the full-length Loqs2 protein coding sequence as well as the dsRNA-GFP region were confirmed. The size of the plasmid was verified by restriction digest using the enzymes Pacl and Sacl. The verified construct was transformed into electrocompetent HT27 E. coli cells which are RNase III mutants. Expression of the Loqs2 protein in HT27 cells was confirmed by Western blot (Figure 2). Briefly, 500 ul of the liquid culture was spun down and the cell pellet was lysed in an extraction buffer (B-PER Bacterial Protein Extraction Reagent, 0.2%Lysozyme,0.3% DNAse I, and lxProtease Inhibitor (EDTA free) for 15 min. Equal parts of protein lysate and Laemeli buffer with Beta mercaptaethanol were mixed and boiled for 3 min prior to being separated on Bis-Tris gel. The protein membrane was probed with xx 1° antibody at 1 :5000 dilution and a 2° anti-flag antibody at 1 :5000.

Flag on the C-terminus: This was a two step cloning process. First the flag tag was removed from the N-terminus by site directed mutagenesis using the primers

Loas2 RemoveF GB forGGGCGGACAAATTCGCCGTCAAATG (SEQ ID NO. 18)

Loas2 RemoveF GB rev: CATGGTGGGTACCTCCTTTGAATTCG (SEQ ID NO. 19)

The ends were ligated back together using the KLD enzyme (New England Biosystems). A flag tag was added to the C-terminus by a second site directed mutagenesis reaction using the primers:

Loqs2 AddF for:

T AC A A AG AC GAT GAC GAC A AGT GAC T GAC T GAT AC A ATCGATTTC T G (SEQ ID

NO. 20)

Loqs2 AddF rev:

GTCgCCgCCgCCgCCgCCgCCTATTGTAGCATTGGTCAATTCGCGC (SEQ ID NO.

21)

The two ends were ligated together using the KLD enzyme (New England Biosystems). The plasmid was transformed into electrocompetent Dh5a E. coli cells. Colonies were screened by Sanger sequencing and the presence of the full length Loqs2 protein coding sequence as well as the dsRNA-GFP region were confirmed. The size of the plasmid was verified by restriction digest using the enzymes Pacl and Sacl . The verified construct was transformed into electrocompetent HT27 E. coli cells which are RNase III mutants. Expression of the Loqs2 protein in HT27 cells was confirmed by Western blot using the protocol stated above. (Figure 2)·

Stable Integration of Loqs2 protein and dsZIKV into symbiotic Enterobacter

The construct of interest was conjugated into Enterobacter Ae073 using the conjugation protocol for Tn7 integration from the Borlee lab at Colorado State ETniversity as follows. E. coli RH03 with plasmid of interest was grown overnight at 37°C in LB supplemented with 400 ug/ml DAP and kanamycin and carbenicillin at 250 rpm, E. coli RH03 containing the pTNS3 transposase was grown overnight at 37 °C in LB supplemented with DAP and carbenicillin at 250 rpm, and the recipient strain of interest, Enterobacter Ae073 was grown overnight at 30 °C in LB. 400 pL of lOmM MgS0 ₄ was mixed with 100 pL of Enterobacter Ae073, 100 pL of E. coli RH03 with the plasmid of interest, and 400 pL of E. coli RH03 pTNS3. The mixture was centrifuged at 7000 x g for 2 minutes, the supernatant was carefully removed as to not disturb the pellet, and the cells were gently resuspended in 1 mL of sterile 10 mM MgS0 ₄ by pipetting. The suspension was centrifuged at 7000 x g for 2 minutes, the supernatant was removed, and the pellet was resuspended in 30 pL of 10 mM MgS0 _4. The entire conjugation was pipetted onto a sterile cellulose acetate membrane (l3-mm diameter, 0.45- pm pore size) on a pre-warmed LB agar plate containing DAP 400 and incubated overnight at 30°C. A disposable inoculating loop was used to remove the conjugation from the membrane filter and the conjugation was washed by being swirled in a microcentrifuge tube containing 1 mL LB medium. The conjugation was centrifuged at 7000 x g for 2 minutes, the supernatant was carefully removed, and the step was repeated with 1 mL LB medium. The pellet was resuspended in 300 pL LB medium, and plated in various dilutions on LB plates with kanamycin, and incubated overnight at 30°C. Individual colonies were selected and re-streaked to isolate colonies on an LB plate with kanamycin and incubated overnight at 30°C. Individual colonies from the streak plates were verified for integration. Integration was verified by colony PCR using ClonelD lx Colony PCR Master Mix (Lucigen) with the following thermocycling conditions: 98°C for 2 minutes, then 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 15 seconds, with a final extension time of 10 minutes. The primers used were Entero glms and pTN7R (see primer table 1). Positive clones were grown up in LB liquid culture containing kanamycin at 30°C and stored as 25% glycerol stocks at -80°C.

Adult mosquito bacterial feeding protocol

35-40 1 week old adult Ae. aegypti female mosquitos were sorted into small adult cages (made of 32 oz wax and cardboard soup cups with mesh lids), and starved overnight. Starved mosquitos were then fed bacteria as follows: HT27 E. coli bacteria containing constructs of interest were grown overnight at 37°C in LB with kanamycin and tetracycline, then washed in fresh LB to remove any antibiotics. Pellets were resuspended in LB broth with no antibiotics and adjusted to have an OD600 of 1.0 +/- 0.07. The cultures were aliquoted into l5ml, then spun down and pelleted. The bacterial pellet was resuspended in l5ml of sterile solution composed of l50mM NaCl, lOmM NaHC03, lmM ATP, 2% sucrose, and 1% liver powder suspension, placed in 2 oz plastic cups covered in nonwoven sterile gauze held in place by a rubber band, and given to the starved adult mosquitos. Adult mosquitos were allowed to feed for 24 hours, after which the bacterial feeding solution was removed and replaced with cotton balls soaked in 10% sucrose.

Mosquito blood feeding protocol

Mosquitos were offered a bloodmeal using the Hemotek artificial feeding system. Each feeder was covered with pork sausage casing, and 2 ml of defibrinated calf blood purchased from Colorado Serum Company was added to each feeder, along with lOOul of ATP. Mosquitos were allowed to feed for 5-15 minutes. Cotton balls soaked in 10% sucrose were added back to the cages until further processing depending on the experimental plan.

Measuring GFP expression in the mosquito

The expression of GFP was assessed in a transgenic line of Ae. aegypti engineered to expression GFP in the midgut following a bloodmeal. The GFP gene is under the control of the carboxypeptidase promoter. The bacteria expressing the protein of interest (Loqs2, loquacious, R2D2) was fed to the mosquitoes following the adult mosquito bacterial feed protocol above. Two control cups of mosquitoes were offered feeding solution with no bacteria, one of which would be offered a bloodmeal later and the other one would not. After bacterial feeding, the adults were allowed to sugar feed for 24hrs and then were starved for 24 hours and then offered a bloodmeal. 20-24 hours after a bloodmeal, mosquitoes were cold anesthetized at 4° and only mosquitoes that bloodied were sorted and harvested. Pools of 5 bloodfed mosquitoes or 5 non- bloodfed mosquitoes from the control group that did not receive any bacteria were ground in Trizol and total RNA was extracted following the published protocol from Invitrogen. Total RNA was converted to cDNA using Superscript™ IV First-Strand Synthesis System (catalog # 18091150, Invitrogen). From each pool of mosquitoes, 500 ng of cDNA was added to the QuantStudio 3D digital droplet PCR master mix (Thermo Scientific) and run on the QuantStudio 3D digital droplet PCR machine using primers to amplify GFP:

LD GFP qPCRl Rev: CCATGTGATCGCGCTTCTCGTTG (SEQ ID NO. 22)

LD GFP qPCRl For: GC AG A AG A AC GGC AT C A AGGT G A AC (SEQ ID NO. 23)

Cloning the eukaryotic Loqs2 mRNA construct Cloning this construct was a 2-steps procedure. The first step of cloning was to insert the Loqs2 sequence downstream of the TuMV IRES region by performing Gibson assembly cloning method using the HiFi enzyme(NEB).

The vector: PSF_OXl l_Tat_TorA_3xHA_DRB4_Ptac_TuMV was used as PCR template to amplify backbone:

TuMV GB for: CAATGCCGCGGGCATAACG (SEQ ID NO. 24)

TuMV GB rev: CATAAGGGACTGACCACCCGGGGATC (SEQ ID NO. 25)

The previously cloned Loqs2 protein expression construct was used as template in PCR to amplify Loqs2

Loas2 tuMV GB forGATCCCCGGGTGGTCAGTCCCTTATGGGGCGGACAAATT

CGCCGTCAAATG (SEQ ID NO. 26)

Loas2 TuMV GB rev:CGTTATGCCCGCGGCATTGTATTGTAGCATTGGTCAATT

CGCGCTG (SEQ ID NO. 27)

The two products were assembled using the HiFi enzyme (NEB) and was transformed in DH5-apha ( E-coli ) bacteria strain for further selection (kanamycin, 50 pg/mL). The 2 ^nd step of cloning was to add HA-tag in C-terminus of Loqs2 by performing PCR (Q5 ^® High-Fidelity PCR Mix, M0492S, NEW ENGLAND BioLabs) with forward primer:

TTCCAGACTACGCATACCCTTACGACGTGCCCGACTATGCGTATCCGTACGATGTGC C AGAC T AT GC AT A AC A AT GC CGC GGGC AT A ACG (SEQ ID NO. 28), and reverse primer: CGTCGTAAGGGTAACCGCCCGATCCACCCGATCCTCCACCTATTGTAGCATTGGTCA ATTCGCGCTG (SEQ ID NO. 29), to add the HA-tag.

The ends were joined back together with the KLD enzyme mix protocol (M0554S, NEW ENGLAND BioLabs) and the product was transformed in DH5-apha bacterial strain and verrifed by Sanger sequencing and restriction digest. The final construct was moved into E-coli HT27 strain (kanamycin50 pg/mL plus Tetracycline 10 pg/mL) for mosquito feeding experiments.

Detection of dsRNA-GFP transcription in the bacteria cells

Total RNA was extracted from the cell pellet of HT27 containing constructs that deliver the Loqs2 protein + dsRNA-GFP or the Loquacious protein + dsRNA-GFP using Trizol and an additional DNase treatment was performed. Total RNA was converted into cDNA using Invitrogen Superscript IV and a PCR reaction was performed using the Q5 enzyme from NEB with an annealing temperature of 69oC using the primers listed below. PCR fragments were visualized by gel electrophoresis on a 1.25% agarose gel.

dsGFP transcript for: C A AGG AC G AC GGC A AC T AC A AG (SEQ ID NO. 38) dsGFP transcript rev : CGATGTTGTGGCGGATCTTGAAG (SEQ ID NO. 39)

Cloning the dsRNA-RACKl construct

ETsing an existing OX19 construct from Oxford genetics previously made in the lab that contained dsRNA-GFP in between two convergent ptac promoters, the dsRNA-GFP sequence was swapped out for a dsRNA-RACKl sequence. The primers:

RACK1 GB for:

CCGGGTCGTCAGCTATCCTGCAGGGAGAAAAAAAAACCCCGCTTCGGCGGGG TTTTTTTTTAETAGGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTG GCGTCGCGTGACAAGACTTTGATC (SEQ ID NO. 30)

RACK1 GB rev:

CGATCTCGCCAAGTAGCTGACTTGACTGGTTAATTAAagaaatcatccttagcgaaagct aa ggattttttttatctgGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGCCGT T GTGTCCCAAGTGGTCGATC (SEQ ID NO. 31) were used to amplify the RACK1 sequence from genomic DNA generated from a pool of 5 females from the MLM colony of Ac. aegypti collected in south Texas. The backbone sequence was amplified using primers:

OXB GB.Fwd: CCAGTCAAGTCAGCTACTTGG (SEQ ID NO. 32)

OXB GB.Rev: GC AGGAT AGCTGACGACC (SEQ ID NO. 33)

The two fragments were joined together by Gibson Assembly using the HiFi enzyme from New England Biosystem. The plasmid was transformed into electrocompetent Dh5a E. coli cells. Colonies were screened by Sanger sequencing and the presence of the full length dsRNA- RACKl region were confirmed. The size of the plasmid was verified by restriction digest using the enzymes Pacl and Sacl. The verified construct was transformed into electrocompetent HT27 E. coli cells which are RNase III mutants.

Expression of RACK1 dsRNA was confirmed by RT-PCR (Figure 7). Total RNA was extracted from a cell pellet of overnight culture of HT27 cells expressing dsRNA-RACKl. Total RNA was converted to cDNA using Superscript™ IV First-Strand Synthesis System (catalog # 18091150, Invitrogen). The RACK1 transcript was amplified using the Q5 polymerase from NEB with the following primers: LD RACK1 RNAdetect for: CGCACTTCATCAGTGACGTTGT (SEQ ID NO. 34)

LD RACK1 RNAdetect rev: CACGATGATCGGGTTCGAGT (SEQ ID NO. 35)

Measuring RACK1 expression in the mosquito

Mosquitoes from the MLM colony (collected in south Texas by collaborators at CSET) were fed HT27 E. coli cells delivering the dsRNA-RACKl or delivering dsRNA-GFP as a control following the adult bacterial feeding protocol described above. Mosquitoes were harvested at 3 days, 5 days, and 7 days post bacterial feed. Total RNA was extracted from pools of 5 individuals. Total RNA was converted to cDNA using Superscript™ IV First-Strand Synthesis System (catalog # 18091150, Invitrogen). 500 ng of cDNA from each sample was added to the QuantStudio 3D digital droplet PCR mix (Thermo Scientific) and run on the QuantStudio digital droplet PCR machine using primers that amply the RACK1 gene:

LD RACK 1 qPCR F or6 : GCACAACGAGGTCATCAAC (SEQ ID NO. 35)

LD RACK 1 _qPCR_rev6 : AGAGACAAACACTGTGGAGG (SEQ ID NO. 36)

Western Blot

Bacterial cell extracts (from 500 mΐ bacterial culture pellet) were prepared in a lysis buffer (B-PER Bacterial Protein Extraction Reagent, 0.2%Lysozyme,0.3% DNAse I, and lxProtease Inhibitor (EDTA free)). The following antibodies were used for immunoblotting: anti-FLAG, or anti-HA (2999S, Cell signaling). Various bacterial extracts were resolved by NuPAGE™ 4-12% Bis-Tris Gel (Invitrogen) and transferred to nitrocellulose membranes. The filters were blocked in Tris-buffered saline (20mM Tris, and l40mM NaCl) containing 0.1% Tween 20 (TBST) with 5% nonfat dry milk, and then incubated with the same blocking solution containing the indicated antibodies at 1 :2500 ratio for 12-16 h at 4°C. The filters were extensively washed in TBST, and bound antibodies were visualized with horseradish peroxidase (HRP)-coupled secondary antibodies (1 :2500).

Northern Blot

Total RNA was extracted from 12 mosquito or a pellet from 2ml of bacterial culture using TRIzol™ Reagent (Invitrogen, Cat: 15596018) followed with chloroform RNA extraction following the protocol in the Invitrogen ETser Guide. The following antibodies were used for northern blotting: AP anti -DIG (MB-7100, Vector Labs). Mosquito ( 1 Opg) and bacterial RNA extracts (lOOng) were resolved by electrophoresis gel (7M urea, 37.5% of 40% polyacrylamide, 0.5X TBE, 0.05%APS, 0.085% TEMED), and then transferred to Nytran SPC 0.45pm membranes. The membrane was later prehybridized with PerfectHyb Plus solution (H7033, SIGMA) at 40°C, and later hybridized with the same solution with DIG-labeled probes (probes were labeled by: 03353575910, Roche DIG Oligonucleotide 3’-End Labeling Kit, 2 ^nd Generation) at the same temperature for 16 hours. The membrane was washed with wash buffers (SSC & SDS). The membrane was blocked in 1% blocking solution with maleic acid buffer (0.1M maleic acid, 0.15M NaCl, PH:7.5) for 30 minutes and then incubated with the same blocking solution containing the indicated antibodies at 1 :20,000 ratio.

Example 6: Analytical Methods.

Protein extraction

Collected mosquitos were stored at -80°C. 100 pL of“I-PER” Buffer (0.1% TritonX-lOO, 8M ETrea, 25mM Tris-HCl, lOOmM NaCl, at pH7, with Protease Inhibitor cocktail) or RIPA buffer (l50mM NaCl, 5mM EDTA pH8, 50mM Tris pH8, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, and protease inhibitors) was added to a tube containing a pool of 3 mosquitos each, and ground with a motorized pestle and allowed to incubate for 5 minutes. The mosquito and buffer mixture were then spun down for 5 minutes at the maximum centrifuge speed and the supernatant was carefully removed from the mosquito debris and approximately 500 pL of protein extract was added to an Amicon® Ultra 0.5 mL centrifugal filter (Millipore, Ireland) with a 3,000 MWCO, and then centrifuged (15,000 rpm x 20 min x RT) to separate intact proteins from signaling peptides. The filtrate was stored at -20°C until processed.

Protein quantitation

Protein concentration was approximated using a NanoDrop (Thermo). Lysis buffer was used to blank the instrument, and absorbance values were collected at 280 nm and used to approximate protein concentration (mg/mL).

Protein digestion

Intact protein samples were reduced, alkylated and digested following the protein digestion protocol from the University of Washington Proteomics Resource with a few modifications (UWPR, 2011). Intact protein samples were reduced by addition of 1 M dithiothreitol (DTT) prepared in 0.1 M ammonium bicarbonate to a final concentration of 50 mM, and allowed to incubate for 25 min at 45°C. Samples were cooled and then alkylated by addition of 0.5 M iodoacetamide (IAA) prepared in 0.1 M ammonium bicarbonate to a final concentration of 55 mM and incubation at room temperature in the dark for 15 min. Samples were then diluted 1 :9 with 0.1 M ammonium bicarbonate, and MS grade trypsin (Fisher) was resolubilized in 0.1 M ammonium bicarbonate added at a ratio of 1 : 100 enzyme: substrate. Protein digestion was performed at 37°C on a shaker plate set to 200 rpm overnight, and the reaction was quenched the following day by addition of glacial acetic acid until pH < 2 was achieved. Peptide mixtures were then concentrated and desalted using Cl 8 ZipTip® pipette tips (Millipore).

Peptide desalting

Peptides were desalted prior to analysis by LC-MS to remove salts and SDS from the lysis buffer. A 10 pL ZipTip® packed with 0.6 pL C18 resin was selected for peptide desalting and concentration. Peptide mixtures resulting from protein digestions were processed as follows. The ZipTip® was conditioned by aspirating with 10 pL 0.1% formic acid three times. Sample was loaded by aspirating the sample five times, and then washed by aspirating with 10 pL 0.1% formic acid three times. Peptides were eluted by aspirating 50 pL of 80% acetonitrile containing 0.1% formic acid five times. Eluted peptides were dried using a CentriVap benchtop vacuum concentrator (Labconco) operated at room temperature for approximately 20-30 min, and then resolubilized in 50 pL 1% acetonitrile supplemented with 0.1% formic acid and 100 fmol/pL Glu'-fibri nopeptide B as an internal standard (ISTD).

LC-MS

Signaling peptides and peptide mixtures were both analyzed by LC-MS/MS using a Synapt G2-Si high definition mass spectrometer (HDMS) coupled to an ACQUITY M-Class ultra-performance liquid chromatograph (UPLC) with an electrospray ionization (ESI) source (Waters). Capillary voltage for sample and LockSpray sprayers was set to 3.6 and 3.0 kV, respectively. Sampling cone and source offset were both set to 40, and source and desolvation temperatures were set to 100 and 200 °C, respectively. Desolvation gas flow rate was 650 L/hr and nebulizer was set to 6.5 bar. Glu^fibrinopeptide (Sigma, US) was prepared at a concentration of 100 fmol/pL in acetonitrile:water (1 : 1 v/v) containing 0.1% formic acid and used as the LockMass solution. LockSpray measurements (Glu'-fibri nopeptide: 785.8426 m/z) were collected every 60 s for both signaling peptide and peptide mixture analysis methods.

Peptide mixtures were separated on an ACQUITY UPLC M-Class HSS T3 Column (100 A pore size, 1.8 pm particle size, 300 pm ID X 100 mm L) (Waters) following a linear gradient using mobile phases (a) water with 0.1% formic acid and (b) acetonitrile with 0.1% formic acid (Table 3 below). The flow rate was maintained at 5 pL/min, and 1 pL injections were made in duplicate. Data were collected by HDMSe in positive polarity (ES+) and high resolution (FWHM = 40,000) modes. Default ion mobility settings were used, and a collision energy ramp of 12-40V was used. Data were acquired in continuum mode, and LockSpray data were collected but not applied during acquisition. The mass range was 300-2000 m/z, and scan time was set to 0.486 s with a 0.014 s interscan delay.

Protein identification

All peptide data were processed using Progenesis QI for proteomics (Nonlinear). Peptide spectra were searched against a custom database generated from ##protein sequences from SnapGene constructs##. The MSe identification workflow was used with default settings for Apex3D parameters, auto-alignment and peak picking. Alignment of peptide samples was conducted automatically using the most suitable fit. Tolerance parameters and ion matching requirements for protein identification were set as follows: 10.0 ppm mass error for peptide ions, 3 fragments per peptide for peptide ion identification, 4% false discovery rate (FDR) and 2 peptides per protein for protein identification. Abundance of both non-conflicting and unique peptides was used for relative quantitation of identified proteins. Abundance values for identified proteins were exported into Excel as .csv files, and were standardized across samples using estimated protein concentration.

TABLE 1A: Exemplary List of additional endosymbiont strains and Biotyper identification

I lPseudomonas putida Ael7l | Endosymbiotic bacteria in mosquito

TABLE 2: Helper Genes.

Table 3. LC gradient for separation of peptide mixtures. Time (min), flow rate (pL/min), % mobile phase A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid), and gradient curve (1-11) values are provided.

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The following references are hereby incorporated in their entirety by reference:

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22. Ford, S.A., Allen, S.L., Ohm, J.R. et al. Selection on Aedes aegypti alters Wolbachia-mediated dengue virus blocking and fitness. Nat Microbiol 4, 1832-1839 (2019) doi : 10.1038/s41564-019-0533-3

SEQUENCE LISTING

SEQ ID NO. 1

DNA

RACK1 dsRNA

Artificial

CGTCGCGTGACAAGACTTTGATCGTCTGGAAGTTGACTCGCGACGATGCCAGCTACGGAA TCCC CCAGAAGCGTCTGTATGGCCACTCGCACTTCATCAGTGACGTTGTCCTCTCGTCGGACGG TAAC TACGCCTTGTCCGGATCGTGGGACAAAACCCTGCGCCTGTGGGATTTGGCTGCCGGAAAG TCCA CCCGCCGTTTTGAAGACCATACCAAGGATGTCCTGTCGGTGGCCTTCTCCGTCGACAACC GTCA AATCGTGTCCGGATCCCGTGACAAGACCATCAAGCTGTGGAACACCCTGGCCGAGTGCAA GTAC ACCATCCAGGAAGATGGCCACAGCGATTGGGTTTCGTGCGTCCGCTTCTCGCCAAACCAC TCGA ACCCGATCATCGTGTCGGCCGGTTGGGATCGCACCGTCAAGGTCTGGAATCTGGCCAACT GCAA GCTGAAGATCGACCACTTGGGACACAACGG

SEQ ID NO. 2

DNA RACK1

A. aegypti

ATGACTGAAACGCTGCAACTCCGCGGCCAGCTTGTTGGCCACTCCGGATGGGTCACC CAGATTG CCACCAATCCGAAGTACCCGGATATGATCCTGTCTTCGTCGCGTGACAAGACTTTGATCG TCTG GAAGTTGACTCGCGACGATGCCAGCTACGGAATCCCCCAGAAGCGTCTGTATGGCCACTC GCAC TTCATCAGTGACGTTGTCCTCTCGTCGGACGGTAACTACGCCTTGTCCGGATCGTGGGAC AAAA CCCTGCGCCTGTGGGATTTGGCTGCCGGAAAGTCCACCCGCCGTTTTGAAGACCATACCA AGGA TGTCCTGTCGGTGGCCTTCTCCGTCGACAACCGTCAAATCGTGTCCGGATCCCGTGACAA GACC ATCAAGCTGTGGAACACCCTGGCCGAGTGCAAGTACACCATCCAGGAAGATGGCCACAGC GATT GGGTTTCGTGCGTCCGCTTCTCGCCAAACCACTCGAACCCGATCATCGTGTCGGCCGGTT GGGA TCGCACCGTCAAGGTCTGGAATCTGGCCAACTGCAAGCTGAAGATCGACCACTTGGGACA CAAC GGATACCTGAACTCGGTTTCCGTGTCGCCCGATGGTTCCCTGTGCACGTCCGGAGGTAAG GACT GCAAGGCCTTCCTGTGGGATTTGAACGATGGCAAGCATCTGCACACCCTGGAGCACAACG AGGT CATCAACGCCTTGTGCTTCTCGCCAAACCGGTACTGGCTGTGCGTTGCCTATGGTCCATC CATC AAGATCTGGGATCTGGCATGCAAGACCATGGTTGAAGAGCTGAAGCCCTCGAAGGCCGAT CCTC CACAGTGTTTGTCTCTGGCCTGGTCCACCGATGGACAGACGCTGTATGCCGGTTATTCCG ATAA CATCATCCGTGTCTGGCAGGTGTCGGTCTCGGCTCGTTAA

SEQ ID NO. 3

Amino Acid

RACK1

A. aegypti

MTETLQLRGQLVGHSGWVTQIATNPKYPDMILSSSRDKTLIVWKLTRDDASYGIPQK RLYGHSH FISDWLSSDGNYALSGSWDKTLRLWDLAAGKSTRRFEDHTKDVLSVAFSVDNRQIVSGSR DKT IKLWNTLAECKYTIQEDGHSDWVSCVRFSPNHSNPI IVSAGWDRTVKVWNLANCKLKIDHLGHN GYLNSVSVSPDGSLCTSGGKDCKAFLWDLNDGKHLHTLEHNEVINALCFSPNRYWLCVAY GPS I KIWDLACKTMVEELKPSKADPPQCLSLAWSTDGQTLYAGYSDNI IRVWQVSVSAR SEQ ID NO. 4

DNA

GFP dsRNA

Artificial

GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG CGCA CCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG ACAC CCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGG GCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC GGCA TCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACC ACTA CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG CACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCG

SEQ ID NO. 5

DNA

Loqs2

A. aegypti

ATGGGGCGGACAAATTCGCCGTCAAATGAATGTTGTGAGAATGGAGACAACGCCCTG CTTAATG GGTACGTCAGTGCTAGTCCCGGTGAGTCGAACCCCCGTCCTGTTCCTACCAATGATGCGT TGCC TATAGAAGAGGCGTTTAAGATAGCTTTGACAGGCAAATCTAATACCAAGAAAATGCCTGT GAGT TTATTATATGAACTTTTAAGCAGAAGAGGGATTACACCTCAATATGATCTGTTACCGCGC GAGG GTGCAGCCCACGAACAAACATTTAGCTACCGCGTCTCGTATCCCGATGGTGATGCCATAG GTAC GGGACAGAGCAAGAAGGAGGCGAAACATGCAGCAGCTAAAGCACTTATCGACAAAATGGG CATA AATGACTCGGCCAATAAACCAGTTGGTAAGAAGACTCCGATCACCGTATTACAGGAAGTA TTGA CTCGTCGCGGTATTTACCCACAATACGACTTCATACAGCCTGACGCAGCCGTTCACGATG GTAC GTTTCGCTATCGTGTCAGTTACCAGGACAAAGAGGCAATGGGAACAGGCAAATCCAAGAA AGAA GCTAAGCAAGCAGCAGCCAAGAGCCTGATAGACAAGCTTGCGGGGGTAGCTTTTTGGGAC ACTC ACTCACATGGTCTGAATTCCAGAGAAGAGACCAAGCAAGATGAGGATGCAGAACCGTCTG CATC CAGTCCCGAAGTCTCGTTACTTTCACGGTCCCATGATGGTGTGCCAGAGGGTTTTACGGA TAAA GCTAAGGGGAAGCGTAATCAGCACACTCAAGTCGACATGTTCGTGTCCCGCAAAAAACAG AAGG CAGGCAATCAGCGCGAATTGACCAATGCTACAATA

SEQ ID NO. 6

Amino Acid

Loqs2

A. aegypti

MGRTNSPSNECCENGDNALLNGYVSASPGESNPRPVPTNDALPIEEAFKIALTDGKS NTKKMPV SLLYELLSRRGITPQYDLLPREGAAHEQTFSYRVSYPDGDAIGTGQSKKEAKHAAAKALI DKMG INDSANKPVGKKTPITVLQEVLTRRGIYPQYDFIQPDAAVHDGTFRYRVSYQDKEAMGTG KSKK EAKQAAAKSLIDKLAGVAFWDTHSHGLNSREETKQDEDAEPSASSPEVSLLSRSHDGVPE GFTD KAKGKRNQHTQVDMFVSRKKQKAGNQRELTNAT I

SEQ ID NO. 7

DNA

Loquacious A. aegypti

ATGTCAAAGACGGATTTACCTGCAGTTGAGGATCTGAACGGACTGGTAAGCGGCGACAAC GGGA ACCCACCGGTGGGGCAGCCAAATTTGAAAAGACCAGTCAGAGTGGCAAAAAAATCCGTAA GCAA GCAAGATGCCCTGCCTATCGAGGAGGCGCTTAAAACAGAATTGACGGGAACAAATAACAT GAAA ACACCGATCTCGGTATTGCAAGAGCTGCTGTCGAGACGCGGGATTACGCCCCAATATGAT CTGA TACAGGTGGAAGGAGCCGTGCATGAACCAACGTTCCGGTATAGAGTAAGCTACCAAGATA AGGA CGCGATGGGAACCGGTAAGTCCAAGAAAGAGGCAAAGCACGCTGCAGCTAAAGCCTTAAT CGAT AAGCTGGCTGGAAACGCCTTCGGAGATACTCAGACAGGTGGATTGAATATCAAAGCGGAG GCCG GCCTGGACGGAGATGACGAGCCTACAGGTAACCCTATCGGGTGGTTGCAGGAAATGTGCA TGGC CCGTCGTTGGCCACCTCCTACATACGAAACGGAGATGGAAGTAGGGCTGCCGCACGAACG TCAA TTCACAATTGCGTGCGCGGTCTTAAAGTATAGAGAAGTTGGTAAGGGTAAGTCAAAAAAA ATAG CAAAGCGCCAAGCGGCCCAACGTATGTGGCAGAGACTTCAGGACCAGCCGCTTGAGCCAA ATCA GATAATACAGATGTTGGACGAGGAAGGTAACGAAGAGCTTAAGGCGGCGAGCATCACTGG TCGG TATGCCGGATTAAAGGATGCTCGGATACCCTCATTAACGACGGGACACGGCCAGAAGGTG TCGC AATTCCACAAGGCTCTGAAGGCCCGCTCTGGGGAAACTCTTCGCCAATTGCAGGTAACGT GCCT GAATGACAAATCCATCGATTTCGTCCAAATGCTTCACGAAATAGCTACAGAGCAGCGTTT TGAA GTGACTTATGTGGATATTGATGAAAAAACCCTTTCTGGGCGGTTCCAATGTTTAGTACAA TTGT CAACCCTTCCCGTGGCGGTCTGCCACGGATCGGGAAGTACTGCAAAGGAAGCCCAAACTG CCGC GGCGCGTAACTCATTAGAGTACTTAAAGATTATGACTAAGACA

SEQ ID NO. 8

Amino Acid

Loquacious

A. aegypti

MSKTDLPAVEDLNGLVSGDNGNPPVGQPNLKRPVRVAKKSVSKQDALPIEEALKTELTGT NNMK TPISVLQELLSRRGITPQYDLIQVEGAVHEPTFRYRVSYQDKDAMGTGKSKKEAKHAAAK ALID KLAGNAFGDTQTGGLNIKAEAGLDGDDEPTGNPIGWLQEMCMARRWPPPTYETEMEVGLP HERQ FTIACAVLKYREVGKGKSKKIAKRQAAQRMWQRLQDQPLEPNQI IQMLDEEGNEELKAAS ITGR YAGLKDARIPSLTTGHGQKVSQFHKALKARSGETLRQLQVTCLNDKS IDFVQMLHEIATEQRFE VTYVDIDEKTLSGRFQCLVQLSTLPVAVCHGSGSTAKEAQTAAARNSLEYLKIMTKTGGG GGGD YKDDDDK

SEQ ID NO. 9

DNA

Feature_26588l

Aedes aegypti

GGAAAGTCCACCCGCCGTTTTGAAGACCATACCAAGGATGTCCTGTCGGTGGCCTTCTCC GTCGACAACC GTCAAATCGTGTCCGGATCCCGTGACAAGACCATCAAGCTGTGGAACACCCTGGCCGAGT GCAAGTACAC CATCCAGGAAGATGGCCACAGCGATTGGGTTTCG

SEQ ID NO. 10

DNA

Feature_266l0l

Aedes aegypti CGAAACCCAATCGCTGTGGCCATCTTCCTGGATGGTGTACTTGCACTCGGCCAGGGTGTT CCACAGCTTG

ATGGTCTTGTCACGGGATCCGGACACGATTTGACGGTTGTCGACGGAGAAGGCCACC GACAGGACATCCT

TGGTATGGTCTTCAAAACGGC

SEQ ID NO. 11

DNA

chymotrypsin-like serine protease (JA15) mRNA (AY957559.2, complete cds)

Aedes aegypti

CTCAGTTCTCATCGTTACATCCAAAGTTCTGTACCGGGTAATGCAATTGGTTCTTGTTTC GCTATTGGCC ACCGCCTTTTTCGGCGCAGTTGCACCATCACCTGCCAGGAGAATCGTCGGAGGCCAATTT GCACAGAATG AGAAACAGTTTCCCTACCAGGTGGCCCTTTTCGAGAAGGATGAGTTCAAGTGCGGAGGAT CGATTATCGC CGAAAAGTGGATTCTAACGGCTGCCCATTGCATCGTGCAGCTTGATGGAAGTCCTACATC AATTGATGTT TTAAAGGTTCATGTCGGTTCACCTCACCTAAAAAGAGGAGGGAAAAAGGTGAAACCTTCA AGGATTATTC CTCACGCTGACTACCCCAAGATCAATTACGACATCGGATTGATTGAGCTAGAAGAGGCCC TCGTTTATGA TAAAATCATCCAAAAAATTGAGCTCTATCGAGGGGAATTACCAGTAAATGTCACAGTTAC AATCTCCGGC CATGGAAAAACCGGAAGTAATGAGCCGATCTCCGAGATGCTCAAGTACAACACATTGACC GTGATGGACG AGACAGAATGTTTTAACCAAATAATTCATTCCGGGTGGAAGAAAATCATGTGTCTGAAGA AGCCTGCGGA TAATAACGGTATTTGCACGGGAGATTCCGGAGGACCTGCCGTGTTCAATGGAAAGCAAGT TGGAGTGGCC AACTTTGTGCTGGGAAAGTGTGGATCGATCCGTACGGATGGATATGCCTTTGTGCCTTTC TTCGTACCCT GGATTGAGGAAACTATGAAACAGCAGTGAGAGATAGTGATTTCAAAAGCCTGGAGTGGAA ATTATCAATA AAAAT GGAGT GG AAAAT AT C AAAAAATC A

SEQ ID NO. 12

DNA

cadherin-87A, transcript variant X5 (LOC110674038) (XM_021837335.1), transcript variant X5, mRNA

Aedes aegypti

TCGTCAGTTTCGTGTTGGCTTGTGACCACGTCGGTTGATATCGTTCGGTGATCGTGCGTT TCGCTTTGCG

TCGTTTTACCGCGTGCCGTTGTTTTATGTTTTTCTTCGGGAAGGTAACGACTCCATA GAGCTTTGCTGGT

GGTTACTTTCTTCGGGTTCCGTGTGAGGCGGGCATCGAAGTGAATGGATATTGTTTT TCCTGTATGTGGC

TTTAAAACCCTCCCATTTGGATACTGCGGGGAGACGAGGAAAAGCGCAGAGCATCAG TTTGACAAAGATT

GAGTGAAAGAAAAACGGGTGAATATCAGTGAAGCCGGGTCGAGAACCGGTTTGGCTG CGAGAAGAATCGG

TCAGTGTTTGGTTTGAGAGTGCGAACCCGACCCGCCGAGGTGGAAGAGCTTAATTTG GTGTAAATTTACC

GTTATCCGTCGCCGTCCGAGTTCCCGGAGTTCGCCGAGTCGAATCTCATCTGCTCGC TCGCAGCCACCGT

CGTCGCTTCGTGGTGTAGCCGACCGAGTTTCATGGTTTGGTGTTTGAAGTGGAGAGC TTCTGGGAGGTCC

AACAGCAGCTGAAGCAATAGATAGTGTGAGAAGTGTGTGAGCCAATTTCTGCTCCAG AAGTGATTCCAAC

CTGCAAAAAAAGAGGAAAAATAGTTCAGTATTGTCTCGGAAGATGGCGTTGAAGTGA AGCAGCGCTGTTG

TCGTCGGCCCATTCTTCATGTCTCTCCGGTGGTTGTCATTGTGGTGCAGTTATGGTG TGTGGCGATGCTG

AGATGGTGATGATGCCATGTCGTTATTGGTTGCATGAATTATGACGGAGAAGTGGTT TATGCCGTGTGCA

GTGTGAGAAAATCGGAGCTGTGCTTTTTGTGTTCCAGTTTGTGTGTGCAATTGTGAA AAAGGGGGGTTTC

TGTGGAAAAGCAAACGAATGCAATTTATGTGCTGTCCAACAGTGTGGAAGCAATTGA AGAAAATTCCAAT

CGTATCGAATCGCAGTGCAGTCACCACAAACAGGTTGTGTTGAAAATTTGAATGGTC AGCAACAAGATTG

GATATATCGAAGTAGCAGAGAAACAGCTTTTTCCACCATTAGTCGTTGTCGTTGAGA TTCATCTTTCGTA

ATTCAATGTACGTTTAAATATTTCCTATTACAACCGTAATTGACATATCTCTAAACT GAACGGAAGTGAA

CTAGAATTGTGTGTGTGTGGCAAGGACGACCAGGCGACGAAGCAGCCGCCATTCAGC AATGATAGCCTCC

ACCCAGAAGCAGCAACAGCGATGGACAGTTTTAATACCGCTCCTAACGATAGGGTTC CTGATTCGGACAT

GTCACTGCAACCTGCCGCCGATTTTCACGCAGGACATGAACAACTTGGCCCTGCCGG AGACAACTCCGGT GGGAAGCGTCGTTTACCGGCTGGAGGGTTACGATCCGGAGGGCGGTAACGTCTCGTTTGG GCTGCTCGGC

TCGGACAACTTTATGGTGGACCCAATCAGTGGGGACGTCAAGGTGATAAAACCGCTG GACCGTGAGGACC

AGGACACCCTCTCCTTCTCGGTGACCATCAAGGATCGCATCAGCACCGCAGGAATCG ATTCCGAGAACGA

CAACGTGGTCAACGTTCCCATCACGATAATCGTCCTGGACGAAAACGACAACCCACC GGAATTTCGCAAT

GTTCCCTACGAAACAGAGGTCCTGGAGGACGCCAAGCCAGGCACCACCGTGTTCAGC GATATCCTGGTTA

CCGATCGGGACACCGTCGGAGATAACCTGATCGTGAACTGTATTCCACAACCGCAGA ACCCGGATGCTTG

CGAAAAGTTCGCCATCGAAACCCTCGAAAGCGGTCAGGATCGACTAACGGCTTCGGT GGTGCTGAAGGGT

CGCCTAGACTACAACGAACGGATGATCTACCAGATTCTGCTGGAGGCTACCGATGGG ATGTTCAACGCCA

CGGCTGGACTGGAGATCCACGTGAAGGATGTTCAGAACAGTGCGCCGGTGTTCCAAG GATCGTTGGCGGC

GGTAATCAACGAGGACAGCAAGATCGGGACGCTGGTGATGATGATCCACGCAAGGGA TGGCGATCGGGGT

CAACCGAGGAAGATTGTCTACGAATTAGTTACGAACCCAATGGATTACTTCTTGCTG GATCGTCAAACGG

GTGAGCTACGCACGGCCAAACCACTCGACAAGGAAGCCCTTCCCGACGACACCGGGT TGATAATCCTGAC

GGTTAAAGCTCGCGAGCTGATCGACGGAGTTCCCGGTAATGACAATCTGACCACGGC AACAACACAAGCG

TCGATCACGATTCGCGATGTGAACGATTCTCCACCGATGTTCAACAAAAAGGAATAC TTCGTATCGCTGT

CGGAGAATACGGCTCCGGGAACGCCACTTCCGATCGAAATGAGCGTTCATGATCCGG ATGTTGGAGAGAA

CGCTGTGTTTTCTCTACGCTTGAATGATGTTTCGGAAGTGTTCGATGTGGAGCCAAA ATTGGTGACGGGA

TCGTCACAGATTAGTATTCGTGTAGCGAATGGTTCGCTGGATTACGAAAACCCTAAC CAACGGAAGTTCA

TCGTATTGGTGATCGCTGAAGAAACCCAGACGAACCCTAAGCTGTCATCGACAGCTA CTTTAACGGTGTC

TATCACCGACTCGAATGACAACCGTCCGATCTTCGAGCAGGACTCGTACTCTACAAC TGTATCGGAAACT

GCTCATCCCGGTCATTTGATAACGACCATCACCGCCAGAGATCTCGACTCAGGTCAT TTCGGCGACCAAG

GAATTCGGTATTCCTTGTCTGGAACGGGAGCCGAACTCTTCAACGTCGACCCGATAA CCGGCGCTATAAC

GGTCGCTGATTGCCCATCCGTAGACAACGACAACAACAAAAGACGTCGTCGGCGACG TCAGATTCCTTCA

TCCGATGAGCTGACTCAAGACTACCCGGATATGAAACGTTTCAACGTGTCAACCGAC GGACGTTCGGGCG

TCCTAGACCGTGGCGTAGACTATATGGCCTACAAGATCTACAACAGTGGCGAATCGA ACGAGTACCGAGA

CGTGAATGTCGTCGCACCTCCAACGGTTTCCAGCAGCTGGGAAACGTCCAGTTTGGA GGAAAGCGACTCC

ACCCCGGCCATCGAGTCGGAAGAATACTTCACGCCATCTAGCACCACCACTCCCATC CACTCGAACGAAA

TCCAGCACCGTTCGGATGTGGGCCCAGGGCGAGCTCCTTGCTTGGACTACGAAAATC AATCGGTGTACTA

TCTGTCCTACAAGGCCACGGATGACGAGGGCCGGGGTCAAACGTCGGTAGTATCGCT CCGGATCACCCTT

CTGGATGCGAACGATTCGCCGCCGGTGTGCGAGAGCCCTCTCTATAGGGCATCGGTC GACGAGGGAGCCA

CCCTATTTGAGCCGCCGCTCGTCATCAAAGCCCGCGATCCGGACGTTATTTCGGAAA TTAATTATCGCAT

AATTGGTAACGAAGCAATTACGCGCCATTTCGAAATCGACAAACGGTCCGGACAGTT GACCATCTCCAAG

AGTACCGCCCTGGACGTGAACCATCTGAAGTCGGAAAACGTGTTCTTCGCCGTGGAG GCAAGCGATGGCC

TCTTCACCACCCTGTGCAACGTGAACATCACCATCCGGGACGTGAACAACCATGCAC CGCAGTTCTCCCG

GGAGCACTATCTTGCCTCGATCGAGGAGAACTTCCCGATTGGCACCCGAGTCGAACG TTTACAGGCAATC

GATTTGGATACCGGCATCAACGCCGAGATCAGGTACCGCATCCAGCAGGGAAGCTTC GATGACTTTGCCA

TCGACAACCAAACCGGGGTGGTGACCATCGCCCGGAAGTTGGACTACGACCGGAGGA ACACCTACCAGAT

GGAAATAGTGGCAGCGGATCTGGGCACCCCAAGTCTGTCGGGGACAACCACCCTGAC GGTGAGCATCATC

AATAGCAACGACAAAGCCCCGTACTTTACGCCGACTACTCAGCGGGCGGAAATATCG GAGGATGCGGAAG

TGGGAACGTTGGTCCACACGCTGGTGGCACTCGATCCGGATGTGGCGTCCAGCGAAG CGTTGGATTATGC

GGCAACGGAACCCATCACGGCCGTTGACAAGGACGGAAAGGAGGTGCGGGACACGGA AGATTTCAAGGAC

ATGTTCCGCATCGATCGGACCGGAAAGGTGTTCGTCAATCGGAAGCTGCAGCGGGAT GATTTTGCGGTGA

TCCGAATCACGGTTCTGGTAACGGACACAACCGCCCCATCGATTCAGCAGGGCGAAG GTCTCCTCATAAT

CACAATCATCGACGTAAATGAAGAGCCACCGCTGTTCGTGCCCCCGTGGACTCCGGC GGATCCCCGCTAC

CGGTTCCAGGTGCTGGAGGAACAACCGATCGGTACCATCCTGACGACGATGCAAGCA ACAGATGCCGACT

CGACCGTCGCCGAGTACCGGATGACAGATAACAGCCATTTCGAGATAAACAACACAA CAGGTCTGATCCG

CACCAAAGCCCGTATCGATTACGAGCAAACGCCAACGATCCAGTTCAACGTCACCGT GGTGGACACCGGA

ATCCCGCAGTTGACGTCCACCGCCGAAGTAACGGTCGACATCATCAACACCAACGAC AACGATCCGGCCT

TCGACGAGCCTGAGTACGAAATGTCCGTCGTGGAAAACGCACCCACCGGAACGGTTG TGGGCATAGTTTC

AGCGCGGGATGCCGACTCGGGACCGTATGGCCAAATCACCTACTCCCTGGTCGGTGA CCACAGTGCTGCC

AGCTTTGCCATCGATCCAGACACCGGAGTTATCACGGTGCGCGACGGCACAACCTTG GACCGTGAACGGA

CAACGGAAATCGGCCTCACTGCCATTGCCACGGATCGGGCCCCGGATGGAACCAGCC GGTCGACCACCGC

ACCGGTTACCATCAAACTGCTGGACGAGAACGACAATGTGCCGACCTTCTCGCAGAA GATTTATCACGCC ACGGTAGCGGAAAATGCGGCACTCAATCCACCGGCAGCAATCTTGCAGGTTTTGGCCACC GATCCGGACG AGGGCGCTGCTGGGGACGTGAAATATAGCATCATCGGTAGCGATATTGAAAACACCTTCC GGCTGGACGC AAACTCGGGCATCCTGTATCCGTACGCCAGTTTGCTGGGACTCGACGGCAACTATCGCAT CCAAATCGAG GCCCGCGATGGCCTAGGATCCGGACCTCACAGCGATCGGGCTGAAATTAAAATTGAAATA CAAAGCATCA ACCAGCATCGTCCGATTTTCATCATGCCGGCACTGTCCAACGCAACGGTGGAAATCCCCG AGAATTTAGC GATGACGGATTATCTCGTGATGACGGTTAAAGCGAACGACAGCGACGAGGGAACGAACGG CAAAGTTTTG TACCATCTGCAGGTCAACAACCAGAACGTCCAGGAAACGGACGAGTTCATCATCAACGAA ATGTCCGGCG AACTGCGCATTCGCAAGCCCCTCAACCGCAAGAAGCAGGCCCGCTTCGAGTTGATCCTGG TGGCCCGGGA CCAGGGTACCCCTGCGTGGTTCGAAACGCTCCGTTTCCTCACCGTACTGCTGGTCGACGT CAACGAAAAC CACCCGGAGTTTCCGGACGCCTCAAACCCCTACAGGTTCTTCATCGCCGAGAACAGTCCT CGGGACATCC GCATCGGTAAAATCCAGGCCTATTACGACACACCCGACCCGAAAATCTACTACTACATGA TGCTCGGCAA CGAGGATGGAGCGTTCTACGTGGACAAAACCACCGGCGATATCTACACCAACAAAACGCT GGACCGCGAG GAAGCGGATGTCTACGCTCTCTATATCAAAGCCAGCAAGAAACAAGACCTGCTGATCACT GAGCGCGATC GGATGATGATGTCGACCAAAAAGCTGGAACGCGATAGCACGGTTGCGAAGGTCTGGATCA CAGTCCTCGA TGTCAACGACAATCCCCCGGTCTTTAAACAGGACGTTTACTACGCTGGCGTAAGCTCCAA GGCTGCCATC AACGAATTGGTGACAATTGTCAATGCGACCGATCGAGATCTGGGCGTGAACTCTACCATG GAACTGTTCA TCAGCGGGTCTTATCTTTACAAATACGGAGCTACGAAGACAACTGGTAGCATAGTTCCAA GTCCGTTCAC TATTTCCAAGGACGGTCGTATAACTACCGCAAACTACATGGCCGAATATAACCAGGACCG TTTCATTCTG GACATTGTAGCAAAAGAGGTGGAATCTCCTGAGCGAGTTGCCACCACCAAAGTCTACGTC TGGATCTTCA ATCCAGAACAACTAGTGCGTGTGATCCTGTCGAGGCCACCCTCGGAAGTTCACATGGAGC GAGATGAGAT CATATCCGAACTTTCGAATGCCACCCAGAAGCTGATTATTGTCGATGAGATTCGATACCA CGTGGACAGC TTGGGTCGCATTCGGATGGATTGGTGCGACATGTACTTCCATGCGATCGATATGAGTTCG CAGACGATCG TGTCGGTAGAGGAGATTCTGCGGGAGATCGACGCCAAATATGATTTCCTACAGGATTACA ATGCCGGCTT TTCGATCGAGAACGTAGTCCCGGCCTACGCAACCAACGTCCAGGACGAGTTCGATTTGGC CCTGGCTGCG ATAATCGCCCTGCTGATAGTGCTGTTTGTCGGTGCCGTAAGCTTCATCGTCCTGTGCTGC TGTCTCAAAC AT T G GG T CAT T AC GAT T C C G AAC G AAAC C AG AAG AAAG G AC G C C T T GAT C AAAAAG C AG AT T AT C G AAG A T T T AAAT AC G AC C G AG AAT C C AC T T T GG AT C GAG C AAAAAC T G AAG C T C T AC G AAG AG C AGG AAC T G AC G ATGCAAGTGTTTTCCGAGCCGGAACTGACGCAACAGCAGCAGCAGCACCACCACCAACAG CAGTTGAACA GCTCGAACAATACTTCGTCGTCGTTGGCCAGCCACCAGAACCAGCACCACCATGTGATGC AACAGCAGGA ACAAGCGTTGGTCCTGGGGCTGGATCGGCGGGATTCGTACCCGGAATTGTCCCAAGGGGG CGGCGATAAC ACGTACGCCACCATCCAGCCACGCAATTATGCGTCCAATCTGAGCTCGGTGCTGATGGGC ACTAGCGGGA TTGGTGGCGGCGGCGGTGGCGGAAGCGGAAACGGTGCGGCCCCGGCAGGCGGACTGAGCG GAGAAATGTC GGATTATGCGACACTGCGGAACAGCAGGGCACCCTCGATGTACGAGTTCCGAGGTTCAAC CTTCCAGGTA CAGCAGCTAAACGGTGGACCCGGCGGTGACCAGCCAGACTACGTGACGGAACTGATTTAA GAGTAAACAA CCTTCGAACAGCATCGAACCGTTTTGACCCAACTCAGCCCCAAAAGTGCAACAGTGGAAC AAACCGTTTT ACGCTCTCGAGATGGACAGAGAAAGAGAGAGCAACATCACTTTTTGGGTTTTTAGCATAG GATATCATCA GGAGACTAGAAAGCGGTTTGGAATTTACAAACCAACCGGAATCGCCGGATTGCCAATTTG GATTTGTAGA AAATGAATGCTCAATGTGTATGACACCCGAATGAAATACTCAAGTGAAGGAAAAGTTCGG AAAGCGATTT TTTAAATTACTGATGAGAGGCACAGATTACAAAACACTCTTTGATAGACAATAAATAGGA GATATCTTAA AAGGATAGTATTTATGACGGAGGAAGCAACATTGAAGAGATAAACGCACCCGGAGAAAAT TGAATCATTC CACACGCGTACTCATTCCGAGTTTAAGTTGTAATTAATTTAAGTTCACAAAAATACATTA ACAGATGACC ACCAGAATCGAATTCGAGCTATCACGACCCGACTCCCCCTTCATTTAAAGGTGCTCGATA GGCAGGGAGC GGACGAGTGGCCATTTACTTCACTTGGATACCTCGGCGGTCTGGGGCCAGCGGCCATTTC GAGCTCATTA TAATTTCTCCCATTTTCTGCCAATTACCAACGAACGTTCGCTCCACCACACTCTCACACG TGGTCGGTAC CGGCACGCGCTTGAGTTCAACAAATGAATGCAATTAAAAATTACGCAAAACGAGATTGGG GGAAAAATTC TGCGCACCAAAAGGCATCAATGTGCAATTTTTCGAGAGAGGCAGGAAGAATATACTGAAA GGGATAAGAG GTCGAATGTGTCGAAATAGTCGAAATCAAGCATTTTTCGAATGGGTTTCCCTACGAAAGG CGGAAATCAC GAAAGGCTCAACTCGTAAAAGCTGAAAATATCAAAATACTGAATGGTATTCAAGTCTTCT CTAGTAAATC TAGTTTCTAGATGTCATCTTGCAATTCAACTAGCCCGAATGACACTAACTTGCAAGCATC TTATCCGAAC TTTATGAACATTCAGCTTTTTTTGTTTCGGCCTTAAGTTGGAATCCTTCGGATGTTACTT TTCTAGGGTT GCACAGCATCCAGTGAACTGACAATGGCAGTTGAAGAGGCACAATTTAATAATTTTGAAA ATTACTTACA AATTCGTTGACATAAAAAAAACTCCGTGAGCTCACTAGAAAATTGTAAAAAATGTCGTTT TGATACAACC TGTATGAAAAATTTGTTTTAACTCAAAACTTATTACGAGTTTCAAAATCAATTCAGTTCA AATCAGCATC AAACGCCTCATTTCGTTCAATACTCTTTCGTTGAGAATATTTGTCTACCGTTCGCATTTG GAATACAACT TTTATGTATTTCGACCTAAAGACCATTCGACCTATTATCGCGATGCGAAACAACCCCCAA CTGTGAAGTG AATATCAGCGCGTGTCGTTCGAAAAAAAAAGACTCAAAAATCCAACCGCCGACGCCAAAC CGTCGAGGTA TCGAGAGTCGAACATTGTAAATAATTAAATCTAGCAAAATGGATGAGAATATTTAAATTA TTAAAAATCA ATTATGTTAACGAGTTTACAGAGACATGCGGGAGGGTGAGGGCTTCGAACAAGGGTCTGA GGGATTGCAT CGCCCTCGGGCTTTAGTTTCGATAGCCAGCCTCGGTGCGATAAAAAAGGGCTCGACCGAT CCAATTCAGC GAGAGCGTCAAGAGTAACTGCCCATTTTGTGAAAGGATTGAAAAAGAGGACGACGCTACG AAAGGACAGC TACCCTCTATCG

SEQ ID NO. 13

Amino Acid

R2D2

Aedes aegypti

MASKPVLSTKTPITELQELCVSKKAPHPLYTFTGEEIDGGNPNSKVFTTSVLALGFTSSG IGRSKKDSKH DAAYKLLKLLFEXGLSDIDVDNDEHFLAVLSXDKVTEVRDICVQRNFEMPEFNCVRSXGP SHAPEFEYEC RIGAIVRRGIHKTKKGAKQAACNEMIKTLQAMPVEDSEMQVQPLNLAAEIDLNEDEHI IRTYRELINSDI KKKLGVKIADRHRFFEEQEEAKISAARRIALDETLNVEDKCTLIPKALGLKFEMKRDNSD LVTIEGRKLF TFELQNSEYDCFIFGKGDKFYESVYKYLKNMLNFDYID

SEQ ID NO. 37

DNA

IRES + 3X HA TAG

Artificial

CAGGAGGAATTAACCATGCAGTGGTGGTGGTGGTGGTGGGGAAAGCTTGCATGCCTGCAG GTCGACTCTA GAAAAATATAAAAACTCAACACAACATACACAAAACGATTAAAGCAAACACAATCTTTCA AAGCATTCAA AGCATTCAAGCAATCAAAGATTTTCAAATCTTTTGTCGTTATCAAAGCAATCACCAACAG GATCCAGGAT CCCCGGGTGGTCAGTCCCTT