WO2020159379A1 - Marqueurs de virulence d'orthoréovirus pisciaire - Google Patents

Marqueurs de virulence d'orthoréovirus pisciaire Download PDF

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WO2020159379A1
WO2020159379A1 PCT/NO2020/050017 NO2020050017W WO2020159379A1 WO 2020159379 A1 WO2020159379 A1 WO 2020159379A1 NO 2020050017 W NO2020050017 W NO 2020050017W WO 2020159379 A1 WO2020159379 A1 WO 2020159379A1
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seq
virus
confirms
mortality
snp
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WO2020159379A8 (fr
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Magnus Andreas DEVOLD
Morten Lund
Håvard AANES
Linda Ramsevik Teigene
Vidar Teis ASPEHAUG
Gordon RICHIE
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Definitions

  • the present invention relates to methods for determining virulence of Piscine orthoreovirus (PRV), and primers and probes to be used in such a method, according to the preamble of the independent patent claims.
  • PRV Piscine orthoreovirus
  • HSMI Heart and Skeletal Muscle Inflammation
  • the virus has spread over the years and in 2014 the virus was delisted from notifiable diseases.
  • the delisting was done because the PRV is ubiquitous in Atlantic salmon, and thus present also in healthy individuals not associated with clinical disease.
  • HSMI is a significant health problem for the aquaculture industry in Norway, but it is not yet possible to differentiate between virulent and less virulent or non-virulent strains of PRV.
  • Reoviruses are non-enveloped icosahedral viruses with double-stranded RNA genomes comprising 10-12 segments and consistent with the genome organization characteristic for members of the family Reoviridae, the genome of the PRV comprises at least 10 RNA segments (GenBank Accession numbers GU994013- GU994022). Palacios et al 2010, identified these 10 different segments as L1 , L2,
  • PRV-2 erythrocytic inclusion body syndrome
  • EP 2482825 describes a known method for determining the presence or absence of piscine reovirus, by using PCR.
  • the genomes of all organisms undergo spontaneous mutations during their continuing evolution, forming variant forms of progenitor genetic sequences.
  • a mutation may result in an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral.
  • a variant that result in an evolutionary advantage may eventually be incorporated in many members of the species and may thus effectively become the progenitor form.
  • Many various variant forms survive and coexist in a species population.
  • the coexistence of multiple forms of a genetic sequence gives rise to genetic polymorphism, including single-nucleotide polymorphisms (SNPs).
  • SNPs single-nucleotide polymorphisms
  • a single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide— A, T, C or G— in the genome (or other shared sequence such as RNA) differs between members of a biological species or paired chromosomes in an organism.
  • SNP single-nucleotide polymorphism
  • AAGCCTA to AAGCTTA two DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide, commonly referred as two alleles.
  • Almost all common SNPs have only two alleles.
  • the genomic distribution of SNPs is not homogenous; SNPs usually occur in non coding regions more frequently than in coding regions or, in general, where natural selection is acting the allele of the SNP that constitutes the most favorable genetic adaptation is predominating.
  • the present invention relates to a method for determining virulence of Piscine Orthoreovirus (PRV) in a biological sample from a fish, comprising a step for detecting whether any of the following single nucleotide polymorphisms (SNPs) are present in the genomic material
  • PRV Piscine Orthoreovirus
  • SNPs as mentioned above are to be understood as defined in table 1 below.
  • the SNP S4-T320C should thus be understood to mean a mutation from T to C in position 320 of the S4 segment.
  • the reference segment of S4 is shown in SEQ ID NO. 4.
  • the method further comprises a step for isolating the genomic material from the biological sample, and possibly a step for sequencing the material before detecting the SNPs.
  • the step for detection of the SNPs may be performed in many ways which will be obvious to a skilled person, and the necessity of isolating and sequencing depends on the detection method.
  • any sequencing is performed by a polymerase chain reaction and use of at least one primer.
  • the sequencing may also be performed by Next Generation Sequencing Methods, such as a method selected from the group consisting of lllumina (Solexa) sequencing, Roche 454 sequencing, Ion Torrent and SOLiD sequencing.
  • the detection of SNPs may be performed by manual or automatic comparing the sequenced genomic material from the sample with the reference.
  • the step of detecting comprises use of a polymerase chain reaction and use of at least one primer and/or probe.
  • each primer or probe used for sequencing or detecting comprise a sequence of at least 10 consecutive nucleotides selected from one of the sequences of a group consisting of SEQ ID NO. 24-26, 28-30, 32-34, 36-38, 40-42, 44-46, 48-50, 52-54, 56-58 and 60-63.
  • primers used for sequencing or detecting are used as primer pairs, selected from a group consisting of the following primer pairs:
  • primers according to SEQ ID NO. 56 and 57 and primers according to SEQ ID NO. 60 and 61.
  • the step of detecting comprises use of probes, preferably in a PCR reaction, wherein binding of a probe comprising a sequence of at least 10 consecutive nucleotides from
  • SEQ ID NO. 26 confirms the virus to have SNP S4-T320C
  • SEQ ID NO. 30 confirms the virus to have SNP S4-G754A
  • SEQ ID NO. 34 confirms the virus to have SNP M2-G551C
  • SEQ ID NO. 38 confirms the virus to have SNP M2-T580A
  • SEQ ID NO. 42 confirms the virus to have SNP M2-G784T
  • SEQ ID NO. 46 confirms the virus to have SNP M2-A1108G
  • SEQ ID NO. 50 confirms the virus to have SNP M3-A280T
  • SEQ ID NO. 54 confirms the virus to have SNP M3-C371T
  • SEQ ID NO. 58 confirms the virus to have SNP M3-A1351G
  • SEQ ID NO. 62 or 63 confirms the virus to have SNP M3-T1687C
  • the invention further relates to a method as described above, wherein the presence of mutation M3-A280T and/or M3-A1351G in SEQ ID NO. 6 confirms the virus to cause high mortality.
  • the invention further relates to a method as described above, wherein the presence of mutations M3-A280T, M3-C1064T, M3-A1351G, M3-T1421C and M3-T1687C in SEQ ID NO. 6 confirm the virus to cause high mortality.
  • the invention further relates to a method as described above, wherein the presence of mutations S4-G754A, M2-G551C, M2-T580A, M2-G784T, M2-A958G, M2- A1108G, M3-A280T, M3-C1064T, M3-A1351G, M3-T1421C, and M3-T1687C in SEQ ID NO. 6 confirm the virus to cause high mortality.
  • the invention further relates to a method as described above, wherein the presence of mutation M3-C371T in SEQ ID NO. 6 confirms the virus to cause moderate to low mortality, and morbidity.
  • the invention further relates to a method as described above, wherein the presence of mutations M3-C371T, M3-C1064T and M3-T1421C in SEQ ID NO. 6 confirm the virus to cause moderate to low mortality, and morbidity.
  • the invention further relates to a method as described above, wherein the presence of mutations S4-G754A, M2-G551C, M2-G784T, M2-A958G, M2-A1108G, M3- C371T, M3-C1064T, and M3-T1421C in SEQ ID NO. 6 confirm the virus to cause moderate to low mortality, and morbidity.
  • the invention further relates to a method as described above, wherein the presence of mutation S4-T320C in SEQ ID NO. 4 confirms the virus to cause high morbidity.
  • the invention further relates to a method as described above, wherein the presence of mutations S4-T320C, M2-G551C, M2-T580A, M2-G784T, M2-A958G, M2- A1108G, M3-C1064T, M3-T1421C and M3-T1687C in SEQ ID NO. 6 confirms the virus to cause high morbidity.
  • the invention further relates to a method as described above, wherein the presence of mutation S4-G754A in SEQ ID NO. 4 confirms the virus to cause high to moderate mortality, and morbidity.
  • the invention further relates to a method as described above, wherein the absence of all SNPs mentioned in table 1 above, confirms the virus to be nonvirulent, and not cause mortality or morbidity.
  • a method according to the above may be carried out, comprising use of a primer and probe comprising a sequence of at least 10 consecutive nucleotides according to the following
  • primers acc. to SEQ ID NO. 24 and 25, and probe acc. to SEQ ID NO. 27, confirm the absence of SNP S4-T320C
  • primers acc. to SEQ ID NO. 28 and 29, and probe acc. to SEQ ID NO. 31 confirm the absence of SNP S4-G754A
  • primers acc. to SEQ ID NO. 32 and 33, and probe acc. to SEQ ID NO. 35 confirm the absence of SNP M2-G551 C
  • primers acc. to SEQ ID NO. 40 and 41 and probe acc. to SEQ ID NO. 43, confirm the absence of SNP M2-G784T,
  • the invention also relates to a primer or probe comprising a sequence of at least 10 consecutive nucleotides selected from the group comprising SEQ ID NO. 24-26, 28- 30, 32-34, 36-38, 40-42, 44-46, 48-50, 52-54, 56-58 and 60-63.
  • the primer or probe may comprise any 10 consecutive nucleotides from SEQ ID NO. 24, or from SEQ ID NO. 25, or from SEQ ID NO. 26 etc.
  • the primer or probe may also comprise, or be identical to, the mentioned sequences.
  • the invention further relates to a method for determining virulence of Piscine Orthoreovirus (PRV) in a biological sample from a fish, comprising the following steps,
  • the invention further relates to a method as described above, wherein the presence of amino acid L in position 94, and/or V in position 451 in SEQ ID NO. 3 confirms the virus to cause high mortality.
  • the invention further relates to a method as described above, wherein the presence of L in position 94, L in position 355, V in position 451 , T in position 474 and P in position 563 of SEQ ID NO. 3 confirms the virus to cause high mortality.
  • the invention further relates to a method as described above, wherein the presence of N in position 252 of SEQ ID NO. 1 , T in position 184, I in position 194, S in position 262, A in position 320 and D in position 370 of SEQ ID NO. 2, and L in position 94, L in position 355, V in position 451 , T in position 474 and P in position 563 of SEQ ID NO. 3 confirms the virus to cause high mortality.
  • the invention further relates to a method as described above, wherein the presence of V in position 124 of SEQ ID NO. 3 confirms the virus to cause moderate mortality, high morbidity.
  • the invention further relates to a method as described above, wherein the presence of V in position 124, L in position 355, and T in position 474 of SEQ ID NO. 3 confirms the virus to cause moderate mortality, high morbidity.
  • the invention further relates to a method as described above, wherein the presence of - N in position 252 of SEQ ID NO. 1 , and T in position 184, S in position 262, A in position 320 and D in position 370 of SEQ ID NO. 2, and V in position 124, L in position 355, and T in position 474 of SEQ ID NO. 3 confirms the virus to cause moderate mortality, high morbidity.
  • the invention further relates to a method as described above, wherein the presence of A in position 107 of SEQ ID NO. 1 , and T in position 184, I in position 194, S in position 262, A in position 320 and D in position 370 of SEQ ID NO. 2, and L in position 355, T in position 474 and P in position 563 of SEQ ID NO. 3
  • the invention further relates to a method as described above, wherein the presence of N in position 252 of SEQ ID NO. 1 confirms the virus to cause moderate mortality, high morbidity.
  • Fig. 1 shows an alignment of PRV protein S4, from GenBank AGR27923 (SEQ ID NO. 1) and from a virulent virus strain (SEQ ID NO. 7-8), AGR27923 is cut 17 amino acids at the end,
  • Fig. 2 shows an alignment of PRV Protein M2 (SEQ ID NO. 2), from GenBank AGR27919 and from a virulent virus strain (SEQ ID NO. 9-10),
  • Fig. 3 shows an alignment of PRV Protein M3 (SEQ ID NO. 3), from GenBank AGR27920 and from a virulent virus strain (SEQ ID NO. 11-13)
  • Fig. 4 shows an alignment of the nucleotide encoding S4, from GenBank KC715687 (SEQ ID NO. 4) and from a virulent virus strain (SEQ ID NO. 14-16) KC715687 is cut 38 nucleotides at 5 end and 105 at 3 end,
  • Fig. 5 shows an alignment of the nucleotide encoding M2, from GenBank KC715683 (SEQ ID NO. 5) and from a virulent virus strain (SEQ ID NO. 17-20) KC715683 is cut with 26 nucleotides at 5 end and 87 at 3 end,
  • Fig. 6 shows an alignment of the nucleotide encoding M3, from GenBank KC715684 (SEQ ID NO. 6) and from a virulent virus strain (SEQ ID NO. 21-23) KC715684 is cut with 83 nucleotides at 5 end and 60 at 3 end,
  • Fig. 7 shows a phylogenetic tree of S4.
  • Fig. 8. shows accumulated mortality associated with the PRV genotypes of virulent virus, wherein Fig 8.A shows HSMI mortality and Fig 8.B. shows mortality of looser fish also called morbidity.
  • the enclosed nucleotide sequences are
  • 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.
  • Tissue samples of heart, head or kidney from HSMI diseased farmed Atlantic salmon were collected from aquaculture farms in Norway and tested for PRV by RT-qPCR.
  • the samples were collected from 11 freshwater sites and 29 seawater sites during the years 2014 to 2016.
  • the samples were collected aseptically on tubes prefilled with RNAIater (Ambion, USA). After sampling, the tubes containing tissue samples were shipped chilled to PatoGen AS with 24-hour delivery service.
  • RT-qPCR assay targeting PRV were performed, the method is validated according to ISO17025 standards and is described elsewhere (Glover et al. 2013).
  • Amino acid changes can be detected by different methods, such as protein sequencing, DNA or RNA sequencing, or by Real Time qPCR SNP assay.
  • the two last methods are easier and more cost effective than protein sequencing.
  • DNA/RNA sequencing by Sanger and by Real Time PCR were used.
  • PRV isolates from 3 freshwater sites and 17 seawater sites where the fish had developed HSMI were selected, the genomic material of the samples were isolated, and the genomic material was sequenced. Further, the amino acid sequences of the sample were isolated and sequenced.
  • virus causing looser fish will be referred to as virus causing morbidity.
  • VT06062012-358 (herein called reference or reference virus), defined as non-virulent, where
  • SEQ ID NO. 1 shows a truncated version of the amino acid sequence of S4, being the outer fiber protein, and
  • SEQ ID NO. 4 shows a truncated version the nucleotide sequence of the S4 segment, encoding the outer fiber protein
  • SEQ ID NO. 2 shows the amino acid sequence of M2 segment being the outer shell protein
  • SEQ ID NO. 5 shows a truncated version of the nucleotide sequence of the M2 segment encoding the outer shell protein
  • SEQ ID NO. 3 shows the amino acid sequence of M3 segment, encoding a non- structural factory protein
  • SEQ ID NO. 6 shows a truncated version of the nucleotide sequence of the M3 segment encoding the non-structural factory protein
  • the genotype groups A and C of the protein are identical.
  • the sequences of A/C and B are enclosed as SEQ ID NO. 7 and 8 respectively.
  • the genotype groups A, B and C of the nucleotide sequence are unique, and enclosed as SEQ ID NO. 14-16.
  • M2 segment there were 4 genotype groups, A, A-2, B and C, where the proteins of A-2 and B, and A and C are identical, sequences of which are enclosed as SEQ ID NO. 9 and 10, respectively.
  • the groups A, A-2, B and C of the nucleotide sequence are unique, and enclosed as SEQ ID NO. 17-20, respectively.
  • the genotype groups of the proteins were unique, the sequences are enclosed as SEQ ID NO. 11-13, showing genotype group A, B and C respectively.
  • the genotype groups A, B and C of the nucleotide sequence are also unique, and enclosed as SEQ ID NO. 21-23, respectively.
  • the result from the epidemiological study showed that the PRV genotype A was associated to higher level of HSMI mortality.
  • the genogroup B was associated with high morbidity (looser fish) and less mortality.
  • the genogroup C was associated with lower mortality and morbidity (looser fish).
  • Accumulated mortality associated with the PRV genotypes is shown in Figure 8, where fig. 8A shows the HSMI mortality and Fig 8.B. shows % mortality that is of looser fish, also called morbidity. The figure also show the the 25 th and 75 th percentiles. A correlation between the genogroups and the mortality from the epidemiological data were confirmed.
  • the lower and upper border of boxes indicates the 25 th and 75 th percentiles, respectively and the centerline indicates the 50 th percentile.
  • the upper and lower whiskers correspond to the highest and lowest value of the 1.5*IQR (inter-quartile range). The differences between genogroups, are reflected in the 13 amino acid changes shown in Table 2.
  • primers and probes proving the presence or absence of the SNPs according to the present invention were developed. If one or more of the SNPs are present, then the virus is virulent and will cause morbidity and/or mortality of a fish upon infection. Based on this, 10 different Real Time SNP assays were designed to detect amino acid changes (Table 4). The probe 1 detect the virulent mutation, and probe 2 detect the reference being nonvirulent.
  • All SNP’s can also be detected by sequencing, for the virulence markers M2-320, M3-355 and M3-474 only sequencing are used. Table 4 Primer pairs and probes (assays) for detection of the SNPs related to the invention. Probe 1 (P1) detect virulent strains of the virus, P2 detect the reference virus.
  • Example 2 Test of assays. To validate that the Real Time PCR SNP assays (Example 2) can differentiate between the different groups, 3 assays was tested on field samples from Example 1. The assays SNP S4-T320C, M3-C371T and M3-A1351G, detecting B, C and A respectively, were used.
  • the assays give a differentiation in Ct values and the probe giving the lowest Ct values indicates which SNP mutation that is most frequent in the biologic sample.
  • the detected SNPs are marked in Bold in table 5 below.
  • Table 5 Three assays tested on biological samples, and Ct values from PCR.

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Abstract

L'invention concerne des procédés pour déterminer la virulence d'orthoréovirus pisciaire (PRV) dans un échantillon biologique prélevé chez un poisson, comprenant la détection de SNP. L'invention concerne en outre des amorces et sondes d'extrémité 5' destinées à être utilisées dans ledit procédé.
PCT/NO2020/050017 2019-01-30 2020-01-28 Marqueurs de virulence d'orthoréovirus pisciaire WO2020159379A1 (fr)

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NO20190110A NO344967B1 (en) 2019-01-30 2019-01-30 Piscine Orthoreovirus Virulence Markers

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WO2022015173A1 (fr) * 2020-07-14 2022-01-20 Patogen As Marqueurs de virulence d'orthoréovirus pisciaire

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WO2019110664A1 (fr) * 2017-12-06 2019-06-13 Intervet International B.V. Utilisation d'une protéine non structurale issue de prv pour protéger contre l'inflammation du cœur et du muscle squelettique

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WO2019110664A1 (fr) * 2017-12-06 2019-06-13 Intervet International B.V. Utilisation d'une protéine non structurale issue de prv pour protéger contre l'inflammation du cœur et du muscle squelettique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022015173A1 (fr) * 2020-07-14 2022-01-20 Patogen As Marqueurs de virulence d'orthoréovirus pisciaire

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CL2021001992A1 (es) 2022-01-28
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WO2020159379A8 (fr) 2020-10-08

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