WO2018203757A1 - Nouveau virus chez les poissons et son procédé de détection - Google Patents

Nouveau virus chez les poissons et son procédé de détection Download PDF

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WO2018203757A1
WO2018203757A1 PCT/NO2018/050118 NO2018050118W WO2018203757A1 WO 2018203757 A1 WO2018203757 A1 WO 2018203757A1 NO 2018050118 W NO2018050118 W NO 2018050118W WO 2018203757 A1 WO2018203757 A1 WO 2018203757A1
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seq
sequences
sequence
virus
nucleic acid
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PCT/NO2018/050118
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Håvard AANES
Linda RAMSEVIK TEIGENE
Magnus Andreas DEVOLD
Vidar Teis ASPEHAUG
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • 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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • 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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to nucleic acid sequences isolated from a novel virus identified in lumpsucker and a method for detection of this virus, according to the preamble of the independent claims.
  • the invention further provides primers and probes, a vector and a host cell, a DNA vaccine, a recombinant protein, a
  • the most common species are the wrasse species Ctenolabrus rupestris, Symphodus melops and Labrus bergylta, but also a large numbers of lumpsucker (Cyclopterus lumpus) are used.
  • the lumpsuckers are particularly useful as they remain active at lower water temperatures.
  • Lumpsuckers are a good and popular solution for salmon lice control, and therefore they are both farmed and captured from the wild and set out in the salmonid cages, with the purpose to control sea lice.
  • a single fish can eat over 300 lice a day. This has made this fish species very popular and there are more than 25 fish farms producing lumpsuckers in Norway, in 2016.
  • “lumpsucker” is meant any species selected from the whole family of Cyclopteridae. The most preferred species is Cyclopterus lumpus.
  • lumpsuckers are keeping the salmonids healthy, they are very valuable to the fish farming industry, even if they are not used for consumption themselves. Therefore it is important to keep them healthy, and known diseases in lumpsuckers are typically of bacterial origin.
  • Furunculosis caused by atypical Aeromonas salmonicida is the most important pathogen, but also different vibrio species causing vibriosis such as Vibrio anguillarum are well known. Species like V.ordalii, V. spectacularis, V. logei, V. wodanis, V. tapetis are also reported, in addition to Pseudomonas anguilliseptica and the recently reported Tenacibaculum maritimum and Pasteurella sp. Previous examinations of captured cleaner fish have not detected viral infections as Viral haemorrhagic septicaemia virus (VHSV), Infectious Pancreatic Necrosis Virus (IPNV) or nodavirus.
  • VHSV Viral haemorrhagic septicaemia virus
  • IPNV Infectious Pancreatic Necrosis Virus
  • Salmonid alfavirus has been reported from one farm. It is demonstrated that IPNV can infect lumpsuckers in laboratory studies. During summer 2016 it was discovered a new disease among farmed lumpsuckers in Norway. Sick fish showed signs such as pale yellow liver with a rubberish texture, anaemic, open gills, and deformities on "sucker cup". The disease has been observed in all stages of the life cycle. In general heavy outbreaks are mainly observed in small fish, and are often observed in relation to handling, such as transfer and/or vaccination. It is not clear how the virus is spreading, high mortality up to 50% can be observed in one tank while the neighbour tank remains healthy. The small fish die rapidly and often the whole tank has to be eradicated. In larger fish the virus seem to cause reduction in appetite and the fish stop eating, the liver loses its vital functions and the fish gets skinny and die. The virus has also been detected in broodfish kidney, but there were no clinical sign of disease.
  • Another object is to develop a vaccine and a further treatment to treat infected fish.
  • the invention relates to an isolated nucleic acid sequence originating from a virus in lumpsuckers having a sequence selected from the group consisting of SEQ ID No 1 - 14 or a sequence complementary to any of SEQ ID No 1 -14.
  • nucleic acid sequence of at least 10 nucleotides wherein said sequence hybridizes specifically to a nucleic acid sequence having a sequence selected from the group consisting of SEQ ID No 15- 53, sequences being complementary to SEQ ID No 15-53, and variants being at least 80 % identical with any of the sequences SEQ ID NO 15-53 and sequences being complementary to SEQ ID NO 15-53.
  • the said nucleotide sequence may be at least 15 or at least 20 nucleotides long.
  • the invention provides a primer or probe comprising a sequence of at least 10 nucleotides, having a sequence selected from the group consisting of SEQ ID NO 15-53, sequences being complementary to SEQ ID NO 15-53, and variants being at least 80 % identical with any of the sequences SEQ ID NO 15-53 and sequences being complementary to SEQ ID NO 15-53.
  • the primer may be 15 nucleotides or 20 nucleotides long.
  • nucleic acid sequence being at least 10 nucleotides long, or the primer or probe, may be at least 90%, preferably 95 % identical with any of the sequences SEQ ID No 15-53, or any sequences being complementary to SEQ ID No 15-53.
  • a method for detection of a virus in a biological sample comprises the following steps:
  • primer pair it is herein meant two primers, one forward primer and one reverse primer, working together in a PCR method to amplify a sequence between the binding site of each primer. This is well known to a skilled person, and it is within his/her skills to find primers suitable to constitute a pair.
  • the primers of the method above are selected from a group consisting for SEQ ID No 28-53.
  • the primer pair may hybridize to a nucleic acid being at least 90%, preferably 95 % or 100 % identical with any of the sequences SEQ ID No 15-53, or any sequences being complementary to SEQ ID No15-53.
  • 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 ).
  • the invention also relates to use of a nucleic acid sequence comprising at the least 10 contiguous nucleotides of any of the sequences 15-53, or 10 contiguous nucleotides being complementary of any of the sequences 15-53, for confirming the presence of the virus in a biological sample.
  • it is used a nucleic acid sequence having the sequence of SEQ ID No 1 , or a sequence being complementary to the sequence of SEQ ID No 1.
  • Another aspect of the invention relates to a vector comprising nucleic acid
  • sequences according to the present invention and host cells comprising said vectors.
  • DNA vaccines comprising a nucleic acid sequence selected from the group consisting of SEQ ID No. 1 -14 and sequences being complementary to sequences of SEQ ID No 1 -14.
  • Another aspect of the invention relates to recombinant proteins encoded by a nucleic acid sequence selected from the group consisting of SEQ ID No. 1 -14ln one aspect the amino acid sequence of the recombinant protein is given in SEQ ID No 54.
  • the present invention also provides a recombinant vaccine comprising at least one of the recombinant proteins according to the present invention.
  • an antibody that recognises and binds to a recombinant protein according to the present invention.
  • the present invention relates to the use of the nucleic acid sequences having a sequences selected from the group consisting of SEQ ID NO 1 -14 and sequences being complementary to SEQ ID NO 1 -14, 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 80% 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 80 % identical thus means that at least 80 % of the nucleotides over the entire sequences which are compared, are identical.
  • a specified percentage of nucleotides can be referred to as e.g. 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.
  • variants thereof used in respect of the nucleic acid sequences and recombinant proteins according to the present invention, is to be understood to encompass nucleic acid sequences and recombinant proteins that only differs from the isolated sequences SEQ ID No. 1 -54 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 coding 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.
  • Such alterations may be introduced to adapt the nucleic acid sequence to the codons preferably used by a host cell and thus to enhance the expression of a desired recombinant protein.
  • 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 codes 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 54, and proteins being encoded by the sequence SEQ ID No. 1 -53.
  • hybridizing under stringent conditions refers to conditions of high stringency, i.e. in term of
  • nucleic acid hybridizations are conducted.
  • 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
  • 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
  • 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 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 vaccines of the invention when administering to a fish, is immunised against the disease caused by the virus.
  • 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 present invention also provides short nucleotide sequences having a length of at least 10 nucleotides. These sequences may be primers or probes useful in
  • 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.
  • primer pair When a primer pair is used, 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.
  • Figure 1 shows the genomic sequence of the Virus (LV) corresponding to SEQ ID No. 1 .
  • Figure 2 shows the genomic sequence of LV1 corresponding to SEQ ID No. 2
  • Figure 3 shows the genomic sequence of LV2 corresponding to SEQ ID No. 3
  • Figure 4 shows the genomic sequence of LV3 corresponding to SEQ ID No. 4
  • Figure 5 shows the genomic sequence of LV4 corresponding to SEQ ID No. 5
  • Figure 6 shows the genomic sequence of LV5 corresponding to SEQ ID No. 6
  • Figure 7 shows the genomic sequence of LV6 corresponding to SEQ ID No. 7
  • Figure 8 shows the genomic sequence of LV7 corresponding to SEQ ID No. 8
  • Figure 9 shows the genomic sequence of LV8 corresponding to SEQ ID No. 9
  • Figure 10 shows the genomic sequence of LV9 corresponding to SEQ ID No. 10
  • Figure 1 1 shows the genomic sequence of LV10 corresponding to SEQ ID No. 1
  • Figure 12 shows the genomic sequence of LV1 1 corresponding to SEQ ID No. 12
  • Figure 13 shows the genomic sequence of LV12 corresponding to SEQ ID No. 13
  • Figure 15 shows the amino acid sequences of SEQ ID No. 1 , corresponding to SEQ ID NO 54.
  • Figure 1 shows the genomic sequence of the novel virus identified in lumpsuckers.
  • Figure 2 shows an important part of the genome, used to isolate and identify lumpsucker probe 1 (probe LV1 ) and forward and revers lumpsucker primers 1 (forward and revers primers LV1 ), as shown in table 2-4 below.
  • Figures 2-14 show corresponding parts used to isolate and identify probes and primers LV2-LV13.
  • Tissue RNA sequencing and virus detection
  • IPNV Infectious Pancreatic Necrosis Virus
  • APD Paramoeba perurans
  • Nucleospora cyclopten atypical Aeromonas salmonicidae
  • Pasteurella sp and Vibrio anguillarum All samples except one, were negative on all tests. Two samples from the same randomly chosen fish, which tested negative on all other tests, were chosen for further analysis.
  • RNA sequencing library preparations 420 to 480 g of total RNA from the tissue samples was used as starting input for each of two RNA sequencing library preparations.
  • the total RNA of each of the samples was treated with RiboMinus TM Eukaryote Kit v2 (Thermo Fischer
  • RNA-depleted RNA was fragmented and libraries were constructed using the Ion Total-RNA Seq Kit v2 (Thermo Fischer Scientific). Each library were bar-coded and further quantified with qRT-PCR.
  • the library templates were clonally amplified on Ion Sphere Particles, loaded into two 530 Chips and sequenced on the Ion Torrent S5 (Thermo Fischer Scientific).
  • the final genome is 10.7 Kbs, and divided into the following sections;
  • BLASTX search of 4 evenly sized parts of the sequence matches different flavivirus proteins (envelope protein, NS3 and NS5).
  • flavivirus proteins envelope protein, NS3 and NS5
  • phylogeny using neighbour joining and Jukes Cantor as the substitution model, and 100 bootstraps, using hepatitis virus C as the outgroup. This analysis revealed that the sequence we obtained was most similar to the sequence of Tamana Bat Virus. Also, phylogenetic analyses cluster the sequence most closely to the flavivirus Tamana Bat Virus.
  • the novel identified virus is probably a Flavi virus.
  • LV is abbreviated for Lumpsucker Virus
  • LV is abbreviated for Lumpsucker Virus
  • MGB minor groove binding
  • 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 probes were found not to form secondary structures or primer dimers, nor to hybridize to any other known sequence.
  • the primers and the probes were manufactured by Thermo Fischer Scientific.
  • RT-PCR assays were performed using the QIAGEN QuantiTech Master Mix. Amplifications were done on an Applied Biosystems 7500 Real-Time PCR machine (Thermo Fischer Scientific) with the following conditions: 30 min at 50 °C, 15 min at 95 °C followed by 45 cycles of 94 °C/15 s and 60 °C/1 minute.
  • LV1 and LV13 represent LV Ct-value.
  • LV1 and LV13 represent LV Ct-value.
  • the fish had clinical signs of disease such as pale yellow liver, pale heart, pale gills, ascites in the abdominal cavity and some wounds in the abdominal region.
  • the LV1 and LV13 Real Time assay detected relatively high amounts of the gene sequence the LV1 and LV13 assays targeted, in all except from one fish. There are multiple factors that may have affected this one fish, the overall trends is clear positive.
  • the farm from M0re og Romsdal had Healthy fish and now signs of disease or clinical symptoms, or LV. This confirms that the virus detected causes the observed symptoms.

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Abstract

L'invention concerne une séquence d'acide nucléique isolée issue d'un virus présent chez les lompes ayant une séquence choisie dans le groupe constitué par SEQ ID No 1-14 et des séquences complémentaires de SEQ ID No 1-14. Un procédé de détection de l'acide nucléique, des amorces, des sondes, un vecteur et une cellule hôte, un vaccin à ADN, une protéine recombinée, un vaccin recombiné, un anticorps et un kit de diagnostic sont en outre décrits.
PCT/NO2018/050118 2017-05-04 2018-05-03 Nouveau virus chez les poissons et son procédé de détection WO2018203757A1 (fr)

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NO20170739A NO344051B1 (en) 2017-05-04 2017-05-04 Novel virus in Fish and Method for detection
NO20170739 2017-05-04

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3733688A1 (fr) * 2016-06-30 2020-11-04 Pharmaq AS Virus de poisson
JP2022520756A (ja) * 2019-02-05 2022-04-01 ファルマック アクティーゼルスカブ 新規魚コロナウイルス
NO20201187A1 (en) * 2020-10-30 2022-05-02 Patogen As Novel fish virus

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