WO2017060662A1 - Procédé de préparation d'un témoin à utiliser dans un dépistage de virus pathogène - Google Patents

Procédé de préparation d'un témoin à utiliser dans un dépistage de virus pathogène Download PDF

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WO2017060662A1
WO2017060662A1 PCT/GB2015/052935 GB2015052935W WO2017060662A1 WO 2017060662 A1 WO2017060662 A1 WO 2017060662A1 GB 2015052935 W GB2015052935 W GB 2015052935W WO 2017060662 A1 WO2017060662 A1 WO 2017060662A1
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virus
rna
nucleotide sequence
pathogenic
sequence
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PCT/GB2015/052935
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English (en)
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Giada MATTIUZZO
Mark Page
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The Secretary Of State For Health
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Priority to PCT/GB2015/052935 priority Critical patent/WO2017060662A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/14011—Filoviridae
    • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
    • C12N2760/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to a method for providing a control for use in a screen for a pathogenic virus, uses, nucleotide sequences, vectors, ribonucleic acids, deoxyribonucleic acids, compositions, kits and methods for manufacture and for detecting the presence or absence of a pathogenic virus.
  • Biological reference materials or standards of known activity or potency are important calibrators for bioassays to permit results to be compared and harmonised within and between laboratories, no matter when or where the bioassays are performed.
  • nucleic acid amplification technology NAT
  • the desired requirements of a NAT reference standard should be that it is safe (non-infectious), represent all the relevant target sequences in equal or known amounts, control for the whole process including nucleic acid extraction and purification and exhibit commutability (behave as close as possible to the clinical sample).
  • HIV Human Immunodeficiency virus
  • Parvovirus B19 Parvovirus B19
  • Hepatitis A B and C.
  • infectious material e.g. HCV
  • Nucleic acid amplification based diagnostic techniques have a crucial role during the ongoing Ebola virus outbreak in West Africa. Reference materials are needed to assess the validity of the assays used, to compare results across assays and to provide guidance to the regulatory agencies in the evaluation of new assays. It is crucially important that Ebola virus NAT reference materials standardise and control the entire process from the extraction to the final amplification and detection reaction. Current in-house and commercial assays utilize plasmids or in vitro RNA transcripts as reference for the assays. Such reference materials do not control for the extraction procedure and may therefore incur false negative results. Wild type virus preparations have been used as controls, but these are limited to those laboratories which have access to bio-containment level 4 facilities.
  • the present invention provides a method for providing a control for use in a screen for a pathogenic virus comprising:
  • RNA ribonucleic acid
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus
  • control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof;
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus
  • the pathogenic virus is a pathogenic RNA virus
  • the invention provides a use of a recombinant RNA virus wherein:
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus
  • control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof;
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus ;
  • RNA virus in the preparation of a positive control for reducing false negatives associated with a pathogenic viral screen and/or for stabilising a control RNA sequence.
  • a method for preparing a recombinant RNA virus comprising:
  • RNA virus a nucleotide sequence encoding one or more Lentiviral or Gammaretroviral protein(s) to form a recombinant RNA virus.
  • a nucleotide sequence comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof operably linked to a promoter, wherein said nucleotide sequence is modified such that:
  • a. it comprises one or more nucleotide modifications, so that the nucleotide sequence is incapable of producing a pathogenic viral protein; and/or b. it lacks one or more regulatory nucleotide sequence essential for translation of the viral genome sequence, so that the nucleotide sequence is incapable of producing a pathogenic viral protein.
  • a vector comprising a nucleotide sequence of the present invention.
  • RNA ribonucleic acid
  • RNA obtainable by a method of the invention or a synthesised RNA structurally equivalent thereto.
  • the invention provides a deoxyribonucleic acid (DNA) obtainable from a RNA of the invention.
  • DNA deoxyribonucleic acid
  • a ribonucleoprotein comprising a RNA of the invention and a viral protein.
  • the invention provides a composition comprising a nucleotide sequence according to the invention, a vector according to the invention, a RNA according to the invention, a DNA according to the invention or a ribonucleoprotein according to the invention.
  • the present invention provides a kit comprising a nucleotide sequence, a vector, a RNA, a DNA, a ribonucleoprotein or a composition according to the invention and instructions for using same.
  • a method for detecting the presence or absence of a pathogenic virus comprising:
  • control sample comprises:
  • RNA extracted from a recombinant RNA virus wherein:
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus
  • control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof;
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus
  • the pathogenic virus is a pathogenic RNA virus
  • step b. comparing a result obtained in step b. for the experimental sample and the control sample.
  • a data-storage medium comprising data obtained by the method of any preceding claim.
  • Figure 1 shows generation of lentiviral particles containing Ebola virus RNA.
  • Ebola virus genes nucleoprotein (NP), viral protein 35 (VP35), glycoprotein (GP), viral protein 40 (VP40) and the polymerase encoding gene L were sequentially cloned into lentiviral vector pSFJenti between the restriction sites Sail and BstEII. Main elements of the lentiviral vector for the production of the viral RNA and incorporation within HIV-like particle are indicated.
  • the graph represents the mean of 5 acquisitions (black line) ⁇ standard deviation (grey shading surrounding black line).
  • C) A representative image of the same preparation at dilution 1 : 10 in PBS analysed by negative staining transmission electron microscopy. The average particle size was 116.02 nm (average of 9 fields).
  • Figure 2 shows performance of the HIV-EBOV RNA preparations in qRT-PCR.
  • Viral RNA extracted from serial dilutions of WHO 3 rd HIV-1 International standard (dilution factor 5, diamond), LVV_NP-VP35-GP high (dilution factor 10, square) and LVV-VP40-L (dilution factor 10, triangle) were processed in duplicate in a quantitative RT-PCR using primers and probes annealing within the U5 region of the HIV LTR.
  • Figure 4 shows a sequence of a vector of the invention (pSF-lenti-NP_VP35_Gp) shown as SEQ ID No. 1.
  • Figure 5 shows a sequence of a vector of the invention (pSF-lenti-VP40-L) shown as SEQ ID No. 2.
  • Figure 6 shows the nucleotide sequence of Ebola Zaire virus gene NP shown as SEQ ID No. 3.
  • Figure 7 shows a polypeptide sequence (SEQ ID No. 4) encoded by SEQ ID No. 3.
  • Figure 8 shows the nucleotide sequence of Ebola Zaire virus gene VP35 shown as SEQ ID No. 5.
  • Figure 9 shows a polypeptide sequence (SEQ ID No. 6) encoded by SEQ ID No. 5.
  • Figure 10 shows the nucleotide sequence of Ebola Zaire virus gene VP40 shown as SEQ ID No. 7.
  • Figure 11 shows a polypeptide sequence (SEQ ID No. 8) encoded by SEQ ID No. 7.
  • Figure 12 shows the nucleotide sequence of Ebola Zaire virus gene GP/sGP shown as SEQ ID No. 9.
  • Figure 13 shows a polypeptide sequence (SEQ ID No. 10) encoded by SEQ ID No. 9.
  • Figure 14 shows the nucleotide sequence of Ebola Zaire virus gene VP30 shown as SEQ ID No. 11.
  • Figure 15 shows a polypeptide sequence (SEQ ID No. 12) encoded by SEQ ID No. 11.
  • Figure 16 shows the nucleotide sequence of Ebola Zaire virus gene VP24 shown as SEQ ID No. 13.
  • Figure 17 shows a polypeptide sequence (SEQ ID No. 14) encoded by SEQ ID No. 13.
  • Figure 18 shows the nucleotide sequence of Ebola Zaire virus gene L shown as SEQ ID No. 15.
  • Figure 19 shows a polypeptide sequence (SEQ ID No. 16) encoded by SEQ ID No. 15. DETAILED DESCRIPTION
  • a seminal finding of the present invention is that a RNA extracted from a recombinant RNA virus as disclosed herein is particularly useful for the preparation of a control for use in a screen for a pathogenic virus (e.g. a diagnostic assay).
  • a pathogenic virus e.g. a diagnostic assay
  • a method for providing a control for use in a screen for a pathogenic virus comprising:
  • RNA ribonucleic acid
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus
  • control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof;
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus
  • the pathogenic virus is a pathogenic RNA virus
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus; b. the control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof; and
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus
  • the recombinant RNA virus for use in the present invention is a Lentivirus or a Gammaretrovirus (preferably the recombinant RNA virus may be a Lentivirus).
  • the recombinant RNA virus for use in a method and/or use of the present invention comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus and further comprises at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof.
  • control as used herein is synonymous with "positive control” and takes its normal meaning in the art.
  • an extracted (e.g. isolated) RNA is used as a positive control a result which is positive for the presence of a pathogenic viral nucleotide sequence in the control sample is indicative of a functional assay (e.g. in a PCR-based assay a positive result may be the presence of an amplicon).
  • a positive control as per the present invention may thereby reduce false negatives associated with a viral diagnostic screen when compared to not using a positive control of the invention.
  • the control RNA sequence allows for the identification of the presence or absence of a pathogenic virus.
  • the presence or absence of a pathogenic virus may be determined using a method for detecting a pathogenic virus herein and include inter alia detection of the presence or absence of a pathogenic viral nucleotide sequence (e.g. using PCR).
  • stabilising a control RNA sequence may mean improving the stability of a control RNA sequence of the invention when compared to an RNA sequence that has not been packaged within a recombinant RNA virus disclosed herein. Stability of a control RNA sequence (e.g. at an ambient temperature) can be determined using any suitable means. For example, RNA stability may be assessed by PCR (e.g. reverse transcriptase-PCR).
  • RNA stability may be determined using gel electrophoresis and determining whether truncations of a RNA are present.
  • a use according to the present invention improves stability of a control RNA of the invention when stored for at least 1 week (preferably for about 2 weeks) at at least 10 °C, 20 °C, or 30 °C when compared to a RNA that is not comprised in a recombinant Lentivirus or Gammaretrovirus.
  • pathogenic virus used herein preferably refers to a pathogenic virus requiring containment level BSL-3 or 4.
  • the pathogenic virus invention may be a pathogenic RNA virus.
  • the pathogenic RNA virus from which the control RNA sequence is obtainable may be the same pathogenic RNA virus being screened for.
  • the pathogenic virus e.g. pathogenic RNA virus
  • may be a virus detailed on the ViPR Virus Pathogen Resource (http://www. viprbrc.org/brc/home. spg?decorator vipr, which is incorporated herein by reference).
  • the pathogenic virus may be one or more selected from the group consisting of: Calciviridae, Coronaviridae, Flaviviridae, Hepeviridae, Picornaviridae, Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae and Rhabdoviridae.
  • An Arenaviridae virus may be one or more genus selected from the group consisting of: Arenavirus, Mammarenavirus, Reptarenavirus, and Unclassified Arenaviridae.
  • a Bunyaviridae virus may be one or more genus selected from the group consisting of: Hantavirus, Nairovirus, Negevirus, Orthobunyavirus, Phlebovirus and Unclassified Bunyaviridae.
  • a Caliciviridae virus may be one or more genus selected from the group consisting of: Lagovirus, Nebovirus, Norovirus, Recovirus, Sapovirus, Vesivirus and Unclassified Caliciviridae.
  • a Coronaviridae virus may be selected from the subfamily Coronavirinae or Torovirinae.
  • a Coronavirinae virus may be one or more genus selected from the group consisting of: Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus and Unclassified Coronaviridae.
  • a Torovirinae virus may be one or more genus selected from the group consisting of: Bafinivirus and Torovirus.
  • a Filoviridae virus may be one or more genus selected from the group consisting of: Cuevavirus, Ebolavirus, Marburgvirus and Unclassified Filoviridae.
  • a Flaviviridae virus may be one or more genus selected from the group consisting of: Flavivirus, Hepacivirus, Pegivirus, Pestivirus and Unclassified Flaviviridae.
  • a Hepeviridae virus may be one or more genus selected from the group consisting of: Hepevirus, Piscihepevirus and Unclassified Hepeviridae.
  • a Paramyxoviridae virus may be one or more subfamily selected from the group consisting of: Paramyxovirinae and Pneumovirinae.
  • a Paramyxovirinae virus may be one or more genus selected from the group consisting of: Aquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Morbillivirus, Respirovirus, Rubulavirus and Unclassified Paramyxoviridae.
  • a Pneumovirinae virus may be one or more genus selected from the group consisting of: Metapneumovirus and Pneumovirus.
  • a Picornaviridae virus may be one or more genus selected from the group consisting of: Aphthovirus, Aquamavirus, Avihepatovirus, Avisivirus, Cardiovirus, Cosavirus, Dicipivirus, Enterovirus, Erbovirus, Gallivirus, Hepatovirus, Hunnivirus, Kobuvirus, Megrivirus, Mischivirus, Mosavirus, Oscivirus, Parechovirus, Pasivirus, Passerivirus, Rosavirus, Sakobuvirus, Salivirus, Sapelovirus, Senecavirus, Teschovirus, Tremovirus and Unclassified Picornaviridae.
  • a Rhabdoviridae virus may be one or more genus selected from the group consisting of: Bracorhabdovirus, Cytorhabdovirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Sprivivirus, Tibrovirus, Tupavirus, Vesiculovirus and Unclassified Rhabdoviridae.
  • a Togaviridae virus may be one or more genus selected from the group consisting of: Alphavirus, Rubivirus and Unclassified Togaviridae.
  • the pathogenic RNA virus may be a positive sense pathogenic RNA virus.
  • a positive sense pathogenic RNA virus may be one or more selected from the group consisting of: a SARS coronavirus, a MERS coronaviruses, a Dengue virus, a Chikungunya virus, a Ross river virus, a Yellow fever virus, a West Nile virus, a Japanese encephalitis virus, a Zika virus, and an ambisense RNA viral sequences (e.g. Lassavirus).
  • the pathogenic RNA virus may be a negative sense pathogenic RNA virus.
  • a negative sense pathogenic RNA may include viruses causing haemorrhagic fever.
  • a negative sense pathogenic RNA virus may be selected from the group consisting of: Ebolavirus spp. Sudan and Bundibugyo, Marburg virus, Hantaviruses, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Nipah virus and Hendra virus.
  • the negative sense pathogenic RNA virus being screened for in the present invention may be a Marburg virus.
  • a Marburg virus for use in the present invention may be a Marburg virus having a genome with GenBank accession number DQ447649.
  • the Ebola virus may be one or more Ebola virus species selected from the group consisting of: Zaire, Bundibugyo, Sudan, Reston and Tai Forest.
  • the Ebola virus may be Ebola virus species Zaire. Ebola Zaire is taught in Gire et al, Science, 2014, 345(6202), 1369-1372 the contents of which is incorporated herein by reference.
  • the Ebola virus screened for may be an Ebola Zaire Makona 2014 virus (e.g. isolate H.sapiens-wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014 Oct 9;371 (15): 1418- 25 incorporated herein by reference).
  • Ebola Zaire Makona 2014 virus e.g. isolate H.sapiens-wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus
  • the Ebola virus screened for may be an Ebola Sudan virus, suitably an Ebola Sudan virus having a genome with GenBank accession number AY729654.
  • a method of the present invention comprises performing RNA extraction on a recombinant RNA virus.
  • extract is synonymous with “isolating” and as used herein means removing a product of interest from one or more contaminants which might be present in a composition comprising said product of interest, preferably with the aim of obtaining a product of interest that is free from said contaminants.
  • extracting or “isolating” as used herein may refer to a degree of purification rather than to absolute purification.
  • extracting may refer to removing at least 5% (suitably at least 10% or 20%) of contaminants.
  • extracting may refer to removing at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of contaminants.
  • extracting refers to removing a RNA comprising a control RNA sequence for use in the invention from one or more recombinant RNA viral proteins, preferably with the aim of obtaining a RNA that is free from said recombinant RNA viral protein(s) and/or further contaminant(s).
  • extracting or “isolating” as used in this context may mean removing at least 5% (suitably at least 10% or 20%) of viral protein and/or further contaminant(s).
  • isolated may refer to removing at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of viral protein and/or further contaminant(s).
  • the term "extracting” or “isolating” may refer to removing an nucleotide, vector, RNA, DNA, ribonucleoprotein and/or composition of the invention from one or more contaminants present in a composition.
  • one or more contaminants may be one or more alternative nucleotides, vectors, RNAs, DNAs and/or ribonucleotides.
  • RNA and/or ribonucleoproteins are commercially available, such as for example the RNeasy kit or the QIAamp ® Viral RNA Mini Kit (both from Qiagen Ltd., UK).
  • the present invention provides a RNA obtainable by a method according to the invention or a synthesised RNA structurally equivalent thereto.
  • RNA has an identical structure to a RNA produced by a nucleotide sequence of the invention.
  • the present invention comprises the use of a control RNA sequence comprising at least 3 kb of contiguous nucleotide sequence of a pathogenic virus or a complement thereof.
  • the term "contiguous nucleotide sequence of a pathogenic virus or a complement thereof" as used herein refers to sequential nucleotides of a reference viral genome sequence.
  • the reference viral genome sequence is preferably identical to the pathogenic virus being detected in a screen for which the control is to be used.
  • at least 3 kb contiguous nucleotide sequence of a pathogenic virus genome sequence suitably means 3 kb of nucleotides that are sequential in the reference pathogenic virus genome sequence.
  • contiguous nucleotide sequence of a pathogenic virus or a complement thereof as used herein is intended to encompass orthologues and/or variants of at least 80% or 85% sequence identity to the reference viral genome sequence.
  • the term “contiguous nucleotides of a viral genome sequence” may encompass orthologues and/or variants of at least 90% or 95% sequence identity to the reference viral genome sequence.
  • the term “contiguous nucleotides of a viral genome sequence” may encompass orthologues and/or variants of at least 98% or 99% or 100% sequence identity to the reference viral genome sequence.
  • RNA sequence of a pathogenic virus or a complement thereof is also intended to encompass sequences comprising a modification (e.g. one or more nucleotide modifications and/or lacking one or more regulatory elements associated therewith in the viral genome sequence) as well as antisense transcripts of a pathogenic viral genome sequence.
  • a control RNA as disclosed herein may be derived from a viral DNA sequence.
  • the RNA of, or for use in, the present invention may be of an opposite sense to a viral DNA or RNA sequence. For example, if a viral DNA or RNA sequence is in a positive sense, the RNA of the invention may be in the negative sense.
  • control RNA sequence of the invention is an RNA sequence exogenous to the recombinant Lentivirus and/or Gammaretrovirus referred to herein.
  • exogenous preferably means that the RNA sequence of the invention is obtainable from a pathogenic virus that is of a different family, genus, species, strain or isolate (more preferably a different family) from the recombinant Lentivirus and/or Gammaretrovirus used in the method.
  • control RNA sequence as referred to herein is contiguous with a Lentiviral or Gammaretroviral sequence.
  • the at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof herein may refer to a nucleotide sequence that has been PCR-derived from genetic material from an RNA virus or may be in vitro synthesised. In some cases restriction sites may be added to the 5' and/or 3' end of said sequences allowing for cloning using basic molecular biological techniques.
  • control RNA sequence comprise between about 3 kb to about 9.5 kb contiguous nucleotide sequence of a pathogenic viral genome sequence.
  • control RNA sequence may comprise between about 4 kb to about 9 kb of a pathogenic viral genome sequence or a complement thereof.
  • control RNA sequence may comprise between about 5 kb to about 9 kb of a pathogenic viral genome sequence or a complement thereof.
  • control RNA sequence may comprise between about 6 kb to about 9 kb of a pathogenic viral genome sequence or a complement thereof.
  • control RNA sequence may comprise at least about 4 kb or at least about 5 kb of a pathogenic viral genome sequence or a complement thereof.
  • control RNA sequence may comprise at least about 6 kb or at least about 7 kb of a pathogenic viral genome sequence or a complement thereof.
  • control RNA sequence may comprise or consist of at least 5 kb of a pathogenic viral genome sequence or complement thereof.
  • control RNA sequence may comprise about 6.7 kb or about 8.3 kb or of a pathogenic viral genome sequence or a complement thereof.
  • control RNA sequence of, or for use in, the present invention may comprise a viral gene or portion thereof.
  • control RNA sequence of, or for use in, the present invention may comprise at least 2, 3, 4 or 5 pathogenic viral genes or portions thereof
  • the pathogenic RNA virus gene or portion thereof may be a positive sense pathogenic RNA virus.
  • a positive sense pathogenic RNA virus gene or portion thereof may be one or more selected from the group consisting of: a SARS coronavirus, a MERS coronaviruses, a Dengue virus, a Chikungunya virus, a Ross river virus, a Yellow fever virus, a West Nile virus, a Japanese encephalitis virus, a Zika virus, and an ambisense RNA viral sequences (e.g. Lassavirus).
  • the pathogenic RNA virus gene or portion thereof may be a negative sense pathogenic RNA virus.
  • the negative sense pathogenic RNA virus gene or portion thereof may be obtainable from a virus causing haemorrhagic fever.
  • a negative sense pathogenic RNA virus gene or portion thereof may be selected from the group consisting of: Ebolavirus spp. Sudan and Bundibugyo, Marburg virus, Hantaviruses, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Nipah virus and Hendra virus.
  • the gene or portion thereof may be obtainable from a Marburg virus gene or portion thereof.
  • a Marburg virus gene or portion thereof may be obtainable from Marburg virus having a genome with GenBank accession number DQ447649.
  • a Marburg virus gene or portion thereof may be selected form the group consisting of: NP, VP35, VP40 and GP.
  • the gene or portion thereof may be an Ebola virus gene or portion thereof.
  • the gene or portion thereof may be obtainable from Ebola Zaire, suitably an Ebola Zaire having a genome with GenBank accession number KJ660346.2.
  • the gene or portion thereof may be obtainable from Ebola Sudan virus, suitably an Ebola Sudan virus having a genome with GenBank accession number AY729654.
  • an Ebola virus gene or portion thereof may be selected from the group consisting of: NP, VP35, VP40, GP/sGP, VP30, VP24 and L.
  • the Ebola virus gene may be NP or a portion thereof.
  • the NP gene of Ebola encodes a RNA binding protein believed to be responsible for genomic packaging.
  • the NP gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 3 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the NP gene may be a nucleotide sequence encoded by SEQ ID No. 3 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the NP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 4 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the NP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 4 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP35 or a portion thereof.
  • the VP35 gene of Ebola encodes a polymerase cofactor in the RNA polymerase transcription and replication complex.
  • the VP35 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 5 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP35 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 5 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP35 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 6 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP35 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 6 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP40 or a portion thereof.
  • the VP40 gene of Ebola encodes a virus assembly and budding promotion factor. Without wishing to be bound by theory, it is believed that VP40 interacts with host proteins of the multivesicular body pathway.
  • the VP40 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 7 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP40 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 7 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP40 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 8 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP40 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 8 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be GP/sGP or a portion thereof. The GP/sGP gene of Ebola encodes a glycoprotein responsible for binding to receptors on target cells.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 9 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 9 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 10 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 10 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP30 or a portion thereof.
  • the VP30 gene of Ebola encodes a transcription anti-termination factor.
  • the VP30 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 1 1 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP30 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 1 1 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP30 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 12 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP30 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 12 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP24 or a portion thereof.
  • the VP24 gene of Ebola encodes a membrane-associated protein believed to prevent the establishment of cellular antiviral state by blocking the interferon-alpha/beta and IFN-gamma signalling pathways.
  • the VP24 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 13 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP24 gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 13 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP24 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 14 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP24 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 14 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be L or a portion thereof.
  • the L gene of Ebola encodes an polypeptide believed to have RNA-directed RNA polymerase, mRNA guanylyl transferase, mRNA (guanine-N(7)-)-methyltransferase and/or poly(A) synthetase activities.
  • the L gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 15 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the L gene or portion thereof may be a nucleotide sequence encoded by SEQ ID No. 15 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the L gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 16 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the L gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 16 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus sequence may be an Ebola virus non-coding sequence or portion thereof.
  • an Ebola virus non-coding sequence or portion thereof may be selected from the group consisting of: an intergenic region, a 5' UTR, and a 3UTR.
  • the term "portion thereof" as used in this context means at least about 200, 400, 600, 700, 800, 1000, 1200 or 1400 bp.
  • the term "portion thereof” means at least about 1000 bp.
  • the recombinant RNA virus for use in a method of the invention may be a Lentivirus and/or a Gammaretrovirus.
  • the recombinant RNA virus for use in a method of the invention may be a Lentivirus.
  • control and/or recombinant RNA virus may be prepared by a Lentiviral or Gammoretroviral (suitably a Lentiviral system).
  • a Lentiviral system as used herein may refer to a system whereby at least 3 kb contiguous nucleotide sequence of a pathogenic virus genome sequence or complement thereof is cloned into a Lentiviral vector (e.g. pSF-lenti, commercially available from Oxford Genetics).
  • a Lentiviral vector e.g. pSF-lenti, commercially available from Oxford Genetics.
  • Such vectors suitably comprise one or more genetic elements for packaging a RNA expressed from said vector into a Lentiviral particle.
  • RNA virus prepared by such a method may then be isolated as per step b. of the method of the invention.
  • preparation of a ribonucleoprotein may be carried out in a host cell.
  • a viral protein may be co-expressed with the nucleic acid comprising at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof.
  • the recombinant RNA virus may be prepared by a method comprising:
  • a Lentiviral vector or Gammaretroviral vector (preferably a Lentiviral vector) comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus genome sequence or complement thereof;
  • RNA virus co-expressing said vector in a host cell with a nucleotide sequence encoding one or more Lentiviral or Gammaretroviral protein(s) to form a recombinant RNA virus.
  • the method further comprises isolating the recombinant RNA virus.
  • a method for providing a control for use in a screen for a pathogenic virus comprising: providing a Lentiviral vector or Gammaretroviral vector (preferably a Lentiviral system) comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus genome sequence or complement thereof;
  • RNA virus co-expressing said vector in a host cell with a nucleotide sequence encoding one or more Lentiviral or Gammaretroviral protein(s) to form a recombinant RNA virus (and optionally isolating said recombinant RNA virus);
  • RNA ribonucleic acid
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus
  • control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof;
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus; and
  • the pathogenic virus is a pathogenic RNA virus; and providing an isolated recombinant RNA virus sequence.
  • the one or more Lentiviral or Gammaretroviral proteins may be selected from the group consisting of a gag protein, pol protein and a rev protein (suitably a Lentiviral gag protein, pol protein and rev protein).
  • a gag protein, pol protein, rev protein or combinations thereof may be encoded for by a Lentiviral and/or Gammaretroviral (suitably a Lentiviral) packaging vector.
  • the term "packaging vector" as used herein is intended to refer to a vector that is different (e.g. a different entity) to a Lentiviral or Gammaretroviral vector as used herein.
  • Lentiviral vector refers to a vector comprising one or more genetic sequence elements derived from a Lentivirus suitable for packaging an RNA of the invention into a ribonucleoprotein (e.g. a recombinant RNA virus) comprising one or more Lentiviral protein(s).
  • a ribonucleoprotein e.g. a recombinant RNA virus
  • Gammaretroviral vector refers to a vector comprising one or more genetic sequence elements derived from a Gammaretrovirus suitable for packaging an RNA of the invention into a ribonucleoprotein (e.g. a recombinant RNA virus) comprising one or more Gammaretroviral protein(s).
  • a ribonucleoprotein e.g. a recombinant RNA virus
  • a Lentiviral or Gammaretroviral vector for use in the present invention comprises one or more selected from the group consisting of: retroviral long terminal repeats (LTR) either wild type or missing the U3 region, packaging signal, Rev responsive element (REV)- required for lentiviral vector, poly purine tract, unique restriction sites for the cloning of the heterologous genes, retroviral long terminal repeats (LTR) either wild type or missing the U5 region and a polyadenylation site.
  • retroviral long terminal repeats either wild type or missing the U3 region
  • packaging signal Rev responsive element (REV)- required for lentiviral vector
  • poly purine tract poly purine tract
  • unique restriction sites for the cloning of the heterologous genes retroviral long terminal repeats (LTR) either wild type or missing the U5 region and a polyadenylation site.
  • a lentiviral vector may be a pSF-lenti vector ((OG269) commercially available from Oxford Genetics Ltd, UK).
  • Lentiviral vectors include OG593 and OG637 also commercially available from Oxford Genetics Ltd, UK as well as those disclosed in GB9517263.1 (which is incorporated herein by reference in its entirety).
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • Gammaretroviral vectors are known in the art and suitable examples include those from Takara Clontech (e.g. Cat. no. 631503-631501-631509) or as described in Soneoka et al Nucleic Acids Res. 1995 Feb 25; 23(4): 628-633 (incorporated herein by reference).
  • the pathogenic virus to be detected is a positive strand pathogenic RNA virus
  • a control RNA sequence comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus may be inserted into a Lentiviral or Gammaretroviral vector in the same orientation as other Lentiviral or Gammaretroviral features (e.g. the promoter and/or LTR).
  • RNA sequence comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus may be inserted into a Lentiviral or Gammaretroviral vector in the opposite orientation with respect to other Lentiviral or Gammaretroviral features (e.g. the promoter and/or LTR).
  • host cell includes any cell that comprises a nucleotide as defined herein or an expression vector as described above and which is used in the production of a RNA or recombinant RNA virus of the invention.
  • a host cell for use in a method of the invention may be a mammalian host cell.
  • a human host cell for example a human cell line.
  • the host cell may be a HEK cell, such as a HEK 293 cell.
  • the host cell may be a HEK 293T-17 cell such as ATCC CRL-1 1268 (commercially available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 201 10 USA).
  • Any means for transfecting a host cell with a nucleotide sequence, DNA or RNA of the invention may be employed, for example by polyethylenimine (PEI), calcium phosphate, Llpofectamine (Life Technologies), Xtreme Transfection reagent (Roche), FuGene (Promega) techniques.
  • PEI polyethylenimine
  • Ca phosphate calcium phosphate
  • Llpofectamine Life Technologies
  • Xtreme Transfection reagent Gibco-treme Transfection reagent
  • FuGene Promega
  • the association of said RNA with a viral protein may be achieved by admixing one or more viral proteins with a RNA of the invention.
  • the association may occur in vivo (e.g. in a host cell).
  • a method for manufacturing a recombinant RNA virus for use in the present invention may comprise expressing one or more RNA viral protein(s), suitably co- expressing one or more viral protein(s) in a host cell or in vitro (preferably in a host cell) to produce a recombinant RNA virus comprising a RNA of the invention and one or more viral protein(s).
  • said method may further comprise isolating said recombinant RNA virus (e.g. using a sucrose gradient).
  • the viral protein may be a Lentiviral protein (e.g. an HIV viral protein) and/or a Gammaretroviral protein.
  • the viral protein may be a structural viral protein (e.g. a nucleoprotein).
  • the viral protein may be a gag protein, pol protein, rev protein or combinations thereof (suitably a Lentiviral gag protein, pol protein, rev protein or combinations thereof).
  • a gag protein, pol protein, rev protein or combinations thereof may be encoded for by a Lentiviral and/or Gammaretroviral (suitably a Lentiviral) packaging vector.
  • the ribonucleoprotein of the invention may be a virus comprising a nucleotide sequence, vector and/or RNA of the invention (suitably Lentivirus or a Gammaretro virus) .
  • the ribonucleoprotein of, or for use in, the present invention may be prepared using a Lentiviral system or a Gammaretroviral system (more preferably a Lentiviral system).
  • RNA, recombinant RNA virus, nucleotide sequence, DNA, ribonucleoprotein, kit or composition of the invention may be incapable of producing a pathogenic viral protein.
  • the invention provides for a nucleotide sequence comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof operably linked to a promoter, wherein said nucleotide sequence is modified such that:
  • nucleotide sequence is incapable of producing a pathogenic viral protein
  • nucleotide sequence may refer to an oligonucleotide sequence or polynucleotide sequence.
  • the nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.
  • the nucleotide sequence may be double- stranded.
  • nucleotide sequence as used herein in reference to the present invention may include genomic DNA, cDNA, synthetic DNA, and/or RNA.
  • nucleotide sequence as used herein refers to DNA.
  • a nucleotide sequence encompassed by the scope of the present invention may be prepared using recombinant DNA techniques (i.e. recombinant DNA).
  • the nucleotide sequence may be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232 which is incorporated herein by reference).
  • RNA e.g. a RNA of the invention
  • incapable of producing a pathogenic viral protein in not intended to exclude the translation of RNA (e.g. a RNA of the invention) transcribed from the nucleotide sequence or vector of the invention into a protein if such a protein has less than 50% sequence identity to a pathogenic viral protein.
  • a protein translated from a RNA e.g. a RNA of the invention
  • a protein translated from a RNA may have less than 1 % sequence identity to a pathogenic viral protein.
  • the nucleotide sequence (suitably at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) may be modified such that it comprises one or more nucleotide modifications, so that the nucleotide sequence (preferably the pathogenic viral genome sequence comprised therein) is incapable of producing a pathogenic viral protein.
  • one or more nucleotide modifications embraces one or more nucleotide modifications when compared to an unmodified nucleotide sequence (preferably an unmodified pathogenic viral genome sequence (e.g. a pathogenic viral reference sequence)).
  • the one or more nucleotide modification(s) may result in the preparation of a nucleotide sequence that is no longer capable of producing a pathogenic viral protein.
  • the one or more nucleotide modification may result in a frame- shift of the pathogenic viral genome sequence with respect to a start codon.
  • Such nucleotide modifications can be modifications of the pathogenic viral genome sequence or modification outside of the pathogenic viral genome sequence (e.g. a modification between the pathogenic viral genome sequence and the operably linked promoter).
  • unmodified nucleotide sequence refers to a reference nucleotide sequence from which a nucleotide sequence of the genome is derivable (e.g. derived).
  • the unmodified nucleotide sequence may be an unmodified pathogenic viral genome sequence.
  • the terms "unmodified pathogenic viral genome sequence” and " pathogenic viral genome sequence capable of producing an viral protein” as used herein refer to an pathogenic viral genome sequence prior to modification. For example if the (e.g. at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) pathogenic viral genome sequence is derived from Ebola (e.g. Ebola Zaire), then the unmodified pathogenic viral genome sequence is the wild-type Ebola (e.g. Ebola Zaire) viral genome sequence prior to modification.
  • Ebola e.g. Ebola Zaire
  • unmodified pathogenic viral genome sequence and "pathogenic viral genome sequence capable of producing a pathogenic viral protein” as used herein refer to a genome sequence obtainable from Ebola. Suitably obtainable from one or more Ebola virus species selected from the group consisting of: Zaire, Bundibugyo, Sudan, Reston and Tai Forest (preferably an Ebola Zaire virus species).
  • Ebola virus species selected from the group consisting of: Zaire, Bundibugyo, Sudan, Reston and Tai Forest (preferably an Ebola Zaire virus species).
  • the terms "unmodified pathogenic viral genome sequence” and “ pathogenic viral genome sequence capable of producing a pathogenic viral protein” as used herein may mean an Ebola Zaire Makona 2014 virus (e.g.
  • the one or more nucleotide modifications may be selected from the group consisting of substitutions, deletions, inversions and insertions.
  • the nucleotide modification may be a substitution.
  • nucleotide modification may be a deletion. In a further embodiment the nucleotide modification may be an inversion.
  • nucleotide modification may be an insertion.
  • the one or more modification may introduce one or more stop codon(s) in the nucleotide sequence.
  • two or more stop codons may be introduced in the nucleotide sequence.
  • three or more (more preferably three) stop codons may be introduced in the nucleotide sequence.
  • the one or more modification may introduce one or more stop codon(s) in the pathogenic viral genome sequence comprised in the nucleotide sequence of the invention.
  • the pathogenic viral genome sequence comprised in the nucleotide sequence of the invention may be introduced in the pathogenic viral genome sequence comprised in the nucleotide sequence of the invention.
  • two or more stop codons may be introduced in the pathogenic viral genome sequence comprised in the nucleotide sequence of the invention.
  • three or more (more preferably three) stop codons may be introduced in the pathogenic viral genome sequence comprised in the nucleotide sequence of the invention.
  • a plurality of stop codons may be inserted sequentially.
  • stop codon has its normal meaning in the art and refers to a three nucleotide sequence that results in translation termination of a RNA comprising said sequence.
  • the stop codon (when present in a RNA sequence) may be one or more selected from the group consisting of: UAG, UAA and UGA.
  • a nucleotide sequence of the present invention may be modified to render one or more start codon(s) non-functional.
  • a viral genome sequence comprised in the nucleotide sequence of the invention may be modified to render one or more start codon(s) non-functional.
  • Rendering one or more start codon(s) non-functional may comprise substituting one or more start codon nucleotides with an alternative nucleotide, deleting the start codon or a portion thereof, inverting the start codon, or inserting one or more nucleotide(s).
  • nucleotide sequence (suitably the at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) may be modified such that it lacks one or more regulatory nucleotide sequences essential for translation of the pathogenic viral genome sequence, so that the nucleotide sequence is incapable of producing a pathogenic viral protein.
  • one or more regulatory nucleotide sequences essential for translation may refer to a start codon and/or a promoter.
  • one or more regulatory nucleotide sequences essential for translation refers to a start codon.
  • start codon refers to a first codon of a RNA translated by a ribosome.
  • start codon may be AUG (when comprised in a RNA sequence).
  • the nucleotide sequence (suitably the at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) may be modified such that the pathogenic viral genome sequence is in an antisense orientation with respect to the promoter when compared to a pathogenic viral genome sequence capable of producing a pathogenic viral protein, so that the nucleotide sequence is incapable of producing a pathogenic viral protein.
  • RNA that is a reverse complement of an pathogenic viral genome sequence capable of being translated to produce a pathogenic viral protein comprises the nucleotides:
  • RNA transcript and ultimately a protein that is structurally and/or functionally equivalent to that of a pathogenic viral genome sequence capable of being translated to produce a pathogenic viral protein.
  • any protein that may be produced may have less than 50% sequence identity to a pathogenic viral protein (preferably less than 25%, 10% or 5% sequence identity to a pathogenic viral protein, more preferably less than 1 % sequence identity to a pathogenic viral protein).
  • a nucleotide sequence that is in an "antisense orientation with respect to the promoter" may be prepared by inverting the orientation of a pathogenic viral genome sequence.
  • the nucleotide sequence (suitably the at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) may be modified such that it comprises one or more nucleotide modifications and modified such that it lacks one or more regulatory nucleotide sequences essential for translation of the pathogenic viral genome sequence, so that the nucleotide sequence is incapable of producing a pathogenic viral protein.
  • the nucleotide sequence (suitably the at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) may be modified such that: a. the pathogenic viral genome sequence is in an antisense orientation with respect to the promoter when compared to a pathogenic viral genome sequence capable of producing a pathogenic viral protein; and
  • nucleotide sequence (suitably the at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereof) modified such that it comprises one or more nucleotide modifications;
  • nucleotide sequence is incapable of producing a pathogenic viral protein.
  • the nucleotide sequence may comprise between about 3 kb to about 9.5 kb contiguous nucleotide sequence of a pathogenic viral genome sequence.
  • the nucleotide sequence may comprise between about 4 kb to about 9 kb of a pathogenic viral genome sequence or a complement thereof.
  • the nucleotide sequence may comprise between about 5 kb to about 9 kb of a pathogenic viral genome sequence or a complement thereof.
  • the nucleotide sequence may comprise between about 6 kb to about 9 kb of a pathogenic viral genome sequence or a complement thereof.
  • the nucleotide sequence may comprise at least about 4 kb or at least about 5 kb of a pathogenic viral genome sequence or a complement thereof.
  • the nucleotide sequence may comprise at least about 6 kb or at least about 7 kb of a pathogenic viral genome sequence or a complement thereof.
  • the nucleotide sequence may comprise or consist of at least 5 kb of a pathogenic viral genome sequence or complement thereof.
  • the nucleotide sequence may comprise about 6.7 kb or about 8.3 kb or of a pathogenic viral genome sequence or a complement thereof.
  • the pathogenic viral genome sequence of the present invention may be obtainable (e.g. obtained) from a pathogenic RNA virus.
  • the pathogenic RNA virus may be a positive sense pathogenic RNA virus.
  • a positive sense pathogenic RNA virus may be one or more selected from the group consisting of: a SARS coronavirus, a MERS coronaviruses, a Dengue virus, a Chikungunya virus, a Ross river virus, a Yellow fever virus, a West Nile virus, a Japanese encephalitis virus, a Zika virus, and an ambisense RNA viral sequences (e.g. Lassavirus).
  • the pathogenic RNA virus may be a negative sense pathogenic RNA virus.
  • a negative sense pathogenic RNA may include viruses causing haemorrhagic fever.
  • a negative sense pathogenic RNA virus may be selected from the group consisting of: Ebolavirus spp. Sudan and Bundibugyo, Marburg virus, Hantaviruses, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Nipah virus and Hendra virus.
  • the pathogenic RNA virus is a negative sense pathogenic virus
  • the RNA virus may be a Marburg virus.
  • a Marburg virus may be one having a genome with GenBank accession number DQ447649.
  • the Ebola virus may be one or more Ebola virus species selected from the group consisting of: Zaire, Bundibugyo, Sudan, Reston and Tai Forest.
  • the Ebola virus genome sequence of the present invention may be obtainable (e.g. obtained) from the Ebola virus species Zaire.
  • Ebola Zaire is taught in Gire et al, Science, 2014, 345(6202), 1369-1372 the contents of which is incorporated herein by reference.
  • the Ebola virus genome sequence of, or for use in, the present invention may be obtainable (e.g. obtained) from an Ebola Zaire Makona 2014 virus (e.g. isolate H.sapiens- wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014 Oct 9;371 (15):1418-25 incorporated herein by reference).
  • an Ebola Zaire Makona 2014 virus e.g. isolate H.sapiens- wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Mag
  • the Ebola virus genome sequence of the present invention may be obtainable (e.g. obtained) from the Ebola virus species Sudan, suitably Ebola Sudan having a genome with GenBank accession number AY729654.
  • nucleotide sequence of the present invention may comprise a pathogenic viral gene or portion thereof.
  • nucleotide sequence of the present invention may comprise at least 2, 3, 4 or 5 pathogenic viral genes or portions thereof
  • the nucleotide sequence of the invention may comprise a pathogenic RNA virus gene or portion thereof of a positive sense pathogenic RNA virus.
  • a positive sense pathogenic RNA virus gene or portion thereof may be one or more selected from the group consisting of: a SARS coronavirus, a MERS coronaviruses, a Dengue virus, a Chikungunya virus, a Ross river virus, a Yellow fever virus, a West Nile virus, a Japanese encephalitis virus, a Zika virus, and an ambisense RNA viral sequences (e.g. Lassavirus).
  • the nucleotide sequence of the invention may comprise a pathogenic RNA virus gene or portion thereof of a negative sense pathogenic RNA virus.
  • a negative sense pathogenic RNA virus gene or portion thereof of a virus causing haemorrhagic fever may be selected from the group consisting of: Ebolavirus spp. Sudan and Bundibugyo, Marburg virus, Hantaviruses, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Nipah virus and Hendra virus.
  • the gene or portion thereof may be obtainable from a Marburg virus gene or portion thereof.
  • a Marburg virus gene or portion thereof may be obtainable from Marburg virus having a genome with GenBank accession number DQ447649.
  • a Marburg virus gene or portion thereof may be selected form the group consisting of: NP, VP35, VP40 and GP.
  • the gene or portion thereof may be an Ebola virus gene or portion thereof.
  • the gene or portion thereof may be obtainable from Ebola Zaire, suitably an Ebola Zaire having a genome with GenBank accession number KJ660346.2.
  • the gene or portion thereof may be obtainable from Ebola Sudan virus, suitably an Ebola Sudan virus having a genome with GenBank accession number AY729654.
  • an Ebola virus gene or portion thereof may be selected form the group consisting of: NP, VP35, VP40, GP/sGP, VP30, VP24 and L.
  • the Ebola virus gene may be NP or a portion thereof.
  • the NP gene of Ebola encodes a RNA binding protein believed to be responsible for genomic packaging.
  • the NP gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 3 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the NP gene may be a nucleotide sequence shown as or encoded by SEQ ID No. 3 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the NP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 4 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the NP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 4 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP35 or a portion thereof.
  • the VP35 gene of Ebola encodes a polymerase cofactor in the RNA polymerase transcription and replication complex.
  • the VP35 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 5 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP35 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 5 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP35 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 6 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP35 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 6 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP40 or a portion thereof.
  • the VP40 gene of Ebola encodes a virus assembly and budding promotion factor.
  • the VP40 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 7 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP40 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 7 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP40 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 8 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP40 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 8 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be GP/sGP or a portion thereof.
  • the GP/sGP gene of Ebola encodes a glycoprotein responsible for binding to receptors on target cells.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 9 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 9 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No.
  • the GP/sGP gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 10 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP30 or a portion thereof.
  • the VP30 gene of Ebola encodes a transcription anti-termination factor.
  • the VP30 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 11 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP30 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 1 1 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP30 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 12 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP30 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 12 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be VP24 or a portion thereof.
  • the VP24 gene of Ebola encodes a membrane-associated protein believed to prevent the establishment of cellular antiviral state by blocking the interferon-alpha/beta and IFN-gamma signalling pathways.
  • the VP24 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 13 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP24 gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 13 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the VP24 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 14 or a polypeptide having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the VP24 gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 14 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus gene may be L or a portion thereof.
  • the L gene of Ebola encodes an polypeptide believed to have RNA-directed RNA polymerase, mRNA guanylyl transferase, mRNA (guanine-N(7)-)-methyltransferase and/or poly(A) synthetase activities.
  • the L gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 15 or a nucleotide sequence having at least 75% identity thereto (suitably at least 80% or 85% identity thereto) or a portion thereof.
  • the L gene or portion thereof may be a nucleotide sequence shown as or encoded by SEQ ID No. 15 or a nucleotide sequence having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the L gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No.
  • the L gene or portion thereof may be a nucleotide sequence encoding a polypeptide shown as SEQ ID No. 16 or a polypeptide having at least 90% identity thereto (suitably at least 95% or 99% identity thereto) or a portion thereof.
  • the Ebola virus sequence may be an Ebola virus non-coding sequence or portion thereof.
  • an Ebola virus non-coding sequence or portion thereof may be selected from the group consisting of: an intergenic region, a 5' UTR, and a 3UTR.
  • the term "portion thereof" as used in this context means at least about 200, 400, 600, 700, 800, 1000, 1200 or 1400 bp.
  • the term "portion thereof” means at least about 1000 bp.
  • the pathogenic viral genome sequence of the invention is operably linked to a promoter.
  • operably linked to a promoter means that the pathogenic viral genome sequence as described herein is situated such that when the promoter is activated the pathogenic viral genome sequence is transcribed. Therefore, the term “operably linked” is intended to exclude scenarios where the pathogenic viral genome sequence described herein is unable to be transcribed upon promoter activation.
  • promoter takes its normal meaning in the art, i.e. a RNA polymerase binding site.
  • the promoter may be any promoter capable of directing expression of a nucleotide sequence or vector of the invention (e.g. in a host cell).
  • the promoter may be any promoter (e.g. an eukaryotic promoter) capable of directing expression of a nucleotide sequence or vector of the invention in a mammalian cell, suitably in a human cell.
  • a promoter of the present invention may be a cytomegalovirus promoter (CMV), an SV40 promoter, an EF1a promoter, a PGK1 promoter, a Ubc promoter, a CAG promoter, a TRE promoter, a UAS promoter, an Ac5 promoter, a Polyhedrin promoter, a CaMKIIa promoter, a GAL1 promoter, a GAL10 promoter, a TEF1 promoter, a GDS promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, an H1 promoter, a U6 promoter or combinations thereof.
  • the promoter of the present invention may be a CMV promoter, preferably a CMV major immediate early promoter.
  • the pathogenic viral genome sequence may be operably linked to a promoter and one or more additional gene regulatory elements.
  • a nucleotide sequence and/or vector of the present invention may comprise one or more additional gene regulatory elements of viral origin.
  • the one or more additional gene regulatory elements are Lentiviral or Gammaretroviral regulatory elements.
  • a Lentiviral or Gammaretroviral vector for use in the present invention comprises one or more selected from the group consisting of: retroviral long terminal repeats (LTR) either wild type or missing the U3 region, packaging signal, Rev responsive element (REV)- required for lentiviral vector, poly purine tract, unique restriction sites for the cloning of the heterologous genes, retroviral long terminal repeats (LTR) either wild type or missing the U5 region and a polyadenylation site.
  • retroviral long terminal repeats either wild type or missing the U3 region
  • packaging signal Rev responsive element (REV)- required for lentiviral vector
  • poly purine tract poly purine tract
  • unique restriction sites for the cloning of the heterologous genes retroviral long terminal repeats (LTR) either wild type or missing the U5 region and a polyadenylation site.
  • a nucleotide sequence of the invention may comprise (or consist of): a. SEQ ID No. 1 or a nucleotide sequence having at least 75% sequence identity to thereto;
  • a nucleotide sequence of the invention may comprise (or consist of): a. SEQ ID No. 1 or a nucleotide sequence having at least 80% sequence identity to thereto;
  • SEQ ID No. 2 or a nucleotide sequence having at least 80% sequence identity to thereto; and/or d. a portion of SEQ ID No. 2 or a portion of a nucleotide sequence having at least 80% sequence identity to thereto.
  • nucleotide sequence of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 85% sequence identity to thereto;
  • nucleotide sequence of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 90% sequence identity to thereto;
  • nucleotide sequence of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 95% sequence identity to thereto;
  • the present invention also provides a vector comprising a nucleotide sequence of the invention.
  • vector as used herein means a construct capable of being transcribed in vivo and/or in vitro.
  • vector e.g. plasmid, cosmid, virus or phage vector, genomic insert, (preferably a plasmid) will often depend on the host cell into which it is to be introduced.
  • the present invention may cover other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.
  • the vector Once transformed into the host cell of choice, the vector may replicate and function independently of the host cell's genome, or may integrate into the genome itself.
  • the vectors may contain one or more selectable marker genes - such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Alternatively, the selection may be accomplished by co- transformation (as described in W091/17243 which is incorporated herein by reference).
  • a vector of the present invention may be a lentiviral vector.
  • the invention provides a method of making nucleotide sequences of the present invention for use in any one of the vectors, host cells, other methods and/or uses of the present invention, by introducing a nucleotide sequence into a replicable vector.
  • methods may further comprise introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • a vector of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 75% sequence identity to thereto;
  • a vector of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 80% sequence identity to thereto; b. a portion of SEQ ID No. 1 or a portion of a nucleotide sequence having at least 80% sequence identity to thereto;
  • SEQ ID No. 2 or a nucleotide sequence having at least 80% sequence identity to thereto;
  • a vector of the invention may comprise (or consist of):
  • SEQ I D No. 1 or a nucleotide sequence having at least 85% sequence identity to thereto;
  • a vector of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 90% sequence identity to thereto;
  • a vector of the invention may comprise (or consist of):
  • SEQ ID No. 1 or a nucleotide sequence having at least 95% sequence identity to thereto;
  • nucleotide sequence and/or vector of the invention may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al., (1981 ) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801 -805.
  • oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
  • the nucleotide sequence and/or vector of the invention may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).
  • the present invention also encompasses sequences that are complementary to the nucleic acid sequences and/or vector of the invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
  • hybridisation shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • the present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
  • variant also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein.
  • the present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
  • the present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
  • polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
  • the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under stringent conditions (e.g. 50°C and 0.2 x SSC). In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under high stringent conditions (e.g. 65°C and 0.1 x SSC).
  • stringent conditions e.g. 50°C and 0.2 x SSC
  • high stringent conditions e.g. 65°C and 0.1 x SSC
  • a nucleotide sequence and/or vector of the invention may be a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.
  • the present invention provides a method for manufacturing a nucleotide sequence and/or vector of the invention comprising: a. providing a nucleotide sequence comprising at least 3 kb contiguous nucleotide sequence of a pathogenic viral genome sequence or complement thereofoperably linked to a promoter;
  • nucleotide sequence i. it comprises one or more nucleotide modifications, so that the nucleotide sequence is incapable of producing a pathogenic viral protein; and/or ii. it lacks one or more regulatory nucleotide sequences essential for translation of the pathogenic viral genome sequence, so that the nucleotide sequence is incapable of producing a pathogenic viral protein.
  • the present invention also provides methods for manufacturing a RNA of the invention.
  • a method for manufacture a RNA comprises synthesising a RNA the equivalent to that encoded by a nucleotide sequence of the invention.
  • a method for manufacturing a RNA comprises expressing a nucleotide sequence or vector according to the invention.
  • expression of the nucleotide sequence or vector can be achieved using any technique known by the person skilled in the art.
  • expression may be carried out in a host cell.
  • methods may comprise transforming a suitable host cell with a nucleotide sequence or vector according to the invention.
  • a host cell for use in a method of the invention may be a mammalian host cell.
  • a human host cell for example a human cell line.
  • the host cell may be a HEK cell, such as a HEK 293 cell.
  • the host cell may be a HEK 293T-17 cell such as ATCC CRL-1 1268 (commercially available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 201 10 USA).
  • the method further comprises isolating said expressed RNA.
  • the present invention provides a RNA (preferably a control RNA) obtainable by a method according to the invention or a synthesised RNA structurally equivalent thereto.
  • the present invention also provides a DNA obtainable from the RNA of the present invention.
  • the DNA may be obtained by standard techniques, such as reverse transcriptase PCR (e.g. as available commercially from ThermoFisher Scientific, USA).
  • a RNA of the invention may be associated with a viral protein to form a ribonucleoprotein.
  • ribonucleoprotein refers to a RNA sequence associated with a protein.
  • the association of RNA and protein may be effected by any suitable means, including, for example, protein-nucleic acid interactions.
  • ribonucleoprotein as used herein may refer to a RNA-protein complex.
  • a nucleic acid of, or for use in, the present invention e.g. comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof;
  • preparation of a ribonucleoprotein may be carried out in a host cell.
  • a human host cell for example a human cell line.
  • the host cell may be a HEK cell, such as a HEK 293 cell.
  • the host cell may be a HEK 293T-17 cell such as ATCC CRL-1 1268 (commercially available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 201 10 USA).
  • RNA with a viral protein may be achieved by admixing one or more viral proteins with a RNA of the invention.
  • the association may occur in vivo (e.g. in a host cell).
  • preparation of a ribonucleoprotein may comprise co-expressing a viral protein (e.g. a viral protein encoded by one or more plasmid(s)) with a nucleic acid of, or for use in, the present invention (e.g. comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof).
  • a method for manufacturing a ribonucleoprotein of the invention may comprise expressing one or more viral protein(s), suitably co-expressing one or more viral protein(s) in a host cell or in vitro (preferably in a host cell) to produce a ribonucleoprotein comprising a RNA of the invention and one or more viral protein(s).
  • said method may further comprise isolating said ribonucleoprotein comprising a RNA of the invention and one or more viral protein(s) (e.g. using a sucrose gradient).
  • the present invention relates to a ribonucleoprotein comprising a RNA of the invention and one or more viral protein(s) as well as its uses.
  • a viral protein may be a Lentiviral protein (e.g. an HIV viral protein) and/or a Gammaretroviral protein.
  • the viral protein may be a structural viral protein (e.g. a nucleoprotein).
  • the viral protein may be a gag protein (preferably a Lentiviral gag protein) or a pol protein (preferably a Lentiviral pol protein).
  • the ribonucleoprotein of the invention may be a virus comprising a nucleotide sequence, vector and/or RNA of the invention (suitably Lentivirus or a Gammaretro virus) .
  • the ribonucleoprotein of, or for use in, the present invention may be prepared using a Lentiviral system or a Gammaretroviral system (more preferably a Lentiviral system).
  • the ribonucleoprotein may be a virus, suitably an RNA virus (preferably a recombinant Lentivirus or Gammaretrovirus).
  • compositions comprising a nucleotide sequence, vector, RNA, DNA and/or ribonucleoprotein of the present invention.
  • a composition of the invention may comprise a buffer, suitably a buffer that stabilises a nucleotide sequence, vector, RNA, DNA and/or ribonucleoprotein of the present invention and/or that is compatible with performing a PCR reaction.
  • a composition according to the present invention may be formulated into a high or low titre composition.
  • a high titre composition may comprise a high concentration of a nucleotide sequence, vector, RNA, DNA and/or ribonucleoprotein of the present invention (preferably a high concentration of a ribonucleoprotein of the present invention).
  • a low titre composition may comprise a low concentration of a nucleotide sequence, vector, RNA, DNA and/or ribonucleoprotein of the present invention (preferably a high concentration of a ribonucleoprotein of the present invention).
  • a high titre composition as used herein may refer to a composition comprising a nucleotide sequence, vector, RNA, DNA and/or ribonucleoprotein of the present invention in a concentration that can be titrated in a qPCR reaction using a standard curve.
  • a low titre composition as used herein may refer to a composition comprising a nucleotide sequence, vector, RNA, DNA and/or ribonucleoprotein of the present invention in a concentration that cannot be titrated in a qPCR reaction using a standard curve e.g. which is at or below the limit of detection sensitivity of qPCR.
  • a composition of the invention may comprise universal buffer (e.g. 10mM Tris-HCI pH 7.4, 0.5% Serum Albumin (e.g. human serum albumin - Bio Produces Laboratory Limited), 0.1 % D-(+)-trehalose dehydrate (Sigma)), preferably an isovolume thereof (e.g. an equal volume of universal buffer).
  • universal buffer e.g. 10mM Tris-HCI pH 7.4, 0.5% Serum Albumin (e.g. human serum albumin - Bio Produces Laboratory Limited), 0.1 % D-(+)-trehalose dehydrate (Sigma)
  • an isovolume thereof e.g. an equal volume of universal buffer.
  • composition may be freeze-dried and/or desiccated.
  • the present invention also provides a kit comprising a nucleotide sequence, a vector, a RNA, a DNA, a ribonucleoprotein or a composition according to the invention and instructions for using same.
  • the kit may comprise one or more further reagents.
  • the kit may comprise one or more nucleic acid detection reagents.
  • the kit may comprise one or more nucleic acid detection reagent selected from the group consisting of: a primer, a probe, a polymerase and a buffer (e.g. a reaction buffer for a polymerase).
  • the invention provides a method for screening for a virus comprising: a. providing a nucleotide sequence, a RNA, a DNA, a ribonucleoprotein, a composition or a kit according to the invention; and
  • the present invention also relates to a method for detecting the presence or absence of a pathogenic virus comprising:
  • control sample comprises:
  • RNA extracted from a recombinant RNA virus wherein:
  • said recombinant RNA virus comprises a control RNA sequence for identification of the presence or absence of a pathogenic virus
  • control RNA sequence comprises at least 3 kb contiguous nucleotide sequence of said pathogenic virus or a complement thereof;
  • the recombinant RNA virus is a Lentivirus or a Gammaretrovirus
  • the pathogenic virus is a pathogenic RNA virus
  • step b. comparing a result obtained in step b. for the experimental sample and the control sample.
  • the method for detecting the presence or absence of a pathogenic virus may refer to detecting the presence or absence of an RNA virus.
  • the pathogenic RNA virus may be a positive sense pathogenic RNA virus.
  • a positive sense pathogenic RNA virus may be one or more selected from the group consisting of: a SARS coronavirus, a MERS coronaviruses, a Dengue virus, a Chikungunya virus, a Ross river virus, a Yellow fever virus, a West Nile virus, a Japanese encephalitis virus, a Zika virus, and an ambisense RNA viral sequences (e.g. Lassavirus).
  • the pathogenic RNA virus may be a negative sense pathogenic RNA virus.
  • a negative sense pathogenic RNA may include viruses causing haemorrhagic fever.
  • a negative sense pathogenic RNA virus may be selected from the group consisting of: Ebolavirus spp. Sudan and Bundibugyo, Marburg virus, Hantaviruses, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, Nipah virus and Hendra virus.
  • the negative sense pathogenic RNA virus may be a Marburg virus.
  • the Ebola virus may be one or more Ebola virus species selected from the group consisting of: Zaire, Bundibugyo, Sudan, Reston and Tai Forest.
  • the Ebola virus may be obtainable (e.g. obtained) from the Ebola virus species Zaire.
  • Ebola Zaire is taught in Gire et al, Science, 2014, 345(6202), 1369-1372 the contents of which is incorporated herein by reference.
  • the Ebola virus may be obtainable (e.g. obtained) from an Ebola Zaire Makona 2014 virus (e.g. isolate H.sapiens-wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014 Oct 9;371 (15):1418-25 incorporated herein by reference).
  • an Ebola Zaire Makona 2014 virus e.g. isolate H.sapiens-wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence
  • test sample refers to a sample to be tested.
  • the experimental sample may be obtained from a test subject (preferably a human test subject).
  • the experimental sample may be obtained from a biological fluid (biofluid) sample or a fraction thereof.
  • the biofluid sample may be a urine sample, a blood sample, a cerebrospinal fluid sample, a lymph sample, combinations thereof or a fraction thereof.
  • the biofluid sample may be a urine sample or a fraction thereof.
  • the biofluid sample may be a saliva sample or a fraction thereof.
  • the biofluid sample may be a blood sample or a fraction thereof.
  • blood as used herein comprises whole blood, blood serum (henceforth “serum”) and blood plasma (henceforth “plasma”).
  • Serum and plasma are derived from blood and thus may be considered as specific subtypes within the broader genus "blood”.
  • Processes for obtaining serum or plasma from blood are known in the art.
  • blood can be subjected to centrifugation in order to separate red blood cells, white blood cells, and plasma.
  • Serum is defined as plasma that lacks clotting factors. Serum can be obtained by centrifugation of blood in which the clotting process has been triggered. Optionally, this can be carried out using specialised centrifuge tubes designed for this purpose.
  • the biofluid sample for use in the present invention may not have undergone any processing or may have only undergone minimal processing after being obtained from a test subject.
  • the term "fraction thereof" when used herein in the context of biofluid fractions refers to a portion of a biofluid fraction obtainable or obtained following processing of a biofluid.
  • a biofluid fraction refers to one or more constituent(s) of a biofluid that has been separated from one or more further biofluid constituent(s).
  • a fraction of a blood sample may be a blood plasma fraction.
  • control sample for use in the present invention may comprise (or consist of) an isolated recombinant RNA extracted from a recombinant RNA virus of the invention, a nucleotide sequence, vector, RNA, DNA, ribonucleoprotein, composition or kit according to the present invention.
  • control sample comprises (or consists of) an isolated recombinant RNA extracted from a recombinant RNA virus of the invention
  • control sample may comprise (or consist of) a DNA obtainable from an RNA of the invention.
  • control sample may comprise (or consist of) an DNA of the present invention.
  • an isolated DNA of the present invention may comprise (or consist of) a RNA of the present invention.
  • an isolated RNA of the present invention e.g. isolated using a total nucleic acid extraction kit (Roche Diagnostics) in a COBAS® Ampliprep Instrument (Roche Diagnostics)).
  • the methods for screening may therefore comprise a sample processing step, wherein the experimental sample and/or control sample are processed to isolate a DNA and/or RNA.
  • the method for detecting the presence or absence of a pathogenic virus may additionally or alternatively comprise a step whereby DNA is obtained from RNA.
  • DNA may be obtained from RNA by a reverse transcriptase PCR step.
  • said DNA may be subsequently isolated.
  • the method may further comprise screening the experimental sample and the control sample for the presence or absence of a pathogenic viral nucleotide sequence.
  • the screening may be carried out using any method in the art suitable for detecting the presence or absence (preferably presence) of a DNA and/or RNA molecule.
  • the screening may be carried out using PCR, suitably quantitative PCR, preferably quantitative reverse-transcriptase PCR (qRT-PCR).
  • PCR suitably quantitative PCR, preferably quantitative reverse-transcriptase PCR
  • kits are available to assist the skilled person to perform qRT-PCR, for example the RNA UltraSense One-Step Quantitative RT-PCR system (available from Life Technologies) and/or Realstar Filovirus Screen RT-PCR Kit 1.0 (Altona Diagnostics). Where qRT-PCR is used, the skilled person will typically use a specially-designed instrument for quantification of results.
  • Suitable instruments include the Mx3005p instrument (Stratagene) and analysis software such as MxPro v.4.1 (Stratagene) or the LightCycler 480 II (Roche Diagnostics) and LightCycler 480 v.1.5.62 software (Roche).
  • the methods of screening may further comprise a step of comparing a result obtained in the screening step for the experimental sample and the control sample.
  • the comparison may be effected using suitable quantification software (e.g. as described above).
  • a result which is positive for the presence of a pathogenic viral nucleotide sequence in the control sample is indicative of a functional assay.
  • a positive result in the control sample and a negative result in an experimental sample provides a user with an indication that a pathogenic viral diagnostic is functional and that the experimental sample does not contain a pathogenic viral nucleotide.
  • the method of the invention reduces the incidence of false negatives.
  • the method for detecting the presence or absence of a pathogenic virus may further comprise recording the output of at least one step on a data-storage medium.
  • the method of the present invention can generate data relating to the experimental sample and/or control sample, such data being recordable on a data-storage medium (for example, a form of computer memory such as a hard disk, compact disc, floppy disk, or solid state drive).
  • data may comprise (or consist of) data relating to detection of the absence or presence of a pathogenic virus nucleotide and/or the absence or presence of a pathogenic virus and/or quantification data related to the absence or presence of a pathogenic virus nucleotide sequence.
  • the invention therefore also provides a data-storage medium comprising data obtained by a method of the present invention.
  • the percent identity is then calculated as: Total number of identical matches
  • Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • conservative amino acid substitutions see below
  • small amino- or carboxyl-terminal extensions such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • Aromatic phenylalanine
  • non-standard amino acids such as 4- hydroxyproline, 6-/V-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues.
  • the polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo- threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro- glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.
  • an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 1 13:2722, 1991 ; Ellman et al., Methods Enzymol.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci.
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
  • related components e.g. the translocation or protea
  • RNA comprising a control RNA sequence extracted from a recombinant RNA virus of the invention is particularly useful as a control (e.g. positive control) in a screen for identification of the presence or absence of a pathogenic virus.
  • the isolated RNA reduces the incidence of false negatives when compared to use of diagnostic screens not incorporating the use of an isolated RNA of the invention.
  • the methods (and/or products of the invention) provide a generic standard to be used for viral diagnostics.
  • this is achieved via the use of a RNA comprising at least 3 kb contiguous nucleotide sequence of a pathogenic virus or a complement thereof.
  • the standard is generic (e.g. for a particular viral species/isolate) since, due to the coverage of the genome, it is not necessary to know with which primers/probes a user will choose to perform a diagnostic assay for the virus.
  • RNA of the invention Use of a recombinant Lentiviral and/or Gammaretroviral for housing a RNA of the invention have advantageously been shown to be stable at ambient temperatures (or higher) (especially when compared to a naked e.g. unpackaged RNA), thus, allowing for ease of handling and/or reduced transmission and/or shipping costs. Similar advantages are associated with the ribonucleoprotein of the invention. Therefore the invention provides for a more accurate and/or reliable control when compared to less stable compositions.
  • a recombinant Lentiviral and/or Gammaretroviral for housing a RNA of the invention allows for the easy establishment of the relative potency of each viral product (e.g. by using Lentiviral and/or Gammaretroviral sequences comprised on the RNA housed therein). Alternatively or additionally this ensures that reference materials are produced in equimolar ratios. Advantageously this enables the relative sensitivity of viral diagnostic assays targeting any region of the viral genome to be established.
  • nucleotide, vector, RNA, DNA or ribonucleoprotein according to the present invention can be used reliably and accurately as a control for pathogenic viral screening.
  • the nucleotide sequence (including the vector, RNA, DNA, ribonucleoprotein, composition or kit derived or obtainable therefrom) of the invention allows for a positive control to determine if a pathogenic viral diagnostic assay is functioning and thus reduces the incidence of false negatives.
  • nucleotide sequence including the vector, RNA, DNA, nbonucleoprotein, composition or kit derived or obtainable therefrom - preferably the nbonucleoprotein and/or RNA and/or DNA
  • an extraction step preferably the nbonucleoprotein and/or RNA and/or DNA extraction step.
  • the method and/or nucleotide sequence (including the vector, RNA, DNA, nbonucleoprotein, composition or kit derived or obtainable therefrom) of the invention suitably allows for the provision of materials for calibrating secondary standards and/or materials for use as in-run controls.
  • the nucleotide sequence (including the vector, RNA, DNA, nbonucleoprotein, composition or kit derived or obtainable therefrom) is safe since the nucleotide sequence (including the vector, RNA, DNA or nbonucleoprotein derived or obtainable therefrom) is incapable of producing a pathogenic viral protein as a result of the modification(s) disclosed herein and is therefore safe and/or non-infectious and/or non-replicative.
  • the products of the invention can be handled (or the methods of the invention carried out) with minimal health risk to a user (e.g. by infection with a pathogenic virus).
  • the present invention advantageously allows for the production of a control for use in diagnostic assays for newly-emerging pathogenic viral strains/isolates.
  • the reference material can be produced using the approach described herein in a relatively short amount of time, advantageously allowing the medical community to react quickly to new pathogenic viral strains/isolates.
  • Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
  • protein includes proteins, polypeptides, and peptides.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.
  • polypeptide proteins and polypeptide are used interchangeably herein.
  • the conventional one-letter and three-letter codes for amino acid residues may be used.
  • the 3-letter code for amino acids as defined in conformity with the lUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
  • Ebola virus gene nucleotide sequences were derived from Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Kissidougou-C15, GenBank accession number KJ660346.2 (Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014 Oct 9;371 (15): 1418-25 incorporated herein by reference). Sequences were modified to contain random stop codons and enzymatic restriction sites at 5' end and 3' end as illustrated in Fig 1A. Each gene was synthesised by GeneWiz Inc.
  • Lentiviral vector pSF- lenti-PGK-FLuc is a customised version of the pSF-lenti (OG269, Oxford Genetics) in which the Cytomegalovirus (CMV) major immediate early promoter (MIEP) in front of the multi cloning site has been removed and the Puromycin resistance gene has been substituted with the reporter gene Firefly luciferase.
  • CMV Cytomegalovirus
  • MIEP Cytomegalovirus
  • MIEP Cytomegalovirus
  • MIEP Cytomegalovirus
  • Each Ebola virus gene was sequentially subcloned from pUC57-Kan plasmid into the pSF-lenti-PGK-Fluc using standard molecular techniques.
  • Lentiviral particles were generated by transfection of 5x10 6 HEK 293T-17 (ATCC CRL- 1 1268) cells in a 10cm dish with a mixture of 18 of FuGene-6 transfection reagent (Promega) and 1.5 ⁇ g of p8.9 (Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997 Sep;15(9):871-5 - incorporated herein by reference) and 2 ⁇ g of pSF-lenti-NP-VP35-GP or pSF-lenti-VP40-L in 200 uL of Optimem (Gibco).
  • DM EM Dulbecco modified essential media
  • PAA foetal calf serum
  • Particles were purified by ultracentrifugation on a 20% sucrose cushion in 50mM sodium phosphate buffer using a SW28 rotor in a Beckman Optima LE-80K Ultracentrifuge for 2 hours at 23,000rpm at 4°C. Pellets were resuspended in an isovolume of universal buffer, composed of 10mM Tris-HCI pH 7.4, 0.5% Human Serum Albumin (Bio Products Laboratory Limited), 0.1 % D-(+)-trehalose dehydrate (Sigma). High titre preparations were obtained by dilution 1 : 10 of the purified particles in universal buffer; low titre preparations were produced by further 1 : 10,000 dilution of the high titre material in universal buffer. All preparations were kept refrigerated overnight and were freeze/dried within 24 hours. Freeze-drying procedure
  • the final preparations were aseptically dispensed in 1 ml_ aliquots into 5 ml_ Schott vials (high titre) or 5 ml_ DIN ampoules (low titre). Stoppers were partially inserted into the containers and the trays of containers loaded onto precooled (-50°C) shelves in a Virtis Advantage Plus freeze dryer (Biopharma Process Systems) for the high titre preparations or in a CS-100 freeze dryer (Serail) for the low titre ones. The same freeze- drying schedule was used for both freeze-driers. For initial freezing, the products were held at -50°C for 5 h, followed by a further 1 h at -50°C at 0.1 mbar to begin primary drying.
  • the temperature was then ramped up to -20°C over 1 hour and held at -20°C for a further 30 h. Secondary drying proceeded by ramping the temperature up to 25°C over 10 h followed by holding at 25°C for a further 15 h at 0.03 mbar.
  • the containers were backfilled with dry nitrogen gas and the stoppers fully inserted into the containers.
  • the ampoules were flame-sealed.
  • the sucrose-purified supernatant preparation of the LVV_NP-VP35-GP particles was 10-fold serially diluted in PBS to achieve a particle concentration of approximately 10 8 particles/mL.
  • Reconstituted vials of the freeze/dried preparations LVV_NP-VP35-GP high and LW_VP40- L high were 10-fold diluted in universal buffer.
  • Samples were analysed using a NanoSight LM10 instrument (Malvern). Samples were injected using a 1 ml_ syringe loaded in the NanoSight syringe pump. Each sample dilution was acquired 5 times per 90-second/time and analysed using Nanoparticle Tracking Analysis 2.3 Analytical software. Before each acquisition 100nm polystyrene latex microspheres, diluted in the same buffer of the samples, were used to calibrate the machine.
  • the four freeze-dried HIV-EBOV RNA preparations were reconstituted by adding 1 mL of molecular-grade water.
  • high titre preparations were 10-fold serially diluted in universal buffer.
  • the 3 rd HIV-1 international standard (NIBSC code 10/152) was reconstituted in 1 mL of molecular water as per Instruction for use, and 5-fold serially diluted in universal buffer prior to extraction.
  • 140 of each sample were extracted using QIAamp viral RNA mini kit (Qiagen) following manufacturer's instructions, and eluted in 60 AE buffer.
  • RNA UltraSense One-Step Quantitative RT- PCR system Life Technologies
  • All the reactions were performed using RNA UltraSense One-Step Quantitative RT- PCR system (Life Technologies), following manufacturer's instructions and adding 10 uL of the extracted viral RNA, 0.2 ⁇ of each primer and 0.1 ⁇ of probe (Table 1).
  • Reactions were run on a Mx3005p instrument (Stratagene) for 30 minutes at 50°C, 10 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C and 90 seconds at 60°C, and analysed using MxPro v.4.1 software.
  • 0.85 mL of each sample was extracted using total nucleic acid extraction kit (Roche Diagnostics) in a COBAS® Ampliprep Instrument (Roche Diagnostics), with an elution volume of 75 ⁇ .
  • Ebola virus np target gene 10 ⁇ of each extract was added to * ⁇ ⁇ - of LIPSGENE ZEBOV kit oligonucleotides (Bioactiva Diagnostica GmbH), 5 ⁇ AB TaqMan polymerase (Applied Biosciences) and 4 ⁇ molecular grade water in a 96-well micro-titre plate (Roche Diagnostics) and run on a LightCycler 480 II (Roche Diagnostics) for 5 minutes at 50°C, 20 seconds at 95°C, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
  • LIPSGENE ZEBOV kit oligonucleotides Bioactiva Diagnostica GmbH
  • 5 ⁇ AB TaqMan polymerase Applied Biosciences
  • 4 ⁇ molecular grade water 4 ⁇ molecular grade water in a 96-well micro-titre plate (Roche Diagnostics) and run on a LightCycler 480 II (Roche Diagnostics) for 5 minutes at 50°C, 20 seconds at 95°C,
  • Viral RNA extracted for the in-house assays was also analysed by droplets digital reverse transcriptase polymerase chain reaction (ddRT-PCR). 2 ⁇ _ of each sample were added to 12.5 ⁇ _ of One-Step RT-ddPCR kit for probes (Bio-Rad) with 0.9 ⁇ of each primer and 0.125 ⁇ of probe (Table 1), 1 ⁇ _ of 25mM manganese acetate and molecular-grade water to a final volume of 25 ⁇ _. To generate the droplets, 20 ⁇ _ of these solutions were pipetted in Droplet Generator DG8 Cartridge (Bio-Rad) together with 70 ⁇ _ of droplet generator oil for probes and loaded in the QX100 Droplet Generator (Bio-Rad).
  • ddRT-PCR digital reverse transcriptase polymerase chain reaction
  • the entire droplet emulsion volume was then loaded in a twin. tec semi-skirted 96-well PCR plate (Eppendorf) and heat sealed with pierceable foil in the PX1TM PCR Plate Sealer and placed in a C1000 TouchTM Thermo Cycler (both from Bio-Rad). Thermal cycling conditions were: 30 minutes at 60°C, 5 minutes at 95°C, followed by 45 cycles of 30 seconds at 94°C and 1 minute at 60°C, and a final step of 10 minutes at 98°C.
  • the droplets were read in a QX100TM droplet reader (Bio- Rad), and analysed using QuantaSoftTM software version 1.7.4.
  • Zaire Makona 2014 Ebola virus sequences encoding nucleoprotein (np), viral protein 35 (vp35), glycoprotein (gp), viral protein 40 (vp40) and / genes were in vitro synthesised and sub-cloned into a lentiviral vector derived from pSF-lenti (Oxford Genetics) using the restriction enzymes as indicated in Figure 1A. Due to the size limitation of the insert that can be cloned into a lentiviral vector (about 9 kilo bases), two vectors were produced containing either np-vp35-gp genes or vp40-l gene sequences.
  • each Ebola virus gene lacks its start codon, and contains 3 nucleotide changes which introduce stop codons. Furthermore, the Ebola virus genes have been sub-cloned in reverse orientation in relation to the CMV promoter, driving the expression of the lentiviral genomic RNA.
  • the two lentiviral vectors carrying Ebola virus genes were sequenced using lllumina sequencing technology (GenBank KT186367 for pSF- lenti-NP-VP35-GP and KT186368 for pSF-lenti-VP40-L).
  • Lentiviral particles containing Ebola virus RNA were produced by transfection of 293T cells with pSF_NP-VP35-GP or pSF_VP40-L together with a packaging plasmid expressing HIV-1 gag and pol genes. Antisense Ebola virus RNA was packaged within the HIV-like particles. To increase biosafety of the system, the long terminal repeats in the lentiviral vectors are defective ( ⁇ 3), an internal promoter is missing and no envelope protein is expressed in the transfected cells, rendering HIV-like particles non-infectious.
  • NTA nanoparticle tracking analysis
  • Universal buffer is a TRIS- HCI solution containing trehalose and human serum albumin; this has been previously used for lyophilised viral NAT reference materials that may be diluted in a number of different clinical matrices (Fryer JF, Heath AB, Anderson R, Minor PD, World Health Organization. Biologicals Unit, Collaborative Study Group.
  • a "high” titre standard obtained by diluting the viral particle stock 1 : 10 in Universal buffer
  • a "low” titre control obtained by diluting the "high” titre preparations 1 :10,000 in the same buffer.
  • the four preparations were filled in 1 mL aliquots and freeze-dried following validated standard operating procedures. The appearance of the final products is a white compact cake.
  • the final freeze-dried preparations were evaluated by quantitative RT-PCR (qRT-PCR) using in-house and commercially available assays.
  • qRT-PCR quantitative RT-PCR
  • two vials for each standard were reconstituted using 1 mL of molecular grade water, and viral RNA extracted using commercially available kits.
  • In-house assays were developed using published primers and probe sequences for the Ebola virus np gene (Trombley AR, Wachter L, Garrison J, Buckley- Beason VA, 1967ling J, Hensley LE, et al. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.
  • RealStar® Ebolavirus RT-PCR kit (Altona Diagnostics)-authorised by the Food and Drug administration for emergency use (Hamburg MA, commissioner of Food and Drugs. FDA letter authorising the emergency use of RealStar Ebolavirus RT-PCR kit 1.0, Altona Diagnostic GmBH. 2014 Nov 16 - incorporated herein by reference), and targeting Ebolavirus / gene, and LIPSGENE ZEBOV kit (Bioactiva Diagnostica GmbH) targeting the np gene.
  • Reconstituted freeze-dried preparations were evaluated after three independent extractions using either in-house or commercially available quantitative RT-PCR probe based kits targeting the Ebola virus np or / gene. Samples were run in duplicate and the results expressed as average of the threshold cycle (Ct) ⁇ standard deviation.
  • HIV-EBOV RNA preparations were also analysed using another NAT-based assay, droplet digital RT-PCR (ddRT-PCR) using the same sets of primers and probe employed for the qRT-PCR; this method is based on Poisson distribution and can provide an absolute copy number in absence of a calibrator.
  • ddRT-PCR droplet digital RT-PCR
  • Each sample was analysed using two sets of primers and probe, one targeting the HIV LTR and one for the Ebola virus gene np.
  • Serial dilutions of the high titre HIV-EBOV RNA standard confirmed the linearity of the assay for both set of primers and probe.
  • Table 3 are summarised the values obtained for the HIV-EBOV RNA preparations in the different assays, expressed in the readout units of each assay. Where results are reported as 'copies/ml_', the relationship to genuine genome equivalence numbers is unknown. Copies reported in one assay are not necessarily equivalent to copies reported in another. Furthermore, there is no conversion factor between International Units/mL or copies/mL. In all cases the ratio between the high titre and the low titre for each preparation is about 10,000-fold, as expected. All the samples are detectable using both qRT-PCR and ddRT- PCR technologies using different set of primers and probe with results in the same order of magnitude. The high concentration preparations were also analysed by NTA in universal buffer. The particle concentrations were about 10 times higher than values estimated with molecular methods (Table 3) suggesting that about 10% of the particles incorporate HIV- EBOV viral genome. Table 3. Examples of the HIV-Ebola virus RNA standard values using different technologi
  • Quantitative RT-PCR was performed in duplicate in three independent experiments using HIV-LTR specific primers and probe; samples were quantified against the 3 rd HIV-1 International standard (assigned value 185 000 lU/mL) run in parallel. The same samples and set of primers and probe were used in a droplet digital RT-PCR (ddRT-PCR) and results expressed as average of two independent experiments run in duplicate. HIV- EBOV RNA preparations were also quantified using Ebola virus NP or L specific primers and probes (qRT-PCR np/l) and copies per mL were inferred using standard curves obtained by serial dilutions of the lentiviral plasmids used to produce the particles.
  • NP-specific primers and probe were also used in a ddRT-PCR. HIV-EBOV RNA high titre were also analysed by NTA. Results are reported as average concentrationistandard deviation calculated on 5 acquisitions of a 1 : 10 dilution in universal buffer. Where results are reported as 'copies/mL, the relationship to genuine genome equivalence numbers is unknown.
  • Nucleic acid amplification based diagnostic techniques have a crucial role during the ongoing Ebola virus outbreak in West Africa. Reference materials are needed to assess the validity of the assays used, to compare results across assays and to provide guidance to the regulatory agencies in the evaluation of new assays. It is crucially important that Ebola virus NAT reference materials standardise and control the entire process from the extraction to the final amplification and detection reaction.
  • the cloned Ebola virus gene sequences were designed to lack the start codon and contained random stop codons, ensuring that full length Ebola virus protein could not be produced.
  • the plasmids used to generate the particles were fully sequenced.
  • freeze-dried HIV-EBOV RNA reference materials were stable at temperatures up to 37°C for 2 weeks, making them suitable for shipping at ambient temperature.
  • the lentiviral packaging system represents a safe, stable and rapid tool to create reference materials for highly pathogenic RNA viruses which can also be employed in the face of future outbreaks.
  • Marburg virus nucleotide sequences for genes np-vp35-gp are derived from Lake Victoria isolate (GenBank acc. number DQ447649; Towner.J.S et al Marburg virus genomics and association with a large hemorrhagic fever outbreak in Angola; J Virol. 2006, 80(13): 6497- 6516). Sequences are modified to contain random stop codons and enzymatic restriction sites at 5' end and 3' to allow for the subcloning. Each gene is synthesised by GeneWiz Inc. and cloned into pUC57-Kan plasmid.
  • Lentiviral vector pSF-lenti-PGK-FLuc is a customised version of the pSF-lenti (OG269, Oxford Genetics). Each Marburg virus gene is sequentially subcloned from pUC57-Kan plasmid into the pSF-lenti-PGK-Fluc using standard molecular techniques. Final plasmid sequences pSF-MARV_NP-VP35-GP are confirmed by sequencing using Nextera XT kit (lllumina) following manufacturer's instructions, and run on an MiSeq 2 ⁇ 251 paired-end v2 Flow Cell (lllumina). Results are analysed using Genious R7 version 7.1.7(Biomatters).
  • Lentiviral particles are generated by transfection of 5x10 6 HEK 293T-17 (ATCC CRL-1 1268 ) cells in a 10cm dish with a mixture of 18 ⁇ _ of FuGene-6 transfection reagent (Promega) and 1.5 ⁇ g of p8.9 and 2 ⁇ g of pSF-MARV-NP-VP35-GP in 200 uL of Optimem (Gibco). After 20 minutes at room temperature, the mixture is added drop-wise to the cells in 8 ml_ of Dulbecco modified essential media (DM EM, Gibco) supplemented with 10% foetal calf serum (PAA) and the cells are cultivated at 37°C with 5% C02.
  • DM EM Dulbecco modified essential media
  • PAA foetal calf serum
  • High and low titre reference material preparation are purified, diluted and freeze-dried as described for Zaire ebolavirus (e.g. in Example 2) and as detailed in the Materials and Methods section "Freeze-drying procedure" herein. Preparations are tested upon reconstitution by adding 1 mL of molecular-grade water. High titre preparations are 10-fold serially diluted in universal buffer.
  • each sample 140 ⁇ of each sample are extracted using QIAamp viral RNA mini kit (Qiagen) following manufacturer's instructions, and eluted in 60 ⁇ AE buffer. All the reactions are performed using RNA UltraSense One-Step Quantitative RT-PCR system (Life Technologies), following manufacturer's instructions and adding 10 uL of the extracted viral RNA, 0.2 ⁇ of primers F1788 (5 -TTATATGCTCAGGAAAAGAGA CAGG-3') and R1864 (5 -CCAATACTGCCAAAGGGATCTTG-3') and 0.1 ⁇ of probe (FAM- CCCATACAGCATCCAGCCGTGAGC-BHQ) as previously described (Trombley A.R. et al.
  • Lentiviral vector containing Ebola Sudan virus np-vp35-gp genes Lentiviral vector containing Ebola Sudan virus np-vp35-gp genes.
  • Ebola Sudan virus nucleotide sequences for genes np-vp35-gp are derived from Sudan ebolavirus strain Gulu (GenBank acc. number AY729654; Sanchez, A. and Rollin.P.E. Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Kenya; Virus Res. 2005, 1 13(1): 16-25). Sequences are modified to contain random stop codons and enzymatic restriction sites at 5' end and 3' to allow for the subcloning. Each gene is synthesised by GeneWiz Inc. and cloned into pUC57- Kan plasmid.
  • Lentiviral vector pSF-lenti-PGK-FLuc is a customised version of the pSF-lenti (OG269, Oxford Genetics).
  • Each Ebola Sudan virus gene is sequentially subcloned from pUC57-Kan plasmid into the pSF-lenti-PGK-Fluc using standard molecular techniques.
  • Final plasmid sequences pSF-SEBOV_NP-VP35-GP are confirmed by sequencing using Nextera XT kit (lllumina) following manufacturer's instructions, and run on an MiSeq 2 ⁇ 251 paired- end v2 Flow Cell (lllumina). Results are analysed using Genious R7 version 7.1.7(Biomatters).
  • Lentiviral particles are generated by transfection of 5x10 6 HEK 293T-17 (ATCC CRL-1 1268 ) cells in a 10cm dish with a mixture of 18 ⁇ _ of FuGene-6 transfection reagent (Promega) and 1.5 ⁇ g of p8.9 and 2 ⁇ g of pSF-SEBOV-NP-VP35-GP in 200 uL of Optimem (Gibco). After 20 minutes at room temperature, the mixture is added drop-wise to the cells in 8 ml_ of Dulbecco modified essential media (DM EM, Gibco) supplemented with 10% foetal calf serum (PAA) and the cells are cultivated at 37°C with 5% C02.
  • DM EM Dulbecco modified essential media
  • PAA foetal calf serum
  • High and low titre reference material preparation are purified, diluted and freeze-dried as described for Zaire ebolavirus (e.g. in Example 2) and as detailed in the Materials and Methods section "Freeze-drying procedure" herein. Preparations are tested upon reconstitution by adding 1 mL of molecular-grade water. High titre preparations are 10-fold serially diluted in universal buffer.
  • each sample 140 ⁇ of each sample are extracted using QIAamp viral RNA mini kit (Qiagen) following manufacturer's instructions, and eluted in 60 ⁇ AE buffer. All the reactions are performed using RNA UltraSense One-Step Quantitative RT-PCR system (Life Technologies), following manufacturer's instructions and adding 10 uL of the extracted viral RNA, 0.2 ⁇ of primers F1051 (5'- CAT GCA GAA CAA GGG CTC ATT C-3') and R1 130 (5'- CTC ATC AAA CGG AAG ATC ACC ATC-3')and 0.1 ⁇ of probe (FAM- CAA CTT CCT GGC AAT-BHQ) as previously described (Trombley A.R. et al.

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Abstract

La présente invention concerne un procédé de préparation d'un témoin à utiliser dans un dépistage de virus pathogène comprenant : a. l'extraction d'acide ribonucléique (ARN) sur un virus à ARN recombinant, où : i. ledit virus à ARN recombinant comprend une séquence d'ARN témoin pour l'identification de la présence ou de l'absence d'un virus pathogène à ARN ; ii. la séquence d'ARN témoin comprend au moins une séquence nucléotidique de 3 kb continue dudit virus pathogène à ARN ou de son complément ; et iii. le virus recombinant à ARN est un lentivirus ou un gammaretrovirus ; et b. la préparation d'une séquence recombinante isolée de virus à ARN. L'invention décrit également les utilisations, les séquences nucléotidiques, les vecteurs, les acides ribonucléiques, les acides désoxyribonucléiques, les compositions, les kits, et les procédés de fabrication et de détection de la présence ou de l'absence d'un virus pathogène.
PCT/GB2015/052935 2015-10-07 2015-10-07 Procédé de préparation d'un témoin à utiliser dans un dépistage de virus pathogène WO2017060662A1 (fr)

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CN107245532A (zh) * 2017-07-19 2017-10-13 湖北出入境检验检疫局检验检疫技术中心 一种鲫鱼冠状病毒hb93的数字rt‑pcr检测引物及应用
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