# Patent

## Title
Alphavirus compositions and methods of use

## Abstract

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

## Background
This application is a continuation in part of and claims priority to International Patent Application number PCT/US2011/066996 filed Dec. 22, 2011 (pending) and U.S. Provisional Patent Application Ser. No. 61/459,989 filed Dec. 22, 2010. Each of the above referenced applications is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under N01-AI-30027 awarded by the National Institutes of Health/National Institute of Allergy and Infectious Disease. The government has certain rights in the invention.


REFERENCE TO SEQUENCE LISTING
A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference.
BACKGROUND
Current classification of the genus Alphavirus includes 29 species that can be classified into nine complexes based on antigenic and/or genetic similarities (Powers et al., J Virol., 2001, 75 (21): 10118-31). Barmah Forest, Ndumu, Middelburg, and Semliki Forest complexes consist of almost exclusively Old World viruses whereas Venezuelan equine encephalitis (VEE), Eastern equine encephalitis (EEE), and Trocara complexes are comprised of New World viruses (id.). Western equine encephalitis (WEE) complex contains both Old World (Whataroa and Sindbis) and New World (Aura) viruses as well as recombinant viruses (WEE, Highland J, Fort Morgan and Buggy Creek) (Powers et al., J Virol., 2001, 75(21):10118-31; Hahn et al., Proc Natl Acad Sci USA. 1988, 85(16):5997-6001; Weaver et al., J. Virol. 1997, 71:613-623). The latter viruses are decedents of a recombinant virus that obtained nonstructural and capsid genes from an EEE-like virus and the remaining genes from a Sindbis-like virus (Hahn et al., Proc Natl Acad Sci USA. 1988, 85(16):5997-6001; Weaver et al., J. Virol. 1997, 71:613-623). Lastly, the aquatic alphavirus, salmonid alphavirus (SAV), consists of two species, salmon pancreatic disease virus (SPDV) and sleeping sickness virus (SDV) that are distantly related to all other alphaviruses (Weston et al., J Virol. 2002, 76(12):6155-63).
Most alphaviruses are maintained in natural cycles between arthropod vectors, mainly mosquitoes, and susceptible vertebrate hosts (Strauss and Strauss. The alphaviruses: gene expression, replication, and evolution. Microbiol. Rev. 1994, 58(3): 491-562). Occasionally, these cycles can spill over into the human and animal populations, and can cause disease. Human infections with Old World viruses such as Ross River (RRV), chikungunya (CHIKV), and Sindbis (SINV) are characterized by febrile illness, rash and polyarthritis (id.). In contrast, infections with New World viruses, Venezuelan equine encephalitis (VEEV), Eastern equine encephalitis (EEEV) and Western equine encephalitis (WEEV), can cause fatal encephalitis. The ability of alphaviruses to infect both invertebrates and vertebrates facilitates a broad host range that enables the viruses to be maintained in ecological niches with sporadic outbreaks in humans and animals. As such, alphaviruses have been shown to either naturally or experimentally infect many vertebrate and invertebrate hosts. Alphaviruses have been shown to infect mosquito species encompassing three genera (Aedes sp., Culex sp., Anopheles sp.) as well as ticks and lice (Griffin. Alphaviruses, In: Fields B N, Knipe D M, Howley P M, editors. Virology. 5th edition. New York, N.Y.: Lippincott-Raven; Pages 1023-68; Linthicum et al., J Med. Entomol. 1991, 28(3):405-9; La Linn et al., J Virol. 2001, 75(9):4103-9). Vertebrate hosts include fish, equids, birds, amphibians, reptiles, rodents, pigs, humans and non-human primates (Griffin. Alphaviruses, In: Fields B N, Knipe D M, Howley P M, editors. Virology. 5th edition. New York, N.Y.: Lippincott-Raven; Pages 1023-68; Burton et al., Science 1966, 154(752):1029-31). Consequently, they can be readily cultured in vitro in many vertebrate and invertebrate cell lines (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). Whereas distantly related fish alphaviruses, which are not known to have arthropod vectors, exhibit a narrow host range that is at least partially due to temperature (Weston et al., Virology 1999, 256(2):188-95; Villoing et al., J Virol. 2000, 74(1):173-83; Graham et al., J Fish Dis. 2008, 31(11):859-68).
The viral factor(s) that underlie the broad host range of mosquito-borne alphaviruses are poorly understood. Host-restricted viruses may provide insight into these factor(s) and provide vector delivery platforms for expression or attenuation in specific hosts. But until the present invention, no mosquito-only alphaviruses were known in the genus Alphavirus. 
SUMMARY
Described herein is a new alphavirus, Eilat virus (EILV), including nucleic acid compositions, protein compositions, viral compositions, and methods of using the same.
Certain embodiments are directed to a recombinant alphavirus expression cassette comprising an alphavirus nucleic acid segment having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1 or a fragment thereof. In certain aspects the expression cassette is incorporated into isolated nucleic acids, expression vectors, or plasmids comprising all or part of an EILV nucleic acid sequence (SEQ ID NO:1). In certain aspects the nucleic acid is a recombinant DNA. The EILV nucleic acids can have at least 80, 85, 90, 95, 98, 99, or 100% sequence identity to SEQ ID NO:1 or 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. In certain aspects, a nucleic acid comprises a nucleotide sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to all or a part of the non-structural protein coding region of EILV (nucleotides 57 to 7304 of SEQ ID NO:1, or any 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 consecutive nucleotide segment thereof, including all values and ranges there between). In a further aspect, a nucleic acid comprises a nucleotide sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to all or a part of the structural protein coding region of EILV (nucleotides 7387 to 11088 of SEQ ID NO:1, or any 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 consecutive nucleotide segment thereof, including all values and ranges there between). In certain aspects, the nucleotide segments specifically bind EILV nucleic acids and distinguish EILV from other alphaviruses under the appropriate conditions. In certain aspects the nucleotide segments are synthetic oligonucleotides. In a further aspect the oligonucleotide is a DNA oligonucleotide or analog thereof. The EILV nucleic acids can be isolated or recombinant nucleic acids or included in a recombinant alphavirus replicon, a virus, an alphavirus, a viral particle, an alphavirus particle, an expression cassette, a host cell, an alphavirus vector, and the like. In still a further aspect, an alphavirus nucleic acid sequence can comprise a heterologous nucleic acid segment. In certain aspects, the heterologous nucleic acid segment can encode a therapeutic protein, an antigen, a toxin, or a marker.
Certain aspects are directed to an isolated, recombinant, and/or purified EILV polypeptide or peptide having at least 85, 90, 95, 98, 99, or 100% amino acid sequence identity to all or part of the amino acid sequence of SEQ ID NO:3 (EILV non-structural polyprotein) or SEQ ID NO:5 (EILV structural polyprotein). The term a “polyprotein” refers to a polypeptide that is post-translationally cleaved to yield more than one polypeptide. “Polypeptide” refers to any peptide or protein comprising a chain or polymer of amino acids joined to each other by peptide bonds. “Polypeptide” refers to both short chains of 100 amino acids or less, commonly referred to as peptides, and to longer chains, generally referred to as proteins. “Polypeptides” may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques, which are well known in the art. In certain aspects, the isolated and/or purified EILV protein has at least 85, 90, 95, 98, 99, or 100% amino acid sequence identity to all or part of the amino acid sequence of an EILV non-structural protein including: an EILV nsP1 (amino acids 1 to 543 of SEQ ID NO:3, or any peptide having 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 consecutive amino acids of SEQ ID NO:3 including all values and ranges there between), an EILV nsP2 (amino acids 544 to 1352 of SEQ ID NO:3, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, or 700 consecutive amino acids of SEQ ID NO:3 including all values and ranges there between), an EILV nsP3 (amino acids 1353 to 1808 of SEQ ID NO:3, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 consecutive amino acids of SEQ ID NO:3 including all values and ranges there between) or an EILV nsP4 (amino acids 1809 to 2415 of SEQ ID NO:3, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 consecutive amino acids of SEQ ID NO:3 including all values and ranges there between). In certain aspects, the isolated and/or purified EILV protein has at least 85, 90, 95, 98, 99, or 100% amino acid sequence identity to all or part of the amino acid sequence of an EILV structural protein including: an EILV C protein (amino acids 1 to 255 of SEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 consecutive amino acids of SEQ ID NO:5 including all values and ranges there between), an EILV E2 protein (amino acids 319 to 739 of SEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 consecutive amino acids of SEQ ID NO:5 including all values and ranges there between), an EILV E1 protein (amino acids 795 to 1233 of SEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 consecutive amino acids of SEQ ID NO:5 including all values and ranges there between), an EILV E3 protein (amino acids 256 to 318 of SEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, 40, or 50 consecutive amino acids of SEQ ID NO:5 including all values and ranges there between), or an EILV 6K (amino acids 740 to 794 of SEQ ID NO:5, or any peptide of 5, 10, 15, 20, 30, or 40 consecutive amino acids of SEQ ID NO:5 including all values and ranges there between). In certain aspects, an immunogenic composition comprises all or part of 1, 2, 3, 4, 5, 6, 7, 8, or 9 EILV proteins. In a further aspect, an immunogenic composition comprises all or part of one or more EILV structural proteins. In another aspect, an immunogenic composition comprises all or part of one or more EILV non-structural proteins.
Other embodiments are directed to alphaviruses comprising all or part of the EILV nucleic acid sequence of SEQ ID NO: 1. In certain aspects the alphavirus is a recombinant alphavirus. Certain embodiments are directed to an alphavirus having a genome comprising (a) an alphavirus nucleic acid segment that is at least 95% identical to a corresponding segment of SEQ ID NO:1 and (b) a heterologous gene. In certain aspects, the alphavirus is chimeric and comprises certain segments of an EILV alphavirus and other segments from a non-EILV alphavirus. A non-EILV alphavirus includes, but is not limited to Ross River (RRV), chikungunya (CHIKV), Sindbis (SINV) Venezuelan equine encephalitis (VEEV), Eastern equine encephalitis (EEEV) and Western equine encephalitis (WEEV). In certain embodiments the chimeric alphavirus only replicates in an arthropod host. Such chimeric alphaviruses can comprise an EILV nucleic acid with 1, 2, 3, 4, or 5 of the structural gene regions coding for C, E3, E2, 6K, or E1 proteins substituted with a corresponding non-EILV region or segment.
Still further embodiments are directed to immunogenic compositions comprising an EILV nucleic acid, EILV polypeptide, EILV virus, or alphavirus comprising all or part of an EILV nucleic acid or all or part of 1, 2, 3, 4, 5, 6, 7, 8, or 9 EILV proteins. Certain aspects are directed to one or more recombinant EILV nucleic acid, recombinant EILV polypeptide, recombinant EILV virus, or recombinant alphavirus comprising all or part of an recombinant EILV nucleic acid or all or part of 1, 2, 3, 4, 5, 6, 7, 8, or 9 recombinant EILV proteins. Certain embodiments are directed to virus like particle comprising a recombinant nucleic acid described herein.
Pharmaceutical formulations, such as vaccines, of the present invention comprise an immunogenic amount of alphavirus or viral particle comprising alphavirus proteins or antigens, as disclosed herein, in combination with a pharmaceutically acceptable carrier.
Other embodiments are directed to alphaviruses as arthropod expression systems. These expression systems include all or part of the EILV nucleic acid sequence of SEQ ID NO:1 and a heterologous gene, e.g., a toxin, for expression in arthropods, e.g., mosquitoes. For example, the heterologous gene can be a gene (e.g., antisense or short interfering nucleotide) that disrupts replication or hinders transmission of an arthropod-borne infectious disease including, but not limited to, alphavirus infections (e.g., Chikungunya), flavivirus infections (e.g., Dengue), and malaria (Plasmodium). See, e.g., Ito et al. “Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite.” Nature 417: 452-455 (2002); Olson K E et al. “Genetically Engineered Resistance to Dengue-2 Virus Transmission in Mosquitoes.” Science 272(5263):884-86 (1996). In still other embodiments, the heterologous gene can be toxic to the arthropod (e.g., a bacterial gene such as from Wolbachia), thus killing or reducing the longevity of the arthropod. See, e.g., Moreira L et al. “A Wolbachia Symbiont in Aedes aegypti Limits Infection with Dengue, Chikungunya, and Plasmodium.” Cell 139:1268-78 (2009).
The term “recombinant” refers to an artificial combination of two otherwise separated segments of nucleic acid, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
An “immunogenic amount” is an amount of alphavirus or viral particles comprising alphavirus antigens sufficient to evoke an immune response in a subject administered an immunogenic composition. An amount of from about 101 to about 1010 plaque forming units (pfu) per dose is believed suitable, depending upon the age and species of the subject being treated. Subjects that can be administered immunogenic amounts of a composition described herein, e.g., EILV virus or host-range restricted EILV-containing alphavirus chimeras, include but are not limited to human, mammal, and other animal subjects (e.g., horse, donkey, mouse, hamster, monkeys), including livestock and laboratory animals. Administration may be by any suitable route, such as intraperitoneal, intravenous, subcutaneous, or intramuscular injection.
The term “alphavirus” has its conventional meaning, and includes the various species of alphaviruses, including the newly identified Eilat virus, as well as Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus, and the like.
The term “alphavirus replicon” is used to refer to a nucleic acid molecule expressing alphavirus nonstructural protein genes such that it can direct its own replication (amplification).
The term “alphavirus replicon particle” refers to a virion or virion-like structural complex incorporating an alphavirus replicon.
Alphavirus-permissive cells are cells that, upon transfection with the viral RNA transcript, are capable of producing viral particles.
The term “expression vector” refers to a nucleic acid that is capable of directing the expression of a sequence(s) or gene(s) of interest. The vector construct can include a 5′ sequence capable of initiating transcription of a nucleic acid, e.g., all or part of an alphavirus. Expressed nucleic acid can code for biologically active alphavirus non-structural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), and an alphavirus RNA polymerase recognition sequence. In addition, the vector construct may include a viral junction region. The vector may also include nucleic acid molecule(s) to allow for production of virus, a 5′ promoter that is capable of initiating the synthesis of viral RNA in vitro from cDNA, as well as one or more restriction sites, and a polyadenylation sequence. In addition, the constructs may contain selectable markers such as Neo, SV2 Neo, hygromycin, phleomycin, histidinol, and DHFR. Furthermore, the constructs can include plasmid sequences for replication in host cells and other functionalities known in the art. In certain aspects the vector construct is a DNA construct.
“Expression cassette” refers to a recombinantly produced nucleic acid molecule capable of directing the expression of one or more proteins. The expression cassette includes a promoter capable of directing the expression of a target protein(s), and a sequence encoding one or more proteins, e.g., proteins comprising alphavirus structural protein(s) or heterologous nucleic acids in the context of alphavirus. Optionally, the expression cassette may include transcription termination, splice recognition, and polyadenylation addition sites. Promoters include the CMV, MMTV, MoMLV, and adenovirus VA1RNA promoters. In addition, the expression cassette may contain selectable markers such as Neo, SV2 Neo, hygromycin, phleomycin, histidinol, and DHFR.
“Virus-like particle” refers to an infective and/or immunogenic non-virus particle containing viral nucleic acids and/or proteins. In certain aspects, viral envelope proteins are included in a lipid bilayer or a viral nucleic acid is encapsulated within a lipid bilayer.
“Antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” The term “antigen” denotes both subunit antigens (i.e., antigens that are separate and discrete from a whole organism with which the antigen is associated in nature), as well as non-replicative, killed, attenuated, or inactivated bacteria, viruses, fungi, parasites or other microbes.
An “immune response” to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. A “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells.
A composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an immunized host.
The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, for example, Erickson et al., J. Immunol. 151:4189-4199, 1993; Doe et al., Eur. J. Immunol. 24:2369-2376, 1994. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by McMichael, A. J. and O'Callaghan, C. A., J. Exp. Med. 187(9):1367-1371, 1998; Mcheyzer-Williams, M. G. et al., Immunol. Rev. 150:5-21, 1996; Lalvani, A. et al., J. Exp. Med. 186:859-865, 1997).
Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



DESCRIPTION OF THE DRAWINGS
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.
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 3′UTR nucleotide size. (B) Cloning strategy of full-length Eilat virus cDNA clone. Endonuclease sites within EILV sequence, and pRS2 restriction sites and sequence provided.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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
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.).
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.).
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
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.
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.
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.
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.
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.
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. 
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
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.
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.
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.
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.
The aqueous suspension in crude or purified form can be dried by lyophilization or evaporation at ambient temperature.
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.
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.
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.
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.
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.
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
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 
A. Results
Virus Isolation.
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.
Genomic Analysis.
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).







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.








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









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 5′UTR 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).
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.
Phylogenetic Analysis.
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).
Serological Analysis.
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.
Rescue of Infectious EILV Clone, In Vitro Characterization and TEM.
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).
In Vitro Host Range.
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).
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 (>107 pfu/mL) 12-hrs-post infection (hpi) with peak titer ranging from 5×107 to 5×108 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).
Analysis of EILV RNA Replication in Vertebrate Cells.
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.
Chimeric Virus Host Range.
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.
B. Materials and Methods:
Viruses and Cells.
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.
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% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, sodium pyruvate (1 mM), and Penicillin (100 units/mL)-Streptomycin (100 μg/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 μg/ml).
Genomic Sequencing.
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.
Cloning and Rescue of Full-Length Infectious EILV Clone.
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 μg of RNA was electroporated into C7/10 cells and supernatants were harvested at 48 hrs post-electroporation and stored at −80° C.
Phylogenetic Analysis.
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.
Serologic Tests.
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.
Transmission Electron Microscopy.
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% CaCl2 in 0.05 M cacodylate buffer pH 7.3, and washed in washed in 0.1 M cacodylate buffer. Cells were scraped, pelleted 1% OsO4 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.
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 (R2×2 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.
RNA Analysis.
C7/10 monolayers were infected with SINV or EILV at MOI of 10, 4 hrs-post-infection cells were be labeled with [3H]uridine (20 μCi/ml) in the presence of dactinomycin (ActD) (1 μg/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).
Plaque Assay.
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 2×MEM with 5% FBS, 2% tryptose phosphate broth solution, 2% Pen-Strep. Cells were incubated at 28° C. in 5% CO2 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.
One-Step Growth Curves.
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 cm2 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.
Infection with EILV-eRFP Construct.
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.
Electroporation of EILV-eRFP RNA in Vertebrate Cells.
EILV-eRFP cDNA was linearized with Not I and in vitro transcribed using SP6 RNA polymerase transcription kit (Ambion). ≈4 μg 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.

## 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.