NO344051B1

NO344051B1 - Novel virus in Fish and Method for detection

Info

Publication number: NO344051B1
Application number: NO20170739A
Authority: NO
Inventors: Håvard Aanes; Linda Ramsevik Teigene; Magnus Andreas Devold; Vidar Teis Aspehaug
Original assignee: Patogen As
Priority date: 2017-05-04
Filing date: 2017-05-04
Publication date: 2019-08-26
Also published as: WO2018203757A1; NO20170739A1

Description

Novel virus in fish and a method for detection

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 recombinant protein, an antibody and a diagnostic kit.

Background

Fish are an increasingly important source of food and income; global annual consumption is projected to rise from 110 million tons in 2010 to more than 200 million tons in 2030. However, the emergence of infectious diseases in aquaculture threatens production and may also impact wild fish populations.

Sea lice (Lepeophtheirus salmonis and Caligus spp.) are one of the major pathogens affecting global salmonid farming industry and have a significant impact in many areas. The annual loss has recently been estimated to €300 million and the aquaculture industry relays heavily on a few chemotherapeutants for lice control.

In Norwegian salmonid farming the use of cleaner fish has become more and more popular to prevent infections by sea lice. The most common species are the wrass 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. By the expression "lumpsucker" is meant any species selected from the whole family of Cyclopteridae. The most preferred species is Cyclopterus lumpus.

As 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. splendidis, 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. Salmonid alfavirus (SAV) 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. This is for instance described in Hjeltnes, B. et al., Fiskehelserapporten 2016,Veterinærinstituttets rapportserie nr.4/2017. A virus causing disease in lump suckers are also described in EP 3263590, which is filed before but published after the priority date of this application.

In general heavy outbreaks are mainly observed in small fish, and are often observed in relation to handling, 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.

There is therefore a need for a rapid method for identification of virus and a tool to monitor the production and avoid outbreak. Another object is to develop a vaccine and a further treatment to treat infected fish.

The invention

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.

According to one aspect, the isolated nucleic acid sequence is selected from the group of SEQ ID No 2-14, and sequences being complementary to SEQ ID NO 2-14.

In another aspect, not part of the claimed invention, it relates to a 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 1-14, sequences being complementary to SEQ ID NO 1-14, and variants being at least 70 % identical with any of the sequences SEQ ID NO 1-14 and sequences being complementary to SEQ ID NO 1-14. Such 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.

The nucleic acid sequence being at least 10 nucleotides long, may have 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 70 % identical with any of the sequences SEQ ID NO 15-53 and sequences being complementary to SEQ ID NO 15-53. Further, the said nucleotide sequence may be at least 15 or at least 20 nucleotides long.

In another aspect the invention provides a primer and/or probe comprising a sequence selected from the group consisting of SEQ ID NO 15-53, and sequences being complementary to SEQ ID NO 15-53. In some aspects the primer may be 15 nucleotides or 20 nucleotides long.

According to another aspect of the invention, a method for detection of a virus 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, in a biological sample is provided. The method comprises the following steps:

a) preparing a sample comprising nucleic acid sequences isolated from a biological sample for a reverse transcription reaction,

b) subjecting the mixture of a) to a polymerase chain reaction with at least one primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID No 15-53, and sequences being complementary to SEQ ID No 15-53, c) determining whether the binding of the nucleotide sequences to nucleotide sequences in the biological sample and amplification of the sequence between them have occurred indicating the presence of virus in the sample tested.

In a preferred embodiment, the primers of the method above are selected from a group consisting for SEQ ID No 28-53.

According to another aspect of the invention, another method for detection of a virus 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, in a biological sample is provided. The method comprises the following steps:

b) sequencing the mixture of a), and

c) comparing the resulting sequence with a sequence selected of the group consisting of SEQ ID No 1-14, sequences being complementary to SEQ ID NO 1-14, wherein 70 % identity verifies the presence of virus in the biological sample tested.

In 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.

In a preferred embodiment, the sequencing of the mixture in the method above is performed by a method selected from the group consisting of Next Generation Sequencing, preferably Illumina (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 any of the sequences 1-53, or nucleotides being complementary of any of the sequences 1-53, for confirming the presence of the virus 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, in a biological sample. In another aspect it is used a nucleic acid sequence having a sequence selected from the group consisting of SEQ ID NO 2-14, or sequences being complementary to the sequences of SEQ ID NO 2-14. In yet another embodiment it is used a nucleic acid sequence having a sequence selected from the group of SEQ ID No 1, or a sequence being complementary to the any of the sequences 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 may be provided comprising a nucleic acid sequence selected from the group consisting of SEQ ID No. 1-14, sequences being complementary to sequences of SEQ ID No 1-14, and variants thereof being at least 70% identical with any of the sequences SEQ ID No.1-14 or sequences being complementary to the sequences of SEQ ID No 1-14, but are not a part of the claimed invention. The identity is preferably based on the entire sequence length of the said isolated nucleic acid sequence.

The nucleic acid sequence of the DNA vaccine may be at least 80%, preferably 90%, more preferably 95 % identical with any of the sequences SEQ ID No 1-14, or any sequences being complementary to 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.1-14. In one aspect the amino acid sequence of the recombinant protein is given in SEQ ID No 54.

A recombinant vaccine comprising at least one of the recombinant proteins according to the present invention, may also be provided, but are not a part of the claimed invention.

Furthermore, according to another aspect of the invention, it is provided an antibody that recognises and binds to a recombinant protein according to the present invention.

Finally, 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 a live recombinant microorganism.

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). 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. The 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. Typically, 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.

The terms "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.

The term "vaccine" as used herein 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. salmonids. Thus, when administering to a fish, the vaccines of the invention 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 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. 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.

Brief description of the figures

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 11 shows the genomic sequence of LV10 corresponding to SEQ ID No.11 Figure 12 shows the genomic sequence of LV11 corresponding to SEQ ID No.12 Figure 13 shows the genomic sequence of LV12 corresponding to SEQ ID No.13, Figure 14 shows the genomic sequence of LV13 corresponding to SEQ ID No.14, and

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 shows corresponding parts used to isolate and identify probes and primers LV2-LV13.

Examples

Tissue:

RNA sequencing and virus detection

Samples from fish were collected from fish with clinical signs of disease (pale yellow liver, pale heart, pale gills, ascites in the abdominal cavity and some wounds in the abdominal region). The samples were tested by Real Time qPCR for known diseases in lumpsucker; Infectious Pancreatic Necrosis Virus (IPNV), Paramoeba perurans (AGD), Nucleospora cyclopteri, 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.

Tissue preparation, RNA isolation and analysis

RNA was extracted from kidney and liver tissue samples using RNAeasy Universal kit (Qiagen) on an automated system at the PatoGen Analyses AS accredited commercial laboratory. The RNA quality and concentration of the samples were assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies) and the Qubit 3.0 Fluorometer (Thermo Fischer scientific) using the RNA 6000 Pico Kit (Agilent) and the Qubit HS Assay kit (Thermo Fischer Scientific).

Library preparation and RNA sequencing with Ion Torrent S5

420 to 480 µg 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 Scientific) to remove ribosomal RNA. The rRNA-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.

Using the Ion Chef and the Ion 520<TM>and Ion 530<TM>kit Chef (Thermo Fischer Scientific), 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).

Description of bioinformatics analysis

Over 20 million reads were generated for two different whole transcriptome analysis samples. BAM-files were converted to FASTQ files using SAMTOOLS fastq command and we applied our in-house pathogen detection pipeline on the reads. Our approach utilizes publically available research tools.

First, reads were first trimmed 25bp in both the 5’ and 3’ end, and short reads (<50 bps) were removed using seqtk (https://github.com/lh3/seqtk). Reads mapping to the Stickleback genome were then removed using Kraken (using Kraken (Wood and Salzberg, 2014), before sequence construction with SPADES using the remaining reads (Bankevich et al., 2012). The resulting scaffold sequences were then tested for complementarity with known sequences using blastn and blastx algorithms against the Swiss-Prot and nt databases, respectively.

Several sequences in both samples which were chosen above, matched partly to members of the flaviviridae family (Cell fusing agent virus, Yellow fever virus, Dengue virus 3 and Tamana bat virus). To extend the length of the sequence we merged both samples and did sequence construction on this merged sample. The final genome were generated by aligning generated sequences, predicting ORFs and performed BLAST searches, with the assumption that long ORFs and complementarity with flaviviruses were indicative of correct sequence and reading frame. Final trimming of the UTRs were done based on read coverage differences.

The final genome is 10.7 Kbs, and divided into the following sections;

Bases 1-169; 5’UTR

Bases 170-10,615; CDS (3482 aa)

Bases 10,616-10,760; 3’UTR

Notably, BLASTX search of 4 evenly sized parts of the sequence matches different flavivirus proteins (envelope protein, NS3 and NS5). To further assess sequence similarity with different flaviviruses, we downloaded representative sequences of flaviviruses, and performed a maximum likelihood 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.

Based on this, the novel identified virus is probably a Flavi virus.

Real Time PCR

To validate if the tentative virus, in the following referred to as LV, could be disease causing we designed a qPCR-assay in the presumptive polymerase region of the sequence using forward and reverse primers (LV1-F and LV1-R table 3 and 4), and a minor groove binding (MGB) probe (LV1-P) labelled with 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 the QIAGEN QuantiTech Master Mix. Amplifications were done on a 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.

Results of the qPCR

To validate if the tentative virus could be disease causing, 17 fish with clinical symptoms and 20 fish without clinical symptoms from 4 different farms along the Norwegian coastline were tested with the LV1- qPCR assay. Extracted total RNA from kidney samples were used in the test.

In farms from Sogn og Fjordane, Nordland and Troms 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. In these tree farms, the LV1 Real Time assay detected relatively high amounts of the gene sequence the LV1 assay 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 Møre 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.

Additional qPCR assays

We have designed 12 additional quantitative Real Time TaqMan assays targeting various regions of the genome, as described above for LV1.

Claims

Patent claims

1. An isolated nucleic acid sequence originating from a virus in lumpsucker, the sequence is selected from the group consisting of SEQ ID No 1-14 and sequences being complementary to SEQ ID NO 1-14.

2. A primer and/or probe comprising a sequence selected from the group consisting of SEQ ID NO 15-53 and sequences being complementary to SEQ ID NO 15-53.

3. A primer and/or probe according to claim 2, wherein said sequence comprises at least 15 nucleotides, or 20 nucleotides.

4. A method for detection of a virus comprising a sequence according to claim 1, in a biological sample, characterized by comprising the following steps:

a) preparing a biological sample comprising nucleic acid sequences isolated from a biological sample for a reverse transcription reaction,

b) subjecting the mixture of a) to a polymerase chain reaction with at least one primer according to claims 2-3, and

c) determining whether the binding of the primers to nucleotide sequences in the sample and amplification of the sequence between them have occurred indicating the presence of the virus in the sample tested.

5. A method according to claim 4, wherein the primers are selected from a group consisting of SEQ ID No 28-53.

6. A method for detection of a virus comprising a sequence according to claim 1 in a biological sample, characterized by comprising the following steps:

b) sequencing the mixture of a), and

c) comparing the resulting sequence with the sequence selected of the group consisting of SEQ ID No 1-14 and sequences being complementary to SEQ ID NO 1-14, wherein identity verifies presence of the virus in the biological sample tested.

7. A method according to claim 6, wherein the sequencing is performed by a method selected from the group consisting in Illumina (Solexa) sequencing, Roche 454 sequencing, Ion Torrent and SOLiD sequencing.

8. Use of any of the SEQ ID No 15-53, or nucleotides being complementary of any of the SEQ ID No 15-53, for establishing the existence of virus comprising a sequence according to claim 1, in a biological sample.

9. Use of SEQ ID No 1, or a sequence being complementary to SEQ ID No 1, for establishing the existence of a virus comprising a sequence according to claim 1, in a biological sample.

10. A vector comprising a nucleic acid sequence according to any one of claims 1-3.

11. A host cell comprising a vector according to claim 10.

12. A recombinant protein encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO 1-14.

13. A recombinant protein having an amino acid sequence according to SEQ ID No 54.

14. An antibody specifically recognizing and specifically binding to a recombinant protein according to claim 12 or 13.

15. Diagnostic kit comprising at least one primer sequence according to any of claims 2-3.