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  Infectious pestivirus pseudo-particles containing functional erns, e1, e2 envelope proteinsUSPTO Application #: 20070072191Title: Infectious pestivirus pseudo-particles containing functional erns, e1, e2 envelope proteins Abstract: The invention relates to the generation and the use of pestivirus pseudo-particles containing native functional E1, E2 envelope glycoproteins assembled onto retroviral core particles. These particles are highly infectious and constitute a valid model of pestivirus virion. (end of abstract) Agent: Stites & Harbison PLLC - Alexandria, VA, US Inventors: Birke Bartosch, Francois-Loic Cosset USPTO Applicaton #: 20070072191 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070072191. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The invention relates to the generation and the use of pestivirus pseudo-particles containing native functional E1, E2 envelope glycoproteins assembled onto retroviral core particles. These particles are highly infectious and constitute a valid model of pestivirus virion. [0002] Pestivirus are single-stranded RNA (ssRNA) enveloped spherical viruses that constitute a genus within the family Flaviviridae, which also includes the genera flavivirus and hepacivirus (human hepatitis C viruses). Several pestiviruses are important mammalian pathogens, especially cattlepathogens, such as the bovine viral diarrhea, the swine fever and the border disease viruses. Pestivirus can cause mucosal diseases (diarrhea), respiratory disease, suppression of an animal's immune system, and severe bleeding disorders. [0003] Pestivirus structural proteins and non structural proteins are expressed from a single polyprotein precursor and individually released in their respective cell compartments upon cleavage by cellular and viral proteases. By analogy with other members of the Flaviviridae, pestivirus genomic organization suggests a virus consisting of a nucleocapsid comprising a viral genome and core protein (C) coated by a lipid envelop containing the two envelope glycoproteins E1 and E2. [0004] The majority of acute bovine viral diarrhea virus (BVDV) infections are caused by noncytopathic viruses. Cattle acutely or persistently infected with BVDV are the primary source of virus. Infected animals shed virus in nasal and oral secretions feces and urine. The primary virus entrance route is probably oral nasally. Other less important routes of entry may include infected semen, biting insects, and contaminated instruments. Following entry and contact with the mucosal lining of the mouth or nose, initial replication occurs in epithelial cells with a predilection for the palatine tonsils. From here, the virus is able to spread systemically through the blood stream. Spread can occur through both free virus in the serum and virus infected leucocytes, particularly lymphocytes and monocytes. Isolation of virus from serum or leucocytes is generally possible between 3 and 10 days post infection. During systemic spread, the virus is able to gain entry to most tissues with a preference for lymphoid tissues. BVDV broadly infects cattle, sheep, goats, and pigs. [0005] Classical swine fever disease (SVF, previously called hog cholera virus) is another member of the family Flaviviridae, genus Pestivirus. SFV is an economically important contagious disease of swine world-wide. The disease occurs in much of Asia, Central and South America, and parts of Europe and Africa. Several countries have eradication programs in force, based on rapid diagnosis and stamping out of infected herds, supplemented by other control measures. Despite these efforts, SFV has still not been eliminated in many countries. Although SFV can replicate in non-porcine cells, porcine kidney cells are used most frequently for virus growth. Virus replication is restricted to the cytoplasm of the cell and does not result in a cytopathic effect. The first progeny virus is released from the cells at 5-6 hours post-infection. Virion assembly occurs on membranes of the endoplasmic reticulum, but performed capsids and budding are not seen. Instead, fully formed virions appear within the cisternae of the endoplasmic reticulum and are released via exocytosis or cell lysis. Pigs and wild boar are the natural hosts of SFV. [0006] Border disease (BD) is a congenital disease of sheep that was first reported in the bordering countries of England and Wales. A similar, but rare condition also occurs in goats. The causative agent of BD, the border disease virus (BDV), is found worldwide in sheep. Five to fifty percent of sheep tested have antibodies against BD virus; meaning that these ewes have either been exposed to, or are carrying the disease. Transmission of the virus occurs via oral and/or intranasal routes in sheep. Persistently infected sheep are the primary virus reservoir. These ewes will shed virus in all excretions and secretions. Lambs of persistently infected ewes are at risk of becoming persistently infected with the BDV, and thereby perpetuating the disease cycle. [0007] The invention describes the formation and use of infectious pestivirus pseudo-particles harboring unmodified E1 and E2 glycoproteins Definitions [0008] The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence that initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. [0009] A "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. [0010] The term "transfection" means the introduction of a foreign nucleic acid (DNA, cDNA or RNA) into a cell so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein coded by the introduced gene or sequence. The introduced gene may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. A host cell that receives and expresses introduced DNA or RNA has been "transformed". [0011] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA sequence, a protein, a virion. In the context of the invention, the host cell is a mammalian cell, preferably a cell from cattle, rabbit, pig, goat, swine. Suitable host cells include for instance epithelial cells, leucocytes, lymphocytes, macrophages, monocytes, primary kidney cells from cattle or pig, and BT cells (ATCC CRL-1390). [0012] As used herein, the term "permissive celf" is meant for a cell that is permissive for a pestivirus infection. [0013] "Pestviruses" are members of the Flaviviridae family. Pestivirus genome encodes a single polyprotein NH.sub.2-C-Erns-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH that is processed co and post-translationally into both structural (N-terminal nucleocapsid protein termed "Core" (C), and proteins Erns, E1 and E2) and non-structural (NS) proteins. The amino-terminal part of the polyprotein is cleaved by host cell proteases and its products, core and envelope (Erns, E1 and E2) proteins, are believed to be the major constituents of pestivirus particles (virions). However, the ectodomain Erns-E1 is thought to be processed upon synthesis, thus releasing the non anchored Erns protein. [0014] Although most cleavages in the polyprotein precursor proceed to completion during or immediately after translation, processing between E2 and p7, a hydrophobic domain found at the carboxy terminus of E2, is incomplete and results in the production of fully processed E2 and uncleaved E2-p7. [0015] In the context of the invention, said pestivitus may be of any specie, genotype, subtype, or variant of perstivirus strains. Preferably, the pestivirus according to the invention is selected from the group consisting of bovine viral diarrhea virus (BVDV), Type I or Type II, swine fever virus (SFV) and border disease virus (BDV). The complete genome sequence of BVDV (Genebank: NC.sub.--001461), SVF (Genbank NC.sub.--002657) and BDV (Genbank: NC.sub.--003679) is shown in SEQ ID No 1, 7 and 13, respectively. [0016] The term "variant" refers to the homologous polynucleotide sequences and corresponding amino acid sequences found in the different pestivirus strains owing to pestivirus hypervariability. [0017] The term "pestivirus-like particles" as used herein refers to non naturally occurring viral particles that comprise an envelope protein of an pestivirus. [0018] The pestivirus pseudo-particles of the invention are infectious for a target cell. The particles of the invention more particularly comprise retroviral core proteins. Such particles may be readily produced by one skilled in genetic engineering techniques. One can for instance refer to EP 1 201 750 that describes production of synthetic retroviral particles expressing an antigen for modulating an immune response. [0019] In the context of the invention, the term "infectious" is used to describe the capacity of the particles of the invention to complete the initial steps of viral cycle that lead to cell entry. However, upon interaction with the host cell, pestivirus-like particles may or may not produce progeny viruses. [0020] The term "an envelope protein of a pestivirus" denotes the native Erns, E1 or E2 glycoprotein: of a pestivirus, or a mutant thereof. [0021] By an "Erns glycoprotein" or "Erns protein" is meant a Erns from any specie, genotype, subtype, or variant of perstivirus strains. The amino acid sequence of BVDV, SFV and BDV Erns protein is shown in SEQ ID No 3, 9, and 15, respectively. [0022] By an "E1 glycoprotein" or "E1 protein" is meant a envelope 1 protein (E1) from any specie, genotype, subtype, or variant of perstivirus strains. The amino acid sequence of BVDV, SFV and BDV E1 protein is shown in SEQ ID No 4, 10, and 16, respectively. [0023] By an "E2 glycoprotein" or "E2 protein" is meant a envelope 2 protein (E2) from any specie, genotype, subtype, or variant of perstivirus strains. The amino acid sequence of BVDV, SFV and BDV E2 protein is shown in SEQ ID No 5, 11, and 17, respectively. Continue reading... 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