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

recombinant vaccine, 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 1 10 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 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. 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. 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.

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.

In another aspect of the 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 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. Further, the said nucleotide sequence may be at least 15 or at least 20 nucleotides long.

In another aspect 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. In some aspects the primer may be 15 nucleotides or 20 nucleotides long.

The above said 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.

According to another aspect of the invention, a method for detection of a virus 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 a primer pair, wherein 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 15-53, sequences being complementary to SEQ ID No 15-53, and variants being at least 80 % identical with any of the sequences, c) determining whether the binding of the primer pair 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. By "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. In a preferred embodiment, the primers of the method above are selected from a group consisting for SEQ ID No 28-53.

In step b) of the method above, 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.

According to another aspect of the invention, another method for detection of a virus 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) 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 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 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. In yet another embodiment 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.

According to yet another aspect of the invention, DNA vaccines are provided 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.

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 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. The term "% 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. The term "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.

The term "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. The skilled person will acknowledge that 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.

Furthermore, the addition of nucleic acid sequences coding polypeptides which facilitates purification may be added without affecting the activity of the resulting recombinant protein.

The skilled person will further acknowledge that also 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. For example, 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. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a recombinant protein with substantially the same functional activity as the protein encoded by the nucleotide sequences SEQ ID No. 1 -53 and thus to be expected to constitute a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the functional activity of the protein. Each of the proposed modifications is well within the routine skills in the art, as is determination of retention of biological activity of the encoded products. Therefore, where the terms "nucleic acid sequences of the invention" or "recombinant protein of the invention" are used in either the specification or the claims each will be understood to encompass all such modifications and variations which result in the production of a biologically equivalent protein.

Thus, 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.

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 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 1 1 shows the genomic sequence of LV10 corresponding to SEQ ID No. 1 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 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 show 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 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.

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 Bioanaiyzer (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 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

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™ and Ion 530™ 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 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 (LV is abbreviated for Lumpsucker Virus), could be disease causing we designed a qPCR-assay in the presumptive polymerase region of the sequence using forward and reverse primers (LV1 and LV13-F and LV1 and LV13-R table 3 and 4), and a minor groove binding (MGB) probe (LV1 and LV13-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 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.

The 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.

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 and LV13- qPCR assay. Extracted total RNA from kidney samples were used in the test.

Table 1 :

37 fish from 4 different localities along the Norwegian coastline were tested for the tentative virus using the LV1 and LV13 qPCR assay.

LV1 and LV13 represent LV Ct-value.

Localization Sample ID Tissue Clinical symptoms LV1 LV13

Sogn og Fjordane FR10096677 Kidney Moribund 18,9 19,8

Sogn og Fjordane FR10096663 Kidney Moribund 13,7 14,5

Sogn og Fjordane FR10096698 Kidney Moribund 14,6 14,5

Sogn og Fjordane FR10096668 Kidney Moribund 17,2 17,2

Sogn og Fjordane FR10096658 Kidney Moribund 17,6 18,4

Sogn og Fjordane FR10096673 Kidney Moribund 12, 1 15, 1

Sogn og Fjordane FR10096682 Kidney Moribund 17,4 17,3

Sogn og Fjordane FR10096688 Kidney Moribund 15,6 16,0 Table 1 :

37 fish from 4 different localities along the Norwegian coastline were tested for the tentative virus using the LV1 and LV13 qPCR assay.

LV1 and LV13 represent LV Ct-value.

Sogn og Fjordane FR10096653 Kidney Moribund 16,2 16, 1

Nordland FR1 1427739 Kidney Moribund 1 1 ,5 14,0

Nordland FR1 1427734 Kidney Dead 33,3 35,6

Nordland FR1 1427736 Kidney Dead 14, 1 14,5

Nordland FR1 1427737 Kidney Moribund

Nordland FR1 1427735 Kidney Dead 24,5 25,0

Nordland FR1 1427738 Kidney Moribund 13,4 15,2

M0re og Romsdal FR10094939 Kidney Healthy

M0re og Romsdal FR10094934 Kidney Healthy

M0re og Romsdal FR10094933 Kidney Healthy

M0re og Romsdal FR10094935 Kidney Healthy

M0re og Romsdal FR10094936 Kidney Healthy

M0re og Romsdal FR10094938 Kidney Healthy

M0re og Romsdal FR10094937 Kidney Healthy

M0re og Romsdal FR10094940 Kidney Healthy

M0re og Romsdal FR10094930 Kidney Healthy

M0re og Romsdal FR10094932 Kidney Healthy

M0re og Romsdal FR10094929 Kidney Healthy

M0re og Romsdal FR10094924 Kidney Healthy

M0re og Romsdal FR10094928 Kidney Healthy

M0re og Romsdal FR10094926 Kidney Healthy

M0re og Romsdal FR10094931 Kidney Healthy

M0re og Romsdal FR10094927 Kidney Healthy

M0re og Romsdal FR10094986 Kidney Healthy

M0re og Romsdal FR10094925 Kidney Healthy

Troms FR10136552 Kidney Healthy

Troms FR10136555 Kidney Moribund 14,4 15,0

Troms FR10136553 Kidney Healthy

Troms FR10136554 Kidney Moribund 14,8 15, 1 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 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.

Additional qPCR assays

We have designed 1 1 additional quantitative Real Time TaqMan assays targeting various regions of the genome, as described above for LV1 and LV13.

Table 2

Probes targeting the sequence.

Name Probe SEQ ID No

LV1 TACATCCAGACATCATTTG SEQ ID No 15

LV2 CACTTTCAGTATCTGGCC SEQ ID No 16

LV3 AGGACTACACAGCCCAC SEQ ID No 17

LV4 C AC G G C AC CTC AC AT SEQ ID No 18

LV5 TTCAGAGGAAATTCG SEQ ID No 19

LV6 CAAAGGCGACACCC SEQ ID No 20

LV7 TCCTGGCGGAGACAA SEQ ID No 21

LV8 AC AC ATG GTC C C C ATAC A SEQ ID No 22

LV9 AAACCTATGGCATTGATC SEQ ID No 23

LV10 CCGCCTGCCCAAA SEQ ID No 24

LV1 1 AAGAGCCTCCCCAGGC SEQ ID No 25

LV12 TCACCTGCCATAATT SEQ ID No 26

LV13 CGCGACTTCAT SEQ ID No 27 Table 3

Primers forward targeting the sequence.

Name Primer Forward SEQ ID No

LV1 CGCAGCCGTCGGAAAC SEQ ID No 28

LV2 GGTCGAAAGGCCTAAATTGGA SEQ ID No 29

LV3 GGGAAAACCCAACTCGTTCTG SEQ ID No 30

LV4 GGGCGCCACTGTTCGA SEQ ID No 31

LV5 AC AG G C ATG G AC CTTTC AAAA SEQ ID No 32

LV6 AG C AG AATG G AG AGC C ACTC A SEQ ID No 33

LV7 GGGTTAGTGCCCCTCATCAA SEQ ID No 34

LV8 CAAGAACACCTTCTACAACCTGAGAA SEQ ID No 35

LV9 CCTGAACTTTTCATACGCAACAAC SEQ ID No 36

LV10 G ATG C C C AG GAG ATG G AG AA SEQ ID No 37

LV1 1 C GAG ATG G C C AAAG G ATC AG SEQ ID No 38

LV12 TG ATG G GAAAC AAG GAG C ACTA SEQ ID No 39

LV13 GATGTCCTGCACAT SEQ ID No 40

Table 4

Primers revers targeting the sequence.

Name Primer Reverse SEQ ID No

LV1 CTAGGGCTTCCAGTGCTGTTG SEQ ID No 41

LV2 GCGGGCCTCAGTGAAGAAA SEQ ID No 42

LV3 TTGCGTGTCCGGCTATCTC SEQ ID No 43

LV4 CTTTGTCTATGGGTGCTAACAGTTG SEQ ID No 44

LV5 TCCATCCTGGGCCTCTTGT SEQ ID No 45

LV6 GGAACCACGGAGTTCGTAAATG SEQ ID No 46

LV7 TTTTGTAGGCCTTTGCTCTGTGT SEQ ID No 47

LV8 TGGAGCATACTGTCCCCTTGTC SEQ ID No 48

LV9 AAGCCGGTGGAACACTCTGT SEQ ID No 49

LV10 CTCTAGCGCAGATGGAATATGGT SEQ ID No 50

LV1 1 GGGCTAATGTCTAAGGGCATCTT SEQ ID No 51 Table 4

Primers revers targeting the sequence.

LV12 GGCAGTTCTTGCAGTGTTTTTCT SEQ ID No 52

LV13 TTCGCCGTAGATT SEQ ID No 53