NO346211B1

NO346211B1 - Piscine Orthoreovirus Virulence Markers

Info

Publication number: NO346211B1
Application number: NO20200829A
Authority: NO
Inventors: Espen Rimstad; Magnus Andreas Devold; Håvard Aanes; Morten Lund; Øystein Wessel
Original assignee: Patogen As; Norges Miljoe Og Biovitenskapelige Univ Nmbu
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2022-04-19
Also published as: EP4182483A1; WO2022015173A1; NO20200829A1; CA3185583A1

Description

Piscine Orthoreovirus Virulence Markers

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.

Background

The aquaculture industry is an important food source and income, and Salmonids in particular are very popular farmed species in many regions of the world. Viral diseases pose a significant threat to the productivity in aquaculture, and thus impact the future global aquaculture production. Heart and Skeletal Muscle Inflammation (HSMI) has become a serious disease in farmed salmonids in several geographical areas. It was discovered in salmon in the sea in Norway in 1999, and is now reported from fresh and seawater sites in for instance UK, Scotland, Canada and Chile. The causal relationship between the PRV and HSMI was described by (Wessel Ø, et al. (2017) (5) Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon. PLoS ONE 12(8):e0183781). The mortality during HSMI outbreaks varies from negligible to 20 %.

According to The National Veterinary Institute 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. Still, 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 (6), identified these 10 different segments as L1, L2, L3, M1, M2, M3, S1, S2, S3 and S4 (Palacios G, et al. (2010) Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus. PLoS ONE 5(7): e11487).

Viruses closely related to PRV from farmed Atlantic salmon have been discovered in association with diseases in other salmonid species. A virus called PRV-2 was demonstrated to be the possible causative agent of erythrocytic inclusion body syndrome (EIBS), a disease that can cause mass mortality in Coho salmon (Oncorhynchus kisutch). Another PRV-like virus was detected in association with a disease outbreak in rainbow trout (Oncorhynchus mykiss) in Norway; the fish displayed signs of circulatory disturbance and histopathological changes resembling HSMI. Furthermore, a PRV strain closely related to the PRV from rainbow trout was found in association with HSMI-like lesions in Coho salmon in Chile. The presence of several PRV variants associated with diseases in salmonids suggests that species adaptation is important for pathogenesis (Wessel 2017) (5). Further, the complete sequence of the Piscine orthoreovirus clone NGS104 lambda 3 is known from GenBank Accession number MT758744.1.

Siah et al 2015 (7) conducted a phylogenetic study of PRV strains from Norway, Canada and Chile, and found that Norwegian strains differed in S1 segment. Wessel et al have shown the genetic relationship of strains from different regions and species, and shows that strains from Atlantic salmon all belong to the same group. Parts of the genome of the Norwegian strain is sequenced and known for instance from Genbank. However, also in Norway there are fish detected positive to PRV without clinical symptoms, and fish detected positive to PRV developing HSMI.

Wessel et al 2020 (4) performed a dose standardized challenge trial comparing six PRV-1 strains including two contemporary Norwegian strains, three historical Norwegian strains predating discovery of HSMI and one Canadian strain. All strains reached peak of infection in blood 3-4 weeks post challenge (wpc), and the two contemporary Norwegian strains presented with lower load of viral protein in blood cells but higher viral load in plasma that the other strains. At 6 wpc the two contemporary Norwegian strains induced histopathological lesions in the heart consistent with HSMI with a significantly higher score than the lesions observed in all three historical Norwegian strains and the Canadian strain. This clearly demonstrated the virulence difference in PRV-1 strains. The data in Wessel et al 2020 is included herein by reference.

Therefore it is important to have diagnostic tools to differentiate between the strains that can cause disease, and those that do not.

The current diagnostic methods are histology, immunohistochemistry, virus propagation, serology, neutralizing assay, sequencing and PCR, wherein PCR gives the fastest result and is the preferred method. 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. Furthermore, often 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).

A single-nucleotide polymorphism (SNP) 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. For example, 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 noncoding 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 invention

The issues set out above are solved by methods, primers and probes according to the characterizing part of the enclosed independent claims. Further advantageous features are stated in the corresponding dependent claims.

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

S1-T126C , S1-C195T, S1-A205G, S1-C206T, S1-T228C, S1-A234T, S1-G253A, S1-C300T, S1-G306A, S1-A321G, S1-C350A, S1-A409G, S1-G466A, S1-T469G, S1-A520G, S1-T618C and/or S1-A652G, in SEQ ID NO 1, M2-G551C, M2-G784T, M2-A1108G and/or M2-A2140G in SEQ ID NO.5,

or the complementary oligonucleotides thereof, wherein the numbering of said positions are in accordance with sequences 1 and 5, respectively, wherein absence of at least one SNP confirms that the virus is non-virulent and will not cause morbidity and/or mortality of a fish upon infection.

The expressions "virulent" and "non-virulent" is, in this patent application, used to express the ability of the virus to cause disease despite host resistance mechanisms, thus if a virus is said to be virulent, it will cause disease or death of the host. In case a virulent virus is confirmed for instance in a fish cage, the fish of the cage should to be treated to avoid sickness and death and to maintain fish welfare. However, if the virus is confirmed to be non-virulent, actions may not be necessary.

The above said SNPs tend to appear in clusters, and if a virus strain has one SNP it is very likely to comprise all the said SNPs of the same segment. Thus, in a preferred embodiment, absence of all above said SNPs in S1 and/or M2 confirms that the virus is non-virulent.

The fact that one may conclude that the virus is non-virulent if at least one of the said SNPs in S1 and/or M2 are absent, should not be interpreted opposite, it may not be concluded that the virus is virulent if one or more, or even all of the SNPs are present. This is, among others, due to the fact that more than one virus may be present in the sample.

In a preferred embodiment, if the virus comprises one or more of the SNPs in segment S1 and/or M2 mentioned above, the method comprises a further step for detecting whether any of the following SNPs are present in the genomic material: L1-G88A, L1-A535G, L1-T551C, L1-C599G, L1-A1028G, L1-C1041A, L1-A1114G, L1-G1468A, L1-A2273G, L1-A2810C, L1-G2884A, L1-A2987G and/or L1-G3796A in SEQ ID NO 10, and/or

L2-G343T, L2-G478A, L2-G1253A, L2-G1320T, L2-G1357A, L2-A2812G, L2-C3146G, L2-G3181A, L2-T3231A, and/or L2-C3440T and/or in SEQ ID NO 16, or the complementary oligonucleotides thereof, wherein the numbering of said positions are in accordance with sequences SEQ ID NO.10 and 16 respectively, wherein absence of at least one SNP confirm that the virus is virulent and will cause morbidity and/or mortality of a fish upon infection.

The above said SNPs tend to appear in clusters, and if a virus strain has one SNP it is very likely to comprise all the said SNPs of the same segment. Thus, in a preferred embodiment, absence of all said SNPs in L1 and/or L2 confirms that the virus is virulent.

The fact that one may conclude that the virus is virulent if at least one of the above said SNPs in segment L1 and L2 is absent, should not be interpreted opposite, it should not concluded that the virus is non-virulent if one or more, or even all of the SNPs in L1 and/or L2 are present. This is, among others, due to the fact that more than one virus may be present in the sample.

In a preferred embodiment, if the virus comprises one or more of the SNPs in segment S1, M2, L1 and/or L2 mentioned above, the method comprises a further step for sequencing the genomic material, and compare the sequences with any one of SEQ ID NO 1-21 to determine which virus strain is in the sample, and thereby the virulence of the virus. If any part of the sequence from the biological sample is identical to SEQ ID NO 14-15, or 20-21, the virus is virulent.

The SNPs as mentioned herein are to be understood as defined in table 1 below.

The SNP S1-T126C should thus be understood to mean a mutation from T to C in position 126 of the S1 segment.

The virus strains also comprise SNPs being unique for one virus stain, and in a preferred embodiment, presence of any of these SNPs are used to confirm that a given strain is present in the biological sample.

The SNP L2-A3852G should thus be understood that the strain CAN BC has a mutation in position 3852 of the L2 segment, from G to A. An alignment of the nucleotide segments of S1, M2, L1 and L2 for CAN BC, NOR1988, NOR1996, NOR1997, NOR2018NL and NOR2018SF are shown in Figure 6, 7, 8 and 9, respectively.

The virus strains also comprise SNPs not causing any changes in amino acids, also called "silent mutations" or "silent SNPs". Some of the silent SNPs are unique for one virus strain and may be used to confirm that a given strain is present or absent, others are present in several strains and may be used to confirm that a group of strains are present or absent. The presence of the different SNPs in the different strains are given in the alignments shown in Figures 6-9.

In an alternative embodiment of the present invention, the following SNPs are used to confirm presence and/or absence of virulent PRV strains:

- S1-T492C, S1-G504T, S1-C507T, S1-G564A, S1-A705G, S1-T735C, S1-A747G, S1-A789G, S1-C900T, S1-T957C,

- M2-T81C, M2-C190T, M2-C213T, M2-A240G, M2-G276A, M2-A282G, M2-C300T, M2-G303A, M2-T363C, M2-C405G, M2-A411G, M2-G420A, M2-T465C, M2-T780C, M2-C783T, M2-T807C, M2-T870C, M2-C897T, M2-C903T, M2-C906T, M2-G918A, M2-A945G, M2-G951A, M2-A961C, M2-C984T, M2-T1005A, M2-C1047T, M2-T1050C, M2-G1071A, M2-C1099T, M2-C1101A, M2-T1143C, M2-G1200A, M2-A1215G, M2-A1218G, M2-G1248A, M2-A1269T, M2-C1545A, M2-C1551T, M2-G1584A, M2-G1590A, M2-C1641T, M2-A1695G, M2-C1752T, M2-A1764G, M2-A1776G, M2-G1848A, M2-A1881G, M2-A1902G, M2-A1941G, M2-G1995A, M2-T2001C,

- L1-T150C, L1-G192A, L1-C312T, L1-G384A, L1-A417T, L1G420A, L1-T435C, L1-G486A, L1-T490C, L1-T498C, L1-C507T, L1-C534T, L1-C678T, L1-A693G, L1-C762T, L1-C774T, L1-A846C, L1-G849A, L1-A879G, L1-T939C, L1-G996A, L1-G1020A, L1A1044G, L1-A1245G, L1-T1269C, L1-C1282T, L1-G1284A, L1-G1308A, L1-T1323C, L1-C1356T, L1-C1368T, L1-C1383T, L1-T1395C, L1-C1396T, L1-A1413G, L1-A1428T, L1-A1434G, L1-A1452G, L1-C1455A, L1-A1512C, L1-C1534T, L1-C1581A, L1-A1599G, L1-A1716G, L1-C1734T, L1-C1746T, L1-1758C, L1-A1881G, L1-G1896A, L1-T1914G, L1-C1953T, L1-G2013A, L1-G2025A, L1-G2055A, L1-T2079C, L1-T2085C, L1-C2088T, L1-C2136T, L1-C2152T, L1-T2196C, L1-C2229T, L1-C2325A, L1-T2337C, L1-T2370C, L1-C2373T, L1-A2394G, L1-T2478C, L1-C2598T, L1-G2667A, L1-G2739A, L1-T2763C, L1-G2769A, L1-C2775T, L1-G2823A, L1-T2835C, L1-C2880T, L1-G2889A, L1-A2940G, L1-C2991T, L1-A3000G, L1-C3027T, L1-G3066A, L1-A3069C, L1-A3081C, L1-C3096T, L1-T3114C, L1-G3144A, L1-C3153A, L1-C3189T, L1-A3201G, L1-G3219A, L1-G3231T, L1-A3279G, L1-G3282A, L1-T3297C, L1-C3303G, L1-C3381T, L1-C3387T, L1-T3438C, L1-T3483G, L1-A3498G, L1-G3639A, L1-C3684T, L1-C3753T, L1-G3816A, L1-T3828C,

- L2-A252G, L2-A264G, L2-T309C, L2-T345C, L2-C363T, L2-T390C, L2-T402A, L2-T408C, L2-C411T, L2-G444A, L2-T474C, L2-T495C, L2-C501T, L2-C531T, L2-T541C, L2-T561C, L2-T564C, L2-G588A, L2-T T702C, L2-C783T, L2-T786A, L2-C810T, L2-A891G, L2-T960C, L2-T981C, L2-C1029T, L2-G1038A, L2-T1065C, L2-A1098T, L2-T1131C, L2-T1152C, L2-T1155G, L2-T1272C, L2-A1281G, L2-C1302T, L2-C1308T, L2-G1347A, L2-A1356G, L2-C1377T, L2-A1383G, L2-C1398T, L2-C1557T, L2-C1581T, L2-T1587C, L2-G1617A, L2-A1620G, L2-T1635C, L2-C1644T, L2-T1695C, L2-A1710G, L2-C1752T, L2-T1773C, L2-G1788A, L2-G1836A, L2-G1863A, L2-A1881G, L2-G1905A, L2-C1911T, L2-C1917T, L2-G1968A, L2-T1989C, L2-G2049C, L2-T2073C, L2-C2109A, L2-T2179C, L2-C2244A, L2-T2331C, L2-T2340C, L2-C2346T, L2-A2382G, L2-T2439C, L2-C2475T, L2-A2490G, L2-T2532C, L2-C2535T, L2-T2559C, L2-T2655C, L2-T2670C, L2-G2721A, L2-T2730C, L2-C2766T, L2-C2769T, L2-C2794T, L2-G2817A, L2-T2829C, L2-G2838A, L2-C2839T, L2-C2847T, L2-G2922A, L2-C2955A, L2-G2973T, L2-A3009G, L2-A3021G, L2-A3036G, L2-A3069G, L2-G3087A, L2-C3102T, L2-C3106A, L2-A3138G, L2-T3177C, L2-C3204T, L2-C3216T, L2-G3225A, L2-A3351G, L2-C3157T, L2-A3402G, L2-C3408T, L2-A3417G, L2-C3447T, L2-C3450T, L2-G4386A, L2-T3537C, L2-T3645C, L2-G3687A, L2-C3761T, L2-G3822A, L2-T3825C, L2-T3837C, L2-C3849T, L2-C3870T, L2-G3879A.

In one embodiment, 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.

In a preferred embodiment, 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 Illumina (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.

In a preferred embodiment of the invention, the step of detecting comprises use of a polymerase chain reaction and use of at least one primer and/or probe.

In one embodiment, the method comprises a first step confirming that a PRV virus is in the sample, before the detection step described above. In another embodiment, the step comprising a polymerase chain reaction is used to confirm that virus is present in the sample.

In a preferred embodiment, each primer or probe used for sequencing or detecting is a sequence of at least 10 consecutive nucleotides selected from one of the sequences of a group consisting of SEQ ID NO.40-55.

In a preferred embodiment, the 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.40 and 41,

primers according to SEQ ID NO.44 and 45,

primers according to SEQ ID NO.48 and 49, and

primers according to SEQ ID NO.52 and 53.

In a preferred embodiment, 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

probe acc. to SEQ ID NO.42, confirms the virus to have SNP L1-G88A

probe acc. to SEQ ID NO.46, confirms the virus to have SNP L2-G1253A

probe acc. to SEQ ID NO.51, confirms the virus to have SNP S1-G466A and S1T469G probe acc. to SEQ ID NO.55, confirms the virus to have SNP M2-G551C.

probe acc. to SEQ ID NO.43, confirms the virus to lack SNP L1-G88A

probe acc. to SEQ ID NO.47, confirms the virus to lack SNP L2-G1253A

probe acc. to SEQ ID NO.50, confirms the virus to lack SNP S1-G466A and S1T469G probe acc. to SEQ ID NO.54, confirms the virus to lack SNP M2-G551C.

The SNPs mentioned above may cause changes in the amino acids and proteins when the genome of the virus is being transcripted and translated, and thus the amino acids in the proteins expressed by the virus may also be used to determine virulence of a virus strain. Table 3 below shows the amino acid differences identified in the PRV strains.

Segment S1 comprises two ORFs, one starting at nt 1, giving protein sigma 3 (σ3), and another ORF starting at nt 45 giving protein P13.

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,

a) isolating amino acid sequences of the sample,

b) sequencing the amino acid sequence,

c) detecting whether any of the following amino acids are present in the amino acid sequences from the sample

- V in position 69, D in position 78, T in position 85, N in position 117, V in position 137, T in position 156, A in position 157, E in position 174, A in position 206 and/or V in position 218 of PRV protein sigma 3,

- A in position 26, M in position 39, T in position 60, I in position 62, V in position 84, Q in position 86, R in position 91, and/or M in position 101 of PRV protein P13, and/or

- T in position 184, S in position 262, D in position 370 and/or D in position 714 of PRV protein my 1,

wherein the sequences and numbering of said positions are given in sequences SEQ ID NO.22-32, respectively,

wherein absence of at least one of the amino acids confirms that the virus is non-virulent and will not cause morbidity and/or mortality of the fish upon infection.

In a preferred embodiment, where the biological sample comprises at least one of the above mentioned amino acids, the method comprises a further step for detecting whether any of the following amino acids are present

- T in position 30, V in position 179, A in position 184, S in position 200, G in position 343, E in position 347, D in position 372, I in position 490, A in position 758, A in position 937, I in position 962, S in position 996, and/or I in position 1266 of PRV protein lamda 3, and/or

- S in position 115, N in position 160, K in position 418, F in position 440, T in position 453, A in position 938, S in position 1049, I in position 1061,E in position 1077, and/or L in position 1147 of PRV protein lamda 2,

wherein the sequences and numbering of said positions are given in sequences SEQ ID NO.33-39, respectively,

wherein absence of at least one of the amino acids confirms that the virus is virulent and will cause morbidity and/or mortality of the fish upon infection.

Reference throughout the specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the subject matter disclosed. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” in various places throughout the specification is not necessarily referring to the same embodiment. Further, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

Description of the diagrams

The invention will now be described in detail with reference to the enclosed Figures and sequences wherein:

Fig. 1 shows an alignment of PRV protein S1σ3 from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 22-25),

Fig. 2 shows an alignment of PRV Protein S1p13, CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 26-29).The reading frame starts at position 45 on the nucleotide sequences for S1,

Fig. 3 shows an alignment of PRV Protein M2μ1, CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 30-31),

Fig. 4 shows an alignment of PRV Protein L1λ3, from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 33-35),

Fig. 5 shows an alignment of PRV Protein L2λ2, from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 36-39),

Fig. 6 shows an alignment of the nucleotide encoding S1 from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 1-4),

Fig. 7 shows an alignment of the nucleotide encoding M2, from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 5-9),

Fig. 8 shows an alignment of the nucleotide encoding L1, from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 10-15),

Fig. 9 shows an alignment of the nucleotide encoding L2, from CAN BC, NOR1988, NOR1996, NOR1997, NOR2018 NL, NOR2018 SF (SEQ ID NO 16-21), and

Fig. 10 shows a phylogenetic analysis of amino acid sequences of PRV proteins from all strains included in the study; NOR2018 SF, NOR2018 NL, NOR1997, NOR1996, NOR1988, CAN BC. Proteins with sequence variation that could be linked to virulence are colored in grey; dark grey used to mark protein variants potentially important for increased virulence, light grey used to mark potentially low virulent versions. PRV-3 NOR060214 (deposited in Genebank as MG253807-MG253816) was chosen as outgroup.

The virulent and non-virulent virus strains included in the study were NOR2018 SF, NOR2018 NL, NOR1997, NOR1996, NOR1988, CAN BC, wherein NOR2018 SF and NOR2018 NL were virulent and NOR1997, NOR1996, NOR1988, and CAN BC were non-virulent.

The enclosed nucleotide sequences are

Example 1

Background:

Wessel (2020) (4) demonstrated clear virulence difference between PRV-1 strains in an experimental challenge trial. The study showed that the strains differ in replication in blood cells, release of virus to plasma and subsequent development of histopathological lesions. Six PRV-1 strains were included in the study; two contemporary Norwegian strains (NOR2018 SF, NOR2018 NL), three historical Norwegian strains (NOR1988, NOR1996, NOR1997) and one Canadian strain (CAN 16-005ND, herein called CAN BC).

The two contemporary Norwegian strains were collected in 2018 from sea sites in the Norwegian counties Sogn og Fjordane (NOR2018 SF) and Nordland (NOR2018 NL), strains collected from a site with mortality of PRV. The three historical Norwegian strains NOR1988, NOR1996 and NOR1997 all originated from sea sites in Norway two, three and eleven years prior to the first description of HSMI.

NOR1988 and NOR1997 have been previously sequenced and had been passaged once through Atlantic salmon [1], whereas NOR1996 was identified in an archived plasma. The Canadian strain (CAN BC) had been previously characterized and failed to induce HSMI in two experimental trials in Canada [2,3]. Briefly, the strain originated from a cohort of healthy Atlantic salmon in Vancouver Island, Canada, with no history of HSMI and had been passaged three times through Atlantic salmon in Canada [2] and once in Norway. The observation from the analysis post challenge were summarized as in table 4 below.

Method:

RNA was isolated from 200 uL of the batches of purified virus from all six strains (100 uL for CAN 16-005ND) using a combination of Trizol LS (Life Technologies) and RNeasy Mini spin column (Qiagen). In brief, the purified virus was mixed with Trizol LS, added chloroform, then separating the phases by centrifugation. The aqueous phase was collected and proceeded with the RNeasy Mini spin column (Qiagen) as recommended by the manufacturer, eluting isolated RNA in 30 μL RNase-free water.

DNA/RNA were sequenced by Sanger and by Real Time PCR. Phylogenetic trees were constructed from the amino acid sequence of the 11 known encoded proteins. SNP’s in the nucleotide sequences, causing changes in amino acids that have impact for virulence were identified.

Results:

Phylogenetic analysis of all PRV strains included in the study was performed on the amino acid (Figure 10) to identifying one or more viral proteins linked to the phenotypical difference observed in the trial.

In the σ3 and p13 protein, both encoded by segment S1, two distinct groups were observed; one encompassing the two historical Norwegian strains NOR1988 and NOR1996 as well as the Canadian strain CAN BC, while the contemporary Norwegian strains NOR2018 SF and NOR2018 NL group together with the NOR1997 strain, with identical amino acid sequence (Figure 10). The same grouping was observed in μ1 encoded by segment M2 (Figure 10).

In λ3 encoded by segment L1 the NOR1997 strain grouped separately from all other strains with 13 unique amino acid sites. Similarly, in λ2 encoded by segment L2, the NOR1997 strain grouped separately with 10 unique amino acid sites (Figure 10).

The results revealed that multiple segments (L1, L2, M2, S1) are linked to virulence and a combination of different mutations and proteins are needed for virulence. The virulent strains (NOR2018 NL and NOR2018 SF) are most likely a product of reassortment events between the historical strains obtaining S1 and M2 from a PRV strain closely related or identical to NOR1997, and the L1 and L2 from a PRV strain closely related or identical to NOR1988.

Example 2:

In order to determine whether a fish is infected by a PRV virulent virus causing mortality and/or morbidity, or a nonvirulent virus, primers and probes proving the presence or absence of the SNPs according to the present invention were developed.

Four different Real Time SNP assays were designed to detect nucleotide and amino acid changes (Table 5).

The 4 assays were tested on field samples on the 6 different isolates, NOR1996 NOR1997 NOR1998, CAN_BC, NOR2018_NL and NOR2018_SF.

The combination of SNP’s indicates virulent and non-virulent group. The assays give a differentiation in Ct values and the probe giving the lowest Ct values indicates whether the SNP mutation that is most frequent in the biologic sample. If the Ct values of both probes are similar, then the test is inconclusive, possibly due to lack of virus, or more than one virus strain being present.

Testing the 4 SNP assays on samples containing either NOR1996, NOR1998, or CAN_BC the absence probe gives the lowest Ct values for S1, M2 L1, and L2 and defines these isolates as nonvirulent. Testing the 4 SNP assays on the NOR1997 the presence probe gives the lowest Ct values for S1, M2, L1 and L2 for NOR1997 and defines NOR1997 as non-virulent. Testing the 4 SNP assays on the NOR2018_NL and NOR2018_SF the presence probe gives the lowest Ct values for S1 and M2, but the absence probe for L1 and L2, and defines thus NOR2018_NL and NOR2018_SF as virulent.

When testing a sample with unknown infection, the first step of the method is to detect whether any of the following single nucleotide polymorphisms (SNPs) are present in the genomic material; S1-T126C , S1-C195T, S1-A205G, S1-C206T, S1-T228C, S1-A234T, S1-G253A, S1-C300T, S1-G306A, S1-A321G, S1-C350A, S1-A409G, S1-G466A, S1-T469G, S1-A520G, S1-T618C and/or S1-A652G, M2-G551C, M2-G784T, M2-A1108G and/or M2-A2140G.

In another embodiment, the sample is tested to confirm PRV infection by standard testing, before the first step given above.

Either SNP assay comprising SEQ ID NO 48-51, for S1-G466A/T469G or SNP assay comprising SEQ ID NO 52-55, for M2 -G551C may be used. If the assay confirms that the SNP was absent in the sample, then it may be concluded that no virulent virus strains were in the sample.

If however, the sample confirmed that the SNP was present in the sample, or the Ct of the probes were similar giving an inconclusive result, then the method comprises a further step for detecting whether any of the following single nucleotide polymorphisms (SNPs) are present in the sample L1-G88A, L1-A535G, L1-T551C, L1-C599G, L1-A1028G, L1-C1041A, L1-A1114G, L1-G1468A, L1-A2273G, L1-A2810C, L1-G2884A, L1-A2987G and/or L1-G3796A and/or

L2-G343T, L2-G478A, L2-G1253A, L2-G1320T, L2-G1357A, L2-A2812G, L2-C3146G, L2-G3181A, L2-T3231A, and/or L2-C3440T.

Either SNP assay comprising SEQ ID NO 40-43, for L1-G88A or SNP assay comprising SEQ ID NO 44-47, for L2-G1253A may be used. If the assay confirmed that the SNP was absent in the sample, then it was concluded that at least one virulent virus strains were in the sample.

References:

1. Dhamotharan, K.; Tengs, T.; Wessel, O.; Braaen, S.; Nyman, I.B.; Hansen, E.F.; Christiansen, D.H.; Dahle, M.K.; Rimstad, E.; Markussen, T. Evolution of the Piscine orthoreovirus Genome Linked to Emergence of Heart and Skeletal Muscle Inflammation in Farmed Atlantic Salmon (Salmo salar). Viruses 2019, 11, doi:10.3390/v11050465.

2. Polinski, M.P.; Marty, G.D.; Snyman, H.N.; Garver, K.A. Piscine orthoreovirus demonstrates high infectivity but low virulence in Atlantic salmon of Pacific Canada. Sci. Rep.2019, 9, 3297, doi:10.1038/s41598-019-40025-7.

3. Garver, K.A.; Johnson, S.C.; Polinski, M.P.; Bradshaw, J.C.; Marty, G.D.;

Snyman, H.N.; Morrison, D.B.; Richard, J. Piscine orthoreovirus from western North America is transmissible to Atlantic salmon and Sockeye salmon but fails to cause heart and skeletal muscle inflammation. PLoS One 2016, 11, e0146229, doi:http://dx.doi.org/10.1371/journal.pone.0146229.

4. Wessel, Ø. Virulensforskjeller hos PRV-1. Oral presentation, Havbruk 2020, Bergen, Norway, June 9 and 10th 2020,

5. Wessel, Ø.; Braaen, S.; Alarcon, M.; Haatveit, H.; Roos, N.; Markussen, T.; Tengs, T.; Dahle, M.K.; Rimstad, E. Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon. PLoS One 2017, 12, e0183781, doi:10.1371/journal.pone.0183781.

6. Palacios, G.; Lovoll, M.; Tengs, T.; Hornig, M.; Hutchison, S.; Hui, J.;

Kongtorp, R.T.; Savji, N.; Bussetti, A.V.; Solovyov, A.; Kristoffersen, A.B.; Celone, C.; Street, C.; Trifonov, V.; Hirschberg, D.L.; Rabadan, R.; Egholm, M.; Rimstad, E.; Lipkin W.I., Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus. PLoS ONE 20105(7): e11487) https://doi.org/10.1371/journal.pone.0011487

7. Siah A, Morrison DB, Fringuelli E, Savage P, Richmond Z, Johns R, et al. (2015) Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast. PLoS ONE 10(11):e0141475. doi:10.1371/journal.pone.0141475

Patent claims

1. 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 S1-T126C , S1-C195T, S1-A205G, S1-C206T, S1-T228C, S1-A234T, S1-G253A, S1-C300T, S1-G306A, S1-A321G, S1-C350A, S1-A409G, S1-G466A, S1-T469G, S1-A520G, S1-T618C and/or S1-A652G, in SEQ ID NO.1, M2-G551C, M2-G784T, M2-A1108G and/or M2-A2140G in SEQ ID NO.5,

or the complementary oligonucleotides thereof, wherein the numbering of said positions are in accordance with sequences SEQ ID NO.1 and 5 respectively, wherein absence of at least one SNP confirm that the virus is non-virulent and will not cause morbidity and/or mortality of a fish upon infection.

2. Method according to claim 1, wherein absence of all SNPs confirm that the virus is non-virulent.

3. Method according to claim 1 or 2, wherein the genomic material contains at least one of the said SNPs, the method comprises a further step for detecting whether any of the following single nucleotide polymorphisms (SNPs) are present in the genomic material L1-G88A, L1-A535G, L1-T551C, L1-C599G, L1-A1028G, L1-C1041A, L1-A1114G, L1-G1468A, L1-A2273G, L1-A2810C, L1-G2884A, L1-A2987G and/or L1-G3796A in SEQ ID NO.10, and/or

L2-G343T, L2-G478A, L2-G1253A, L2-G1320T, L2-G1357A, L2-A2812G, L2-C3146G, L2-G3181A, L2-T3231A, and/or L2-C3440T in SEQ ID NO.16,

or the complementary oligonucleotides thereof, wherein the numbering of said positions are in accordance with sequences SEQ ID NO.10 and 16 respectively, wherein absence of at least one SNP confirm that the virus is virulent and will cause morbidity and/or mortality of a fish upon infection.

4. Method according to claim 3, wherein the absence of all SNPs confirm that the virus is virulent.

5. Method according to claim 3 or 4, wherein the sample contains at least one of the said SNPs in S1, and/or M2, and in addition at least one of the SNPs in L1 or L2, the method comprises a further step for sequencing and comparing the sequence with any one of SEQ ID NO.1-21, wherein identity with any one of SEQ ID NO.14-15, or 20-21, confirms the virus is virulent and will cause morbidity and/or mortality of a fish upon infection.

6. Method according to any one of the preceding claims, wherein the step of detecting and/or sequencing is performed by a polymerase chain reaction and use of at least one primer, each primer comprises a sequence of at least 10 consecutive nucleotides selected from one of the sequences of a group consisting of SEQ ID NO.

40-41, 44-45, 48-49 and 52-53.

7. Method according to claims 6, wherein primers are used as primer pairs, selected from a group consisting of the following primer pairs:

primers according to SEQ ID NO.40 and 41,

primers according to SEQ ID NO.44 and 45,

primers according to SEQ ID NO.48 and 49, and

primers according to SEQ ID NO.52 and 53.

8. Method for determining virulence of Piscine Orthoreovirus (PRV) in a biological sample from a fish, comprising the following steps,

a) isolating amino acid sequences of the sample,

b) sequencing the amino acid sequence,

c) detecting whether any of the following amino acids are present in the amino acid sequences from the sample:

wherein absence of at least one of the amino acids confirms that the virus is nonvirulent and will not cause morbidity and/or mortality of the fish upon infection.

9. Method according to claim 8, wherein the biological sample comprises at least one of the above mentioned amino acids in the proteins sigma 3, P12 and/or my1, the method comprises a further step for detecting whether any of the following amino acids are present

- T in position 30, V in position 179, A in position 184, S in position 200, G in position 343, E in position 347, D in position 372, I in position 490, S in position 758, A in position 937, I in position 962, S in position 996, and/or I in position 1266 of PRV protein lamda 3, and/or

- S in position 115, N in position 160, K in position 418, F in position 440, T in position 453, A in position 938, S in position 1049, I in position 1061, E in position 1077, and/or L in position 1147 of PRV protein lamda 2,

Claims

Patent claims

1. Method for determining the virulence of Piscine Orthoreovirus (PRV) in a biological sample from a fish, comprising the step of detecting whether any of the following single nucleotide polymorphisms (SNPs) are present in the genomic material

S1-T126C , S1-C195T, S1-A205G, S1-C206T, S1-T228C, S1-A234T, S1-G253A, S1-C300T, S1-G306A, S1-A321G, S1-C350A, S1-A409G, S1- G466A, S1-T469G, S1-A520G, S1-T618C and/or S1-A652G, in SEQ ID NO.1, M2-G551C, M2-G784T, M2-A1108G and/or M2-A2140G in SEQ ID NO.5 ,

or the complementary oligonucleotides thereof, the numbering of positions being in accordance with sequence SEQ ID NO.1 and 5 respectively,

as the absence of at least one SNP confirms that the virus is non-virulent and will not cause morbidity and/or mortality in a fish upon infection.

2. Method according to claim 1, wherein the absence of all the SNPs confirms that the virus is non-virulent.

3. Method according to claim 1 or 2, in that the genomic material contains at least one of said SNPs, the method comprises a further step for detecting whether one of the following single-nucleotide polymorphisms (SNPs) is present in the genomic material L1-G88A, L1-A535G, L1-T551C, L1-C599G, L1-A1028G, L1-C1041A, L1-A1114G, L1-G1468A, L1-A2273G, L1-A2810C, L1-G2884A, L1-A2987G and/or L1-G3796A in SEQ ID NO 10, and/or

L2-G343T, L2-G478A, L2-G1253A, L2-G1320T, L2-G1357A, L2-A2812G, L2-C3146G, L2-G3181A, L2-T3231A, and/or L2-C3440T in SEQ ID NO 16,

or the complementary oligonucleotides thereof, the numbering of positions being in accordance with sequence SEQ ID NO 10 and 16 respectively,

the absence of at least one SNP confirming that the virus is virulent and will cause morbidity and/or mortality in a fish upon infection.

4. Method according to claim 3, the absence of all the SNPs confirming that the virus is virulent.

5. Method in accordance with claim 3 or 4, in that the sample contains at least one of the mentioned SNPs in S1 and/or M2, and in addition at least one of the SNPs in L1 or L2, the method comprises a further step for sequencing and comparison of the sequence with any of SEQ ID NO.1-21, identity with any of SEQ ID NO.14-15, or 20-21, confirming that the virus is virulent and will cause morbidity and/or mortality in a fish in case of infection.

6. Method according to one of the preceding claims, in that the step of detecting and/or sequencing is carried out by a polymerase chain reaction and the use of at least one primer, each primer comprising a sequence of at least 10 subsequent nucleotides selected from one of the sequences in a group consisting of SEQ ID NO.40-41, 44-45, 48-49 and 52-53.

7. Method in accordance with claim 6, in that primers are used as primer pairs, selected from a group consisting of the following primer pairs:

primers in accordance with SEQ ID NO.40 and 41,

primers in accordance with SEQ ID NO.44 and 45,

primers in accordance with SEQ ID NO.48 and 49, and

primers in accordance with SEQ ID NO.52 and 53.

8. Procedure for determining the virulence of Piscine Orthoreovirus (PRV) in a biological sample from a fish, comprising the following steps,

a) isolate amino acid sequences from the sample,

b) sequencing the amino acid sequence,

c) detecting whether any of the following amino acids are present in the amino acid sequence from the sample:

- A in position 26, M in position 39, T in position 60, I in position 62, V in position 84, Q in position 86, R in position 91, and/or M in position 101 of PRV protein P13, and/ or

whereas the sequences and numbering of the mentioned positions are given respectively in sequence SEQ ID NO.22-32,

the absence of at least one amino acid confirming that the virus is non-virulent and will not cause morbidity and/or mortality in the fish, upon infection.

9. Method according to claim 8, in that the biological sample comprises at least some of the above-mentioned amino acids in the proteins sigma 3, P12 and/or my1, the method comprises a further step to detect whether any of the following amino acids are present

- T in position 30, V in position 179, A in position 184, S in position 200, G in position 343, E in position 347, D in position 372, I in position 490, S in position 758, A in position 937 , I in position 962, S in position 996, and/or I in position 1266 of PRV protein lamda 3, and/or

whereas the sequences and numbering of the mentioned positions are given respectively in sequence SEQ ID NO.33-39,

the absence of at least one of the amino acids confirming that the virus is virulent and will cause morbidity and/or mortality in a fish upon infection.