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  • 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
• • A—HUMAN NECESSITIES
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to a novel virus, and the use of this virus for vaccines and prophylactic treatment for the disease caused by the virus.
• the invention also relates to nucleic acid sequences, amino acid sequences, primers and primer pairs, probes, diagnostic kits and methods for diagnostic according to the preamble of the independent patent claims
• the novel virus was isolated in a salmon farm with high losses caused by disease.
• the virus shows unique characters and 28% similarity to unclassified Fisavirusl , that has previously been identified in Carp ( Cyprinus carpio) (Reuter 2016).
• the sequence of Fisavirus is 8712 NT long and relates to Posavirus (Porcine stool associated virus), and it is assumed that the host of Fisavirus could be the
• Nematoda phylum (Reuter 2016). Nematodes (Nematoda) belong to the most frequent and the most important parasites of fishes in the freshwater, brackish-water and marine environments throughout the world (Moravec 2007). Small numbers of nematodes often occur in healthy fish, but high numbers cause illness or even death (Roy P. Yanong). Thus it is expected that the novel virus may have a broad range of hosts among fish and shellfish, such as shrimp, carp, tilapia, seabass and salmon, among others.
• the invention relates to the new virus isolated from Atlantic salmon.
• the virus comprises a ribonucleic acid genome having at least 70% sequence identity with SEQ ID NO 2 or 3, and variants and fragments thereof.
• the virus of the invention further comprises a ribonucleic acid genome having at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO 2 or 3, and variants and fragments thereof.
• the invention relates to an isolated nucleic acid sequence originating from the virus.
• the sequence is selected from the group consisting of SEQ ID No 2 and 3 and sequences complementary to any of SEQ ID No 2 and 3, and variants thereof being at least 70% identical.
• the isolated nucleic acid sequence may further be at least 80%, preferably 90%, more preferably 95 % identical with any of the sequences SEQ ID No 2 or 3, or any sequences being complementary to SEQ ID No 2 or 3.
• nucleic acid sequence having a sequence selected from the group consisting of SEQ ID No 4-12, and sequences being complementary to SEQ ID No 4-12
• a nucleic acid may be used as a primer for instance for PCR or a probe for instance for identification of a nucleic acid sequence or gene.
• a method for detection of a virus in a biological sample comprises the following steps:
• each primer of said primer pair comprises at least 10 nucleotides and hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID No 2-12, sequences being complementary to SEQ ID No 2-12, and variants being at least 70 % identical with any of the sequences,
• the primers of the method above are selected from a group consisting for SEQ ID No 2-12, preferably SEQ ID NO 7-12.
• the primers may further hybridize to a nucleic acid being at least 80%, preferably 90%, more preferred 95 % or 100 % identical with the sequences SEQ ID No 2-12, or any sequences being complementary to SEQ ID No 2 12
• Another method for detection of a virus in a biological sample comprises the following steps:
• step c) of the method above 80 %, 90%, 95% or 100 % identity may be required to confirm the presence of virus in the biological sample.
• the sequencing of the mixture in the method above is performed by a method selected from the group consisting of Next Generation Sequencing, preferably lllumina (Solexa) sequencing, Roche 454 sequencing, Ion Torrent or SOLiD sequencing (Goodwin S, et al., (2016) Coming of age: Ten years of next-generation sequencing technologies. Nature reviews, Genetics, 17, 333-351).
• Next Generation Sequencing preferably lllumina (Solexa) sequencing
• Roche 454 sequencing sequencing
• Ion Torrent or SOLiD sequencing SOLiD sequencing
• the invention also relates to use of a nucleic acid sequence comprising at the least 10 contiguous nucleotides of any of the sequences 2-12, or 10 contiguous nucleotides being complementary of any of the sequences 2-12, for confirming the presence of the virus in a biological sample.
• a nucleic acid sequence having a sequence selected from the group of SEQ ID No 2 and 3 and sequences being complementary to the any of the sequences SEQ ID No 2 and 3, for confirming the presence of the virus in a biological sample.
• the methods and uses above may be used to confirm that the virus is present in the biological samples, but it may also be used to confirm that the virus is not present in the biological samples. Further, it may be used to monitor a fish population, for instance for disease/sickness control. Information in this regard, desirable with methods for identifying other diseases and/or sicknesses, may be valuable information as to if or when the fish population should be treated.
• the biological samples to be analysed are preferably from dead fish, but may also be samples removed from live fish without harming the fish.
• DNA vaccines comprising a nucleic acid sequence selected from the group consisting of SEQ ID No. 2 and 3, sequences being complementary to sequences of SEQ ID No 2 and 3, and variants thereof being at least 70% identical with any of the sequences SEQ ID No. 2 and 3 or sequences being complementary to variants of SEQ ID No 2 and 3.
• the nucleic acid sequence of the DNA vaccine may further be at least 80%, preferably 90%, more preferably 95 % identical with any of the sequences SEQ ID No 2 and 3, or any sequences being complementary to SEQ ID No 2 and 3.
• Another aspect of the invention relates to recombinant proteins encoded by a nucleic acid sequence selected from the group consisting of SEQ ID No. 2 and 3, and variants thereof being at least 70 %, 80 %, preferably 90%, more preferably 95 % identical with any of the sequences SEQ ID No. 2 and 3.
• amino acid sequence of the recombinant protein is given in SEQ ID No 1.
• the present invention also provides a recombinant vaccine comprising at least one of the recombinant proteins according to the present invention.
• the immunogenic composition may further comprise at least one excipient, additive or adjuvant, and may be administrated orally, by immersion or by intraperitoneal or intramuscular injection.
• the immunogenic composition may be a monovalent vaccine or combined with other relevant antigens.
• the invention also relates to use of the immunogenic composition described above in the manufacture of a vaccine for the treatment or prophylaxis of infection in an animal.
• the invention also relates to use of the immunogenic composition described above in the manufacture of a functional feed or additive to a functional feed for the treatment or prophylaxis of infection in an animal.
• an antibody that recognises and binds to a recombinant protein according to the present invention.
• the invention also relates to challenge methods using a virus as defined above for testing susceptibility of animals, with the purpose of testing for instance efficacy of a vaccine or functional feed, or resistance towards disease.
• the invention also relates to use of the virus in breeding programs to obtain animals that is resistant to the virus as defined above.
• the invention also relates to a diagnostic kit, which may be used to decide whether a sample comes from an organism being infected by the virus according to the invention.
• the kit may comprise at least one primer sequence selected from the group consisting of SEQ ID NO 4-12, sequences being complementary to SEQ ID NO 4-12, and variants being at least 90 % identical with any of these sequences.
• the diagnostic kit may in addition or instead comprise an antibody or a recombinant protein.
• the present invention relates to the use of the nucleic acid sequences having a sequences selected from the group consisting of SEQ ID NO 2 and 3, sequences being complementary to SEQ ID NO 2 and 3, and variants thereof being at least 70 %, 80 %, preferably 90%, more preferably 95 % identical with any of these sequences, for the preparation of DNA vaccine, recombinant vaccine or a live recombinant microorganism.
• the present invention relates to isolated nucleic acid sequences and variants thereof being at least 70% identical with the isolated nucleic acid sequences.
• % identity is to be understood to refer to the percentage of nucleotides that two or more sequences or fragments thereof contains that are the same.
• at least 70 % identical thus means that at least 70 % of the nucleotides over the entire sequences which are compared, are identical.
• a specified percentage of nucleotides can be referred to as e.g. 70% identical, 80% identical, 85% identical, 90% identical, 95% identical, 99% identical or more over a specified region when compared and aligned for maximum correspondence. The skilled person will acknowledge that various means for comparing sequences are available.
• nucleic acid sequences and recombinant proteins are to be understood to encompass nucleic acid sequences and recombinant proteins that only differs from the isolated sequences SEQ ID No. 1 , 2, and 3 by way of some amino acid or nucleotide additions, deletions or alteration that have little effect, if any, on the functional activity of the claimed sequences.
• modifications of a protein encoding nucleotide sequence may be introduced which does not alter the amino acid sequence, e.g. the substitution of a nucleotide resulting in that the triplett affected by the substitution still codes for the same amino acid.
• nucleic acid sequences coding polypeptides which facilitates purification may be added without affecting the activity of the resulting recombinant protein.
• alterations of the nucleic acid sequence resulting in modifications of the amino acid sequence of the recombinant protein it encodes may have little, if any, effect on e.g. the proteins' ability to induce protection against the virus if the alteration does not have any impact on the resulting three dimensional structure of the recombinant protein.
• a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
• the present invention encompasses recombinant proteins and variants thereof which differ in respect of amino acid substitutions, addition or deletions compared with the protein of SEQ ID No 1 , and proteins being encoded by the sequence SEQ ID No. 2 or 3.
• the primers and probes according to the present invention will hybridize under stringent conditions with the sequence in question.
• the term“hybridizing under stringent conditions” refers to conditions of high stringency, i.e. in term of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur only between nucleic acids having a high frequency of complementary base sequences. Stringent hybridization conditions are known to the skilled person (see e.g. Green M. R., Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th edition, 2012).
• stringent hybridization The precise conditions for stringent hybridization are typically sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
• stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C. for longer probes, primers and oligonucleotides.
• Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
• antigen when used in connection with the present invention is to be understood to refer to a recombinant protein or fragment thereof according to the invention being able to induce protection against the virus in fish or to be able to bind to an antibody which recognise and bind to the virus.
• vaccine refers to a material that can produce an immune response that blocks the infectivity, either partially or fully, of an infectious agent, which in respect of the present invention is the virus affecting fish such as e.g.
• the immunising component of the vaccine may be e.g. DNA as in a DNA vaccine, RNA as in a RNA vaccine, a recombinant protein or fragment thereof according to the present invention, or a live recombinant microorganism.
• the vaccines may be administered by immersion, orally or by intraperitoneal or intramuscular injection.
• the vaccines may be monovalent vaccine to protect against the virus, or combined with other antigens to multivalent vaccines.
• the present invention also provides short nucleotide sequences having a length of at least 10 nucleotides. These sequences may be primers or probes useful in polymerase chain reaction techniques to be used as diagnostic tools.
• the terms "primer” and “probes” as used herein refers to an oligonucleotide either naturally occurring or produced synthetically which is significantly complementary to a virus target sequence and thus capable of hybridizing to nucleic acid sequences of the present invention.
• a “primer pair” or “primer set” it is generally one forward and one reverse primer, and the sequence between the primers will be multiplied during a PCR. This is well known to a skilled person, and he/she would know which primers constitute a suitable pair.
• the amplified sequence may be labelled to facilitate detection, e.g. using fluorescent labels on a probe, or other label means well known to the skilled person.
• FIG 1 shows the cumulated mortality of infected fish
• Figure 2 shows the amino acid sequence encoded by SEQ ID No. 3, the amino acid sequence is corresponding to SEQ ID No 1 ,
• FIG. 3 shows the genomic sequence of the novel virus (PV) corresponding to SEQ ID No. 2,
• Figure 4 shows the genomic sequence of the Codon DNA Segment of the novel virus (PV), corresponding to SEQ ID No. 3,
• Figure 5 shows the genomic sequence of PV1 P corresponding to SEQ ID No. 4
• Figure 6 shows the genomic sequence of PV2P corresponding to SEQ ID No. 5
• Figure 7 shows the genomic sequence of PV3P corresponding to SEQ ID No. 6
• Figure 8 shows the genomic sequence of PV1 F corresponding to SEQ ID No. 7
• Figure 9 shows the genomic sequence of PV2F corresponding to SEQ ID No. 8
• Figure 10 shows the genomic sequence of PV3F corresponding to SEQ ID No. 9,
• Figure 11 shows the genomic sequence of PV1 R corresponding to SEQ ID No. 10,
• Figure 12 shows the genomic sequence of PV2R corresponding to SEQ ID No. 11 .
• Figure 13 shows the genomic sequence of PV3R corresponding to SEQ ID No. 12.
• an embodiment signifies that a particular feature, structure or property specified in connection with an embodiment is included in the least in one embodiment.
• the expressions "in one embodiment”, “in a preferred embodiment” or “in an alternative embodiment” different places in the description does not necessarily point to the same embodiment. Further, the different features, structures or properties may be combined in any suitable way in one or more of the embodiments.
• Example 1 disease outbreak, virus sequencing and detection
• the fish showed symptoms of severe anaemia.
• RNA from one gill sample was used as starting input for the RNA sequencing library preparation.
• the RNA from the sample was treated with
• RiboMinus TM Eukaryote Kit v2 (Thermo Fischer Scientific) to remove ribosomal RNA.
• the rRNA-depleted RNA was fragmented and library was constructed using the Ion Total-RNA Seq Kit v2 (Thermo Fischer Scientific).
• the library was bar-coded and further quantified with qRT-PCR. Using the Ion Chef and the Ion 520TM and Ion 530TM kit Chef (Thermo Fischer
• the final genome is 8,713 bps, and divided into the following sections;
• PV1-F and PV1-R table 4 and 5 forward and reverse primers
• MGB probe PV1-P
• 6-FAM 6-FAM
• the primers were designed using the software Primer Express 3.0.1 (Thermo Fischer Scientific). Secondary structures and the possibility of primer dimers were tested using the online software IDT OligoAnalyzer 3.1 , and the specificity of the primers and the probe were checked using NCBI’s Blastn.
• the primers and the probe were found not to form secondary structures or primer dimers, nor to hybridize to any other known sequence.
• the primers and the probe were manufactured by Thermo Fischer Scientific.
• the RT-PCR assay was performed using TaqMan® Fast Virus 1-Step Master Mix. Amplifications were done on a QuantStudio 5 Real Time PCR systemThermo Fischer Scientific) with the following conditions: 5 min at 50 °C, 20 sec at 95 °C followed by 45 cycles of 95 °C/3 sec and 60 °C/30 seconds. Results of the qPCR
• the PV1 Real Time assay detected relatively high amounts of the gene sequence the PV assay targeted. There are multiple factors that may have affected the fish, however the overall trends is clear positive, and samples from the gills of all fish having clinical symptoms, were positive with Ct values ranging from 16,2 to 29,8.
• the gill tissue from fish with no symptoms of disease were in general negative to PV1 by Real Time assay, except for two fish that had Ct values of 35,4 and 37 which show that there are very small amount of virus detected in these fish. This confirms that the virus detected causes the observed symptoms of anaemia. Additional qPCR assays
• SEQ ID No. 2 was amplified by PCR and cloned into a pET system using standard system. E. coli host cells were transformed, and recombinant protein expressed. Additionally, SEQ ID No. 2 was amplified by PCR and cloned in to a eukaryotic vector. An eukaryotic host cell was transformed, and a recombinant protein expressed. Recombinant protein was purified using standard techniques.
• Recombinant proteins described herein were formulated in a suitable vaccine that could be added excipients for example but not limited liposomes, water in oil emulsions.
• the vaccine will be used to produce an immune response in Salmonoids.
• Any variants of SEQ ID No 2 that has a conservative amino acid substitution may be used, and are included in this description.
• any open reading frames expressing protein from the novel virus or any variant of a novel virus having about 70% sequence identity to the open reading frame described herein may be used to produce a formulation and immune response.
• the efficacy of the vaccine based on recombinant protein from example 2 were injected to salmon in doses on 0,025 to 0,1 ml/fish. After an immunization period of 5-10 weeks, vaccine efficacy was measured in an intraperitoneal challenge of vaccinated and non-vaccinated control fish. The fish were challenged with the novel virus either directly from homogenate or from virus propagated in cell lines.
• Tissue samples at from 2 to 8 weeks post challenge were analysed by histology and PCR screening. Mortality was observed after vaccination, but Relative Percentage Survival was more than 50% which confirms protection.
• SEQ ID No. 2 or SEQ ID No. 3 and parts, fragments and variants thereof will be cloned into a vaccine vector suitable to be used as a DNA vaccine.
• the resulting vector will be injected intramuscularly to salmon. After a suitable immunization period the vaccinated and none vaccinated fish will be challenged and evaluated as described in example 3.
• Juvenile Atlantic salmon were challenged to a suspension of the virus, in freshwater, under standard conditions. High mortality was observed. 4-10 weeks post challenge survived fish were sampled for histology and PCR, and showed clear symptoms of infection.
• FAST fusion-associated small transmembrane protein

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