Alphavirus compositions and methods of use

Abstract

Embodiments are directed compositions related to Eilat virus and uses thereof.

Claims

1. A recombinant alphavirus expression cassette comprising (i) an alphavirus nucleic acid segment having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of at least 100 consecutive nucleotides of SEQ ID NO: 1 and (ii) a heterologous nucleic acid segment.

2. The expression cassette of claim 1, wherein the alphavirus nucleic acid segment has a nucleic acid sequence that is the nucleic acid sequence of at least 100 consecutive nucleotides of SEQ ID NO: 1.

3. The expression cassette of claim 1, wherein the expression cassette is comprised in a pRS2 plasmid backbone.

4. A host cell comprising an expression vector of claim 1.

5. The expression cassette of claim 1, wherein the heterologous nucleic acid segment encodes heterologous alphavirus structural proteins.

6. An alphavirus produced by the cell of claim 4.

7. An immunogenic composition comprising the alphavirus of claim 6.

8. A method of stimulating an immune response in a subject comprising administering an effective amount of an immunogenic composition of claim 7.

Description

DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

(2) FIGS. 1A-1B. (A) Diagram of the EILV genome. Amino acid size of each protein is provided, as well as the intergenic region, 5 and 3UTR nucleotide size. (B) Cloning strategy of full-length Eilat virus cDNA clone. Endonuclease sites within EILV sequence, and pRS2 restriction sites and sequence provided.

(3) FIGS. 2A-2B. (A) Alignment of putative nsP1 conserved sequence element (CSE) within the genus Alphavirus. Nucleotides identical to EILV are displayed with dots. (B) Alignment of putative polyprotein cleavage sites within the genus Alphavirus. Amino acids identical to EILV are displayed with dots.

(4) FIGS. 3A-3C. (A) Alignments of putative subgenomic promoter (A) and 3 CSE (B) within the genus Alphavirus. Nucleotides identical to EILV are displayed with dots. (C) Phylogenetic tree of representative Alphavirus species generated from concatenated nonstructural and structural genes nucleotides by using Bayesian method. Mid-point rooted tree is shown with posterior probabilities on major branches.

(5) FIG. 4. Alignments of putative E1 fusion peptide and ribosomal binding site within the genus Alphavirus. Amino acids identical to EILV are displayed with dots.

(6) FIGS. 5A-5B. Phylogenetic trees of representative Alphavirus species generated from structural (A) and nonstructural (B) gene nucleotides by using Bayesian method. Mid-point rooted trees are shown with posterior probabilities on major branches.

(7) FIGS. 6A-6B. Complement fixation (A) and hemagglutination-inhibition (B) tests with Eilat virus and other alphavirus antigens and hyperimmune mouse ascitic fluids (MIAF). * Reciprocal of heterologous titer/reciprocal of homologous titer.

(8) FIGS. 7A-7B. In vitro characterization of Eilat virus. EILV plaque size 3 days post infection on C7/10 cells in a 6-well plate (A). Synthesis of virus-specific RNA in C7/10 cells infected with EILV and SINV, analyzed by agarose gel electrophoresis (B). Lane 1=mock-infected cells, lane 2=SINV, lane 3=EILV.

(9) FIGS. 8A-8B. Eilat virus particle morphology by cryo-EM and TEM. EILV virions embedded in vitreous ice (A). Virions budding from the surface of C7/10 cells (B).

(10) FIGS. 9A-9B. Growth kinetics of Eilat virus on representative invertebrate (A) and vertebrate (B) cell lines. Monolayers were infected at MOI of 10. Supernatants were collected at indicated intervals post-infection and titrated on C7/10 cell monolayers. Each data point represents the average titer of samples taken from triplicate infections.

(11) FIGS. 10A-10C. Illustrates the infectivity of EILV across various host cells. (A) Diagram of an EILV marker construct. (B) Results of infection of insect cell lines with a virus comprising the EILV marker genome, light and fluorescent image. (C) Results of infection of vertebrate cell lines with a virus comprising the EILV marker genome, light and fluorescent image.

(12) FIGS. 11A-11B. Illustrates results of electroporation of an EILV encoding ribonucleic acid across various host cells. (A) Diagram of an EILV marker construct. (B) Light image and fluorescent image of various cell lines four days post electroporation.

(13) FIGS. 12A-12B. Illustrates the infectivity of EILV/SIN structural construct across various host cells. (A) Diagram of an EILV/SIN marker construct. (B) Results of infection of cell lines with a virus comprising the EILV/SIN marker genome, light and fluorescent image.

(14) FIGS. 13A-13B. Illustrates the infectivity of EILV/EEEV structural construct across various host cells. (A) Diagram of an EILV/EEEV marker construct. (B) Results of infection of cell lines with a virus comprising the EILV marker genome, light and fluorescent image.

DESCRIPTION

(15) The genus Alphavirus in the family Togaviridae is comprised of small, spherical, enveloped viruses with a genome consisting of single strand, positive-sense RNA approximately 11-12 kb in length (Kuhn R J. Togaviridae: The viruses and their replication, In: Fields B N, Knipe D M, Howley P M, editors. Virology. 5th edition. New York, N.Y.: Lippincott-Raven; Pages 1001-22). The genome contains two open reading frames: the 5 two-thirds of the genome encodes four nonstructural proteins (nsP1, nsP2, nsP3, and nsP4); and the 3 one-third of the genome encodes for structural proteins (Capsid, E2, E1). Alphaviruses enter susceptible cells via receptor-mediated endocytosis and replicate in the cytoplasm of infected cells (id.). Following internalization, low endocytic pH induces a conformational change that exposes E1 fusion peptide and results in the release of the nucleocapsid (id.).

(16) Since the genome of alphaviruses are capped at the 5 end and have a poly A tail at the 3 end, the viral RNA serves as mRNA for translation of nonstructural proteins (id.). The resulting polyprotein is sequentially cleaved into four proteins that are responsible for RNA replication, modification, and proteolytic cleavage (id.). Non-structural proteins facilitate the synthesis of negative and positive strands as well as the transcription of subgenomic mRNA encoding structural proteins (id.). Following translation, E1 and E2 are processed and glycosylated, and E1/E2 heterodimers are inserted into the host plasma membrane (id.). Capsid proteins interact with one genomic RNA copy to form the nucleocapsid, which interacts with the cytoplasmic tail of E2 protein to initiate virion budding from host cell membranes to commence another infectious cycle (id.).

(17) Representative examples of alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69), Venezuelan equine encephalomyelitis virus (ATCC VR-923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375), all of which are incorporated herein by reference.

I. Eilat Virus

(18) Described herein is a new alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes collected in the Negev desert of Israel. Phylogenetic analyses places EILV as a sister to the Western Equine Encephalitis (WEE) antigenic complex within the main Glade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions are spherical, roughly 60-70 nm in diameter and bud from the plasma membrane of mosquito cells in culture. EILV readily infects a variety of insect cells with little overt cytopathology. However, in contrast to all other alphaviruses, EILV does not infect various mammalian and avian cell lines at 37 C. Evolutionarily, these findings indicate that EILV lost its ability to infect vertebrate cells. Thus, one use of EILV is in reverse genetic studies to assess the determinants of alphavirus host range. The EILV genome (SEQ ID NO:1) includes a 5 promoter, a non-structural protein (nsPs) coding segment (SEQ ID NO:2), an intergenic region containing a subgenomic promoter (SEQ ID NO:6), a structural protein (sPs) coding region (SEQ ID NO:4), 3 promoter, and a poly-A tail.

(19) In one embodiment, the present invention provides an isolated nucleic acid comprising a coding segment having at least 80, 85, 90, 95, 98, 99, or 100% sequence identity to SEQ ID NO:1 or a fragment thereof. A fragment can be any 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000 consecutive nucleotide segment thereof, including all values and ranges there between.

(20) In some embodiments, the coding segment comprises a non-structural EILV coding region, e.g., SEQ ID NO:2, or a fragment thereof. In some embodiments, the coding segment encodes a non-structural EILV protein, or fragment thereof, e.g., nsP1, nsP2, nsP3, and/or nsP4. In one embodiment, all four of nsP1, nsP2, nsP3, and nsP4 are encoded.

(21) In some embodiments, the coding segment comprises a structural EILV coding region, e.g., SEQ ID NO:4, or a fragment thereof. In some embodiments, the coding segment encodes a structural EILV protein or fragment thereof, e.g., C, E1, E2, E3, and/or 6K. In some embodiments, the coding segment encodes structural EILV protein C, E1, and/or E2.

(22) In one embodiment, the present invention provides a chimera encoding at least one EILV protein or fragment thereof and a heterologous gene. In one embodiment, the chimera encodes at least one structural EILV protein; in another, it encodes at least one non-structural EILV protein. The heterologous gene can be, e.g., a therapeutic protein, an antigen, a toxin, or a marker. An antigen can be, e.g., a structural protein of another virus, e.g., a non-EILV alphavirus, e.g., VEEV, EEEV, or WEEV. In one embodiment, the heterologous gene encodes C, E1, and/or E2 of a non-EILV alphavirus. In one embodiment, the chimera encodes all three of C, E1, and E2 of a non-EILV alphavirus. Alternatively, the antigen can be a non-viral antigen. Such viral and non-viral antigens are useful in the manufacture of immunogenic compositions, and in methods for eliciting immune response in mammals including humans as discussed below.

(23) In another embodiment, the heterologous gene is selected for selective expression in arthropods. Particular genes of interest include those that disrupt replication or hinder transmission of an arthropod-borne infectious disease and/or reduce the lifetime of the arthropod. Particularly useful hosts for such host-selective expression systems include mosquitoes (Aedes sp., Culex sp., Anopheles sp.). In one embodiment, the heterologous gene is expressed in Aedes sp., e.g., Aedes albopictus or Aedes aegypti.

(24) In certain embodiments, the isolated nucleic acid is incorporated into an alphavirus vector capable of replicating in arthropods, e.g., Aedes sp., but not in mammals, e.g., humans.

II. Pharmaceutical Compositions

(25) Certain embodiments are directed to pharmaceutical or immunogenic compositions comprising an alphavirus nucleic acid, alphavirus vector, alphavirus particle, alphavirus protein, or alphavirus virus, in combination with a pharmaceutically acceptable carrier, diluent, adjuvant, or recipient.

(26) Briefly, the compositions described herein may be formulated in crude or purified forms. To produce virus in a crude form, virus-producing cells may first be cultivated in a bioreactor, wherein viral particles are released from the cells into the culture media. Virus may then be preserved in crude form by adding a formulation buffer to the culture media containing the virus to form an aqueous suspension. Within certain embodiments, the formulation buffer is an aqueous solution that contains one or more saccharide, high molecular weight structural additive, and buffering component in water. The aqueous solution may also contain one or more amino acids.

(27) The virus or viral particle can be formulated in a purified form. More specifically, before adding the formulation buffer, the crude virus or viral particle described above may be clarified by passing it through a filter and then concentrated, e.g., by a cross flow concentrating system (Filtron Technology Corp., Nortborough, Mass.). DNase can be added to the concentrate to digest exogenous DNA. The digest is then filtered to remove excess media components and to establish the virus or viral particle in a more desirable buffered solution. The filtrate may then be passed over an affinity column, e.g., Sephadex S-500 gel column, and a purified virus or viral particle is eluted. A sufficient amount of formulation buffer is then added to this eluate to reach a desired final concentration of the constituents and to minimally dilute the virus or viral particle. The aqueous suspension may then be stored, e.g., at 70 C., or immediately dried. The formulation buffer may be an aqueous solution that contains one or more saccharide, high molecular weight structural additive, and/or buffering component in water. The aqueous solution may also contain one or more amino acids.

(28) Crude virus or viral particle may also be purified by ion exchange column chromatography. Briefly, crude virus or viral particles may be clarified by passing it through a filter, followed by loading the filtrate onto a column containing a highly sulfonated cellulose matrix. The virus or viral particle may then be eluted from the column in purified form by using a high salt buffer, and the high salt buffer exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. A sufficient amount of formulation buffer is then added to the purified virus or viral particle and the aqueous suspension is either dried immediately or stored, e.g., at 70 C.

(29) The aqueous suspension in crude or purified form can be dried by lyophilization or evaporation at ambient temperature.

(30) In certain aspects, the aqueous solutions used for formulation are composed of a saccharide, high molecular weight structural additive, a buffering component, and water. The solution may also include one or more amino acids. The components act to preserve the activity of the virus or viral particle upon freezing and lyophilization or drying through evaporation. A saccharide can be lactose, or other saccharides, such as sucrose, mannitol, glucose, trehalose, inositol, fructose, maltose, or galactose. In addition, combinations of saccharides can be used, for example, lactose and mannitol, or sucrose and mannitol.

(31) The high molecular weight structural additive aids in preventing viral aggregation during freezing and provides structural support in the lyophilized or dried state. Within the context of the present invention, structural additives are considered to be of high molecular weight if they are greater than 5000 m.w. In certain aspects, a high molecular weight structural additive is human serum albumin. However, other substances may also be used, such as hydroxyethyl-cellulose, hydroxymethyl-cellulose, dextran, cellulose, gelatin, or polyvinylpyrrolidone.

(32) The amino acids, if present, function to further preserve viral or viral particle integrity upon cooling and thawing of the aqueous suspension. A preferred amino acid is arginine, but other amino acids such as lysine, ornithine, serine, glycine, glutamine, asparagine, glutamic acid or aspartic acid can also be used.

(33) The buffering component maintains a relatively constant pH. A variety of buffers may be used, depending on the pH range desired, preferably between 7.0 and 7.8. Suitable buffers include phosphate buffer, citrate buffer, and tromethamine.

(34) In certain aspects, a viral or viral particle formulation can contain a neutral salt to adjust the final formulation to an appropriate iso-osmotic salt concentration. Suitable neutral salts include sodium chloride, potassium chloride or magnesium chloride.

(35) The lyophilized or dehydrated viruses can be reconstituted using a variety of substances, such as water. In certain instances, dilute salt solutions that bring the final formulation to isotonicity may also be used.

III. Examples

(36) The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Eilat Virus, a Newly Identified Host Restricted Alphavirus

(37) A. Results

(38) Virus Isolation.

(39) EILV was one of 91 viruses collected during a survey of the Negev desert in Israel between 1982-84 (Muriu et al., Malar J. 2008, 7:43). Mosquitoes were collected from many parts of the desert including in the city of Eilat and the isolation was from a pool of Anopheles coustani (Fornadel et al., Vector Borne Zoonotic Dis. 2011, 11(8):1173-9). Preliminary characterization showed that the virus was unable to grow in mammalian cells but could grow to high titers in insect cells.

(40) Genomic Analysis.

(41) The sequence of EILV was determined by 454 sequencing. EILV genomic sequence was translated and compared with Sindbis virus to determine the length of each gene product. A schematic of EILV genome is shown in FIG. 1. The length of untranslated regions (UTRs), intergenic region, and each gene product is similar to that of other alphaviruses. The coding region nucleotides and deduced amino acids of EILV were compared with other members within the genus. The nucleotide and amino acid identity of EILV with other members ranged from 57%-43% and 58% to 28%, respectively (Table 1).

(42) TABLE-US-00001 TABLE 1 Comparison of nucleotide and amino acid identity of structural and nonstructural coding regions of alphaviruses. Upper diagonal displays percent amino acid identity; lower diagonal contains percent nucleotide identity. EV TROV AURAV WHATV SINV WEEV EEEV VEEV CHIKV EV 52 43 58 44 37 36 37 49 TROV 53 43 57 43 38 38 39 51 AURAV 55 57 47 65 38 39 51 41 WHATV 57 56 61 55 39 39 39 53 SINV 56 57 61 70 39 39 52 40 WEEV 52 54 55 58 57 70 46 40 EEEV 51 53 53 54 54 64 47 41 VEEV 51 54 54 55 54 58 60 41 CHIKV 52 53 53 55 54 53 54 54 RRV 51 53 54 55 55 55 55 54 62 UNAV 52 54 54 54 55 53 54 53 62 SFV 53 53 54 55 55 54 55 54 62 MIDV 52 53 54 55 54 53 54 54 60 BFV 52 52 53 54 53 53 54 53 56 NDUV 51 52 53 54 53 53 53 53 58 SESV 50 51 52 52 51 52 52 52 54 SPDV 43 44 44 44 45 44 45 45 45 RRV UNAV SFV MIDV BFV NDUV SESV SPDV EV 48 39 28 39 38 49 47 28 TROV 52 39 28 39 38 51 47 29 AURAV 41 41 30 40 39 41 37 21 WHATV 54 41 31 41 40 53 49 30 SINV 41 41 31 41 39 41 37 22 WEEV 40 40 30 40 39 40 38 21 EEEV 41 40 30 40 40 40 38 21 VEEV 40 40 30 40 39 40 38 22 CHIKV 66 48 36 46 42 58 53 30 RRV 49 38 47 42 60 53 30 UNAV 64 59 46 42 44 39 22 SFV 65 66 36 33 33 29 29 MIDV 62 61 63 59 44 39 22 BFV 57 56 58 58 42 39 22 NDUV 58 57 59 59 57 53 29 SESV 54 54 54 54 54 54 29 SPDV 46 45 46 45 45 43 43
In both analyses, EILV had the highest identity to Whataroa virus (WHATV) and lowest identity to SPDV. Amino acid comparison of individual protein was also performed (Table 2). EILV polymerase, nsP4, displayed the highest amino acid identity with other alphaviruses, whereas nsP3 had the least. Overall, EILV proteins shared greater identity with Aura (AURAV), WHATV and STNV than other members. The putative cleavage sites for the polyproteins were also compared (FIG. 2B). The nsP4 cleavage site was the most conserved within the genus even amongst the distantly related aquatic alphaviruses, SESV and SPDV. The cleavage sites of EILV non-structural and structural proteins had a greater identity with Trocara (TROV), AURAV, WHATV and STNV.

(43) TABLE-US-00002 TABLE 2 Comparison of individual EILV proteins within the genus Alphavirus. Percent amino acid identities are shown. Virus nsP1 nsP2 nsP3 nsP4 capsid E3 E2 6k E1 Trocara 64 58 30 72 49 41 34 41 46 Aura 73 60 36 74 53 46 36 36 47 Whataroa 72 65 36 74 50 42 43 40 49 Sindbis 71 65 34 77 53 45 40 45 50 WEE 57 49 29 68 43 44 42 38 49 EEE 56 50 29 69 43 42 36 40 47 VEE 56 51 29 68 40 47 34 39 45 Chikungunya 56 53 34 69 44 43 36 44 42 Ross River 60 52 30 69 41 45 35 25 42 Una 58 53 32 71 42 45 36 28 43 Semliki Forest 58 53 36 69 42 53 35 28 43 Middelburg 59 53 37 70 42 46 37 32 42 Barmah Forest 58 52 35 70 45 42 32 34 42 Ndumu 60 52 32 70 42 50 33 18 42 SES 53 51 28 65 44 47 30 42 42 SPD 41 38 21 52 31 25 24 26 36

(44) The four conserved sequence elements (CSE) were also compared. First CSE is located in the 5 UTR that serves as a core promoter for RNA synthesis and is structurally conserved. Utilizing mFold EILV 5UTR could form hairpin structures similar to that of SINV (data not shown). The second CSE is a 51 nt sequence within nsP1 gene which likely functions as a replication enhancer. EILV nsP1 CSE shared identity with AURAV, WHATV and SINV (FIG. 2). Similar to 5 CSE, EILV nsP1 CSE was able to form similar hairpin structures as SINV (data not shown). The third CSE is the 24-nt subgenomic promoter that serves as the promoter for transcription of the subgenomic RNA. EILV subgenomic CSE shared significant identity with WEEV and EEEV (FIG. 3A). Lastly, the 3 CSE is a 19-nt element located immediately before the poly-A tail, which serves as the promoter for negative strand synthesis. EILV 3 CSE was almost identical to AURAV, EEEV, VEEV and SFV (FIG. 3B).

(45) Lastly, the putative E1 fusion peptide and ribosomal binding site in capsid of EILV were also compared. EILV E1 fusion peptide was identical to WHATV and shared significant identity with SINV, WEEV, EEEV, VEEV and CHIKV (FIG. 4). Whereas the ribosomal binding site showed greater sequence divergence, with greater identity with AURAV and SINV (FIG. 4). Many of the amino acid differences in the EILV ribosomal binding site were present in other viruses.

(46) Phylogenetic Analysis.

(47) Neighbor joining, maximum likelihood and Bayesian methods were utilized to determine the relationship of EILV within the alphavirus genus. Trees were generated using full-length, non-structural and structural nucleotide alignments. The full-length and structural nucleotide analysis utilizing all three methods placed EILV sister to the WEE complex (FIG. 5A, FIG. 3C, and data not shown) with high posterior and bootstrap support. The analysis of the non-structural alignment showed some inconsistency. The neighbor joining method placed EILV sister to WEE complex where as Bayesian and maximum likelihood analyses placed EILV within the WEE complex basal to WHATV (FIG. 5B, and data not shown).

(48) Serological Analysis.

(49) Both complement fixation (CF) and hemagglutination inhibition (HI) tests were also performed to determine the antigenic relationship of EILV with the genus. In CF test, EILV antigen did not cross react with sera against most members and had minimal cross reactivity with TROV, AURAV, SINV, EEEV, and VEEV (FIG. 6A). In HI test, EILV anti-sera minimally cross-reacted with TROV, SINV, WEEV, and EEEV (FIG. 6B). Purified EILV did not hemagglutinate and EILV anti-sera yielded high background with homologous antigen therefore these data were removed from the analysis.

(50) Rescue of Infectious EILV Clone, In Vitro Characterization and TEM.

(51) EILV cDNA clone was constructed utilizing standard molecular techniques. Virus did not cause any overt cytopathology on C7/10, however at lower cell density EILV infected cells grew at a slower rate (data not shown). EILV formed 3-4 mm plaques 3 days post infection on C7/10 cells (FIG. 7A). RNA analysis of EILV infected C7/10 demonstrated that EILV could produce similar RNA species as SINV, indicating the synthesis of genomic RNA as well as expression of subgenomic RNA (FIG. 7B). TEM analysis of EILV virions showed that the virions are spherical in shape, roughly 60-70 nm in diameter and bud from the plasma membrane (FIGS. 8A-8B).

(52) In Vitro Host Range.

(53) Representative vertebrate (Vero, BHK-21, 293, NIH 3T3, and DEF) and invertebrate (C6/36, C7/10, Cu. tarsalis, and P. papatasi) cell lines were used to determine the in vitro host range of EILV. SINV was used as a positive control as it has been shown to have a broad in vitro host range (Way et al., J Gen Virol. 1976, 30(1):123-30; Sarver and Stollar, Virology. 1977, 80(2):390-400; Igarashi, J Gen Virol. 1978, 40(3):531-44).

(54) Both SINV and EILV were able to infect Cu. tarsalis, P. papatasi, C6/36, and C7/10 cells (FIG. 9A and data not shown). EILV grew rapidly to high titers (>10.sup.7 pfu/mL) 12-hrs-post infection (hpi) with peak titer ranging from 510.sup.7 to 510.sup.8 pfu/mL at 48-hpi. Although both SINV and EILV were able to infect all four invertebrate cell lines, the infection did not yield any overt cytopathology (data not shown). All vertebrate cell lines were readily infected by SINV and showed extensive cytopathology at 12-hpi (data not shown). Whereas EILV was unable to infect any of the vertebrate cell lines tested and no overt cytopathology was observed (FIG. 9B and data not shown). The initial EILV inoculum decayed significantly by 72-hpi and was barely above the limit of detection at 96-hpi. These results were also confirmed by infection with EILV-eRFP at MOI of 10 (FIG. 10). EILV infected invertebrate cells expressed eRFP 24-hrs-post-infection, whereas no fluorescent signal was observed in any vertebrate cell lines up to 4-days-post-infection (FIGS. 10B-10C).

(55) Analysis of EILV RNA Replication in Vertebrate Cells.

(56) To ascertain whether the host range restriction was present at the RNA levels, EILV-eRFP clone was in vitro transcribed and electroporated into vertebrate and invertebrate cells. EILV-eRFP was unable to replicate in vertebrate cells up to 4 days-post-electroporation, whereas it readily replicated in invertebrate cells (FIGS. 11A-11B). This indicates that the EILV RNA itself in incapable of replication in vertebrate cells.

(57) Chimeric Virus Host Range.

(58) Representative vertebrate and invertebrate cell lines were used to determine the in vitro host range of EILV/SIN and EILV/EEEV chimeras (FIG. 12 and FIG. 13). The chimeras were EILV backbones having the structural proteins substituted with sindbis virus (SIN) or eastern equine encephalitis virus (EEEV) structural protein genes. The chimeric virus maintained the EILV host range, i.e., arthropod specific replication.

(59) B. Materials and Methods:

(60) Viruses and Cells.

(61) Eilat and Sindbis (Eg 339) viruses were obtained from Arbovirus Reference Center at the University of Texas Medical Branch. Both viruses were amplified on C7/10 cells and stored at 80 C.

(62) Vero, baby hamster kidney (BHK-21), human embryonic kidney (HEK-293), Duck embryo fibroblast (DEF), mouse fibroblast (NIH 3T3), and Aedes albopictus (C6/36 and C7/10) cell lines were obtained from the American Type Culture Collection. Culex tarsalis and Phlebotomus papatasi cells were obtained from the Arbovirus Reference Center at the University of Texas Medical Branch. Cell lines were propagated under conditions of 37 C. or 28 C. and 5% CO.sub.2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, sodium pyruvate (1 mM), and Penicillin (100 units/mL)-Streptomycin (100 g/ml). C6/36, C7/10 and Culex tarsalis media was additionally supplemented with 1% tryptose phosphate broth solution (Sigma). Phlebotomus papatasi cells were maintained in Schneider's media (Sigma) supplemented with 10% fetal bovine serum and Penicillin (100 units/mL)-Streptomycin (100 g/ml).

(63) Genomic Sequencing.

(64) EILV genome was sequenced by 454 sequencing (Roche Diagnostics Corp.). Briefly, viral RNA was extracted using TRIzol LS (Invitrogen), DNase I (Ambion) treated and cDNA was generated by reverse transcription utilizing Superscript II system (Invitrogen) using random hexamers linked to an arbitrary 17-mer primer sequence. The cDNA was RNase H treated and randomly amplified by PCR with random hexamer linked 17-mer primer. Products were purified (Qiagen) and ligated to specific adapters for sequencing on the 454 Genome Sequencer FLX (454 Life Sciences) without fragmentation. The removal of primer sequences, redundancy filtering, and sequence assembly was performed by utilizing software programs at the GreenePortal website (available on the WorldWideWeb at tako.cpmc.columbia.edu/Tools/). Sequence gaps were filled by using primers based on pyrosequencing in both directions with ABI PRISM Big Dye Terminator 1.1 Cycle Sequencing kits on ABI PRISM 3700 DNA Analyzers (Perkin-Elmer Applied Biosystems). The terminal sequences for each virus were amplified using the Clontech Smarter RACE kit (Clontech). Full-length genome was verified by classical dideoxy sequencing using primers designed from the draft sequence to create products of 1,000 basepairs (bp) with 500 bp overlaps.

(65) Cloning and Rescue of Full-Length Infectious EILV Clone.

(66) EILV cDNA clone was constructed utilizing standard molecular techniques. Briefly, EILV RNA was obtained by infecting C7/10 cells. Viral RNA was isolated from cell culture supernatant using the QIAamp Viral RNA Mini kit (Qiagen). cDNA was produced by using random hexamers and Superscript III (Invitrogen). cDNA fragments were amplified with Phusion DNA polymerase (New England BioLabs) and EILV primers. Amplified PCR products were purified using QIAquick Gel Extraction Kit (Qiagen). Fragments were cloned into pRS2 (FIG. 1B). The full-length cDNA clone was confirmed by ABI PRISM Big Dye sequencing (Applied Biosystems). EILV infectious clone was rescued using standard techniques. EILV cDNA was linearized with Not I and in vitro transcribed using SP6 RNA polymerase transcription kit (Ambion). Approximately 4 g of RNA was electroporated into C7/10 cells and supernatants were harvested at 48 hrs post-electroporation and stored at 80 C.

(67) Phylogenetic Analysis.

(68) Phylogenetic analysis was performed. Alphavirus sequences were downloaded from GenBank, aligned in SeaView utilizing the MUSCLE algorithm (Edgar, 2004, Nucleic Acids Research, 32:1792-97; Gouy et al., 2010. Molecular Biology and Evolution, 27:221-224). The sequences were aligned by deducing the amino acid sequence from open reading frames (ORFs) aligned and then returned to nucleotide sequences for subsequent analyses. Sequences were aligned as deduced amino acids (aa) from ORFs and then returned to nucleotide sequences for most analyses. The two ORFs were concatenated, and the C-terminus of nsP3 and the N-terminus of the capsid genes were removed from the analysis as these sequences display significant sequences divergence and produce poor alignments. Following manual adjustments, the complete alignment was split into non-structural and structural protein ORFs. Three analyses were performed; neighbor joining (NJ), maximum-likelihood (ML), and Bayesian. NJ analysis was performed utilizing PAUP* version 4.0 (Swofford, 1998, PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4. Sunderland, Mass.: Sinauer Associates). Trees were generated utilizing p-distance algorithm and the robustness of NJ phylogeny was evaluated by bootstrap resampling of 1,000 replicates. ML and Bayesian analyses were performed utilizing the PHYLIP package and Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) in MrBayes v3.1.2, respectively (Felsenstein, 1989, Cladistics, 5, 164-166; Ronquist and Huelsenbeck, 2003, Bioinformatics 19, 1572-74). Model test in PAUP was used to identify the best-fit nucleotide substitution model, GTR+I+G model (Posada and Crandall, 1998, Bioinformatics 14, 817-18). The robustness of ML and Bayesian phylogeny was evaluated by booststrap resampling of 100 and five million generations, respectively.

(69) Serologic Tests.

(70) Complement fixation (CF) and Hemagglutination-inhibition (HI) tests were performed using microtechniques described previously (Beaty et al., 1989, Arboviruses. Schmidt N J, Emmons R W, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, Sixth Edition, Washington D.C.: American Public Health Association, 797-855). CF was performed using two full units of guinea pig complement and titers were recorded as the highest dilutions giving 3 or 4 fixation of complement on a scale of 0 to 4. HI tests were performed using immune sera and acetone-extracted ascitic fluids. Antigens and anti-sera were obtained from the Arbovirus Reference Center at the University of Texas Medical Branch.

(71) Transmission Electron Microscopy.

(72) Thin section and cryo-electron (cryo-EM) microscopy were performed as described previously (Ito and Rikihisa, 1981. Techniques for electron microscopy of rickettsiae. Burgdorfer W, Anacker R L, eds. Rickettsiae and Rickettsial Diseases. New York: Academic Press, 213-27; Travassos da Rosa et al., Am J Trop Med Hyg. 2001, 64(1-2):93-7; Sherman and Weaver, J Virol., 2010, 84(19):9775-82). Briefly, monolayers of C7/10 cells infected at MOI of 10, 24 hours-post-infection, cell were fixed in 1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% trinitrophenol and 0.03% CaCl.sub.2 in 0.05 M cacodylate buffer pH 7.3, and washed in washed in 0.1 M cacodylate buffer. Cells were scraped, pelleted 1% OsO.sub.4 in 0.1 M cacodylate buffer, en bloc stained with 1% uranyl acetate in 0.1 M maleate buffer pH 5.2, dehydrated in ethanol and embedded in Poly/Bed 812 (Polysciences). Ultrathin sections were cut on Reichert Ultracut S ultramicrotome, stained with 2% aqueous uranyl acetate and 0.4% lead citrate, and examined in a Philips 201 electron microscope at 60 kV.

(73) For cryo-EM, virus was amplified on C7/10 cells at MOI of 0.5. 48-hrs-post-infection supernatants were harvested and clarified by centrifugation at 2,000 g for 10 min. Virus was precipitated overnight at 4 C. by adding polyethylene glycol and NaCl to 7% and 2.3% (wt/vol) concentrations, respectively. Virus was pelleted by centrifugation at 4,000 g for 30 min at 4 C. and precipitate was resuspended in TEN buffer (0.05 M Tris-HCl [pH 7.4], 0.1 M NaCl, 0.001 M EDTA). Virus was loaded onto a 20-to-70% continuous sucrose (wt/vol) gradient in TEN buffer and centrifuged at 270,000 g for 1 hr. Following centrifugation, visible virus band was harvested using a Pasteur pipette and centrifuged 4 times through an Amicon Ultra-4 100-kDa-cutoff filter (Millipore) and resuspened in 1 mL of TEN buffer. The purified virus was applied to the holey films (R22 Quantifoil; Micro Tools GmbH; or C-flat; Protochips), blotted with filter paper, and plunged into liquid ethane cooled in a liquid nitrogen bath. Frozen grids were stored under liquid nitrogen and transferred to a cryo-specimen 626 holder (Gatan, Inc.) under liquid nitrogen before being loaded into a JEOL 2200FS electron microscope, equipped with an in-column energy filter (omega type) and a field emission gun (FEG) operating at 200 keV.

(74) RNA Analysis.

(75) C7/10 monolayers were infected with SINV or EILV at MOI of 10, 4 hrs-post-infection cells were be labeled with [.sup.3H]uridine (20 Ci/ml) in the presence of dactinomycin (ActD) (1 g/ml) for 3 hrs. Following labeling total cellular RNA was isolated by TRIzol (Invitrogen), denatured with glyoxal in dimethyl sulfoxide and analyzed by agarose gel electrophoresis using previously described conditions (Gorchakov et al., J Virol., 2004 78(1):61-75).

(76) Plaque Assay.

(77) Virus titration was performed on freshly confluent C7/10 cell monolayers in six-well plates. Duplicate wells were infected with 0.1-ml aliquots from serial 10-fold dilutions in growth medium, 0.4 mL of growth media was added to each well to prevent cell desiccation, and virus was adsorbed for 2 hrs. Following incubation, the virus inoculum was removed, and cell monolayers were overlaid with 3 mL of overlay containing 1:1 mixture of 2% tragacanth and 2MEM with 5% FBS, 2% tryptose phosphate broth solution, 2% Pen-Strep. Cells were incubated at 28 C. in 5% CO.sub.2 for 3 days for plaque development, the overlay was removed, and monolayers were fixed with 3 mL of 10% formaldehyde in PBS for 30 mins. Cells were stained with 2% crystal violet in 30% methanol for 5 min at RT and excess stain was removed under running water and plaques will be counted.

(78) One-Step Growth Curves.

(79) Growth curves were performed on representative vertebrate and invertebrate cell lines in triplicates. Three independent dilution curves of EILV and a single dilution of STNV virus stocks were performed to obtain a MOI of 10. Each replicate was used to infect 50% confluent monolayers in 25 cm.sup.2 flasks. Virus was adsorbed in 1 ml of growth medium for 2 hrs at 37 C. or 29 C. with occasional rocking to prevent cell desiccation. After the inoculum was removed, monolayers were rinsed five times with 12 ml of PBS to remove unbound virus, and 5 ml of growth medium was added to each flask. 0.5-ml aliquot were taken immediately after as a time hr 0 (T0) sample and replaced with 0.5 ml of fresh medium. Flasks were placed at 37 C. or 28 C. and further samples were taken at 12, 24, 48, 72, and 96 hrs-post-infection. All samples were flash frozen in ethanol-dry ice and stored at 80 C for titration.

(80) Infection with EILV-eRFP Construct.

(81) EILV construct encoding enhanced red fluorescent protein (eRFP) under control of subgenomic promoter was constructed utilizing standard cloning techniques. Representative vertebrate (293-HEK, Vero, BHK-21, DEF, NIH 3T3), and invertebrate cell lines (C6/36, C7/10 Culex tarsalis and Phlebotomus papatasi) cell lines were infected at an MOI of 10. Light and fluorescent microscopy images were obtained at 24 hour intervals post infection.

(82) Electroporation of EILV-eRFP RNA in Vertebrate Cells.

(83) EILV-eRFP cDNA was linearized with Not I and in vitro transcribed using SP6 RNA polymerase transcription kit (Ambion). 4 g of RNA was electroporated into representative vertebrate (293-HEK, Vero, BHK-21, DEF, NIH 3T3), and invertebrate cell lines (C6/36, C7/10). Light and fluorescent microscopy images were obtained at 24 hour intervals post infection. SINV-eGFP replicon was utilized as positive control.