WO2013059168A2 - Espirito santo virus and methods for detecting and preventing infection with the same - Google Patents

Abstract

A new Birnavirus, Espirito Santo Virus (ESV), is described. The RNA and polynucleotide sequences encoding ESV are disclosed. In certain aspects, methods for detecting ESV and reagents for the same are provided. Also provided are anti-ESV antibodies and antigenic compositions comprising ESV and polypeptides therefrom.

Description

DESCRIPTION

ESPIRITO SANTO VIRUS AND METHODS FOR DETECTING AND PREVENTING

INFECTION WITH THE SAME [0001] This application claims the benefit of United States Provisional Patent

Application No. 61/550, 129, filed October 21, 201 1, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0002] The present invention relates generally to the fields of molecular biology, virology, and immunology. More particularly, it concerns the identification and characterization of a new Birnavirus, Espirito Santo Virus (ESV).

2. Description of Related Art

[0003] Commercial scale production of many protein reagents and pharmaceuticals currently employs mammalian cell culture systems. Likewise, a variety of viral vaccines are produced by mammalian cell culture. Insect cell culture is a significantly less expensive system for vaccine and protein production. Accordingly insect cell systems are currently being explored for production of new vaccines, such as vaccines to dengue virus as well as for commercial protein production. However, insect cells, like mammalian cells, may harbor viral contaminants that must be identified and eliminated.

[0004] The only common contaminants of mosquito cell lines are the small densoviruses, a 20 nm virus belonging to the Parvoviridae family (Barreau et al, 1996). Among them, a new densovirus has been recently isolated and characterized from Aedes albopictus C6/36 cells that have been chronically infected (Chen et al, 2004). Aedes albopictus, along with Aedes aegypti, is considered to be one of the most important dengue virus vectors, with an even higher susceptibility to dengue than that of A. aegypti (Mitchell, 1995). The Aedes albopictus (C6/36) cell line has become very important in the study of arboviruses because of its wide range of susceptibility to different viruses and its ability to produce plaques with a number of them (Davey et al., 1973; Suitor et al., 1969). The unexpected detection of viruses in invertebrates, such as mosquitos that may carry arboviruses, is an interesting phenomenon. It has been described in early studies showing that some Aedes albopictus cell lines developed in the late sixties presented contamination with multiple viruses, such as parvovirus, togavirus, and orbivirus-like particles (Hirumi et ah, 1976). These persistent and innocuous viral infections have been described to be common in insects and crustaceans as single, dual, or multiple co-infections (Kanthong et al, 2010). However, there remains a need to identify viral contaminants in tissue culture systems and to establish methods for detecting the same.

SUMMARY OF THE INVENTION

[0005] In a first embodiment an isolated polynucleotide molecule is provided comprising a nucleic acid sequence selected from the group consisting of: (a) a sequence encoding a polypeptide at least about 80% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; (b) a sequence exhibiting at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (c) a sequence that hybridizes to the nucleic acid sequence complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof, under conditions of 1 x SSC and 65 °C; (d) a sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (e) a sequence comprising at least 15 contiguous nucleic acids of SEQ ID NO: 1 or SEQ ID NO: 3; and (f) the compliment of any one of (a)-(e). In certain aspects, a polynucleotide comprises a nucleic acid sequence encoding a polypeptide at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In further aspects, a polynucleotide molecule comprises a nucleic acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In still further aspects, a polynucleotide molecule of the embodiments comprises a nucleic acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 3 over a segment of about or at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 11, 1 12, 113, 114, 1 15, 1 16, 117, 118, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, or 3400 nucleotides.

[0006] In still further aspects, a polynucleotide molecule of the embodiments further comprises a label. For example, the label can be a radioactive, colorimetric, enzymatic (e.g., a light producing enzyme), fluorescent or affinity label (e.g., biotin), or a sequence tag. [0007] In yet a further embodiment, a polynucleotide of the embodiments is comprised in an expression vector. For example, an expression vector can provide expression of a viral nucleic acid sequence, such as SEQ ID NO: 1 or 3. In still further aspects, an expression vector provides expression of a viral polypeptide, such as a pVP2 (SEQ ID NO: 5), VP4 (SEQ ID NO: 6), VP3 (SEQ ID NO: 7), or VP 1 (SEQ ID NO: 4) polypeptide or a variant thereof as described herein.

[0008] In a further embodiment there is provided an isolated virus particle comprising a polynucleotide molecule (e.g., an RNA molecule) of the embodiments. For example, the virus particle can comprise an RNA polynucleotide about or at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to an RNA encoded by SEQ ID NO: 1 and an RNA polynucleotide about or at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to an RNA encoded by SEQ ID NO: 3. In certain aspects, the virus particle is attenuated or inactivated. For example, the virus particle can be heat inactivated, radiation inactivated, or chemically inactivated (e.g., by formalin treatment). Thus, in some aspects, an antigenic composition is provided comprising a virus particle of the embodiments in a suitable carrier. [0009] In a further embodiment the invention provides an isolated or recombinant polypeptide encoded by a polynucleotide molecule described herein. In certain aspects, a polypeptide is provided comprising an amino acid sequence selected from the group consisting of: (a) a sequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and (b) a sequence comprising at least 20 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. For example, a polypeptide of the embodiments can comprise a sequence about or at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In certain aspects, a polypeptide of the embodiments comprises about or at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 11, 1 12, 1 13, 1 14, 115, 116, 1 17, 1 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0010] In yet a further embodiment there is provided an antigenic composition comprising a polypeptide of the embodiments. In certain aspects, a polypeptide of the embodiment can be coupled with a further antigen (e.g., an antigenic polypeptide, glycoprotein, or carbohydrate). In still further aspects, an antigenic composition further comprises additional elements, such as an adjuvant, an immunomodulator, a preservative, and/or an additional antigen. In certain aspects, an antigenic composition may be defined as a vaccine composition. [0011] In still a further embodiment there is provided an isolated antibody wherein the antibody specifically binds a polypeptide described herein. For example, an antibody can be an antibody that specifically binds to a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. Thus, an antibody of the embodiments can be defined as an ESV VPl, ESV VP2, ESV VP3, or ESV VP4-binding antibody. An antibody according to the embodiment can, for example, be a polyclonal antibody, a monoclonal antibody, a single domain antibody, or an antigen-binding antibody fragment, such as a Fab, Fab2, or ScFv. In still further aspects, an antibody can be defined as a human or humanized antibody.

[0012] In still a further embodiment, a method of determining virus contamination in a sample is provided comprising detecting the presence of a polynucleotide or a polypeptide of the embodiments (e.g., an ESV polypeptide or polynucleotide), to determine the presence of virus contamination. For instance, the sample can be a patient sample, a pharmaceutical composition, or a cell culture sample. Examples of cell culture samples include, without limitation, samples from avian, insect, and mammalian cell cultures. For instance, an insect cell culture sample can be from a Aedes albopictus, Aedes aegypti, Drosophila (e.g., S2), Spodoptera frugiperda (e.g., Sf9 or Sf21), or Trichoplusia ni (e.g., Tn5Bl-4) cell culture. Pharmaceutical compositions can also be assessed for the presence of contaminating virus; in particular, compositions comprising vaccine formulations or recombinant proteins produced in cell culture systems can be tested. Patient samples for use according to embodiments include, but are not limited to, samples of serum, saliva, tissue, urine, or stool of a patient. [0013] Thus, certain aspects of the embodiments concern detecting a polynucleotide

(e.g., a viral polynucleotide) of the embodiments. For example, a polynucleotide can be detected by reverse-transcription PCR (RT-PCR), by nucleic acid sequencing, or by nucleic acid hybridization (e.g., Northern blot, in situ hybridization, dot blot, or array hybridization).

[0014] Embodiments of the invention provide a primer pair comprising a first polynucleotide that hybridizes to the sequence of SEQ ID NO: 1 and a second polynucleotide that hybridizes to the complement of SEQ ID NO: 1 wherein said first and second polynucleotide amplify a portion of SEQ ID NO: 1 under PCR conditions. Likewise, in certain embodiments a primer pair is provided comprising a first polynucleotide that hybridizes to the sequence of SEQ ID NO: 3 and a second polynucleotide that hybridizes to the complement of SEQ ID NO: 3 wherein said first and second polynucleotide amplify a portion of SEQ ID NO: 3 under PCR conditions. In certain aspects, a first polynucleotide and/or a second polynucleotide is labeled (e.g., by a radioactive, colorimetric, enzymatic, fluorescent, or affinity label). Polynucleotide molecules for use as a primer pair can, in some aspects, be between about 18 and 100, 18 and 75, 18 and 50, or 20 and 40 nucleotides in length. [0015] In some aspects, a method is provided for detecting a polynucleotide comprising: (a) subjecting a sample to PCR or RT-PCR in the presence of a primer pair of the embodiments; and (b) detecting a nucleic acid amplification from the sample to detect a polynucleotide. For example, in certain aspects, the PCR or RT-PCR is quantitative (e.g., real time PCR) and detecting a polynucleotide in a sample can comprise quantifying a polynucleotide in a sample.

[0016] Further aspects of the embodiments concern detecting a polypeptide of the embodiments (e.g., a viral polypeptide). For example, the polypeptide can be directly detected by protein sequencing or mass spectroscopy. In some aspects, a polypeptide is detected using an antibody that specifically binds to the polypeptide (e.g., an ESV VP1, ESV VP2, ESV VP3, or ESV VP4-binding antibody). For example, such an antibody can be used to detect a polypeptide by an immunoblot (e.g., a dot blot or Western blot), an ELISA, or an immunofluorescence assay.

[0017] In still further embodiments there is provided a method for producing ESV comprising inoculating a culture of cells with ESV and growing the cells under conditions permissive for ESV production. In certain aspects, the cells are coinfected with a second virus, such a Flavivirus (e.g., dengue virus).

[0018] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0019] 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 herein "another" may mean at least a second or more. [0020] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0021] 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 preferred 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 specific embodiments presented herein.

[0023] FIG. 1: Electron microscopy of purified viral particles and infected mosquito cells. (A) TEM of purified viral particles after negative staining with uranyl acetate. Sample population shows icosahedral symmetry and substantial homogeneity. At higher magnification particles show distinct surface structure (inset). (B) Thin section of a virus infected A. albopictus C6/36 cell where a paracrystal array of particles can be observed. (C) Ultra-thin section of a C6/36 infected cell showing the cytopathic effects characterized by extensive vacuolization and segregation of the chromatin. (D) Cryoelectron micrograph of the purified particles confirming the icosahedral symmetry emphasized by the ability to detect the particles in different orientations and the protrusions on the surface of the particles. Bars: A-(500 nm); B-(l μιη ); C-(2 mm); D-(500 nm).

[0024] FIG. 2: The polypeptides were separated by NuPAGE system and stained by SYPRO Ruby red. The estimated molecular weights of four viral proteins are indicated as 120 KDa, 48 kDa, 43 kDa, and 27 kDa.

[0025] FIG. 3A-B: (A) Schematic representation of the gene organization of genome segment A of ESV and comparison with its DXV (Drosophila X virus), IPNV (infectious pancreatic necrosis virus), and IBDV (infectious bursal disease virus) homologs. (B) Schematic representation of segments A and B of the ESV genome. Lines represent UTRs, boxes represent ORFs, numbers above the ORFs indicate the first nucleotide involved in the initiation codon. Numbers in parentheses indicate the amino acids.

[0026] FIG. 4: (A) Pairwise distances of ESV, DXV, IPNV, and IBDV based on the nucleotide sequence and deduced amino acid sequences of the VP2. (B) Cladogram representing phylogenetic relationships of ESV and other members of the birnavirus family based on deduced amino acid sequences of VP2 capsid protein.

[0027] FIG. 5A-B: Mass spectrometry analysis of the peptides from (A) segment A (SEQ ID NO: 2) and (B) segment B (SEQ ID NO: 4) ORFs. The complete sequences of the ESV proteins are shown. Highlighted in gray are the sequences that were confirmed by mass spectrometry.

[0028] FIG. 6: Three-dimensional reconstruction and surface organization of ESV (A) along the 5-fold and (B) along the 3-fold axis, respectively. The particle size is -700 A and shows 260 trimeric protrusions that extend -45-50 A from the viral surface. (B) Cross- section of the reconstruction along the 5-fold axis, most of the internal map densities corresponding to the nucleocapsid and RNA are at different contour levels and were excised for clarity. (D) Close views of the E protein trimers where areas of extra density on the pentamer trimers can be seen extending (arrows) on the surface. The extra density gives the virion a diameter of 750 A from vertex to vertex. Trimer dimensions are -80 A at the triangular edge. (D-bottom) Top view of one of the 12 pentamers and adjacent hexamers.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

[0029] Emerging methods for vaccine and commercial scale protein production have taken advantage of much lower cost insect cell systems rather than conventional mammalian cell culture. It was likewise envisioned that insect cell systems could be a less likely source of possible inadvertent viral contamination. Arbovirus vaccines in particular lend themselves to production in insect cell systems, given the natural insect vectors of these viruses. However, the inventors have now identified a novel virus that was a contaminant in dengue virus preparations grown in insect cell culture. The virus structure was clearly unrelated to dengue virus (see FIGs. 1 and 6) and the viral proteins also exhibited a distribution that was distinct from dengue (FIG. 2). Further analysis of the new virus by sequencing of viral nucleic acids and mass spectroscopy analysis of viral proteins revealed a novel birnavirus, distantly related to Drosophila X virus (DXV) (FIGs. 4 and 5). The new virus was named Espirito Santo virus after the region in Brazil that was the source of the dengue virus strain in cell culture when ESV was first isolated. Interestingly, the virus was found to grow most efficiently in the presence of dengue virus coinfection, and the presence of ESV in turn severely hampered dengue virus replication.

[0030] The identification of ESV contaminants in insect cell culture highlights the need for monitoring aberrant virus replication in these systems. Thus, embodiments of the invention provide methods for detecting ESV and related viruses in samples, such as cell culture samples and products (e.g., recombinant proteins and vaccines produced from such cell culture systems). Likewise, ESV may infect animals, and methods are provided for detecting ESV and related viruses in patients, such as in blood samples. For example, ESV can be detected by nucleic acid hybridization or RT-PCR to detect the presence of the viral RNA. The presence of virus can likewise be determined by detecting viral proteins, for example, by ELISA using antibodies specific for one or more of the viral polypeptides. In still further aspects, it is contemplated that ESV infection of animals may have clinical significance. Thus, antigenic compositions are provided that can used to stimulate an anti- ESV immune response in a subject. II. Birnaviruses

[0031] The birnavirus family is a distinct double stranded RNA (dsRNA) family of viruses that infects animal species from vertebrates to mollusk, fish as well as insects. The family is grouped into three main genera according to its hosts, the genera Aquabirnavirus, Avibirnavirus, and Entomobimavirus (Dobos et al, 1979; Kelly et al, 1982). They include viruses with a bi-segmented dsRNA genome encapsidated within single-shelled, unenveloped icosahedral particles (Villanueva et al., 2004). These viruses are approximately 70 nm in diameter and exhibit 260 spikes in a proposed T = 13 organization (Bottcher et al, 1997; Coulibaly et al, 2010). Members of Aquabirnavirus and Avibirnavirus genus include the infectious pancreatic pecrosis virus (IPNV) and infectious bursal disease virus (IBDV), respectively, which are two well-characterized viruses of economic importance to aquaculture and poultry industries (Abdel-Alim et al, 2003; Christie et al, 1988). These members account for much of the current knowledge about the structure and function of the birnaviruses. The prototype species of the genus Entomobimavirus is the Drosophila X vims (DXV), which was first isolated as a contaminant in experiments studying the insect mabdovirus Sigma (Dobos et al, 1979). Drosophila X vims stands as the only known member of Entomobimavims (Chung et al, 1996). The bimavimses are characterized by two dsRNA segments A and B that make up their genome (Dobos et al, 1979) and that exhibit a strong degree of conservation with regards to structure (Chung et al, 1996). The size difference between the large (segment A) and smaller (segment B) genome is the least in the case of DVX, where the segment A is 3360 bp and segment B is 3423 bp (Chung et al, 1996). The sizes of IBDV genome segments A and B have been reported to be 3261 bp and 2800 bp, respectively (Azad et al, 1985), and those of IPNV are 3097 bp and 2855 bp (Azad et al, 1985; Dobos et al, 1979). The structural proteins of birnaviruses generally fall into three size classes (large, medium, and small), which are present in different relative proportions. The minor high molecular weight VPl polypeptide is encoded by genome segment B (Nagy and Dobos, 1984a), does not undergo posttranslational cleavage, and is the virion-associated RNA polymerase (MacDonald and Dobos, 1981). Polypeptides VP2, VP3, and VP4 are encoded by genome segment A. VP2 and VP3 form the outer and inner layers, respectively, of the virions and internally, VP3 forms a ribonucleoprotein complex with the genomic RNA (Luque et al, 2009). VPl is found both free and covalently attached to the genomic RNA (Dobos and Roberts, 1983). Other than the studies here, there has been no description of entomobirnaviruses infecting mosquitos or mosquito-derived cells lines.

III. Nucleic Acids

[0032] In certain embodiments, the present invention concerns isolated and recombinant polynucleotides, such as polynucleotides encoding an ESV genome (e.g., SEQ ID NOs: 1 and 3), encoding an ESV polypeptide, or capable of hybridizing to an ESV polynucleotide sequence.

[0033] As used in this application, the term "polynucleotide" refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide. The skilled artisan will recognize that while the ESV genome itself is comprised of RNA, the DNA sequences of SEQ ID NOs: 1 and 3 encode the RNA molecules corresponding to the ESV genomic sequences. [0034] In this respect, the term "gene," "polynucleotide," or "nucleic acid" is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those of skill in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid may comprise a contiguous nucleic acid sequence of: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1 100, 1500, 2000, 2500, 3000, or more nucleotides, nucleosides, or base pairs, including all values and ranges there between, of a polynucleotide described or referenced herein. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0035] In particular embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode an ESV polypeptide. The term "recombinant" may be used in conjunction with a polynucleotide or polypeptide and generally refers to a polypeptide or polynucleotide produced and/or manipulated in vitro or that is a replication product of such a molecule.

[0036] The nucleic acid segments used in the present invention can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits, such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0037] In certain other embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors that include within their sequence a nucleic acid sequence encoding a contiguous amino acid sequence from SEQ ID NO: 2 (ESV VP 1), SEQ ID NO: 4 (the ESV segment A polyprotein), SEQ ID NO: 5 (ESV pVP2), SEQ ID NO: 6 (ESV VP4), SEQ ID NO: 7 (ESV VP3), or any other nucleic acid sequences encoding ESV polypeptide sequence. [0038] In certain embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence of this invention using the methods described herein (e.g., BLAST analysis using standard parameters).

[0039] The invention also contemplates the use of polynucleotides that are complementary to all the above described polynucleotides. In one aspect, these may be used as probes for the detection of viral nucleic acids.

Vectors [0040] Polypeptides and polynucleotides of the invention may be encoded by a nucleic acid molecule comprised in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. A nucleic acid sequence can be "heterologous," which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which it is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al, 2001 ; Ausubel et al, 1998, both incorporated herein by reference). In addition to encoding ESV sequences the vector can encode other polypeptide sequences, such as a one or more other bacterial peptide, a tag, or an immunogenicity enhancing peptide. Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al, 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.

[0041] The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.

[0042] A "promoter" is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0043] Naturally, it may be important to employ a promoter and/or enhancer that effectively direct the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al, 2001, incorporated herein by reference). The promoters employed may be constitutive, tissue- specific, or inducible and in certain embodiments may direct high-level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides. [0044] The particular promoter that is employed to control the expression of peptide or protein-encoding polynucleotides of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human, or viral promoter.

[0045] In embodiments in which a vector is administered to a subject for expression of the protein, it is contemplated that a desirable promoter for use with the vector is one that is not down-regulated by cytokines or one that is strong enough that even if down-regulated, it produces an effective amount of an ESV polypeptide for eliciting an immune response. Non-limiting examples of these are CMV IE and RSV LTR. Tissue-specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters.

[0046] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.

[0047] In certain embodiments of the invention, the use of internal ribosome entry site (IRES) elements is used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). IV. Polypeptides

[0048] As used herein, a "protein" or "polypeptide" refers to a molecule comprising at least ten amino acid residues. In some embodiments, a wild-type version of a protein or polypeptide, such as an ESV polypeptide, is employed; however, in many embodiments of the invention, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A "modified protein" or "modified polypeptide" or a "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.

[0049] In certain embodiments the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 985 amino molecules or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, but also they might be altered by fusing or conjugating a heterologous protein sequence with a particular function (e.g., to provide a detectable tag, for enhanced immunogenicity, for purification purposes, etc.).

[0050] As used herein, an "amino molecule" refers to any amino acid, amino acid derivative, or amino acid mimic known in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.

[0051] Accordingly, the term "proteinaceous composition" encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid.

[0052] Proteinaceous compositions may be made by any technique known to those of skill in the art, including (i) the expression of proteins, polypeptides, or peptides through standard molecular biological techniques, (ii) the isolation of proteinaceous compounds from natural sources, or (iii) the chemical synthesis of proteinaceous materials. The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0053] Amino acid sequence variants of ESV polypeptides, such as VP1, VP2 (or pVP2), VP3, VP4 and other polypeptides of the invention can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the invention may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the polypeptide, as compared to wild-type. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% including all values and ranges there between, identical to any sequence provided or referenced herein, e.g., SEQ ID NO: 2 or 4-7. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substituted amino acids.

[0054] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. Insertional mutants typically involve the addition of material at non-terminal points in the polypeptide. This may include the insertion of one or more residues. Terminal additions, called fusion proteins, may also be generated. These fusion proteins include multimers or concatamers of one or more peptide or polypeptide described or referenced herein.

[0055] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0056] Proteins of the embodiments may be recombinant, or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacteria containing such a variant may be implemented in compositions and methods of the invention. Consequently, a protein need not be isolated.

[0057] The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0058] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity (e.g., immunogenicity) where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0059] It is contemplated that in compositions of the invention, there can be between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per milliliter. The concentration of protein in a composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/mL or more (or any range derivable therein). Of this, about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be an ESV peptide or polypeptide, and may be used in combination with other peptides or polypeptides, such as other bacterial peptides and/or antigens. [0060] The present invention contemplates the administration of ESV polypeptides or peptides to effect a preventative therapy or therapeutic effect against the development of a disease or condition associated with infection by ESV. In certain aspects, combinations of ESV antigens are used in the production of an immunogenic composition that is effective at treating or preventing ESV infection. IV. Antibodies and Antibody-Like Molecules

[0061] In certain aspects of the invention, one or more antibodies or antibody-like molecules (e.g., polypeptides comprising antibody CDR domains) may be obtained or produced which have specificity for an ESV or ESV polypeptide (e.g., SEQ ID NOs: 2 or 4- 7). These antibodies may be used in various diagnostic or therapeutic applications described herein.

[0062] As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent, such as IgG, IgM, IgA, IgD, and IgE, as well as polypeptides comprising antibody CDR domains that retain antigen binding activity. Thus, the term "antibody" is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments, such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and polypeptides with antibody CDRs, scaffolding domains that display the CDRs (e.g., anticalins), or a nanobody. For example, the antibody can be a VHH (i.e., an antigen-specific VHH) antibody that comprises only a heavy chain. For example, such antibody molecules can be derived from a llama or other camelid antibody (e.g., a camelid IgG2 or IgG3, or a CDR-displaying frame from such camelid Ig) or from a shark antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

[0063] "Mini-antibodies" or "minibodies" are also contemplated for use with the present invention. Minibodies are sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et al, 1992). The oligomerization domain comprises self-associating a-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992); Cumber ei al. (1992).

[0064] Antibody-like binding peptidomimetics are also contemplated in the present invention. Liu et al. (2003) describe "antibody-like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods. Likewise, in some aspects, antibodylike molecules are cyclic or bicyclic peptides. For example, methods for isolating antigen- binding cyclic peptides (e.g., by phage display) and for using the such peptides are provided in U.S. Patent Publn. No. 20100317547, incorporated herein by reference. In some embodiments, a scaffolding polypeptide can be a "molecular affinity clamp." See, for example, U.S. Patent Publn. Nos. 201 10143963 and 20110045604, incorporated herein by reference.

[0065] Alternative scaffolds for antigen binding peptides, such as CDRs are also available and can be used to generate ESV polypeptide-binding molecules in accordance with the embodiments. Generally, a person skilled in the art knows how to determine the type of protein scaffold on which to graft at least one of the CDRs arising from the original antibody. More particularly, it is known that to be selected such scaffolds must meet the greatest number of criteria as follows (Skerra, 2000): good phylogenetic conservation; known three- dimensional structure (as, for example, by crystallography, NMR spectroscopy, or any other technique known to a person skilled in the art); small size; few or no post-transcriptional modifications; and/or easy to produce, express, and purify. [0066] The origin of such protein scaffolds can be, but is not limited to, the structures selected among: fibronectin (see, e.g., U.S. Patent Publn. No. 20090253899, incorporated herein by reference) and preferentially fibronectin type III domain 10, lipocalin, anticalin (Skerra, 2001), protein Z arising from domain B of protein A of Staphylococcus aureus, thioredoxin A, or proteins with a repeated motif such as the "ankyrin repeat" (Kohl et al. , 2003), the "armadillo repeat," the "leucine-rich repeat," and the "tetratricopeptide repeat". For example, anticalins or lipocalin derivatives are a type of binding protein that have affinities and specificities for various target molecules and can be used as ESV-specific binding agents. Such proteins are described in US Patent Publication Nos. 20100285564, 20060058510, 20060088908, 20050106660, and PCT Publication No. WO2006/056464, incorporated herein by reference.

[0067] Scaffolds derived from toxins, such as, for example, toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal NO synthase (PIN) may also be used in certain aspects.

[0068] Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production. The invention provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit, and chicken origin.

[0069] "Humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies, and fragments thereof. As used herein, the term "humanized" immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor." A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. Methods for Generating Antibodies

[0070] Methods for generating antibodies (e.g., monoclonal antibodies and/or monoclonal antibodies) are known in the art. Briefly, a polyclonal antibody is prepared by immunizing an animal with ESV, an ESV polypeptide or fragment thereof in accordance with the present invention and collecting antisera from that immunized animal.

[0071] A wide range of animal species can be used for the production of antisera. Typically the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig, or a goat. The choice of animal may be decided upon the ease of manipulation, costs, or the desired amount of sera, as would be known to one of skill in the art. It will be appreciated that antibodies of the invention can also be produced trans genically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750, 172, and 5,741,957.

[0072] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions, or vectors encoding such adjuvants.

[0073] Adjuvants that may be used in accordance with the present invention include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, gamma-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated. MHC antigens may even be used. Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants, and/or aluminum hydroxide adjuvant. [0074] In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA), low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/ Mead, NJ), cytokines such as gamma-interferon, IL-2, or IL-12, or genes encoding proteins involved in immune helper functions, such as B-7. [0075] The amount of immunogen composition used in the production of antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen, including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous, and intraperitoneal. The production of antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.

[0076] A second, booster dose (e.g., provided in an injection), may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs. [0077] For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography, among others.

[0078] MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide, peptide, or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.

[0079] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, rodents, such as mice and rats, are used in generating monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used in generating monoclonal antibodies. The use of rats is well known and may provide certain advantages (Goding, 1986, pp. 60-61). Mice (e.g., BALB/c mice) are routinely used and generally give a high percentage of stable fusions.

[0080] The animals are injected with antigen, generally as described above. The antigen may be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster administrations with the same antigen or DNA encoding the antigen may occur at approximately two- week intervals.

[0081] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils, or lymph nodes, or from a peripheral blood sample. Generally, spleen cells are a rich source of antibody-producing cells that are in the dividing plasmablast stage. Typically, peripheral blood cells may be readily obtained, as peripheral blood is easily accessible.

[0082] In some embodiments, a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10⁷ to 2 x 10⁸ lymphocytes.

[0083] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non antibody producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83).

[0084] Methods for generating hybrids of antibody producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

[0085] Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^"6 to 1 x 10^"8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

[0086] The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

[0087] This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

[0088] The selected hybridomas would then be serially diluted and cloned into individual antibody producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils, such as pristane (tetramethylpentadecane), prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

[0089] Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase and DHFR gene expression systems are common approaches for enhancing expression under certain conditions. High expressing cell clones can be identified using conventional techniques, such as limited dilution cloning and Microdrop technology. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.

[0090] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.

[0091] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. In one embodiment, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination, which further increases the chance of finding appropriate antibodies. Target- binding (e.g., ESV polypeptide-binding) single domain antibodies can also be isolated by use of display libraries, see for example, U.S. Patent Appln. No. 201 10183863, incorporated herein by reference. Ribosome expression libraries for isolation of target-binding Ig coding sequences are also described in U.S. Patent Appln. No. 20040161748; 20070299246 and 20080293591, each incorporated herein by reference.

[0092] Another embodiment of the invention for producing antibodies according to the present invention is found in U.S. Patent No. 6,091,001, which describes methods to produce a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination is disclosed. The method involves first transfecting an antibody-producing cell with a homology-targeting vector comprising a lox site and a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin loci of the genomic sequence that is to be converted to a modified region, so the first lox site is inserted into the genomic sequence via site-specific homologous recombination. Then the cell is transfected with a lox-targeting vector comprising a second lox site suitable for Cre-mediated recombination with the integrated lox site and a modifying sequence to convert the region of the immunoglobulin loci to the modified region. This conversion is performed by interacting the lox sites with Cre in vivo, so that the modifying sequence inserts into the genomic sequence via Cre-mediated site-specific recombination of the lox sites.

[0093] Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.

V. Examples

[0094] The following examples 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 that follow represent techniques discovered by the inventor 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 - Characterization of the unknown virus

[0095] A sample of dengue-2 virus was obtained from Fiocruz Institute, Brazil. This sample was isolated in Aedes albopictus c6/36 cells and at its third passage it was plaque purified. The selected 44/2 clone virus produces uniformly sized plaques. This sample was found to carry an unknown viral contaminant, which grew in c6/36 mosquito cells. The virus was named Espirito Santo Virus (ESV) after the state in Brazil where the original dengue sample was collected. However, ESV does not produce plaques and therefore cannot be plaque purified from the dengue sample. Five days post-infection, the virus was purified and concentrated using isopycnic ultracentrifugation in iodixanol gradients (Optiprep, Sigma, St. Louis MO). The virus was spun to equilibrium in 35%- 12% iodixanol gradients overnight at 76,000 x g in a SW28 rotor at 4 °C. The visible band was collected and diluted in PBS then layered over a second gradient (20%-35%) and run for 3 h at 90,000 x g in a SW 28.1 rotor at 4 °C. For viral protein analysis, the sample was further purified in CsCl gradients (25%-37%) as previously described (Comps et al , 1991). For cryo-EM these samples were cross linked with buffered glutaraldehyde 1.5% in 20 mM HEPES pH 7.5 at room temperature. After 10 minutes the reaction was stopped with 100 mM Tris pH 8.0.

[0096] ESV samples harvested from mosquito cells and purified in iodixanol gradients were analyzed first by cryo-EM. Briefly, samples were subjected to negative staining with 1% uranyl acetate to assess the optimal concentration and sample homogeneity for cryo-EM. Thin sections of infected cells were performed as previously described (Mariante et al, 2006). The specimen was prepared for cryo-EM by applying ESV sample on to holey carbon EM grids prepared according to the method described by Fukami et al (1965). The grids were vitrified in liquid ethane as previously described (Dubochet et al, 1988). These grids were then placed in a 300 keV FEI TF30He Polara G2 electron cryo- microscope. A total of 199 focal pairs were subsequently recorded with the EMMENU4/EMTO OLS automation package (TVIPS) at 39,000x microscope magnification using an F415 4k x 4k CCD (TVIPS) for a final pixel size of 2.29 A per pixel.

[0097] Observation of ultra-thin sections of infected mosquito cells revealed particles that did not resemble dengue virus in both structure and size. After negative staining with uranyl acetate, a predominant shape was identified, consisting of obvious icosahedral particles (FIG. la-b), with a distinct lattice structure on the surface (FIG. lA-inset). In thin sections of infected mosquito cells, ESV virions appeared icosahedral in shape, unenveloped with a diameter between 68-72 nm. In the cytoplasm of heavily infected cells, complete virus particles associated to form paracrystalline arrays (FIG. IB). The average EM-measured diameter for the particles in negative stains was 70±2 nm. Curiously, there was only one homogeneous population of particles in the preparations with no evidence of the presence of dengue-like particles according to its previously described size and overall structure. However, when performing immunofluorescence with mAb against dengue-2 and flavivirus envelope protein, dengue 44/2-infected cells showed positive staining for the viral proteins. In addition, when cells were infected with ESV, cytopathic effects, such as extensive vacuolization and chromatin segregation to the nuclear periphery, could be observed (FIG. 1B-C). Virions that were purified from the infected mosquito cells appeared to be non- enveloped and icosahedral when analyzed by negative-staining, and the presence of protruding spikes on the virus surface was evident. This size range and structural characteristics were further confirmed by the observation of particles embedded in vitreous ice and examined by cryo-EM (FIG. ID described in detail below). Example 2 - Virus-specific Polypeptides

[0098] SDS-PAGE analysis was performed to estimate the molecular weight of the ESV capsid proteins. Briefly, SDS polyacrylamide gel electrophoresis was performed with the NuPAGE^® (4%-12%) electrophoresis system (Invitrogen). Viral proteins were disrupted in the dissociation buffer, containing NuPAGE^® reducing agent according to manufacturer's instructions. The gel was run at 200 V until the dye marker reached the bottom. The gel was then fixed in 50% methanol with 7% acetic acid for 30 minutes. Staining of viral proteins was performed using SYPRO Ruby Red (Molecular Probes, CA). High-Range Rainbow Marker was used as molecular standard (GE Healthcare, NJ).

[0099] The electrophoresis of the viral proteins revealed a profile of four major protein bands of approximately 120 kDa, 48 kDa, 43 kDa, and 27 kDa, respectively. These polypeptides are likely to represent VP 1, VP2, VP3, and VP4 (FIG. 2) according to the molecular weight range of VPs in birnaviruses. In some samples additional protein bands around 17 kDa and 54 kDa could be seen when the sample was not purified in CsCl gradients. This could be a result of protein degradation of the stored sample, mosquito cell proteins, or even dengue viral proteins. Previous studies on DXV with pulse-chase experiments and peptide mapping showed that posttranslational cleavage of a 67 kDa polypeptide gives rise to a 49 kDa protein, which undergoes a slow maturation-associated cleavage to generate the 45 kDa final product (Nagy and Dobos, 1984a; Nagy and Dobos 1984b). As shown in FIG. 2, the cleavage of a polypeptide in the 45-55 kDa range seems to be complete, since only one product can be found in that molecular weight range. The protein sizes were not only in agreement with the size range expected for members of the birnavirus family, but also seemed to be most closely related to the sizes described for DVX (Table 1). It presents pVP2 and VP2 as the 49 kDa and 45 kDa proteins, respectively. A protein with a molecular mass corresponding to VP5 does not seem to be present in the virion in sufficient amounts to be stained by Coomassie blue or SYPRO Ruby Red staining.

[00100] Table 1: Comparison of the Birnaviruses genome sequences and proteins.

(IPNV); and U60650 (DXV), incorporated herein by reference.

Example 3 - ESV sequencing and de novo assembly

[00101] To characterize the virus genome, viral nucleic acid was extracted from virus purified using CsCl gradients to exclude possible flavivirus particles. Purified virus was pelleted at 240,000 x g in a SW55Ti (Beckman Coulter, Fullerton CA) rotor for 1 hour. The pelleted virus was resuspended in lysis buffer (100 mM Tris-Cl pH7.0, 20 mM EDTA, 1% SDS) at 37°C for 20 minutes. Then, the virus was treated twice with warm solid phenol and once with pure chloroform. RNA in solution was then precipitated in 100% isopropanol at - 80°C overnight and resuspended on the following day in water. Purified RNA was run on a 1% agarose gel (SeaKem-GTG-agarose, Lonsa MA) and stained with ethidium bromide. The electrophoresis results indicated a bi-partite genome that had an approximate size of 3.2 Kb. [00102] Purity of samples prepared by RNA extraction of gradient purified virus was determined using an Agilent Bioanalyzer. Libraries of purified RNA were prepared using the TruSeq Sample Prep Kit with the following adjustments: mRNA enrichment was not done, instead purified RNA was directly fragmented and the processed cDNA PCR products were gel purified on a 2% agarose gel. The -350 bp products were extracted using a Qiagen gel extraction kit. Sequencing was performed on the Illumina^® GAIIx using 72 bp single read conditions. 42,993,210 fastq files were generated with -74% of those reads passing the quality control filter. In order to perform the De Novo assembly, the fastq files from the Illumina^® data were imported into Velvet (Zerbino and Birney, 2008) and the Velvet module used to produce sequence nodes. This file was then imported into the Lasergene^® seqman module and used to assemble the contig files and the final assembled sequences of segments A and the B of the virus genome.

[00103] The resulting nucleotide sequence of ESV was determined to be 3250 nt for segment B and 3430 nt for segment A (Table 1). The resulting sizes of the bi-partite genome are in agreement with the average size for other birnaviruses, particularly DXV (Chung et ah, 1996; Nagy and Dobos, 1984). The complete nucleotide sequence of the viral dsRNA was deposited into the GenBank with accession numbers (JN589003) for segment A and (JN589002) for segment B.

Example 4 - ESV genome organization [00104] One large open reading frame (ORF) was found on each segment, spanning from nucleotides 97-3093 on segment B and from nucleotides 59-3158 on segment A. In addition, a small putative ORF was also detected to be capable of encoding a 27 kDa polypeptide of 260 amino acids in segment A. The polyprotein ORF encompasses most of the genome segment A in the case of ESV as well as in other members of Birnavirus. The large ORF in segment B encodes the VP1, which has been described to represent the viral RNA dependent RNA polymerase. The genomic arrangement of ESV therefore resembles that of other members of the Birnavirus family. Expression of the ESV genes during its replication is likely to involve polycistronic expression of the large polypeptide from the primary ORF of segment A, which in this case is capable of encoding a 120 kDa polypeptide (1050 aa). This large ORF should be processed into VP2, VP3, and VP4 as it occurs with DXV and other family members. The putative small polypeptide ORF likely represents the VP5; however, it could not be detected by SDS-PAGE. Further sequence analysis demonstrated that the viral bisegmented RNA presented approximately 70% homology to the DXV RdRp that resulted from the 3200 bp segment B (Table 1) using BLASTn. These results indicate that the newly identified ESV virus is indeed a member of the birnavirus family. The putative protein of 27 kDa of ESV is larger than VP5 of IPNV and IBDV, as is also the case of DXV, in which it was described as a non-structural protein. In this study this protein was not confirmed since there is no start codon. However, there is a single mutation G1920A that would express this protein by creating a start codon. Since this is likely to be a non-structural protein, its presence would be rather difficult to confirm. Furthermore, there may not be a NS/VP5 in ESV that is expressed regularly. Example 5 - ESV sequence comparisons with other birnaviruses

[00105] The ESV putative RdRp nucleotide sequence obtained was compared with all nucleotide sequences in the NCBI database using BLAST. The deduced amino acid sequence of the ESV segments A and B were compared to those of DXV, IPNV, and IBDV, which represent three different genera of birnaviruses. Interestingly, no high degree of similarity was found for RdRp or other viral proteins between ESV and other members of the birnavirus family or any other known virus. When comparing the nucleotide sequences, ESV RdRp and Drosophila X virus segment B putative RNA-dependent RNA polymerase VP1 gene (AF196645.2) shares approximately 71% similarity, while ESV VP 1 shared approximately 4% with IPNV. Segment A of ESV has 72% identity (0.0 E-value/ 1170 total score) for the Drosophila X virus polyprotein gene (U60650.1). And it was found to have 72% identity (2e-04 E-value / 59.0 total score) with Infectious pancreatic necrosis virus strain Reno segment A VP2 structural protein (AY026345.1). Comparison of the genomic relationships of the different birnaviruses based on the nucleotide sequences of the VP2 coding region was also performed. According to the sequence analysis and pairwise alignment of the ESV and birnaviruses (FIG. 4A), a cladogram was constructed using parsimony analysis to describe the phylogenetic relationship of these viruses (FIG. 4B). This was based on the deduced amino acid similarities of the polyprotein encoded by the large ORF (Clustal W method with DNAStar, Lasergene). Branch lengths were drawn proportional to the number of deduced amino acid differences using the Clustal W method. Among the virus-specific polypeptides pVP2 exhibits the highest level of amino acid conservation among members or the birnavirus family (Chung et ah, 1996) and therefore it is a good candidate protein for comparisons. The phylogenetic and sequence analysis revealed that ESV is more closely related to DXV than any other member of the family Birnavirus and therefore may constitute another member of the genus Entomobirnavirus.

Example 6 - Mass spectrometry analysis of ESV proteins

[00106] The cDNA and translated protein products were validated by matching peptide sequences generated from the same virus using Mass spectrometry analysis. Briefly, a small volume (100 μΐ) of purified virus in PBS corresponding to 50 μg of total protein was digested in solution with endoproteinase Lys-C. Prior to digestion, the solution was heated at 80°C for 15 min to facilitate protein denaturation. Following heating, 7 M GuHCl solution in 50 mM phosphate buffer was added to the virus sample in a 7: 1 ratio, yielding a final concentration of 1 M GuHCl. A 1 μg/μl endoproteinase Lys-C solution was prepared in water and 10 μΐ of the Lys-C solution was added to the virus sample and incubated for 24 h at 37°C. The Lys-C digest was stored at -20°C until used. An aliquot of the Lys-C digested virus sample corresponding to 1 μg of digested protein was subjected to LC/MSE analysis using a nanoAcquity UPLC (Waters Corp, Milford MA) coupled to a Q-Tof Premier mass spectrometer (Waters Corp.). A gradient of 2%-40% MeCN in water containing 0.1% formic acid was used to elute peptides over a period of 1 hr from a 75 μιη id x 25 cm analytical column packed with 1.7 μιη BEH particles into the nanolockspray source of the Q-Tof Premier. The Q-Tof Premier was operated in the LC/MSE mode of operation (Silva et al. , 2006) with alternating scans of normal and elevated collision energies to provide both intact precursor and product ion data for all peptides. Alternating normal and elevated collision energy scans were acquired at a rate of 1 Hz. In addition, a 600 fmol/μΐ solution of glu- fibrinopeptide B was infused into the nanolockspray source to allow for post-acquisition "lockmass" correction of observed ions masses.

[00107] LC/MSE spectra were processed and database searched using Proteinlynx Global Server 2.4 (PLGS) software (Waters Corp.). Protein sequences corresponding to the predicted ORFs for ESV were formatted and appended to the Uniprot-Sprot protein database (523, 151 entries) for database searching (available on the World Wide Web at uniprot.org). A variable modification for methionine oxidation was used for searching, and 1 missed Lys-C cleavage site was allowed. Other parameters included for searching were as follows: minimum fragment ions per peptide, 3; minimum fragment ions per protein, >7; minimum number of peptides per protein, 1. Because the data were to be subsequently loaded into the Scaffold program (Proteome Software, Portland, OR) a false positive rate (FPR) of 100% was utilized for the PLGS searches to insure a sufficient number of random matches in the search results needed for proper functioning of the statistical algorithms within Scaffold. The Scaffold plugin within PLGS was used to export database search results from PLGS that could be directly imported into Scaffold. Protein and peptide identifications from PLGS were loaded into Scaffold for visualization and determination of peptide and protein probability scores. For declaring a protein successfully identified, a minimum of two matching peptides and minimum protein and peptide probability scores of 95% and 90%, respectively, were required. [00108] The two predicted protein sequences correspond to Segment A Polyprotein

(1030 amino acids) and RNA-dependent RNA polymerase (RdRp) (998 amino acids) of ESV. Twenty-seven proteins were identified in the sample with high confidence corresponding primarily to growth media components and other expected sample component proteins. In addition, high quality, multi-peptide matches were obtained to the predicted ESV proteins from ORFs in segments A and B. Segment A Polyprotein was observed with 19 unique peptide matches corresponding to 50% amino acid sequence coverage, with a protein probability score of 100% (FIG. 5a). RdRp of ESV was observed with 14 unique peptide matches corresponding to 24% amino acid sequence coverage, with a protein probability score of 100% in Scaffold (FIG. 5b). Identification of these peptide sequences by mass spectrometry confirms expression of these two ORF's corresponding to Segment A Polyprotein and RdRp in ESV. The peptide sequences identified against these two proteins were unique to ESV in the database and were not matched to other protein components in the samples.

Example 7 - ESV Coin faction with Other Viruses [00109] Observations of DENV-2 44/2 infected cells demonstrated that ESV grows to a higher number of particles (observed by TEM) when co-infecting with dengue-2. Co- infection controls with Sindbis virus and mock infected C3/36 cells confirmed that ESV could only grow when cells were infected with dengue-2 strain 44/2. Consequently, the origin of the new virus was apparently from the dengue 44/2 sample isolated in Brazil, although the precise origin of the virus remains unclear. The ESV virus could be isolated from infections with this dengue strain but not from infections with other dengue strains studied, such as DENV-2 16803. The number of p.f.u of ESV could not be determined, because a plaque assay for the virus has not been established. Interestingly, when analyzing coinfected cells, dengue proteins could be detected by immunofluorescence, while no particle that resembles dengue virus could be observed by electron microscopy. The presence of dengue-2 in the samples was confirmed by RT-PCR analysis, immunofluorescence of infected cells with DENV-2 specific polyclonal and monoclonal antibodies, and Western blot of virus E protein with a DENV-2-specific antibody.

Example 8 - Three-Dimensional Structure of ESV

[00110] The 3D structure of ESV virus was determined from images obtained by cryo-EM to a resolution of 13 A (FIG. 6). Briefly, 5928 virus images were boxed out from all CCD images using e2boxer.py in the EMAN2 image processing package (Tang et al, 2007). Once boxed out, the CTF parameters for each particle set was determined using ctfit in EMAN and these parameters used to CTF correct the images for subsequent processing. The virus images were then centered and classified using EMAN (Ludtke et al, 1999). The initial refinement in EMAN classified the data into 123 class averages through 8 iterations and produced a 3D structure with 13 A resolution based on a Fourier Shell Correlation (FSC) of 0.5 between two maps produced from two separate halves of the data. Three additional refinement iterations with 212 class averages produced a 12 A 3D map similarly based on an FSC of 0.5.

[00111] The reconstruction revealed an icosahedral shaped particle with pentameric protrusions on each 5-fold vertex (FIG. 6A-D). The overall features of the structure are shown in FIG. 6, which displays the surface rendering of the cryo-EM reconstruction along the 5-fold (A) and 3-fold (C) axis of symmetry, and includes the protrusions on the viral surface (spikes). These protrusions have 3-fold rotational symmetry (FIG. 6D) and each extends approximately 45 A from the virus surface, giving the particle an average cross sectional diameter ranging from 700 to 750 A (FIG. 6A,C). A closer look at the cryo-EM structure reveals that the 5-fold to 5-fold distance measures 750 A while the 3-fold to 3 -fold distance measures 700 A (FIG. 6D). The envelope proteins appear to be organized in 260 trimers with 60 surrounding the 5 -fold vertices and 200 organized in hexamers making up the faces and edges of the icosahedron (FIG. 6A-C). These hexamers are -130 A apart from center to center. The spikes that form the 5-fold arrays at the vertices are the most prominent structures on the virus surface, though they look structurally similar to those in six fold arrays they appear to have extra density at their tips (FIG. 6D), protruding to a distance of 20-25 A above the trimers of the 3-fold axes. (FIG. 6C, D). The 12 pentamers and 120 hexamers that make up the surface of the virus are seen organized in a characteristic pattern expected for an external shell of 780 protein subunits and a T = 13 quasiequivalent organization (FIG. 6). Such an icosahedral lattice is a skewed lattice with handedness, in which a 5-fold axis is reached from its neighboring 5-fold axis by stepping over three six- coordinated positions and taking a left or a right turn. Interestingly, no tubular forms were observed in the samples, as commonly observed with IBDV and DXV. By using the structure of VP2 from IPNV, previous reports have described a model of the T = 13 capsid of IPNV virion (Coulibaly et al, 2010; Luque et al, 2007) by superimposing the VP2 trimer onto the previously described structure of IBDV (Coulibaly et al, 2005). This forms a model that assumes that the intertrimer interactions forming the IPNV are similar to those observed with the IBDV particle. The most notable differences among the birnavirus structures are at the spike-spike contacts between adjacent trimers in the T = 13 shell (concave x convex). Unfortunately, comparisons cannot be inferred from the structure of ESV because of the large difference in resolution from its cryo-EM reconstruction when compared to the crystal structure of VP2 from IBDV and IPNV.

Example 8 - Discussion

[00112] In recent years, considerable interest has been shown in insect cell lines in which viruses can both survive and disseminate. Viruses of public health and veterinary importance receive the most attention, although bacteriophages and plant viruses have also been studied (Kelly et al, 1982). Aedes albopictus is one of the most important vectors of emergent infectious diseases, such as dengue fever and yellow fever, worldwide (Mitchell, 1995). So far, there is not an effective method of controlling the population of mosquitoes that transmit these diseases, which is one of the main reasons for their prevalence in the tropical areas. In this research, a novel dsRNA virus with characteristics of the birnavirus family has been isolated in mosquito cells from a dengue-2 virus strain sample originated from a dengue fever patient from Espirito Santo, Brazil. It is important to point out that it is unclear at what point the sample became contaminated with ESV. Furthermore, none of the laboratories involved have had projects involving birnaviruses and in the decades of work with these insect cell lines a contaminating virus has never been identified in spite of many years of study involving electron microscopy. The cytopathic observations and the unique biochemical and structural characteristics of the purified virus led to the conclusion that ESV was an undescribed virus. Espirito Santo Virus (ESV) appears to share morphological features with members of the birnavirus family, such as infectious pancreatic necrosis of trout (Cohen et al, 1973), infectious bursal disease of poultry (Harkness et al, 1975), and especially Drosophila X virus (DXV). Accordingly, it is an unenveloped single capsid with a diameter of approximately 70 nm and a suggested triangulation number T = 13. Even though, no three dimensional structure has been reported for other entomobirnaviruses, such as DXV, there are structures described and modeled for other genera, such as IBDV (Avibirnavirus) and IPNV (Aquabirnavirus) (Bottcher et al, 1997; Coulibaly et al, 2005; Luque et al, 2007). In addition to these structural characteristics, the genetic organization of the genome segment A of ESV is similar to other birnaviruses (DVX, IPNV, and IBDV) belonging to different genera (Chung et al, 1996). Like most of the birnaviruses, ESV contains four viral polypeptides, with VP2 and VP3 being the major capsid components. There is a significant variance among members regarding the viral protein sizes (Table 1); however, ESV proteins remained within the size range common for all birnaviruses. Most of these properties correspond to the essential characteristics of the Birnaviridae family as defined by Dobos et al 1979 (Dobos, 1979; Dobos et al, 1979).

[00113] In order to further characterize ESV and establish its relationship to the Birnaviruses, the viral RNA was sequenced and compared. When analyzing the relationship of viruses and comparing their genomes, sequences from the RNA-dependent RNA polymerases (RdRp) are generally the most useful because they are conserved and occur in most viral genomes (Bruenn, 1991 ; Burenn, 2003). In addition, the active domain of RdRp is highly homologous among the viruses belonging from different species to different families. RdRp is a genome-linked protein located inside the viral capsid and thus is not exposed to host immune pressure, compared to the capsid proteins VP2 or VP3 (Zhang and Suzuki, 2003). This might be another explanation for the reason that the RdRp is conserved. Sequence analysis based on the RdRp provided a higher degree of similarity with DXV (-70%), but failed to show any significant relationship with other birnaviruses. In fact, this similarity is much less pronounced when considering structural proteins. In previous studies, among all of the specific polypeptides analyzed, pVP2 has been reported to exhibit the highest level of amino acid conservation between DXV, IBDV, and IPNV (Chung et al, 1996). Even though, the amino acid sequence identity among the VP2 of these viruses is still quite low. It is possible that, as it has been previously described for DXV (Chung et al, 1996), the differences in their amino acid sequences are not reflected in a largely altered secondary structure and the folded polypeptides may have similar functionality. The morphology of these viruses is remarkably similar, except for the clear absence of tubular forms in ESV electron microscopy preparations. Several invertebrate viruses have been isolated from insect cells and other arthropod hosts. However, only one genus of the birnaviruses has been described to be present infecting insects, the entomobirnavirus DXV. Structural variations observed among these viruses suggest that entomobirnaviruses, such as ESV from C6/36 cells, have a capsid structure with a similar overall architecture but with underlying differences in the trimer contacts (Coulibaly et al, 2010) that unfortunately cannot be further analyzed at the resolution obtained. This variance, even though slight among some members, is probably responsible for host range, tissue tropism, and differences in virulence within the birnaviruses (Coulibaly et al, 2010; Luque et al, 2007).

[00114] In studies with flaviviruses, observations demonstrated that ESV grows to a higher number of particles only when co-infecting with DENV-2 44/2. The ESV virus could be isolated from this dengue strain but not from other dengue strains the inventors work with. Previous studies have described similar effects on virus growth during co-infections with different viruses (Rhode, 1978), suggesting that it may be caused by interfering with viral replication or competition for cell surface receptors. It has been reported that infection of A. albopictus mosquitoes and the C6/36 cell line with a densovirus greatly reduces their susceptibility to DENV-2 (Burivong et al, 2004). Natural dual or multiple infections have also been reported to occur in mosquitoes infected with dengue and densoviruses (Burivong et al, 2004; Wei et al, 2006). Wei et al (2006), obtained results that suggest that the infection with DENV-2 can stimulate the production of latent C6/36 densoviruses in mosquitoes and prevent the growth of dengue particles. However, this mechanism of interaction was not well understood. Consequently, the presence of antigens in the cytoplasm of infected cells cannot be equated with the presence of DENV-2 viral particles. This phenomenon seems to be in accordance with a previous report where dengue viral particles were not seen in dual co-infections of AalDNV and DENV-2 (Kanthong et al, 2008). The fact that mosquito cells may carry persistent viral infections without cytopathic effects as previously described (Burivong et al, 2004; Chen et al, 2004) raises the possibility that insect cell lines may be infected with other unknown viruses. The existence of unknown latent viruses in cell cultures could present serious complications in experimental studies involving arboviruses. Such contamination would be even more serious in the case of vaccine development, where an unknown and therefore undetected virus could affect the growth of the target virus. Furthermore, if these two viruses can coexist in the same cells for long periods of time it may be an indication that there may be an opportunity for genetic exchange (Kanthong et ah, 2010). This may have significant medical and epidemiological implications for arboviruses. Further studies are needed in order to answer questions concerning the nature of ESV and the possible interactions with other viruses.

* * *

[00115] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS;

1. An isolated polynucleotide molecule comprising a nucleic acid sequence selected from the group consisting of:

(a) a sequence encoding a polypeptide at least 80% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7;

(b) a sequence exhibiting at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;

(c) a sequence that hybridizes to the nucleic acid sequence complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof, under conditions of lx SSC and 65 °C;

(d) a sequence of SEQ ID NO: 1 or SEQ ID NO: 3;

(e) a sequence comprising at least 15 contiguous nucleic acids of SEQ ID NO: 1 or SEQ ID NO: 3; and

(f) the compliment of any one of (a)-(e).

2. The polynucleotide of claim 1, comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

3. The polynucleotide of claim 2, comprising a nucleic acid sequence encoding a polypeptide at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

4. The polynucleotide of claim 3, comprising a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 2.

5. The polynucleotide of claim 3, comprising a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 4.

6. The polynucleotide of claim 1, comprising a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

7. The polynucleotide of claim 6, comprising a nucleic acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

8. The polynucleotide of claim 7, comprising a nucleic acid sequence of SEQ ID

NO: 1.

9. The polynucleotide of claim 7, comprising a nucleic acid of SEQ ID NO: 3.

10. The polynucleotide of claim 1, comprising a nucleic acid sequence with at least 15, 20, 25, 30, 35, 40, 45 or 50 contiguous nucleic acids of SEQ ID NO: 1 or SEQ ID NO: 3.

1 1. The polynucleotide of claim 1, comprising a nucleic acid sequence that hybridizes to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, or a fragment or complement thereof, under conditions of lx SSC and 65 °C.

12. The polynucleotide of claim 1, further comprising a label.

13. The polynucleotide of claim 12, wherein the label is a radioactive, enzyme, fluorescent, or affinity label.

14. An isolated virus particle comprising a polynucleotide molecule of claim 1.

15. The isolated virus particle of claim 14, wherein the nucleic acid is an RNA.

16. The isolated virus particle of claim 14, comprising an RNA polynucleotide at least 80%) identical to an RNA encoded by SEQ ID NO: 1 and an RNA polynucleotide at least 80% identical to an RNA encoded by SEQ ID NO: 3.

17. The isolated virus particle of claim 14, wherein the virus has been inactivated.

18. The isolated virus particle of claim 17, wherein the virus has been chemically inactivated.

19. An antigenic composition comprising the isolated virus particle of claim 16.

20. A polypeptide encoded by a polynucleotide molecule of claim 1.

21. A polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a sequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and

(b) a sequence comprising at least 20 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

22. The polypeptide of claim 21, comprising an amino acid sequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

23. The polypeptide of claim 22, comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

24. The polypeptide of claim 23, comprising the amino acid sequence of SEQ ID

NO: 2.

25. The polypeptide of claim 23, comprising the amino acid sequence of SEQ ID

NO: 4.

26. The polypeptide of claim 21, comprising at least 20 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

27. The polypeptide of claim 26, comprising at least 25, 30, 35, 40, 35, 50, or 100 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

28. An isolated antibody wherein the antibody specifically binds to a polypeptide of claim 21.

29. The antibody of claim 28, wherein the antibody is a polyclonal antibody.

30. The antibody of claim 28, wherein the antibody is a monoclonal antibody.

31. The antibody of claim 28, wherein the antibody is a Fab, Fab2, ScFv, or a single domain antibody.

32. The antibody of claim 28, wherein the antibody is a human or humanized antibody.

33. The antibody of claim 28, wherein the antibody binds to ESV VP2, ESV VP3, or ESV VP4.

34. The antibody of claim 28, wherein the antibody binds to ESV VP 1.

35. An antigenic composition comprising a polypeptide of claim 21.

36. The antigenic composition of claim 35, further comprising an adjuvant.

37. The antigenic composition of claim 36, wherein the polypeptide is coupled to an antigen.

38. A method of determining virus contamination in a sample comprising detecting the presence of a polynucleotide molecule of claim 1 or a polypeptide of claim 21, to determine the presence of virus contamination.

39. The method of claim 38, wherein the sample is a patient sample, a pharmaceutical composition, or a cell culture sample.

40. The method of claim 39, wherein the sample is an insect cell culture sample.

41. The method of claim 38, wherein said detecting comprises detecting the presence of a polynucleotide molecule of claim 1.

42. The method of claim 41, wherein said detecting comprises RT-PCR, sequencing, or nucleic acid hybridization.

43. The method of claim 38, wherein said detecting comprises detecting the presence of a polypeptide of claim 21.

44. The method of claim 43, wherein said detecting comprises an immunoblot, ELISA, immunofluorescence, or mass spectroscopy.

45. A primer pair comprising a first polynucleotide that hybridizes to the sequence of SEQ ID NO: 1 and a second polynucleotide that hybridizes to the complement of SEQ ID NO: 1 wherein said first and second polynucleotide amplify a portion of SEQ ID NO: 1 under PCR conditions.

46. A primer pair comprising a first polynucleotide that hybridizes to the sequence of SEQ ID NO: 3 and a second polynucleotide that hybridizes to the complement of SEQ ID NO: 3 wherein said first and second polynucleotide amplify a portion of SEQ ID NO: 3 under PCR conditions.

47. The primer pair of claims 45 or 46, wherein the said first polynucleotide or said second polynucleotide is labeled.

48. The primer pair of claims 45 or 46, wherein said first polynucleotide and said second polynucleotide are between about 18 and 50 nucleotides in length.

49. A method of detecting a polynucleotide comprising:

(a) subjecting a sample to PCR or RT-PCR in the presence of the primer pair of claims 45 or 46; and

(b) detecting a nucleic acid amplification from the sample to detect a polynucleotide.